plenary lecture memorial lectures...tawara memorial lecture (march 18, 9:00–10:00, room c) 3sl11c...

Plenary LectureMemorial Lectures

S2 The 91st Annual Meeting of the PSJ, March 16 − 18, 2014, Kagoshima

Plenary Lecture

(March 16, 14:00–15:00, Room A)

1SL04A Life Science in Japan—Past 50 years and futureMitsuhiro Yanagida(Okinawa Institute of Science and Technology Graduate University)

Arimura Memorial Lecture

(March 17, 11:05–12:05, Room B)

2SL05B Challenge to novel bioactive peptidesKangawa Kenji(National Cerebral and Cardiovascular Center Research Institute)

The Memorial Lecture for W.Trautwein

(March 16, 11:05–12:05, Room A)

1SL01A Pluripotent Stem Cells: From Physiology to Regener-ative MedicineHescheler, Juergen(Institute of Neurophysiology, University of Cologne, Germany)

Duetotheirabilitytoreproducetheembryonicdifferentiationofallcellularphenotypes,embryonicandinducedpluripotentstem(ES,iPS)cellsrepresentanidealtooltostudythephysiologyofembryogenesisunderinvitroconditionsaswellastoprovideanewsourceforcellu-larreplacementtherapy.WecultivatedESandiPScellsinthreedi-mensionalcellaggregates(embryoidbodies),wheretheydifferentiateintoderivativesofallthreegermlayers.Toselectonlyonelineage,e.g.thecardiaclineage(cardiomyocytes,CMs),andtoallowtheiden-tificationofthetransplantedcells,transgenicESoriPScellswereusedcontainingavectorwithtwocloningsitesforenhancedgreenfluores-centprotein(EGFP)andpuromycinresistanceforselectionunderthe?-MHCpromoter.Basedonthepioneeringadvancesincardiacelec-trophysiologyprovidedduringProf.Trautwein’serathislecturewilldemonstratehowourknowledgeonfundamentalquestionsonCMsincludingrhythm,actionpotentialgenerationandcalciumchannelregulationcanbeansweredusingsuchESoriPScellderivedCMs.Second,inordertodemonstratetheabilityforregenerativemedicineandtissuerepair,CMscellswereinjectedintothecryoinfarctedleftventricularwallofadultwildtypemice.UsingaslicingtechniqueofthehearttheelectrophysiologicalmaturationoftransplantedCMswillbedemonstrated.Third,reprogrammingoffibroblastsfrompatientswithLQT3orCPVTsyndromebyectopicexpressionofOct4,Sox2,c-MycandKlf4resultedingenerationofiPScellsfordiseasemodeling.NoCOI.

The 91st Annual Meeting of the PSJ, March 16 − 18, 2014, Kagoshima S3

Hagiwara Memorial Lecture

(March 16, 11:05–12:05, Room A)

1SL03FHigher-order motor areas in the frontal cortex of pri-matesTanji, Jun(Brain Science Center, Tohoku University)

Motorareasrostraltotheprimarymotorcortexdevelopgreatlyinprimates.It isgenerallyagreedthatatleastfivemotorareasexistmedially(SMA,pre-SMA,SEF,CMArandCMAc),andthreeareaslaterally(PMd,PMv,andFEF),constitutinghigher-ordermotorareas.Firstandforemost,itisveryimportanttoreconfirmanatomicalfoun-dationsestablishedasthebasisforarealdifferentiation;eachoftheeightareasischaracterizedbydistinctanatomicalconnectivity,bothcortico-corticalandcortico-subcortical.Differencesinafferentandef-ferentconnectivityprovideplausibleexplanationsforarea-uniquelesioneffectsreportedinanimalstudiesandclinicalsignsobservedamongbrain-damagedpatients.Althoughalloftheseareasinfluencelimboroculomotoractions,rolesinselectingmusclesandindeterminingthemagnitudesofmuscleactivityaremuchlesssignificantthanfortheprimarymotorcortex.Conversely,premotorareasaremorecrucialintheselectionofactionbasedonsensoryinformationandonbehav-ioral-context.Electricalstimulationisbynomeanssuitablefordetect-ingfunctionalrolesplayedbyeachofhigher-ordermotorareas;stim-ulusartifactsaretotallyremotefromnaturally-occurringbehavior.Itisnowestablishedthatfunctionalpropertiescharacterizingeachareacouldberevealedonlyunderbehavioralconditionsspecificallycallingforthepreferentialuseofindividualareas.Accumulationofanalyticalstudieshavebeguntoreveal“raisond’être”foreacharea.NoCOI.

Hagiwara Memorial Lecture

(March 17, 9:00–10:00, Room C)

2SL06C Mechanisms of Hearing—studies from hair cells to the cortex—Ohmori, Harunori(Department of Physiology, Faculty of Medicine, Kyoto University)

ProfessorHagiwaramadeinitiativesinvariousfieldsofneurobiology,andalsointhefieldofauditionusingthelaterallineorganhaircellsof themudpuppy (1975). InthisHagiwaramemorial lecture, Iwillsummarizethreetopicsinthehearingresearchesconductedinmylaboratoryusingthechick,since1982aftermy2yearsstayinHagi’slaboratory.Thefirsttalkisonthehaircelltransductionandtrans-mission,whichdemonstratedthathaircellmechano-electricaltrans-ductionisconductedthroughgatingofionchannelsandthatglutamateisreleasedastheafferentneuro-transmitterfromhaircells.Thesecondtalkisaboutthebrainstemauditoryinformationprocess-ing,particularlythetiminginformation.Iwillsummarizedistinctivetonotopicspecializationsinmorphologyandelectrophysiologyinthenucleusmagnocellularis(NM)thatextractstiminginformationfromtheauditorynervefibersandinthenucleuslaminaris(NL)thatpro-cessesinterauraltimedifference(ITD)bythecoincidenceofexcitato-ryinputsfrombilateralNM.FurthermoreIwilltalktherolesofinhi-bitionintheITDprocessinginNLwherethesoundleveldependenttonicinhibitionandthephasicinhibitionthatfollowsipsilateralNMspikeco-operatetomaketheITDprocessingtoleranttoawiderangeofsound level. Thethirdtalk isonaphotometricpatchelectrode(PME)methodology,whichusesapatchelectrodeasalightguide.ThemethodisstilldevelopingandweareapplyingthePMEtotheendofexploringtheneuralactivityinvivobyrecordingbothelectricalandopticalsignalssimultaneouslyfromdeepbraintissuessuchasNM,theinferiorcolliculusandtheField-L(theavianauditorycortex).NoCOI.


Tawara Memorial Lecture

(March 18, 9:00–10:00, Room C)

3SL11C Breathing and EmotionHomma, Ikuo(Tokyo Ariake Univ. of Medical and Health Sciences)

Breathingisabasicphysiologicalfunction.Weinhaleoxygenforen-ergymetabolismandcontrollingcarbondioxidetomaintainacid-basebalance.However,besidesmetabolicbreathing,wehavebehavioralbreathing.Breathingcanchangeinresponsetochanges invariousemotions.Therefore,finalrespiratorymotoroutput,whichwecanseefromthechestwallmovements,isdeterminedbycomplexinteractionbetweenmetabolicandbehavioralbreathing.Thesefactorsareyield-edinthebrainstemandcorticalstructures.Descendingspinalpathwaysformetabolicandbehavioralbreathingaredifferentandstudiesshowedthecorticalprojectionstobrainstemrespiratoryneurons,indicatingthatthebehavioral influencesarisingfromhighercentersmodifymetabolicbreathingpattern.Emotionsexistasaninnatementalandphysicalpartofthebody,butemotionsaremuchmorecomplexandsubdividedintonumerousdimensions.Thelimbicsystemisactivatedwhenthereisachangeinemotionandvariouschangesoccurinthebody.Thetightlinkbetweenemotionandbreathingwasobservedinourstudyonanxietyandrespiration.Inourexperiment,wetestedwhatwecall “anticipatoryanxiety”byconnectinganelectrodetosubjectsandtellingthemthat inthenexttwominutes,anelectricshockwillbeadministered.Duringthistimeanincreaseinrespirato-ryratewasobserved.Wealsotestedtraitanxietyscoresandfoundthattherewasapositivecorrelationbetweenthetraitanxietyscoreandincreaseinrespirationrate.Wealsofoundthatneuralactivation,whichwassynchronizedwithrespiration,occurredintheamygdalaexaminedbyEEGdipoletracingmethod.Theactivitiessynchronizedwithrespirationwerealsoobservedintheamygdalainthelimbic-brain-stem-spinalcordpreparationofnewbornrat.Iwillreviewtherela-tionshipbetweenemotionandrespiration.NoCOI.

Named after Tawara Memorial Lecture

(March 18, 11:05–12:05, Room D1)

3SL12D1 Promotion of medical technology through integration of physiology and other fieldsKajiya, Fumihiko(Kawasaki Univ Med Welf)

TheAmericanInstituteforMedicalandBiologicalEngineering(AIM-BE),acollaborativeorganizationrepresentingacademia,industryandgovernment,highlighted28medicaltechnologiesasthekeyinnovationsofthe20thcenturytotheir“HallofFame”in2005andsubsequentyears.InrelationtoDr.SunaoTawara’sgreatdiscoveryofthe“Car-diacConductionSystem”,abetterunderstandingofcardiacelectricalfunctioncontributedtodevelopmanyessentialmedicaldevicessuchaselectrocardiogram(ECG),cardiacdefibrillatorandthepacemakerwithrecentbreakthroughsincludingGenomicsintheHall.Virtually,everypersonhasbenefittedfromthesekeyinnovationsinreceivingbetterhealthcare.Thesecreativeandinnovativeachievementshavebeenmadeinmostcasesthroughclosecooperationamong“physiolo-gy”,andothermedical,scientificandengineeringfields,sinceoneofthefundamentalparadigms inphysiologicalresearch is integration.Accordingtothe“NationalIndustrialTechnologyStrategy”reportofJapan,theMedicalEngineeringTechnologyIndustrialStrategyCon-sortium(METIS)wasorganizedtofosterthedevelopmentofinnova-tivemedicaltechnologyin2001.ThescopeofMETISincludesnotonlymedicaldevicesbutalsoabroadspectrumofscienceandengineeringinmedicine.Nowwedreamthatmutualunderstandingandcoopera-tionbetweenphysiologyandotherfieldscandevelopmedicaltechnol-ogyfurther.FrommyexperienceinMETISasaco-chairfor10years,Iwouldliketoemphasizethepromotionofmanypossiblecontributionsintheinterdisciplinaryfusionbetweenphysiologyandotherfieldstofurtheradvancemedicaltechnologies.NoCOI.

Special Lectures for Kagoshima Meeting

Educational Lecture


1SL02D1 March 16, 9:00–10:00, Room D1Physiology as a science for elucidating the mecha-nism of behaviorYamamoto, Takashi(Graduate School, International University of Health & Welfare)

Behavioriscomposedofbodilyandmentalactivitiesoccurringinanorganismforacertainpurpose.Whilephysiologicalstudycanhelprevealtheunderlyingmechanismsofavarietyofbehaviors,thebasisofmanydailybehaviorsremainspoorlyunderstood.OwingtotheinfluencebythelateProfessorYojiroKawamura,asafounderofOralPhysiology,Ihavebeenworkinginthefieldofingestivebehaviorwithspecialreferencetochemicalsensessuchastasteandsmellbyseek-ingreasonsforsimplequestions,suchaswhysugarsaresweetandsodiumchlorideissalty?Whysomefoodsarepalatableandothersareaversive?Whysomepeopleeat fastandothersslow?Whysomepeoplearefatandothersarelean,etc.Iwouldliketoshowsomedataaddressingthesequestions,namely,theimportanceofthechemotop-icorganizationinthecortexfortastequalityrecognition,thebasolat-eralnucleusofamygdalaandtherewardsystemforthehedonicshiftoftastepalatabilityintheconditionedtasteaversionparadigmandtheinvolvementofbrainsubstancessuchasbeta-endorphin,dopamineandorexin forpalatability-inducedfeedingbehavior. Inaddition,acertainodor,Osmanthusfragrance,attenuatesfoodintakebyinfluenc-ingtheexpressionof feeding-relatedneuropeptidesandoneofthespices,wasabi,maybeeffectiveinpreventingobesityanddiabetesmellitus.Suchphysiologicalstudiesasansweringthepopularquestionsregardingcommonhumanbehaviorsarenotonlyscientificallyimport-anttosolvebutalsowouldattractyoungpeopletophysiologyaswellasscienceitself.NoCOI.

3SL13E March 18, 11:05–12:05, Room EAdaptation of neuromuscular properties to micrograv-ity environment Ohira, Yoshinobu(Doshisha University)

Chronicexposuretospaceenvironmentcausessomeseriousrespons-esinphysiologicalproperties,duetogravitationalunloading,radiation,mentalstress,etc.Removalofweight-bearingactivityinducesremark-ableeffectsontheskeletalmusclesandbonesresponsibleformain-tainingpostureandgroundsupport.Forexample,chronicunloadingofmuscles,suchasbedrestinhumansandhindlimbsuspensionandactualspaceflightinanimals,resultsinmusclefiberatrophyandashifttowardafastermyosinheavychainprofile,particularlyinmusclescomposedpredominantlyofslowfiberssuchassoleusandadductorlongus.Theseresponsesarecloselyrelatedtoadecreaseintheneu-romuscularactivitylevels,i.e.,mechanicalloading,neuralactivation,and/ormetabolicfactors.Inhibitionoftensiondevelopmentcausedbypassiveshorteningofmusclefibersorsarcomeresplaysoneofthemajorrolesinadvancedbreakdownand/orshiftofthedistributionofproteins inskeletalmuscles. Further,unloading-relatedchangesinmuscularproperties, includingmobilizationpatterns,are influencedbyinhibitionofafferentinput.However,activationofafferentinputalsostimulatestheinvasionofThelper17cellsatL5segmentallevelofspinalcord,whichcausesneuroimmunedisease.Iwouldliketodiscussabouttheneuromuscularresponsesandrolesofafferentinputonthephysiologicaladaptationtogravitationalunloading.NoCOI.

2SL09F March 17, 11:05–12:05, Room FComputer simulation of basic cardiac cell functions for learning Cardiac Physiology at levels of cell and tissueNoma, Akinori; Amano, Akira(Biosimulation Laboratory, College of Life Sciences, Ritsumeikan University)

Togreatlyfacilitateunderstandingofdynamicpropertiesofcardiaccells,weareaimingatpreparingacomputationalpackageofeducationalsimulationsoftwares,whicharelargelybasedondetailedbiophysicalcardiacmyocytemodels.Thecellmodelsincludethemembraneexcitation,intracellularionconcentrations,Ca2+dynamicsatthesarcoplasmicreticulum,thecontractionofmyofilaments,andtheoxidativephosphorylationinmitochondria.Thecellmodelsareforthesinoatrialnodepacemaker,atrial,andventricularcells.Theionchannelaswellasiontransporters,suchasNa/KpumpandtheNa/Caexchanger,incorpolatedinthecellmodels,canbeexaminedbyreconstructingthevoltageclamprecords,singlechan-nelrecordings,orbyapplyingdifferentionconcentrations,toseetheirtime-andvoltage-de-pendentactivationaswellasinactivationifany.Thecellmodelscanbeconnectedtoformaone-dimensionalarrayofcellsconnectedwiththegapjunctionalchannelstosimulatetheAPpropagationundervariousphysiologicalandpathophysiologicalconditions.Therelation-shipbetweenthegapjunctionchannelconductanceandtheAPpropagationvelocitycanbevisualizedonthegraphics.Principalmechanismsunderlyingthepacemakershift,theonedirectionalconductionaswellasthere-entrycanbedemonstratedinappropriatecellarrays.Theabnormalmembraneexcitation,suchasthedelayedafterdepolarizationortheearlyafter-depolarizationcanbedemonstratedbyapplyingvariousinterventionstotheventric-ularcellmodels.　Theventricularcellmodelscanalsobeusedtosimulatethefunctionatthetissueandorganlevels.Theabnormalexcitationpatternscanbetriggeredinatwodimensionalarrayofmodelcells.ThecardiaccyclecanbesimulatedbyassumingasimpleLaplaceheart,after-loadandpre-load.Thepressure-volumecurveortrajectrycanbeex-aminedatvariouspre-loadorafter-loadbywhichmaximumelastance(Emax)canalsobeevaluated.Therelationbetweentheconductionvelocityandtheforcelengthrelationofcellcanbeexaminedbyusingtheonedimensionalfibermodelcombinedwiththecirculationmodel.Ifectopicfocusesofmembraneexcitationwereassumedinaheartmodelofaringshape,modificationsofcardiacoutputwereexamined.NoCOI.

Invited Lectures


2SL07D1 March 17, 11:05–12:05, Room D1Channel Function Reconstitution and Re-animation: Single-Channel Studies in the Post-Crystal AgeOiki, Shigetoshi(Department of Molecular Physiology and Biophysics, Faculty of Medical Sciences, University of Fukui)

Themostessentialchannelpropertiesforthephysiologicallyrelevantfunctionaretheselectiveionpermeationandthegating.Electrophys-iologicalmethodscombinedwiththesite-directedmutagenesishaverevealedtherelevantfunctionaldomains,andunderlyingmechanismsofthesemolecularfeatures.Thestrategyofionchannelstudywasdramaticallychangedaftercrystalstructureofthepotassiumchannelwasresolvedin1998.Inthepost-crystalage, functional featuresofchannelproteinshavebeendiscussedwiththestructuralterms,andone imaginestheactivechannelbybreathing life intothe“frozen”crystal(re-animation).Inthiscontext,experimentalmethodstodetectmotionsofchannelproteinsarecrucially important,andherewepresentsingle-moleculemeasurementmethodsappliedtotheKcsApotassiumchannel.ThediffractedX-raytrackingmethodrevealedthatthechannelundergoesthetwistingconformationalchangearoundthechannelaxis,thus,allowingforonandoffofthechannelcurrent.Usingatomicforcemicroscopy,wecapturedthestructureofmem-brane-embeddedchannels,inwhichopengatewasresolvedexclusive-lyintheactivatedcondition.Moleculardynamicssimulationdemon-stratedpermeatingionsandvigorousfluctuationsoftheopengate.Recently,wefoundthattheKcsAchannelundergoesclusteringanddispersiononthemembranewithconcurrentgatingconformationalchanges.Theseresultsserveanintegratedpictureoftheactivechan-nel,andprovideinsightsintoprocessesunderlyingthephysiologicalfunctionofthechannelonthecellmembrane.NoCOI.

2SL08E March 17, 11:05–12:05, Room ERole of Cerebellum in Acquisition and Storage of Mo-tor MemoryNagao, Soichi(RIKEN Brain Science Institute)

Thecerebellumisnotasmallbrain.Actually,ahalfnumberofneuronsofthewholebrainarecontainedinthecerebellum.Thecerebellumreceivesinputsfromwideareasofthebrainthroughmossyfibers,andprojecttoallareasofthebrainviacerebellarorvestibularnuclei.Asaprincipleofcerebellarfunction,Marr(1969),Albus(1971)andIto(1970)independentlyproposedtheirhypothesisthatthecerebellumcontrolsmotorsystemthroughlearningusingtheerrorsignalsmedi-atedthroughclimbingfibers.The long-termdepression (LTD)ofparallelfiber-Purkinjecellsynapses(Itoetal.,1982)isthebasicsyn-apticmechanismforcerebellarmotorlearningcontrol.BasedontheMarr-Albus-Itohypothesis,Iwilloverviewtheroleofthecerebelluminacquisitionandstorageofmotormemoryrevealedbythestudyofadaptationofocularreflexes,referringtomultipledistributionsofmotormemoriesinthecerebellarcortico-nuclearcircuitry.Iwillalsoaddresshowthecerebellumcontributestovoluntarymovementandcognitivefunctionsthroughcerebro-cerebellarnetworkloops.NoCOI.

3SL10B March 18, 11:05–12:05, Room BSystems Neurophysiology of the Control of Eye and Head Movements—Functional Synergies and Com-mon Coordinates—Shinoda, Yoshikazu(Tokyo Medical and Dental University)

HereIwillreviewneuralmechanismsunderlyingvoluntaryandves-tibularcontrolofeyeandheadmovementsbasedonneurophysiolog-icalandanatomicalcircuitanalysisinhighermammals.Eyemovementsarehierarchicallycontrolledbythefrontaleyefield(FEF),superiorcolliculus(SC)andbrainstemsaccadegenerators.Inhibitionplaysanimportantroleateachlevel;saccadedynamicsdeterminedbyinhibi-toryburstneurons,theirsuppressionbyinhibitoryomnipauseneuronsandcommissuralinhibitionbetweentheSCs.TheFEFisinvolvedinaswitchingfromsaccadeinitiationtosuppressionviatheSCandtheseneurons.BothSCandvestibularsystemscontrolsimilar functionalsynergiesofeyeorneckmusclesandusethecommoncoordinatesystem(semicircularcanalcoordinates).TheseCNScircuitsprovideabasistoreduceredundantdegreesoffreedomoftheeye-headcontrolsystemandcontrolbotheyeandheadmovements(gaze)simultane-ously.Studyingonbrainfunctionswithoutunderstandingtheunder-lyingneuralcircuitsislikebuildingacastleonsand.However,sinceintroductionofmolecularbiologyintoneuroscience,neurophysiologistsengagedinunravelingofneuralcircuitsinhighermammalshavebeenonthevergeofextinction.Althoughnewly-developedapproachessuchasoptogeneticsandimagingarepowerfulandpromising,itisessentialtoelectrophysiologicallyidentifyinput-outputconnectionsoftheneu-ronsrecordedormanipulatedforourunderstandingthebrainfunctions.Untilthependulumswingsbackagain,we,systemsneurophysiologistswillhavetomanagetosurvivethedifficultstatusquo.NoCOI.

3SL14F March 18, 9:00–10:00, Room FVoltage gated calcium channels as therapeutic tar-gets for pain Zamponi, Gerald W.(University of Calgary)

Voltagegatedcalciumchannelsareimportantmediatorsofdepolar-izationinductedcalciumentryintoneurons,therebytriggeringmanyimportantphysiologicalfunctionssuchasneurotransmitterreleaseandtheshapingofneuronalfiringproperties.ThisisparticularlyevidentintheafferentpainpathwaywhereN-typeandT-typecalciumchan-nelscontributetosynaptictransmissionindorsalhornsynapses,withT-typechannelsalsoregulatingafferentfiberexcitability. Underchronicpainconditions,T-typeandN-typechannelsareupregulatedinprimaryafferentneurons,therebycontributingtopain.Converse-ly,blockofT-typeorN-typechannelsviapharmacologicalmeans,orviaactivationofopioidreceptorsmediatesanalgesia.Inmypresenta-tion,IwilldiscusshowN-typechannelscanbeusedaspharmacolog-icaltargetsinchronicpain,bothviadirectinhibitionandviaactivationofGproteincoupledreceptors.IwillpresentevidencethatassociationofN-typechannelswithcertainreceptorscontributestomorphinetolerance.IwillthendescribemechanismunderlyingtheaberrantupregulationofT-typecalciumchannelsinchronicneuropathicandinflammatorypain,andpresentmeansbywhichthesemechanismscanbeexploitedtowardsthedevelopmentofnoveltherapeuticstrat-egies.NoCOI.


3SL15H1 March 18, 9:00–10:00, Room H1The unit glomerular response. Protein sensors of membrane potentialCohen, Lawrence B.1; Braubach, Oliver1; Choi, Yunsook2; Storace, Doug2; Sung, Uhna2; Tombaz, Tuce1(1Yale Univerisity, 2Korea Institute of Science and Technology, Seoul)

Understandingtherolesofdifferentneurontypesrequiresfluorescentproteinindicatorsthatreportactivitywithhighspatio-temporalreso-lution.TheFPvoltageArcLightconsistsofthevoltagesensingdomainoftheCionaintestinalisvoltage-sensitivephosphataseandsupereclip-ticpHluorinA227D.ThefluorescenceofArcLightdecreasesby35%inresponsetoa100mVdepolarization;aboutfivetimeslargerthanpreviouslyreportedsignals.ArcLightreportsodor-evokedelectricalactivityintheinvivomammalianolfactorybulbusingboth2-photonandwide-fieldimaging.WhenthesignalsfromArcLightwerecom-paredtothosefromthecalciumsensorGCaMP3,ArcLight,butnotGCaMP3,hadsufficientlyfastkineticstoclearlydistinguishactivityelicitedbyindividualinspirations.Itisdifficulttoselectivelyactivateasingleglomerulususingodorstimulation.Westudiedtransgenicmiceinwhichchannelrhodopsin-2isselectivelyexpressedinsensoryneuronswhichprojecttheiraxonstoasingleglomerulus.Laserpulsesreliablyactivatedthetargetglomerulusandevokedcalciumresponsesin120-250juxtaglomerularneurons.Mostneurons(~95%)respondedwithincreased intracellularcalcium;theseONcellsclusterednearthetargetglomerulus.Othercells(~5%)respondedwithdecreasedintra-cellularcalcium;theseINHIBITEDcellsweremorewide-spread.Sup-portedbyNIHDC005259andtheWorldClassInstituteprogramoftheNationalResearchFoundationofKorea.NoCOI.


Joint Forum for JPS Editorial Committee, Panel for Research Ethics,

and Academic Research Board

(March 18, 12:10–13:30, Room F)

3F1F-1 JPSの現状と展望

石川義弘

横浜市立大学・医学部JournalofPhysiologicalSciences編集長

3F1F-2 生理学論文における倫理

蔵田　潔

弘前大学・医学部日本生理学会研究倫理委員会委員長

3F1F-3 The Journal of Physiologyの新しい取り組み、査読プロセス、そして論文作成時の注意点

久保義弘

生理学研究所DeputyEditor-in-Chief (Asia/Pacific)&SeniorEditor“TheJournalofPhysiology”

3F1F-4 Online open-access journalの現状と将来性について: Molecular Brainの取り組みを例に

宮川　剛

藤田保健衛生大学　総合医科学研究所／生理学研究所AssociateEditor“MolecularBrain”

Luncheon Seminars


Luncheon Seminar 1

(March 16, 12:15–13:00, Room D1)

1LS1D1 脳卒中後遺症に対する促通反復療法（川平法）と効果的併用療法Megumi Shimodouzono(Rehabilitation and Physical Medicine, Kagoshima University)

Luncheon Seminar 2

(March 16, 12:15–13:00, Room E)

1LS2E 新しい遺伝子治療ウイルスベクターの開発と基礎・臨床への応用Ken-ichiro Kosai(Gene Therapy and Regenerative Medicine, Kagoshima University)

Luncheon Seminar 3

(March 17, 12:15–13:00, Room B)

2LS3B 排便障害におけるトランスレーショナルリサーチ―腸管運動機能検査装置を用いた病態の把握と治療方法の開発―Hiroshi Matsufuji(Kagoshima University of Pediatric surgery)

Luncheon Seminar 4

(March 17, 12:15–13:00, Room D1)

2LS4D1 より大いなるヒトの生存の原理と戦略を俯瞰する―漢方薬の論理と効果―Ikuro Maruyama(System Biology in Thromboregulation, Kagoshima University)


Luncheon Seminar 5

(March 17, 12:15–13:00, Room E)

2LS5E 特異なてんかん発作を呈する自己抗体関連脳炎・脳症〜NMDAR抗体、LGI-1抗体を中心に〜 Osamu Watanabe(Neurology and Geriatrics, Kagoshima University)

Luncheon Seminar 6

(March 18, 12:15–13:00, Room D1)

3LS6D1 指宿でのがん粒子線治療 Yoshio Hishikawa(Medipolis Proton Therapy and Research Center)

Luncheon Seminar 7

(March 18, 12:15–13:00, Room E)

3LS7E 黒酢の抗酸化作用Kazuto Saito(National Institute of Fitness and Sports in Kanoya)

Hiroshi and Aya Irisawa Memorial Award Symposium


Hiroshi and Aya Irisawa Memorial Award for Excellent Paper in the Journal of Physiological SciencesMultiple functions and roles of

serotonin signaling

(March 18, 9:00–11:00, Room B)

3S44B-1Electrophysiological Properties of GABAergic Cells in Mouse Dorsal Raphe NucleusGocho, Yoshihiro; Saitow, Fumihito1; Yanagawa, Yuchio2; Sakai, Atsushi1; Suzuki, Hidenori1(1Department of Pharmacology, Nippon medical school, 2Dept Genetic and Behavior Neurosci, Gunma University)

Thedorsalraphenucleus(DRN)istheoriginofthecentralserotonin(5-HT)systemandhasbeenthoughttobecomposedofheterogeneousneuronalgroupsthatdifferinexpressionofneurotransmitterssuchas5-HT,GABAandglutamate.Tounderstand5-HTsystem,character-izationofthetypesofcellsintheDRNisnecessary.Weperformedelectrophysiologicalrecordingsinacuteslicesofglutamatedecarbox-ylase67-GFPknock-inmice.WeutilizedthismousetodiscriminatevisuallyGABAergic(GFP(+))cellsfromnon-GABAergic(GFP(-))cells.AmongGFP(-)cells,serotonergiccellscouldbedistinguishedbyim-munoreactivity fortryptophanhydroxylase.Putatively,remainingGFP(-)cellsmightbeglutamatergic.Therefore,atleast,propertiesofthreetypesofcellswereexplored.Comparedwithserotonergiccells,GFP(+)cellsdisplayedanarrowerhalf-heightwidthoftheactionpo-tentials.The input-outputrelationshipcurveofGFP(+)cellsweresteeperthanthatofserotonergiccells.Next,weexaminedpostsynap-ticresponsesmediatedbyactivationof5-HTreceptors.Variouscurrentresponseswereelicitedby5-HTandspecific5-HTreceptoragonistsinGABAergiccells.Theseresultssuggestedthatmultiple5-HTre-ceptorsubtypessuchas5-HT1A,5-HT2A/2Cand5-HT7receptorswereoverlappinginGABAergiccells,andtheircombinationmightbeimplicatedinthecontrolof5-HTcells.Understandingthepostsynap-tic5-HTfeedbackmechanismsmaycontributetoelucidatethe5-HTsystemandfacilitatetodevelopnoveltherapeuticapproaches.NoCOI.

3S44B-2The serotonin/GAD67—positive neurons in the rat dorsal raphe nucleus are transiently expressed during the weaning period and preferentially activated by the novel environment stressYoshida, Takayuki(Department of Neuropharmacology, Hokkaido University Graduate School of Medicine)

Theserotonergic (5-HTergic)systemarisingfromthedorsalraphenucleus (DRN) is implicatedinvariousphysiologicalandbehavioralprocesses,includingstressresponses.TheDRNiscomprisedofsev-eralsubnuclei,thelateralwing(DRL)thedorsal(DRD)orventral(DRV)parts.Furthermore,subsetsof5-HTergicneuronsareknowntocoex-pressGABA.However,theproperties,distributions,andinvolvementinstressofGABA-containing5-HTergicneuronsremainunknown.Inthisstudy,wecharacterizedfunctionalpropertiesofGAD67-express-ing5-HTergicneurons (5-HT/GAD67neurons)andcomparedtheirpropertieswiththoseofother5-HTorGAD67-positiveneurons.Weconfirmed5-HT/GAD67neuronswereselectivelydistributedintheDRL,butnot intheDRDorDRV.Unexpectedly,TheyexpressedplasmalemmalGABAtransporter1,but lackedvesicular inhibitoryaminoacidtransporter.Byusingpatch-clamprecording, the inputresistanceandfiringfrequencyof5-HT/GAD67neuronsweresignifi-cantlylowerthanthoseof5-HTneurons.Asrevealedbyc-Fosimmu-nohistochemistry,neuronsintheDRL,particularly5-HT/GAD67neu-rons,showedhigherresponsivenesstoexposuretoanopenfieldarenathanthose intheDRDandDRV.Bycontrast,exposuretocontextualfearconditioningstressshowednosuchregionaldifferenc-es.Thesefindings indicatethat5-HT/GAD67neuronsconstituteauniqueneuronalpopulationwithdistinctiveneurochemicalandelec-trophysiologicalpropertiesandhighresponsivenessto innocuousstressor.NoCOI.

3S44B-3Birth regulates sensory map formation in the brain through serotonin signalingKawasaki, Hiroshi(Graduate School of Medical Sciences, Kanazawa University)

Althoughthemechanismsunderlyingthespatialpatternformationofsensorymapsinthebrainhavebeenextensivelyinvestigated,thosetriggeringsensorymapformationduringdevelopmentare largelyunknown.Hereweshowthatthebirthofpupsinstructivelyandse-lectivelyregulatestheinitiationofsomatosensorymapformationinthecerebralcortexbyreducingserotoninconcentration.Wefoundthatpretermbirthacceleratedsomatosensorymapformation,whileitdidnotaffectwhiskerlesion-inducedbarrelstructuralplasticity.Wealsofoundthatserotoninwasselectivelyreducedsoonafterbirth,andthatthereductionofserotoninwastriggeredbybirth.Thereductionofserotoninwasnecessaryandsufficient fortheeffectofbirthonsomatosensorymapformation. Interestingly, theregulatorymecha-nismsdescribedherewerealsousedinthevisualsystem,suggestingthattheyareutilizedinvariousbrainregions.Ourresultsshedlightonhithertounidentifiedrolesofbirthandserotonininsensorymapformationduringdevelopment.NoCOI.


3S44B-4Abnormal serotonin in a CNV mouse model for au-tismsTakumi, Toru(RIKEN BSI)

Recentadvanceinneurosciencerevealsthatpsychiatricdiseasesmaybebasedonsynapticabnormality.Autismspectrumdisorders(ASD)arechildpsychiatricillnessesthatarecharacterizedbyimpairmentsinsocialinteractionsandverbalandnon-verbalcommunications,andpervasivestereotypicbehavior.Mostoftheircausesareunknown,whereasthereareseveralknowncausesofASD,insyndromiccases(fragileXsyndrome,tuberoussclerosis,Rettsyndrome,etc.),chromo-somalabnormality(15q11-13duplication,otherCNVs),andraremuta-tions.AmongCNVsandraremutations,celladhesionmoleculesthatare involved insynapse formationandotherrelatedmoleculestosynapticfunctionsareparticularlyintriguing.ThecauseofASDmightbeconsideredasabnormalityinpostnataldevelopmentofsynapses.Wegeneratedamousemodel (patDp/+) forduplicationofhumanchromosome15q11-13usingachromosome-engineeringtechnique.ThepatDp/+miceshowabnormalsocialbehavior,abnormalityinserotoninsignalingandlevels,alteredspinemorphologyandexcitatory/inhibi-toryimbalance.NoCOI.

Award Presentations (Oral)


Promotion Award of the Physiological Society of Japan for Young Scientists

3S58H1-2Large-scale imaging of circadian rhythm in the mam-malian master clockEnoki, Ryosuke(Hokkaido Univ, Grad Sch of Med, Photonic Bioimaging, Sapporo, Japan)

3O6F-5On-demand biosynthesis by diacylglycerol lipaseα is the major source of 2-arachidonoylglycerol, the endo-cannabinoid that mediates retrograde signalingHashimotodani, Yuki; Purpura, Dominick P.(Dept Neurosci, Albert Einstein Col. Med., NY)

3O7J-1Overexpression of Leukocyte Kv1.3-Channels Pro-motes Renal Fibrosis in Rats with Advanced Chronic Renal FailureKazama, Itsuro(Department of Physiology I, Tohoku University Graduate School of Medicine, Sendai, Japan)

Hiroshi and Aya Irisawa Memorial Promotion Award

for Young Physiologists

1S14D1-4 The C1q complement family complements glutama-tergic synapses on cerebellar Purkinje cellsKakegawa, Wataru(Department of Physiology, Keio University School of Medicine, JST-CREST, Saitama, Tokyo, Japan)

3S48F-4Calcium Homeostasis Modulator (CALHM): A novel ion channel family encoding voltage-gated ATP re-lease ion channels involved in non-synaptic neuro-transmission from taste cellsTaruno, Akiyuki(Department of Molecular Cell Physiology, Kyoto Prefectural University of Medicine, Kyoto, Japan)

2S34K-4Mitochondria-endoplasmic/sarcoplasmic reticulum Ca crosstalk and cellular functionTakeuchi, Ayako(Integr. Physiol. Fac. Med. Sci. Univ. Fukui)

Symposia


Symposium 01Regulation of the cardiac gap junction

and its pathological aspects

(March 16, 9:00–11:00, RoomA)

1S1A-1Downward remodeling of cardiac gap junction con-nexin 43 and arrhythmogenesisImanaga, Issei(Fukuoka University, Hara-Doi Hospital)

Dysfunctionofthecardiacgapjunctionwhichfundamentallycontrib-utestoelectricalcell-to-cellcouplingisregardedasoneofessentialfactorsgeneratingarrhythmias.Thefunctionofthegapjunctionde-pendsontheupwardordownwardregulationofconnexin(Cx)whichcomposesthegapjunctionchannel.Incardiacmuscletissue,Cx30.2,Cx40,Cx43andCx45areexpressedshowingregionalspecificity.Cx43ispredominantlyexpressedintheventriculartissue.AsuppressionofthePKA-mediatedphosphorylationofCx43 inducedbyhypoxiawhichleadstointracellularCaoverloadandacidosiscausesscant~heterogenousexpressionofCx43atthegapjunctionareaintheinter-calateddiskandadecreaseinelectricalresistanceofthegapjunction.AnactivationofPKApreventsdeteriorativeeffectsofhypoxiaonCx43.AnaugmentationofthePKC-mediatedphosphorylationofCx43inducedbyPKCεactivatorsuchasAngiotensinIImakessparseex-pressionofCx43at thegap junctionarea, lateralization,annuralprofileandinternalizationofthegapjunction,adecreaseinamountofCx43proteinandanincreaseelectricalresistanceofthegapjunction.ProteolyticdegradationofCx43isacceleratedwhenthePKCε-medi-atedphosphorylationofCx43isaugmented.Thedownwardremodel-ingofCx43maketheheartmoresusceptibletotheventriculartach-yarrhythmia.Onthecontrary,anaugmentationofthePKA-nediatedphosphorylationofCx43inducedbyPKAactivatorsorcyclicAMPanalogsisfollowedbyanincrementofexpressionandamountofCx43.TheupwardremodelingofCx43makestheheartlowsusceptibilitytoarrhythmias.NoCOI.

1S1A-2A Connexin40 Mutation Associated with a Malignant Variant of Familial Cardiac Conduction DefectMakita, Naomasa1; Seki, Akiko2(1Department of Molecular Physiology, Nagasaki University, 2Department of Cardiology, Tokyo Women’s Medical University)

Familialcardiacconductiondefect(CCD)isahereditaryarrhythmiacharacterizedbyprogressiveconductiondisturbancesintheHis-Pur-kinjesystem.CCDhasbeenlinkedtogenessuchasSCN5Athatin-fluencecardiacexcitabilitybutnottogenesthatinfluencecell-to-cellcommunication.InordertoexplorewhethermutationsinconnexingeneswouldassociatewithclinicalcasesofCCD,wescreened156probandswithCCD.Inadditionto12sodiumchannelmutations,wefoundagermlineGJA5 (connexin40 [Cx40])mutation (Q58L) in1family.HeterologousexpressionofCx40-Q58L inconnexin-deficientneuroblastomacellsresultedinmarkedreductionofjunctionalcon-ductance (Cx40-wild type [WT], 22.2±1.7 nS, n=14; Cx40-Q58L,0.56±0.34nS,n=14;P<0.001)anddiffuselocalizationofimmunoreactiveproteinsinthevicinityoftheplasmamembranewithoutformationofgapjunctions.HeteromericcotransfectionofCx40-WTandCx40-Q58Lresultedinhomogenousdistributionofproteinsintheplasmamem-braneratherthaninmembraneplaquesin50%ofcells;well-definedgapjunctionswereobserved inothercells.Junctionalconductancevaluescorrelatedwiththedistributionofgapjunctionplaques.Muta-tionCx40-Q58Limpairsgapjunctionformationatcell-cellinterfaces.Thisisthefirstdemonstrationofagermlinemutationinaconnexingenethatassociateswithinheritedventriculararrhythmiasandem-phasizestheimportanceofCx40innormalpropagationinthespecial-izedconductionsystem.NoCOI.

1S1A-3Alterations of connexin expression and cardiac ar-rhythmogenesisHonjo, Haruo1; Ohkusa, Tomoko2; Kodama, Itsuo1; Kamiya, Kaichiro1(1Dept. Cardiovasc. Res., Res. Inst. Environ. Med., Nagoya Univ., Nagoya, Japan, 2Dept. Med., Yamaguchi Univ. Sch. Med., Ube, Japan)

Gapjunction(GJ)channelsprovideapathwayforintercellularcom-municationbetweenadjacentcells.Coordinatedpropagationofelec-tricalimpulseintheheartdependsoncurrentflowthroughGJchan-nels.Alterations intheexpressionandorganizationofGJsubunits,connexins(Cxs),mayleadtocardiacarrhythmiasthroughcell-to-celluncouplingandmodificationofimpulsepropagation.Wedemonstratedthatcardiachypertrophyinducedbypressureoverloadinratsisas-sociatedwithdislocationofimmunelabeledCx43GJsfromtheinter-calateddiscatthesiteofend-to-endcontacttothelateralborderofventricularmyocytes.SomeofthedisplacedCx43GJsappearasin-tracellularannularprofilesinelectronmicroscopy,probablyreflectingGJdegradationduringrapidremodeling.Inaddition,ahamstermod-elofcardiomyopathy,UM-X7.1,showsareductionofventricularCx43expressionandimmunolabeling,atthestageofheartfailure,whichismediatedbyadecreaseinnuclearβ-catenin,atranscriptionalactivatorofCx43.Opticalmappinginperfusedheartsrevealsan increase inspatialheterogeneityoftheventricularactionpotentialandslowingofimpulsepropagationwithmarkeddistortionofthewavefront.UM-X7.1hamstersshowanincreasedvulnerabilitytosustainedventricu-lartachycardiaandfibrillation,whicharecharacterizedbymultiplerotorsandwavebreaks. Inconclusion,alterationsofCxexpressionassociatedwithcardiachypertrophyandheartfailuremayprovidestructuralsubstratesfortachyarrhythmias.NoCOI.


1S1A-4Role of connexins on cardiac arrhythmiaSeki, Akiko1; Nishii, Kiyomasa2; Shibata, Yosaburo3; Kobayashi, Yasushi2; Hagiwara, Nobuhisa1(1Department of Cardiology, Tokyo Women's Medical University, 2Department of Anatomy and Neurobiology, National defence medical college, 3Fukuoka Prefectural University)

ComparedtothemajorcardiacgapjunctionproteinCx43,relativelylittlestudieshavebeencarriedoutonCx45.ToclarifythefunctionalimportanceofCx45intheheart,weinvestigatedelectrophysiologicalcharacteristicsofCx43/Cx45mutantheartswithtargeteddisruptioninCx43orCx45orboth,togetherwithregionspecificdeletionofCx45inthemyocardiumandintheendothelium.Inadult,atamoxifen-in-ducibleCre-LoxsystemwasusedtocircumventembryoniclethalityoftheCx45-/-mice.Inthispresentation,wewilldiscusstheroleofCx45incardiacdevelopmentandintheconductionsystem.NoCOI.

Symposium 02Regional variation and functional

overlapping of taste sensor molecules expressed in the oral cavity, gut and brain

(March 16, 10:05–12:05, Room D1)

1S2D1-1Development of the basal cells in taste buds and taste cell differentiationMiura, Hirohito1; Nakayama, Ayumi1; Scott, Jennifer K.2; Tomonari, Hiroshi1; Ooki, Makoto1; Barlow, Linda A.2; Harada, Shuitsu1(1Department of Oral Physiology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan, 2Department of Cell and Developmental Biology, School of Medicine, University of Colorado, Aurora, CO, USA.)

Inmammals,tastebudsconsistof50–100elongatedcellsandasmallnumberofbasalcells,andaremaintainedbycontinuouscellrenewalthroughoutlife.Theround-shapedbasalcellsoftastebudsareassumedtobeinaprematurestate,whiletheelongatedcellsincludefunction-altastecellsthatareclassifiedintothreecelltypes,I,IIandIIIorig-inallybasedontheirmorphologicalcharacteristics.Wehaveprevious-ly reported Sonic hedgehog (Shh), an evolutionally conservedmorphogen,isspecificallyexpressedinthebasalcellsintastebuds.BrdUincorporationstudiesshowedthatShh(+)basalcellsareinapostmitoticstateandhaveashortlifetime,leadingthehypothesisthatShh(+)basalcellsarepostmitoticimmediateprecursorsofothercellsintastebuds.Recently,weshowedShh(+)basalcellsgiverisetoallelongatedcelltypes,typeI,IIandIII,usingCre/loxPsystem.Here,wewilldiscussthedevelopmentandcellfateofthebasalcellsintastebuds.Also,regionaldifferencesintastecelldifferentiationwithintheoralcavitywillbediscussed.NoCOI.

1S2D1-2Sweet taste signaling in the oral cavity and the gut: functional roles of leptin, endocannabinoids and GLP-1Yoshida, Ryusuke; Jyotaki, Masafumi; Takai, Shingo; Shigemura, Noriatsu; Ninomiya, Yuzo(Section of Oral Neuroscience, Graduate School of Dental Science, Kyushu University, Fukuoka, Japan)

Bothtastecellsintheoralcavityandenteroendocrinecellsinthegutdetecttastesubstances in foodsanddrinksandtheirsignalsplayimportantrolesintheregulationoffoodintakeandnutrientabsorption.Recentstudieshavedemonstratedthattheyshareavarietyofmole-culesinvolvedintastesignaldetection,hormonalsecretionandnutri-entabsorption.Forexample,enteroendocrinecellsexpresssweettastereceptorT1R2+T1R3,whichisoriginallyfoundintastecells.ActivationofT1R2+T1R3isshowntoleadtofacilitationofglucoseabsorptioninthegut.Inconstant,tastecellsexpressglucosesensors,SGLT1andGLUTs,andpeptidehormones,GLP-1andCCK,whichareknowntofunctioninthegut.Howeveritremainsunclearwhetherthesemole-culeswouldbeinvolvedintastesignaldetectionandtransmissionintheoraltastesystem.Regardingsweettaste,werecentlyfoundthatsweettastesensitivitiesoftastecellsaremodulatedbyorexigenicandanorexigenicmediatorssuchasleptinandendocannabinoids,whichregulatefoodintakebyactingonhypothalamicreceptors.Butfunc-tionalrolesofleptinandendocannabinoidsintheguttastesystemstillremainunclear.Inthistalk,wewillfocusonleptin,endocannabinoidsandGLP-1andwillreporttheirfunctionalrolesinsweettastesignal-ingintheoralandguttastesystems.NoCOI.


1S2D1-3Characterization of taste-like cells in the gastrointes-tinal tractIwatsuki, Ken(Institute of Molecular and Cellular Biosciences, The University of Tokyo, Japan)

Recentstudieshavedemonstratedthattastesignalingmoleculesaredistributednotonlyinthegustatoryepitheliumbutalsoinothertissuesincludingthegastrointestinal(GI)tract,airways,testesandbrain.Thesesignalingmoleculesarethoughttoparticipate indetectingsweet,umamiandbittercompounds.InordertoidentifyT1R2-expressingcellsinvivo,T1R2-LacZknock-inmousewasgeneratedandexaminedforLacZexpressionintheGItract.WefoundthatLacZexpressionwasseenintheoralepithelium,duodenum,smallintestine,largein-testinebutnotinthestomach.MostofLacZpositivecellsexpressedchromograninAwhichisamarkerofenteroendocrinecells.WehavefurtheridentifiedthatthesecellsalsoexpressproteinsnecessaryfortastesignaltransductionsuchasPLCbeta2andIP3R3.SomeofLacZpositivecellsalsoexpressedglucagon-likepeptide1(GLP-1).Interest-ingly,unliketastecellsintheoralcavity,enteroendocrinecellsthatexpressT1R2rarelyexpressalpha-gustducin.Furthermore,therewasaclearmorphologicaldifferencebetweenenteroendocrinecellsandalpha-gustudicinpositivecells.Therefore,oneassumptioncouldbemadethatthereareatleasttwosubsetsoftaste-likecellsintheGItract;enteroendocrinecellsthatexpresssweettastereceptorsandbrushcellsthatexpressalpha-gustducin.Insummary,wehaveiden-tifiedtwoseparatepopulationsofcells thatseemtohavedistinctfunctionindetectingnutrientsintheGItract.NoCOI.

1S2D1-4GLP-1 may be involved in sweet specific taste trans-mission from taste cells to gustatory nerve fibersTakai, Shingo1; Yasumatsu, Keiko1; Inoue, Mayuko1; Iwata, Shusuke1; Yoshida, Ryusuke1; Shigemura, Noriatsu1; Drucker, Daniel J.2; Margolskee, Robert F.3; Ninomiya, Yuzo1(1Sect. Oral Neurosci., Grad. Sch. Dental Sci., Kyushu Univ., Fukuoka, Japan, 2Dept. Med., Mt. Sinai Hosp., Lunenfeld Tanenbaum Res. Inst., Univ. Toronto, Toronto, Canada, 3Monell Chemical Senses Center, Philadelphia, USA)

Recentstudiesdemonstratedthattastebudcellsexpressseveralgutpeptides,suchasGLP-1,NPY,andglucagon,andsecretthesepeptidesinresponsetovarioustastestimuli.Interestingly,thesecretionpatternsofpeptidesarecorrelatedwithtastequalities,suggestingthepossibil-itythatthesegutpeptideswouldcontributetotastequalitycoding.Inthisstudy,wereportthatmicegeneticallylackingofGLP-1recep-torshoweddecreasedbehavioralresponsestosweetcompoundsinshort-time licktestandreducedsweettasteresponses inchordatympaninerverecordings.Additionally,wemeasuredGLP-1concen-trationsreleasedfromsingletastebuds,andcellsinresponsetovar-ioustastestimulibyusingELISAmethod.Asaresult,wefoundthatGLP-1issecretedfromasubsetofsweetresponsivecellsbysweettastestimulationinaconcentrationdependentmanner.Furthermore,itisfoundthati.v.injectionofGLP-1producedtransientincreaseofneuralactivitiesinasubsetofsweetspecificsinglenervefiberswith-outaffectingthoseofothertastefibers.Moreover,wefoundthathumansweettastesensitivity isassociatedwithtwoSNPsitesexisting inGLP-1receptorgene.AllthesefindingssuggestthatGLP-1maybeinvolvedinnormalsweettastesignaltransmissioninmiceandhuman.NoCOI.

Symposium 03New aspects of molecular mechanisms

of ENaC-mediated Na+ homeostasis and body fluid regulation

(March 16, 8:40–10:20, Room E)

1S3E-1Hormonal modulation of salty and sweet taste sensi-tivitiesShigemura, Noriatsu; Yoshida, Ryusuke; Yasumatsu, Keiko; Ohkuri, Tadahiro; Iwata, Shusuke; Takai, Shingo; Jyotaki, Masafumi; Niki, Mayu; Sanematsu, Keisuke; Ninomiya, Yuzo(Section of Oral Neuroscience, Graduate School of Dental Sciences, Kyushu University, Fukuoka, Japan.)

Informationderivedfromtastereceptorcells,suchassweet,bitter,salty,sourandumamiisimportantforevaluatingthequalityoffoodcomponents.Modulationofthegustatoryinformationinfluencesfoodpreferenceandintake.Evenso,themolecularmechanismsformodu-latingtastesensitivityarepoorlyunderstood.Ourrecentstudieshavedemonstratedthatsaltyandsweettastesensitivitiesareaffectedbyhormones.AngiotensinII(AngII),whichplaysimportantrolesinthemaintenanceofsodiumhomeostasisinvariousorgansincludingbrainandkidney,modulatesperipheralsaltandsweettastesensitivitiesbyactingonAngIItype1receptorco-expressedwithαENaC(anami-loride-sensitivesalttastereceptor)orT1r3 (asweettastereceptorcomponent) intastecells,andthismodulation influences ingestivebehaviorinmice.Leptin(ananorexigenicmediator)andendocannabi-noids(orexigenicmediators),whichreciprocallyregulatefoodintakeviacentralnervoussystems,modulatepalatabilityoffoodsbyalteringsweettasteresponses.Thusperipheralmodulationsoftasteinformationbythesehormonescriticallyinfluencefoodintake,andmayplayim-portantroles inregulatingsodiumandenergyhomeostasis.Under-standingbasicprinciplesaboutthesetastemodulationsmayleadustodesignnovelstrategiestomaintainandimprovethequalityoflifeinhuman.NoCOI.


1S3E-2An essential role of p38 on ENaC trafficking in aldo-sterone-stimulated Na+ reabsorption in renal epitheli-al A6 cellsNiisato, Naomi1,3; Marunaka, Yoshinori1,2,3(1Department of Molecular Cell Physiology, Kyoto Prefectural University of Medicine, Kyoto, Japan, 2Department of Bio-Ionomics, Kyoto Prefectural University of Medicine, Kyoto, Japan, 3Institution for Food Education and Health, Heian Jogakuin University, Kyoto, Japan)

AldosteroneisanimportanthormonetoregulateepithelialNa+chan-nel(ENaC)-mediatedNa+reabsorptioninthedistalnephronforcon-trollingECFvolumeandbloodpressure.Inourpreviousstudy,wefoundp38playsanessentialroleontheregulatorymechanismofENaC-mediatedNa+reabsorptionbyaldosterone.Inhibitionofp38bySB202190(aspecificp38inhibitor:SB)abolishedthealdosterone-stim-ulatedNa+reabsorption (INa)withremarkablereductionofapicalENaCconductance.Inthisstudy,weinvestigatearoleofp38onthesurfaceexpressionofENaCthroughexocyticandendocyticpathways.PretreatmentwithMG132(aproteasomeinhibitor)mostlyrecoveredtheSB-inducedreductionofENaCproteincontent,ENaCsurfaceex-pressionandINa,althoughMG132slightlyincreasedthemintheab-senceofSB.Ontheotherhand,wedetectedthatp38regulatedAkt/PKBactivation,whichwascorrelatedtoENaCubiquitylationthroughtheSGK1-Nedd4-2-dependentpathway.Indeed,suppressionofp38andAkt/PKBdecreasedNedd4-2phosphorylationandENaCproteinabun-dance.Thesesuggestthatthep38-mediatedsignalpathwayisessen-tialtosuppressubiquitylation-dependentENaCdegradationcontrib-utingtothestimulationofNa+reabsorptionbyaldosteone.SupportedbyJSPS(19590212,20390060),SSRF(0837,1035)andFFPR.NoCOI.

1S3E-3Regulations of ENaC Expressions by Nedd4L and tu-bular Renin-angiotensin SystemsImplications for sodi-um sensitivity and hypertensionIshigami, Tomoaki1; Umemura, Masanari2; Araki, Naomi1; Minegishi, Shintaro1; Yamana, Hisako1(1Yokohama City University Graduate School of Medicine Department of Cardiorenal Medicine and Medical Science, Yokohama, Japan , 2Yokohama City University Graduate School of Medicine Cardiovascular Research Institute)

Itwassuggestedthatprimarymolecularabnormalitiesexisted intubularsodiumtransportsbymoleculargeneticalanalysesforhumanhereditaryandfamilialhypertensionbyR.C..LiftonandJ.M.Lalouel.WehaveanalyzedmolecularpathophysiologicaldissectionsforsodiumsensitivityandhypertensionfocusingonNedd4Lwhichisubiquitinligase forepithelialsodiumchannels located indistalnephronandtubularrenin-angiotensinsystems(RAS)consistedofangiotensinogeninproximaltubulesandrenininconnectingtubules.Bydetailedge-neticalanalysesforhumanandrodents,wediscoveredmoleculardi-versitiesofNedd4Lgene(JHG2002,BBRC2006)anddifferentNedd4LisoformswithC2domainregulatingENaCexpressionsinvivo(Hyper-tension2008).UsingNedd4LC2domainknock-outmice,wealsodis-coveredoralsodiumsensitiveenhancementsofsodiumreabsorptionviaENaCinthedistalnephron(ISHR,2013).TubularRASareregu-lated independentlyfromsystemicRASandenhancedbysodiumdependentmanner inmicesodiumsensitivemodelofhypertension(Nephron,2012).Takentogether,weproposedthatcomprehensiveunderstandingforintegratedtubularendocrinogolicalmodelsofbothtubularRAsystemsandENaC-Nedd4Lsystemsisimportanttoreg-ulatepathophysiologicalbasisofsodiumsensitivityandhypertension.NoCOI.

1S3E-4ENaC-dependent and -independent roles of serine proteases in the aldosterone-induced hypertension and renal fibrosisKakizoe, Yutaka; Kitamura, Kenichiro(Department of Nephrology, Kumamoto University Graduate School of Medical Sciences)

Aldosteronehasanimportantroleintheregulationofbloodpressurebymodulatingtheactivityofepithelialsodiumchannel(ENaC)inthekidney.ItinducesamolecularweightshiftofγENaCfrom85to70kDathatisnecessaryforthechannelactivation,andserineproteaseprostasinplaysacriticalroleinthisshift.Asyntheticserineproteaseinhibitorcamostatmesilate(CM),whichsuppressestheprostasinac-tivity,blockstheproteolyticcleavageofγENaCinducedbyaldosteroneandconcomitantlyitincreasesurinarysodiumexcretion,indicatinganatriureticeffectofserineproteaseinhibitors.Ontheotherhand,recentstudiessuggestedthataldosteronecauseskidneyinjuryindependentofitshemodynamiceffect.However,theprecisemechanismsofthesedirecteffectsremaintobeelucidated.Weisolatedaserineproteasefromratkidneythatwasactivatedbyco-administrationofaldosteroneandahigh-saltdiet. ProteinpurificationandLC-MS/MSanalysesrevealedthiscandidateproteaseasplasmin.TreatmentofratrenalfibroblastcellswithplasminincreasedmRNAexpressionofprofibrot-icandinflammatorygenesandtheyweresignificantlysuppressedbyCM.Furthermore,CMamelioratedtherenal interstitialfibrosis inratsinducedbyaldosteroneandahigh-saltdiet.Ourcurrentstudiessuggestthatserineproteaseshavepivotalrolesinthealdosterone-in-ducedhypertensionandrenalfibrosisandthatserineproteaseinhib-itorcouldbeanewstrategyforthetreatmentofkidneydiseasescausedbyaldosteroneinhumans.NoCOI.

Symposium 04Role of STIM/Orai for physiological

function

(March 16, 10:25–12:05, Room E)


1S4E-1The roles of STIM/Orai and TRPC1/C6 channels in cell cycle progression of bone marrow stromal cellsIchikawa, Jun; Inoue, Ryuji(Department of Physiology, Fukuoka University School of Medicine)

Toquestionhowtheproliferativepotentialandcellcycleprogressionofadultstemcellsarecontrolledisanattractivethemeforregenera-tivemedicineandbioengineering.However,theimplicationsofnon-volt-age-gatedCa2+entrychannelsthereinarepoorlyelucidated.Inthisstudy,weinvestigatedtherolesofSTIM/OraiandTRPCchannelsonthecellcycleprogressionofbonemarrowstromalcells(BMSCs)whichincludemesenchymalstemcells.CulturedBMSCsweresynchronizedintheG1,S,G2orMphases.IntheSphase,theexpressionlevelsofSTIM,OraiandTRPC1weresignificantlyenhanced.Ontheotherhand,thatofTRPC6wasgreatlyreducedintheSphaseandenhancedintheG1phase.Store-operatedCa2+entry(SOCE)wassignificantlyenhancedintheSphasebutsuppressedintheG1phase.Flowcyto-metricanalysisrevealedthatsiRNAknockdownofSTIM/OraiorTRPC1/C6expressiondifferentiallyaffectedthedistributionofcellcyclestages.ToseekaroleofTRPC6channelsforcellcycleprogres-sion,weevaluatedtherestingmembranepotential (RMP)ofcells.siRNAknockdownofTRPC6producedasignificantnegativeshiftofRMP.RMPoftheSphasewasdeeperthanoftheothercellcyclestages.Chemicaldepolarization/hyperpolarizationclampexperimentsabrogatedthedifferences inthemagnitudeofSOCEbetweencellcyclestages.Fromtheseobservations,wesuggestthatTRPC6-medi-atedchangesinRMPmayalterdrivingforceforCa2+influxattheG1andSphase,andmodulateSOCEviaSTIM/OraiandTRPC1.NoCOI.

1S4E-2Immune regulation by store-operated calcium entryOh-hora, Masatsugu1(1Division of Molecular Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan, 2Japan Science and Technology Agency (JST), Precursory Research for Embryonic Science and Technology, Fukuoka, Japan)

T-cellreceptor(TCR)signalingdeterminesTcellfateinthymicde-velopment.CalciumsignalingdownstreamofTCRplaysacrucialroleinthedevelopmentofTcells.Store-operatedcalciumentry istheprimarymechanismforcalciuminfluxinTcells.Althoughithasbeenwidelyassumedthatstore-operatedcalciumentryisessentialforTcelldevelopmentinthethymus,thereisnodirectevidenceforthisassumption.Toaddressthisquestion,wehaveanalyzedmicelackingSTIM1andSTIM2,thetwokeymediatorsofstore-operatedcalciumentry.Weshowherethatunexpectedly,lackofstore-operatedcalciumentryresultsinnoeffectonpositiveselectionofconventionalTCRαβ+Tcelldevelopmentandamildimpairmentofnegativeselection.Incontrast,store-operatedcalciumentryspecificallycontrolsthedevel-opmentofself-reactiveagonist-selectedTcells,suchasregulatoryTcells.AbsenceofSTIM1andSTIM2leadtoasevereimpairmentofthepost-selectionproliferationandfunctionalmaturationofagonist-se-lectedTcells.Thisdefectwascausedby inefficientexpressionofNFATtargetgenesincludingcytokinereceptors.Consistently,doubledeletionofNfat1andNfat2inTcellswithLck-Cretransgenicmiceledtoapartialbutspecificreductioninthepopulationofagonist-se-lectedTcells.ThesefindingsindicatethatSTIM-mediatedstore-op-eratedcalciumentry,leadingtoefficientNFATactivation,iscriticalforthepostselectionmaturationofagonist-selectedTcells.NoCOI.

1S4E-3Role of STIM1- and Orai1-mediated Ca2+ entry in epi-dermal keratinocyte physiologyNumaga-Tomita, Takuro1(1Inst Adv Studies, Kyushu Univ, Fukuoka, Japan, 2NIEHS, NIH, RTP, NC, USA)

Theuppermostthinlayeronthesurfaceoftheskin,calledtheepider-mis,isresponsibleforthebarrierfunctionoftheskin.Theepidermishasamultilayeredstructureinwhicheachlayerconsistsofkeratino-cytes(KCs)ofdifferentdifferentiationstatus.Intracellularandextra-cellularCa2+isknowntoplayimportantrolesinKCdifferentiation.However,themolecularmechanismsunderlyingCa2+regulationofKCdifferentiationarestill largelyunknown.Store-operatedCa2+entry(SOCE)isamajorCa2+influxpathwayinmostnon-excitablecells.Inthisstudy,weanalyzedthecontributionofSOCEtoKCgrowthanddifferentiationinthehumankeratinocytecellline,HaCaT.KnockdownofeitherSTIM1orOrai1,essentialcomponentsofSOCE,stronglysuppressedSOCEandCa2+-switch-inducedCa2+responses,resultinginimpairedexpressionofkeratin1,anearlyKCdifferentiationmarker.Furthermore,lossofeitherSTIM1orOrai1suppressednormalgrowthofHaCaTcellsinlowCa2+andinhibitedthegrowtharrestinresponsetoaCa2+switch.TheseresultsdemonstratethatSOCEplaysmultiplecrucialrolesinKCdifferentiationandfunction.WehaveinvestigatedthephysiologicalrelevanceofSTIM1invivowithepidermis-specificSTIM1knockoutmice(STIM1-epKO).TheeffectofSTIM1deficiencyonskinwoundhealingwasevaluated.Strikingly,STIM1-epKOmiceshowedfasterwoundhealingcomparedtoWT,whichisconcomitantwithfewerinflammatoryinfiltrates,suggestingthatSTIM1inepider-misisalsoimportantfortheregulationofhumoralfactorsproductionuponskininjury.NoCOI.

1S4E-4Identification of Stim1 as a candidate gene responsi-ble for stress-induced hypertension in the stroke-prone spontaneously hypertensive ratOhara, Hiroki; Isomura, Minoru; Nabika, Toru(Department of Functional Pathology, Shimane University School of Medicine, Izumo, Japan)

Thesympatheticnervoussystem(SNS)playsan importantrole inregulatingthebloodpressure(BP)inresponsetovariousenvironmen-talstimuli.Bothinhumansandinrodents,exaggeratedSNSactivityhavebeenimpliedtobeaputativecauseofhypertension.Genesinflu-encingsympatheticactivityare,therefore,importantwhenconsideringthegeneticsusceptibilitytohypertension,especiallyinthecontextofgene-environmentalinteraction.Thestroke-pronespontaneouslyhy-pertensiverat(SHRSP)iswellknowntohaveanexaggeratedsympa-theticresponsetostress,whichmaybecausallyrelatedtoitsseverehypertension.Recently,basedontheresultsofphysiologicalanalysisofcongenicstrainsestablishedbetweenSHRSPandnormotensiveWistar-kyoto(WKY)rat,werevealedthatagene(orgenes)responsi-ble forexaggeratedsympatheticresponsetostress inSHRSPwaslocated ina1.2-Mbpfragmentonchromosome(Chr)1.AmongthegeneslocatedintheChr1region,thewhole-genomesequenceanalysisofSHRSPandWKYidentifiedanonsensemutation inthestromalinteractionmolecule1(Stim1)geneofSHRSP.Thenonsensemutationfound inSHRSPresulted inatruncationof46aminoacidsattheC-terminalendoftheratSTIM1thatwasindeedshownbywesternblotanalysis.STIM1playsakeyroleinstore-operatedcalciumentry(SOCE)process.WehypothesizethatabnormalSOCEactivityinducedbythemutatedformofSTIM1resultsinexaggeratedsympatheticresponsetostressinSHRSP.NoCOI.


Symposium 05 Challenge of synaptologists into

understanding of the structure and function of the neural network

(March 16, 9:00–11:00, Room F)

1S5F-1Retrograde signaling regulates synapse elimination in developing brainUesaka, Naofumi1; Uchigashima, Motokazu3; Mikuni, Takayasu1; Hirai, Hirokazu2; Watanabe, Masahiko3; Kano, Masanobu1(1Dept. of Neurophysiol., Grad. Sch. of Med., Univ. Tokyo, Tokyo, Japan, 2Dept. of Neurophysiol., Grad. Sch. of Med., Gunma Univ., Maebashi, Japan, 3Dept Anat., Grad Sch Med, Hokkaido Univ, Sapporo, Japan)

Neuronsformexuberantsynapseswithtargetcellsearlyindevelop-ment.Then,necessarysynapsesareselectivelystrengthenedwhereasunnercessaryconnectionsareweakenedandeventuallyeliminatedduringthecourseofpostnataldevelopment.Thisprocessisknownassynapseelimination,andisanimportantsteptoshapeinitialredundantneuralcircuitsintofunctionallymaturecircuits.Moreover,thedisrup-tionislikelylinkedtomentaldisorderandbraindysfunction.Climbingfiber(CF)toPurkinjecell(PC)synapsesinthecerebellumprovideanexcellentmodel tostudytheprocessofsynapseelimination.PaststudiesidentifiedseveralmoleculesrequiredfortheCF-PCsynapseselimination.However,wecannotstillunderstandthemolecularmech-anismsofsynapseselimination.Toefficiently identifymoleculesre-quiredforsynapseelimination,weplannedthefollowingapproach.First,wedevelopedan invitroculturesystemwhichenableustoeasilyperformgeneticmanipulations.Second,usingtheculturesystemandalentivirus-mediatedknockdowntechnique,wescreenedmoleculesfortheCF-PCsynapseelimination.Third,weevaluatedthefunctionofthescreenedmoleculesinvivo.Inthispresentation,wearegoingtointroducescreenedmoleculesandtheirroleinsynapseelimination.NoCOI.

1S5F-2Cerebellar ataxia and synaptic abnormalityHosoi, Nobutake; Hirai, Hirokazu(Department of Neurophysiology, Gunma University Graduate School of Medicine, Maebashi, Japan)

Staggerermutationcausesafunctionallossofatranscriptionalfactor,Retinoid-relatedOrphanReceptorα(RORα),whichisabundantlyex-pressedincerebellarPurkinjecells.Thehomozygousstaggerermutantmice(sg/sg)showcerebellarhypoplasiaandsevereataxia.Thesg/sgmiceserveasanimportantextrememousemodelfortheherediataryspinocerebellarataxiatype1(SCA1),becauseRORαintereactsindi-rectlywithamoleculecalledataxin1,whosepolyglutamineexpansionleadstoSCA1pathogenesis,andRORαdepletionisstronglycorrelat-edwithseverityofthedisease.Morphologicalanalysesofsg/sgcere-bellumhavebeenperformedextensively,butfunctionalsynapticab-normalitieshavenotbeenexaminedindetail.Inthispresentation,weshowthatmetabotropicglutamatereceptortype1(mGluR1)signalingiscompletelydisruptedatparallelfiber-Purkinjecell(PF-PC)synapsesinSCA1-relatedsg/sgmice,althoughAMPA-receptormediatedfastsynaptictransmission(fastEPSC)isrelativelypreserved.Sg/sgmicelackedmGluR1-mediatedslowsynapticresponsescompletely,andmodulationofthefastEPSCcausedbymGluR1-mediatedendocanna-binoidreleasefromPCswasalsoabolishedatsg/sgPF-PCsynapses.Inmorerealisticdiseasemodelmice,SCA1transgenicmicewithprolongedglutaminerepeatsof theataxin1gene,wepreliminarilyobserved impairmentofCa2+signalingmediatedbymGluRinPCs.Thesedataindicatethatsg/sgandSCA1micehavecommondeficien-cyinmGluR1signalingandwehypothesizethatdisruptionofmGluR1signalingisamajorsharedmechanismofcerebellarataxiabetweensg/sgandSCA1mice.NoCOI.

1S5F-3Regulation of the state of neuronal maturation by ex-citation/inhibition balance in adult hippocampusKobayashi, Katsunori1,2(Department of Pharmacology, Nippon Medical School, Tokyo, Japan, 2JST,CREST, Kawaguchi, Japan)

Theaberrantoralteredstateofneuronalmaturationhasbeenimpli-cated inthepathophysiologyofpsychiatricdisordersandcellularmechanismsofantidepressantdrugs.Wehaveshownthattheseroto-nergicantidepressantfluoxetinecanreversethestateofmaturationofthegranulecellsinthedentategyrusoftheadultmousehippocam-pus.Thisdematurationisinducedinalargepopulationofthedentategranulecellsandcausessignificantchangesinphysiologicalpropertiesofthegranulecells,includingenhancedexcitatorysynapticinput,in-creasedsomaticexcitabilityandreducedfrequencyfacilitationattheoutputsynapse.Therefore,thegranulecelldematurationissupposedtohaveasubstantialimpactonthefunctioningoftheneuronalcircuitinthedentategyrus.Werecentlyfoundthatelectroconvulsivestim-ulation(ECS)canalsoinducethegranulecelldematuration.ECS-in-duceddematurationcanbepartiallyreversedbyenhancingsynapticinhibition,suggestingapotentialimportanceofexcitation/inhibitioninthemaintenanceofdematuredstate.Ourresultsuggeststhatthestateofmaturationofneuronsintheadultbrainisdynamicallyregu-latedbyneuronalactivity.Thereciprocalrelationshipbetweenthestateofneuronalmaturationandcircuitfunctioningmayplayacriticalroleinsustainedeffectsofantidepressanttreatmentonthebrain.NoCOI.


1S5F-4Distinct pH and buffering capacity between glutama-tergic and GABAergic synaptic vesicles in hippocam-pal neuronsEgashira, Yoshihiro1; Takase, Miki1; Yanagawa, Yuchio2; Takamori, Shigeo1(1Laboratory of Neural Membrane Biology, Graduate School of Brain Science, Doshisha University, kyoto, Japan, 2Department of Genetic and Behavioral Neuroscience, Gunma University Graduate School of Medicine, Gunma, Japan)

ExocytoticreleaseofneurotransmitterssuchasglutamateandGABA,firstrequirestransportofneurotransmitterintosynapticvesicles(SVs),whichcriticallydependsonaprotonelectrochemicalgradientgener-atedbythevacuolarH+-ATPase.BiochemicalanalysesusingisolatedSVshavedemonstratedthattransportofglutamateandGABAexhib-itsadifferentialdependenceonthepHgradientacrosstheSVmem-brane.However,whethergradientitselfisdifferentbetweentheves-iclepopulationsremainsunknown.ByusingculturedhippocampalneuronsderivedfromtheVGAT-Venustransgenicmousebrainandlentivirusexpressionofsynaptophysin-mOrange2whichissensitivetotheluminalpHoftheSVs,wefoundthatGABAergicSVsexhibit~0.7unithigherluminalpHand<2-foldlowerbufferingcapacity(BC)comparedtoglutamatergicSVs.TheseremarkabledifferencesimplydistinctsusceptibilitiesofglutamatergicandGABAergictransmissiontoalterationsinpHandBCunderpathophysiologicalconditions.More-over,sinceacidificationofthesynapticcleftbyprotonreleasefromSVshasbeenimplicatedinmodulationofpresynapticcalciumchannelsandionotropicneurotransmitterreceptors,ourdatademonstratethatglutamatergicSVscanreleaseapproximately5timesmoreprotonsthanGABAergicSVs,therebyimplyingmoredynamicpHdependentsynapticmodulationatglutamatergicsynapses.NoCOI.

1S5F-5Nociceptive Amygdala is Actively Involved in Fear LearningWatabe, Ayako M; Sato, Masaru; Kato, Fusao(Dept Neurosci, Jikei Univ Sch Med, Tokyo, Japan)

Pavlovianfearconditioningisanassociativeformoflearningdepend-ingonamygdalasynapticplasticity.Thecentralamygdala(CeA)isknowntoplaypivotalrolesinthisassociativelearning(Wilenskyetal,2007;Ciocchietal,2010;Lietal.,2013).ThecapsularpartoftheCeA,orCeC,receivesaversiveinformationfromtwodistinctpathways:directpathwayarisingfromthedorsalhornviatheexternalpartofthelateralparabrachialnucleus(lPB)andindirectpathwayfromthebasolateralnucleusoftheamygdala(BLA)thatcarrieshighlyprocessedpolymodalsignals.SuchduplexorganizationwouldmaketheCeCstrategicallywellpositionedforconvergenceunderlyingthisassociativelearning,aconcepthoweverremaininghypothetical.Hereweprovidetwosetsofevidenceindicatingthatthesepathwaysareintegratedatasingleneuronlevel,promotingsynapticplasticityandareinvolvedinfearlearning.First,wefoundthatfearconditioningitselfpotentiatesexcitatorytransmissionatbothlPBandBLAsynapses.Interestingly,thesynapticweightsofthetwopathwayswerecorrelatedinfear-con-ditionedmice,suggestingaheterosynaptic interaction.Second,wefoundthatatransientpharmacological inactivationofthebilateralexternallPBduringconditioningsignificantlyattenuatedfearlearning.Onthebasisoftheselinesofevidence,weproposethattheCeCisactivelyinvolvedinfearlearningviaservingasaconvergencesiteforaversivesignalsofdistinctoriginsandpathways.Synapticplasticityofthesepathwaysmayregulatethesensitivityofemotionalcircuitstofutureaversivesignals.NoCOI.

Symposium 06Maintenance of Glucose Homeostasis

through Tissue-tissue Interaction

(March 16, 8:40–10:20, Room G)

1S6G-1Dynamic regulation of metabolite flow between adi-pose tissues and liver by insulinMiki, Takashi(Department of Medical Physiology, Chiba University Graduate School of Medicine, Chiba, Japan)

Insulinplaysakeyroleintheregulationofcarbohydrateandenergyhomeostasisandinsulinresistanceisthefundamentalpathophysiologyofdiabetesmellitus.Wefoundthatamouseharboringaloss-of-func-tionmutationininsulinreceptor(mIRmouse)developsoverthyper-glycemiaonlyunderhighfatdiet(HFD).mIRmiceunderHFD(mIR/HFD)exhibitedseverelyimpairedglucoseloweringeffectofinsulinasassessedby intraperitoneal insulin tolerancetest, suggestingthatgluconeogensis is increased in liver. Asexpected,wefoundthatglucose-6-phosphatase(G6pc)expressioninliverandgluconeogensisfromglycerolin vivowasincreasedinmIR/HFD.Glycerolisgener-atedthroughlipolysisoftriacylglycerolinwhiteadiposetissues(WAT)andinsulinpotentlysuppresseslipolysisbyinactivatingphosphoryla-tionofhormonesensitivelipase(HSL).Notably,phospho-HSLlevelsweresignificantlyincreasedinWATofmIR/HFD.Inaddition,trans-plantationofwild-typeWATintomIR/HFDmarkedlyamelioratedthehyperglycemia,suggestingthatunsuppressedlipolysisinWATandincreasedgluconeogensisfromglycerolinlivertriggersthedevelop-mentofdiabetesmellitus.SinceinsuliniscriticalinsuppressingbothlipolysisinWATandgluconeogensisinliver,defectiveinsulinsignal-inginmIRmiceandconsequentialincreaseinglycerolflowfromWATinto livercontributetoarise inbloodglucose levelsunderHFD.Accordingly,maintenanceofdynamicflowofmetabolitesbetweenmultipleorgansiscriticalinthemaintenanceofglucosehomeostasis.NoCOI.


1S6G-2Role of adipose tissue inflammation in ectopic fat ac-cumulation and insulin resistanceSuganami, Takayoshi1,3; Ogawa, Yoshihiro2(1Department of Organ Network and Metabolism, Tokyo Medical and Dental University, Tokyo, Japan, 2Department of Molecular Endocrinology and Metabolism, Tokyo Medical and Dental University, Tokyo, Japan, 3JST PRESTO)

Obesitymaybeviewedasachroniclow-gradeinflammatorydiseaseaswellasametabolicdisease.Indeed,macrophagesinfiltrateintoobeseadiposetissueto induce inflammatorypathways, thereby inducinginsulinresistanceandectopicfataccumulation.Inresponsetonutri-tionalconditions,lipidmetabolisminadiposetissueistightlyregulatedbyinsulinandthesympatheticnervoussystem.Adipocytesincreasetheirsize(hypertrophy)andnumber(hyperplasia)duringthecourseofobesitytostoretriglycerideeffectively inadiposetissue.Whenadiposetissuecannotmeetthedemandofstoringexcessiveenergy,triglycerideisaccumulatedinnon-adiposetissuesasectopicfat,whichmay leadto insulinresistance inthe liverandskeletalmuscleandinsufficient insulinsecretion inthepancreas (lipotoxicity).Notably,chronicinflammationiscapableofinducinginsulinresistance,lipolysis,andinterstitialfibrosisinadiposetissue,allofwhichmayreducethelipid-storingfunction.Recently,wefoundthatMacrophage-inducibleC-type lectin (Mincle),apathogensensor forpathogenic fungiandMycobacteriumtuberculosis,playsacriticalroleinadiposetissuefi-brosis,therebyregulatingectopicfataccumulationintheliver.Under-standingthemolecularmechanismunderlyingadiposetissuefibrosismayofferanoveltherapeuticstrategytopreventortreatobesity-in-ducedmetabolicderangement.NoCOI.

1S6G-3Central insulin action induces IL-6-mediated suppres-sion of hepatic glucose productionInoue, Hiroshi; Kimura, Kumi(Department of physiology and metabolism, Brain/Liver Interface Medicine Research Center, Kanazawa University, Japan)

Centralinsulinactionisnotonlyinvolvedintheregulationofenergymetabolismviatheregulationoffoodintakeandheatproduction,butalsoengaged inglucosemetabolismbyregulatinghepaticglucoseproduction(HGP).Severalpapershavedemonstratedthathyperpolar-izationofAgRPneuronsinducedbytheactivationofPI-3Ksignaling/KATPchannelsinthehypothalamusparticipatesinthesuppressionofHGPmediatedbycentral insulinaction.Thecentral insulinac-tion-mediatedsuppressionofHGPresultsfromdecreasedgeneexpres-sionofenzymesinvolvedinhepaticgluconeogenesis,andbothincreasedIL-6expression inhepaticKupffercells inducedbycentral insulinactionandassociatedactivationofhepaticSTAT3playanimportantroleinthesuppressionofgeneexpressionofhepaticgluconeogene-sis-relatedenzymes.Recently,wefoundthatincreasedplasmahistidineresultedinhepat-icSTAT3activation.Intravenousandintracerebroventricularadmin-istrationofhistidinealsoactivatedhepaticSTAT3andaugmentedthesuppressionofHGPbyinsulin.TheincrementinHGPinhibitionbyhistidinewasblockedby inhibitinghistamineH1receptors inthecentralnervoussystem.Therefore,histidineactivateshepaticSTAT3andsuppresseshepaticglucoseproductionviacentralhistamineaction.Theseresultsthus indicatethatIL-6-STAT3signaling inthe livercontributestocentralactionofinsulinandhistamine,leadingtothesuppressionofHGP.NoCOI.

1S6G-4Role of the ventromedial hypothalamus in leptin-in-duced glucose metabolic regulation in skeletal mus-cle and the liverMinokoshi, Yasuhiko; Toda, Chitoku(Div Endocrinol Metab, Natl Inst Physiol Sci, Okazaki, Japan)

Leptinisanadipocyte-derivedhormonethatplaysanimportantroleinglucosemetabolisminmammals.Treatmentwithleptinamelioratesdiabetes in lipodystrophicmiceandhumansaswellas insulindefi-cient-diabetesinrodents.Wepreviouslyshowedthatinjectionofleptinintotheventromedialhypothalamus(VMH)increasesglucoseuptakebyskeletalmuscle(mainlytheredtype),brownadiposetissue,andtheheart,butnotthatbywhiteadiposetissue,throughactivationofthemelanocortinreceptor (MCR) intheVMH.Wenowshowthatsignalingbyextracellularsignal-regulatedkinase (ERK)and itsup-streamkinaseMEKinmediatestheleptin-inducedincreaseinglucoseutilizationaswellasitsinsulinsensitivityinthewholebodyandinred-typeskeletalmuscleofmicethroughactivationoftheMCRintheVMH.Incontrast,activationofsignal transducerandactivatoroftranscription3(STAT3),butnotthatoftheMEK-ERKpathway,intheVMHbyleptinenhancestheinsulin-inducedsuppressionofen-dogenousglucoseproductioninanMCR-independentmanner,withthiseffectof leptinoccurringonly inthepresenceofan increasedplasmaconcentrationofinsulin.Giventhatleptin,butnotMCRagonistMelanotan-II,requires6hto increasemuscleglucoseuptake, thetransientactivationofMEK-ERKpathwayintheVMHbyleptinmayplayanroleintheinductionofsynapticplasticityintheVMH,result-ingintheenhancementofMCRsignalinginthenucleus,andleadingtoanincreaseininsulinsensitivityinred-typemuscle.NoCOI.

Symposium 07Advances in targeted genome editing

and its application to physiology

(March 16, 10:25–12:05, Room G)


1S7G-1Targeted genome editing using highly-active TALENs and CRISPR/Cas9 Yamamoto, Takashi(Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-hiroshima, Japan)

Genomeeditingwithengineerednucleasesisanemergingtechnologythatenablesmanipulationoftargetedgenesinmanyorganismsandcelllines.Todate,twotypesofengineerednucleaseshavebeende-veloped.Zincfingernucleases(ZFNs),whichfirstemergedin1996,havealongandsuccessfulhistoryofgenomeediting.However,theconstructionofZFNsishighlylaboriousandthesuccessrateofactiveZFNconstruction isvery low.Transcriptionactivator-likeeffectornucleases(TALENs),ontheotherhand,havebeenrecentlydevelopedasamoreuser-friendlyengineerednuclease,becauseTALENsareeasytoconstructin-houseandthetargetsitemaybeselectedinanygene.Usingtheseengineerednucleases, targetedgenedisruptionshavebeenachievedsuccessfullyinculturedcellsandvariousorgan-isms.Furthermore,genecorrectionortargetedgeneadditionhavebeenreportedmainlyinculturedcells,andalsoinseveralorganisms(Ochiaietal.,2012).Surprisingly,athirdgenomeeditingtechnology,knownasCRISPR/Cassystem,suddenlyemergedlastyearandeffi-cientgenomeeditingusingthissystemwasreportedinESandiPScells. Inthisway,genomeeditingtechnology isnowbecomingthemostexcitingfieldinlifesciences.Inthispresentation,Iintroducetherecentresearchadvancesingenomeeditingusingengineerednucle-asesandCRISPR/Cas9.IshowtheconstructionsystemforhighlyactiveTALENsthatwenamedPlatinumTALENsandresultsofgenomeeditinginvariousorganismsusingPlatinumTALENs.NoCOI.

1S7G-2Rapid and highly efficient in vivo genome editing in miceAida, Tomomi1; Sakuma, Tetsushi3; Usami, Takako2; Ishikubo, Harumi1; Imahashi, Risa1; Yamamoto, Takashi3; Tanaka, Kohichi1(1Laboratory of Molecular Neuroscience, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan, 2Laboratory of Recombinant Animals, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan, 3Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan)

Gene-targetedknockoutorknockinmicehavedramaticallyimprovedourunderstandingofthefunctionsofgenesin vivo.However, thegenerationoftargetedmicereliesongenetargetinginembryonicstem(ES)cells,whichisatime-consuming,laborious,andexpensiveprocess.Therecentgroundbreakingdevelopmentofgenomeeditingtechnol-ogieshasenabledthetargetedalterationofanysequenceinanycellororganism.Thesetechnologieshavenowbeendirectlyappliedtomousezygotes(in vivogenomeediting),therebyprovidingnewavenuesforsimple,convenient,highlyefficientandultra-rapidproductionoftargetedmicewithoutEScellsortargetingvectors.Wehavegener-atedseveralknockinmouselinescarryingpreciselymodifiedhumansinglenucleotidevariantsaswellasknockoutmouselinesbyin vivogenomeeditingwithhighlyactivePlatinumTALENs(transcriptionactivator-likeeffectornucleases)andoligoDNAdonor.Becausetheefficiencyofourmethodishigh,severalgermline-competentknockout/knockinfounderscanbeobtainedwithinamonthbysinglemicroin-jection.Takentogether,in vivogenomeeditingtechnologyrevolution-izesmousegenetargetingandprovidesexcitingopportunitiesforthephysiologicalresearchtofreelygenerategene-targetedmice.NoCOI.

1S7G-3OptogeneticsTanaka, Kenji F.(Department of Neuropsychiatry, School of Medicine, Keio University, Tokyo, Japan)

Optogeneticshasproventobeapowerfultoolcapableofmanipulatingtheactivityofaspecificpopulationofcellsinacomplexmulticellularorganism.Thisapproachisenthusiasticallypursuedinrecentneuro-sciencefieldandthecausalrelationshipbetweenneuralactivityandbehaviorisfinallystartingtobecomeunveiled.However,moststudiesutilizevirusmediatedgenetransferfortheinductionoflight-sensitiveproteins,suchaschannelrhodopsin-2(ChR2),andsuchmethodinevi-tablyintroducesvariabilityofexpressionbetweentrials.Therefore,transgenicapproachhas longbeensought,however,satisfyingthedemandsofthespecificityaswellastheabundanceofexpressionweredifficult.Here,weestablishedKnockin-mediatedENhancedGeneEx-pressionbyimprovedtetracycline-controlledgeneinductionsystem(KENGE-tet).WefoundthathighlevelsoftTA-mediatedtranscriptioncanbeachievedbyknockingintetO-ChR2cassetteintoalocusatahousekeepinggene,beta-actin.WecrossedthistetO-ChR2knockinmousewith7differenttTAlinesandinallcasesthelevelofChR2expressionwashighenoughtoallowmanipulationofcellactivity.Thistechnological improvement,KENGE-tet,enablesustoexpandtherepertoireofoptogeneticallytargetedcellsandwouldprovideresourc-esbeneficialtotheneurosciencefield;furthergenerationofnewlinesofeithertetOortTAlinewouldaddevenmorerepertoireforscientiststochoosefrom.Webelievethatexpandingrepertoirewillbeofgreatinteresttothelargecommunityofneuroscientistswhoareactivelypursuingthecausalrelationshipbetweencellularactivityandbehav-iorofcomplexorganism.NoCOI.

1S7G-4The problems and limitations associated with geneti-cally engineered animalsTanaka, Kohichi(Lab of Mol Neurosc, Medical Research Insitute, Tokyo Medical & Dental University (TMDU), Tokyo, Japan)

Genemodificationtechnologiesplayacriticalroleinmanyfieldsofresearch,ragingfromphysiologytoneurosciencetogenetics.Althoughgeneticallyengineeredmicehavebecomeinvaluabletoolsforresearch-erstryingtofindouttheinvivofunctionsofindividualgenes,genet-icallyengineeredmicehavetheirlimitations.Thepotentialproblemsandlimitationsofgenetargetingtechnologyareasfollows.About15%ofconventionalknockoutmicearedevelopmentallylethal.Knockoutmicemaynotshowanyobservablechange.Thegeneticbackgroundofaknockoutstraincanhaveaninfluenceonanyobservedphenotype.Somegenes,suchasgenesonYchromosome,aredifficulttoknockout.Acomplicatedcompensatoryresponsetothegene’sabsencecanmaskthephenotypesofknockoutmice.Conditionalgenetargetingisanapproachtoovercomingthesediffi-culties.However,thereexistdebateoverthetissuespecificityandtheefficacyofconditionalknockoutmice.NoCOI.


Symposium 08Progress of Retinal Research

for Neural Circuits

(March 16, 8:40–10:20, Room H1)

1S8H1-1Neuronal circuit for topographically focal facilitation of visual response in the avian retinaUchiyama, Hiroyuki1; Matsutani, Shinji2; Ohno, Hiroshi1; Yamaoka, Seiya1; Mizokami, Takuya1(1Dept Informatics & Biomedical Engineering, Grad Sch, Kagoshima Univ, 2Dept Functional Morphology, Sch Nursing, Kitasato Univ)

Isthmo-optic (IO)neurons intheavianmidbrainsendtheiraxonstopographicallytothecontralateralretina.BecauseIOneuronsvigor-ouslyfirepriortoonsetofgoal-directedheadmovementsinapartic-ulardirection,andlesionofthoseneuronsimpairsvisualtargetselec-tionforreachingwithbeak,IOneuronsarethoughttocentrifugallysendvisual-selection-relatedsignalinordertoenhanceretinalvisualresponsefocallyandtransiently.TheIOneuronsmakesynapticcon-tactswithaspecificclassofretinal intrinsiccells, IOtargetcells(IOTCs).IOTCshaveanaxonofvariouslengthsthatextendshorizon-tallyinthejunctionbetweentheinnernuclearlayer(INL)andtheinnerplexiformlayer (IPL),andterminateatstratum1oftheIPL.BecauseIOTCsdonotcontactdirectlywiththeretinalganglioncells(RGCs),someretinal intrinsiccells (specificallyamacrineorbipolarcells)mustrelaythecentrifugalsignalstoRGCsintheIPL.Basedontheimmunohistochemicalexaminationatthelightmicroscopiclevel,wesupposethatthebipolarcellthatisimmunoreactiveforproteinkinaseC (PKC-BC) isaprobablecandidateofthetargetofIOTCs.AxonterminalsofIOTCsappeartoterminatemainlyonabasalportionofthehorizontalaxonalbranchesofPKC-BCsatstratum1oftheIPL.ThusitispossiblethatthecentrifugalsignalmaytransientlyimprovesynaptictransmissionefficacybetweenPKC-BCsandRGCs,perhapsthroughasignaltransductioncascadewithinPKC-BCs.NoCOI.

1S8H1-2Visualization of the glutamate release in the mouse retinaOhkuma, Mahito1; Horio, Kayo1; Kaneda, Makoto2; Yosida, Sachiko3; Fukuda, Atsuo4; Miyachi, Ei-ichi1(1Dept. of Physiol., Sch. of Med., Fujita Health Univ., Aichi, Japan, 2Dept. of Physiol., Nippon Medical School, Tokyo, Japan, 3Dep. Env. & Life Sci., Toyohashi Univ. Tech., Aichi, Japan, 4Dept. of Neurophysiol., Hamamatsu Univ. Sch. of Med., Shizuoka, Japan)

Glutamateisaneurotransmitterofphotoreceptorsandbipolarcellsintheretina.Becauseoftheimportanceofglutamatergiccircuitsintheretina,manyresearcherschallengedmeasurementofglutamatereleasefromtheiraxonterminals.Glutamatereleasefrombipolarcellshasbeenwellexaminedbythebioassaysystemandthatfromphotore-ceptorshasbeendetectedbythefluometricmethod.However,detec-tionofglutamatereleaseinthesestudieshasbeenlimitedatthesinglecelllevel.Inthepresentexperiment,weappliedtheenzyme-linkedphotoassaytechniqueforslicepreparationofthemouseretinatodetectglutamatereleaseatthecircuitlevel.Inthissystem,anappli-cationofglutamateinducedanincreaseinfluorescentsignals,whileanapplicationofacetylcholine,GABAorglycinedidnot.Whenhigh-Kstimulationwasusedtoevokeglutamaterelease,twotypesofincreaseinfluorescentsignalsweredetected.Anincreaseinfluorescencesignalneartheoutersegmentofphotoreceptorswasglutamate-independent.Intheinnerretina,anincreaseinfluorescentsignalwasglutamate-de-pendent.Applicationof100µMacetylcholinedidnotproduceanyincreaseinfluorescentsignals.Theenzyme-linkedphotoassaytech-niqueisusefultomeasuretheglutamatereleaseatthecircuitlevelinthemouseretina.NoCOI.

1S8H1-3Pathway-dependent modulation of cholinergic amac-rine cells in the mouse retina. Ishii, Toshiyuki; Kaneda, Makoto(Dept. Physiol., Nippon Med. Sch., Tokyo, Japan)

Cholinergicamacrinecellsplayapivotalrolefortheformationofdi-rectionselectivity,whichisamanifestfeatureofthedendriticcompu-tation intheretina.However, the input-outputrelationshipof thecholinergicamacrinecellshasnotbeenelucidated.Inthisstudy,weexaminedhowsignalsofpurinergic,glycinergicandglutamatergiccircuitsareprocessedincholinergicamacrinecellsinthemousereti-na.Thepurinergic inputsweredominant intheOFF-cholinergicamacrinecells,while theglycinergic inputsweredominant intheON-cholinergicamacrinecells.BothON-andOFF-cholinergicamacrinecellsreceivedglutamatergic inputsequally.Purinergic inputsweremediatedbyP2X2-purinoceptorsandglycinergicinputsweremediat-edby,glycinereceptors.Glutamatergic inputsweremediatedbyAMPA/KAandNMDAreceptors.Inthepreviousstudy,wehavereportedthatthemodulatoryactionoffiringactivitybyP2-purinocep-tor-mediatedsignalsisdistinctbetweenON-andOFF-ganglioncells.Theretinaisaskedtoadjustitsvisualactivitytothewiderangeoflightintensity,i.e.moonlightleveltoshinyseasidelevel.Toachievethis,retinaisthoughttoshiftthethresholdofON-orOFF-pathwayindependently.Thepresenceofpathway-specificinputstocholinergicamacrinecellsmight favor forthepathway-specificadjustmentofdynamicrangetoachievethebestvisualacuityinthemouseretina.NoCOI.


1S8H1-4Coding of unstationary images by retinal ganglion cellsMatsumoto, Akihiro; Tachibana, Masao(Department of Psychology, Graduate School of Humanities and Sociology, The University of Tokyo, Tokyo, Japan)

Visualperceptionisstable.However,retinalimagesarenotstabilizedbutchangepositionbymovementsofeyes,head,andbody.Itisnotevidenthowunstationaryretinalimagesarecodedbyretinalganglioncells.Applyingthemulti-electrodearraytotheisolatedgoldfishretina,werecordedspikedischargesfrommultipleganglioncells.DynamiclightstimulipresentedonaCRTdisplaywereprojectedontoawidearea(visualangle:~80degree)oftheretinathroughoptics.Alargesquaretargetwaspresentedwithorwithoutasurroundingback-ground.Tomimicthemovementofretinalimages,thetargetandthebackgroundwasjitteredorrapidlymovedtogetherorindependently.Basedonthereceptivefieldprofileestimatedbythespike-triggeredaverage,weclassifiedganglioncells intosixsubtypes (Fast/Slow,transient/medium/sustained).Wefound insomesubtypesthatthelatencyofthefirstspikeevokedbytherapidlymovingtargetwasshorterwhenthetargetmovedtogetherwiththesurroundingback-groundthanwithoutbackground.Especially,theFast-transientcellsfiredsynchronouslybeforetheleadingedgeofthetargetreachedtheirreceptivefields.Cross-correlationanalysisrevealedthat functionalconnectivitywasnewlyestablishedamongtheFast-transientcellsandotherspecificsubtypesunderthisstimuluscondition.Theseresultssuggestthateyemovementsmaychangethefiringpropertiesandfunctionalconnectivityofretinalganglioncells,andthatmultiplesubtypesofretinalganglioncellsmaysendvisualinformationcooper-ativelytothebrain.NoCOI.

Symposium 09Retinal Signaling to the Brain:

Modulation by Light, Correlation with Behavior, and Restoration of Vision

(March 16, 10:25–12:05, Room H1)

1S9H1-1Retinal Ganglion Cell Spikes Are Not All-or-None EventsIshida, Andrew; Ogata, Genki; Stradleigh, Tyler; Hayashida, Yuki; Oi, Hanako; Partida, Gloria(NPB-OVS, University of California, Davis)

Spikefrequencyandtimingarecommonlyusedtomeasurephysio-logicalresponses.Thistreatsspikesasdigitalsignalsandassumesthatstimulialteronlytheprobabilitythatspikesoccur.Wehaveexploredotherpossibilitiesinthevertebrateretina,wheresomeinterneuronsreleasemodulatoryneurotransmitters(e.g.,biogenicamines)andtheoutputneurons (retinalganglioncells,RGCs)areequippedwithawidervarietyofionchannelsthanareneededtogeneratespikes.Wehavefoundthatdopaminereceptoragonistsreducetheamplitudeofvoltage-gatedsodiumcurrentinRGCsandthat,consistentwiththis,dopaminealterstheamplitudeanddurationofRGCspikes.Wehavealsofoundthatdopamineactivatesamixtureofclassicalandnon-clas-sicalreceptorsinRGCsand,consistentwiththis,thatdopaminerecep-toractivation (byfull-field illumination)elevatesacyclicnucleotide(cAMP)andactivatesacalcium/calmodulin-dependentproteinkinase(CaMKII).TheseresultsareofinterestbecausecAMPregulatessomeionchannelsinRGCs;cAMPfuelsaproteinkinase(PKA)whichmod-ulatesother ionchannels inRGCs; thecAMP-andPKA-regulatedchannelsgateathyperpolarizedanddepolarizedmembranepotentials,respectively;and illumination inducestheappearanceofactivatedCaMKIIinRGCnuclei.Theseresults,alongwithresultspublishedbyotherlaboratories,showthatthatRGCspikesarenotall-or-noneeventsand indicatethat lightmayregulatespikesatdifferentmembranepotentialsandspeeds.Some,ifnotall,oftheseeffectscouldaltertheabilityofRGCstocommunicatewiththebrain.NoCOI.

1S9H1-2Neural representation of visual information for escape behaviorIshikane, Hiroshi1,2(Department of Psychology, Senshu University, 2Center for Psychological Science, Institute for the Development of Social Intelligence, Senshu University)

Recentstudieshaveshownthatretinalcircuitsareinvolvedinmoreadvancedinformationprocessingthanthatpreviouslypostulatedbyvisionscientists.Variousextractedstimulusfeaturescanberepresent-edbythespiketrainsofretinalganglioncells,althoughitishardtodemonstrateiftheobservedretinalcodesaredecodedandutilizedinthenextstage.Infrogretina,ithasbeenshownthatsynchronizedoscillationsamongdimmingdetectors(class-4neurons)encodevisualinformationforescapebehavior.Pharmacologicalmanipulationsofthestrengthofsynchronizedoscillationscausechangesinthefrog’sescapebehavior.However,itisstillunclearwhatvisualfeaturesareencodedbyretinaloscillations.Toelucidatethisissue,thestimulusdependenceofsynchronizedoscillationswasinvestigated.Thestrengthofsynchro-nizedoscillationsdependedbothonthecontinuityandthesizeofvi-sualstimuli.Furthermore,spikedischargeswererecordedfromdim-mingdetectorsaswellastheotherthreetypesofretinalganglioncellsinordertoidentifytheretinalcodethattriggeredthefrog’sescapebehavior.Theresultsofthebehavioralandelectrophysiologicalexper-imentssuggestedthattheactivitiesofclass-1and-2neuronsdonotplayafunctionalroleintriggeringescapebehavior.Bothsynchronizedoscillationsamongdimmingdetectorsandthedirectionalactivitiesofclass-3neuronsmayencodevisualinformationforescapebehavior.NoCOI.


1S9H1-3Microstimulation for Visual Prosthetic InterfacesHayashida, Yuki; Kameda, Seiji; Ishikawa, Naohiro; Takeuchi, Kouzou; Tanaka, Hiroki; Okazaki, Yuka; Yagi, Tetsuya (Graduate school of Engineering, Osaka University)

Visualprosthesesutilizemicrostimulationstothevisualpathwaybywhichperceptiblephosphenescanbeinducedinblindpatients.Pre-viousstudiessuggestedthatthenumberofstimulatingelectrodesasmuchasseveralhundredisrequiredforprovidingacertaindegreeofusefulvisualsensation,althoughelectricalcurrentdeliveredfromeachelectrodecanspatiallydiffuseinthetissue,activatingamassofneuronsatatime.Thus,thecortical-basedprosthesescanbeadvan-tageousbecauseoftherelativelylargeareaavailableforimplantingmultipleelectrodeswithenoughspacing.Besidesthediffusivestimuluscurrent,ourexperimentswithavoltage-sensitivedyeimaginginthemouseorratvisualcortexinvivohaveshownthatneuralresponsestoevensingle-electrodestimulationsignificantlyvariedwiththepa-rameterssuchaspulseamplitude,inter-pulseinterval,dutyratioofpulses inatrain.Moreover,neuralresponses inducedatspatiallyseparatedtwostimulationsitescan interactnonlinearlywitheachother.These impliedthecomplexrelationshipbetweenpatternsofelectricalstimulationandtheresultingneuralactivities,whichhaveremainedlargelyunrevealed.Inordertoenablesystematicanalysesonsucharelationshipinpreclinicalanimalexperiments,werecentlydevelopedaCMOS-VLSIchipinwhich64channelcurrent-pulsestim-ulatorswereimplementedinanimplantablesize.Thecurrentpulsesgeneratedbythestimulatorwefabricatedwereabletoinduceneuralresponsesintherodentvisualcorticalareas.Thus,ourstimulatorchipisexpectedtobeuseful for furtheranimalstudies,and inturnforoptimizingthedesignofvisualprostheticinterfaces.NoCOI.

1S9H1-4Application of optogenetic technologies to vision—Restoring vision for blind patients—Tomina, Hiroshi; Sugano, Eriko; Murayama, Namie; Tabata, Kitako; Takahashi, Maki; Saito, Takehiko; Nishiyama, Fumiaki; Tamai, Makoto(Dept. of Chemistry and Bioengineering, Iwate University)

Channelrhodopsin2(ChR2)derivedfromtheunicellulargreenalgaeChlamydomonasreinhardtiihasuniquecharacteristicsasalight-acti-vatedcation-selectivechannel.Thisspecificfeatureallowsustogen-eratephotosensitiveneuronsbythetransferofasinglegene,ChR2,toneuronalcells.ThediscoveryofChR2andadvancesintechniqueshasintroducedthefieldofoptogeneticsinneuroscience.Anewstrat-egyforrestoringvisionincludesthetransductionoftheChR2geneintoretinalganglioncells(RGCs)orON-bipolarcellsingeneticallyblindmiceandrats.Theseanimalsaremodelsforretinitispigmentosa(RP),whichisaretinaldegenerativediseaseassociatedwithalossofpho-toreceptors,arisingfrommutationsingenesrelatedtothephototrans-ductionpathwayintheretina.Thisprogressivephotoreceptordegen-erationfinally leads toblindness.Gene therapyusingChR2 is apotentialmethodforrestoringvisiontoRPpatients.However, thevisualfunctionofChR2lackssensitivitytowavelengthsover540nm.VChR1derivedfromVolvoxcatericandetectlongerwavelengthsthanthoseforChR2andisthusanothercandidategeneforrestoringvision.WegeneratedmodifiedVolvoxchannelrhodopsin-1(mVChR1),whichisachimeraofVolvoxchannelrhodopsin-1andChlamydomonaschan-nelrhodopsin-1anddemonstratedincreasedplasmamembraneinte-grationanddramaticimprovementinitschannelproperties.Wein-troducethevisualfunctionofblindratstransducednewlydevelopedmVChR1.NoCOI.

Symposium 10Renal tubular transport function in the 21st century: insights from transgenic

and knockout animals

(March 16, 10:05–12:05, Room H2)

1S10H2-1Regulation of water and electrolytes transport in kid-ney tubule cells: Vasopressin V1a receptor and calci-um sensing receptorYasuoka, Yukiko; Kawahara, Katsumasa(Dept of Physiology, Kitasato-univ. Sch, of Med, Sagamihara, Japan)

AQP2intheprincipalcells (PC)ofkidneycollectingducts (CD) isstimulatedbythevasopressinV2receptoraxisandismodifiedbytheV1areceptoraxis(Bankir.2001).Moreover,luminalhigh[Ca2+]causesdownregulationofAQP2expression inPC (Renkemaetal.2009)throughactivationofcalciumsensingreceptorsignalpathway(CaSR-Ca2+-PKCaxis).SinceYasuokaetal.(2013)foundthatV1aRonlylocal-izedintheintercalatedcelltypeA(IC-A)alongtheCD,wehavein-vestigatedtheamountofAQP2expressioninWTandV1aR-/-mice.TheluminalexpressionofAQP2significantlyincreasedbothinWTandV1aR-/-duringdehydration,althoughtheir levelsofexpressionsignificantlydecreasedinV1aR-/-vscontrol.ThissuggeststhatV1aRinIC-AmaintainsandstimulatestheexpressionofAQP2throughunknowncellularpathways.Ontheotherhands,wefoundthatCaSR,geneticallysameasCaSRinTAL,only localized inthebasolateralmembraneofintercalatedcelltypeB(IC-B)throughtheCD(Yasuokaetal.2011).We investigatedtheurineconcentrationandbodypHmetabolisminmicefedcalciumandacid/basediets.ThelevelsoftheCaSRmRNAandproteinexpressioninIC-Bwereincreasedandde-creased,respectively,duringalkali-andacid-loading.CaSRofIC-Bmaycontributetoalkalisecretiontopreventurolithiasiscausedbyhyper-calciuria.V1aRandCaSR,respectively,maycontributetowaterandelectrolytestransportbymaintainingthebasalexpressionofAQP2andbystimulatingalkalisecretioninkidneycollectingducts.NoCOI.


1S10H2-2Analysis of urate transporter gene-overexpressed mice for the study of renal tubular epithelial transport of urateAnzai, Naohiko1; Jutabha, Promsuk1; Kimura, Toru2; Fukutomi, Toshiyuki2(1Department of Pharmacology and Toxicology, Dokkyo Medical University School of Medicine, Tochigi, Japan, 2Department of Pharmacology, Kyorin University School of Medicine, Tokyo, Japan)

Urate,theionizedformofuricacid,isabreakdownproductofingest-edandendogenouslysynthesizedpurinenucleotidesinhumans.Renaluratehandlingplaysanimportantroleinthedeterminationofserumuratelevel.Wehaveidentifiedaurate-anionexchanger,URAT1,lo-calizedattheapicalsideandavoltage-drivenurateeffluxtransporter,URATv1,expressedatthebasolateralsideofrenalproximaltubules.BothURAT1andURATv1arefundamentaltorenaluratereabsorp-tionbecausetheloss-of-functionmutationsofthesetransporterscausehypouricemia.Sincerecentgenome-wideassociation (GWA)studieshaverevealedcloseassociationsbetweenserumurate levelsandsinglenucleotidepolymorphisms(SNPs)mainlyintwotransporter-re-lated genes such as SLC2A9 (GLUT9/URATv1), and SLC22A12(URAT1),wehaveconstructedkidney-specifictransgenic(Tg)miceforURAT1orURATv1.EachtransgenewasunderthecontrolofthemouseUrat1promotersothattransgeneexpressionwasdirectedtothekidney.PlasmaurateconcentrationsinURAT1andURATv1Tgmicewerenotsignificantlydifferentfromthatinwild-type(WT).UrateexcretioninURAT1TgmicewassimilartothatinWT,whileURATv1TgmiceexcretedmoreuratecomparedwithWT.OurresultssuggestthatincreasedURATv1functioninthekidneyscanleadtoincreaseduratereabsorptionandmaycontributetothedevelopmentofhyper-uricemia.NoCOI.

1S10H2-3CARS microcopy and high-speed AFM: two emerging measurement techniques useful for epithelial trans-port physiologySohma, Yoshiro; Yamashita, Hayato; Yu, Ying-Chun; Yasui, Masato(Department of Pharmacology, Keio University School of Medicine, Tokyo, Japan)

Recentgreatadvancesinthenonlinearopticalscienceandthenano-technologygiveusnovelkindsofinformationinthebiomedicalsciencefield.Inthissymposium,wewillintroduceourchallengesfortheap-plicationof twoemergingmeasurementtechniques,CoherentAn-ti-stokesRamanScattering(CARS)microcopyandHigh-SpeedAtom-icForceMicroscopy(HS-AFM),totheepithelialtransportphysiology.TheCARSlaser-scanningmicroscopygivesusH2OimagesincellsandtissuesfromtheO-Hstretchvibration-specificscattering.Applyingthistechniqueto3DcystsformedbyMDCKcells,wesucceededtomakeadirectobservationofH2O/D2OexchangeprocessacrosstheMDCKepitheliumanddeterminedthediffusionalwaterpermeabilityof luminalandbasolateralmembranesbyanalyzingthetime-lapseimagedatawithacomputersimulationmodel.TheHS-AFMhasenabled, forthefirsttime,directvisualizationofdynamicstructuralchangesanddynamic interactionsoccurring inindividualbiologicalmoleculesinasolution,whichhasbeenimpossiblewithothertechniques.TheHS-AFMsucceededto imagedynamicstructuralchangesinmembraneproteinsincludingCFTRchannelsandalsovisualizethebinding/unbindingprocessofautoantibodiestohumanaquaporins.Weexpectthatthesetwotechniqueswilltakeanewturnintheepi-thelialtransportphysiology.NoCOI.

1S10H2-4An attempt to create xenotransplantation-competent cloned piglets by the next-generation KO system CRISPR/Cas9Sato, Masahiro1; Miyoshi, Kazuchika2; Nagao, Yozoh2; Nishi, Yohei2; Ohtsuka, Masato3; Nakamura, Shingo4; Sakurai, Takayuki5; Watanabe, Satoshi6(1Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima, Japan, 2Lab Anim Reprod, Fac Agri, Kagoshima Univ, 3Div Basic Mol Sci Mol Med, Sch Med, Tokai Univ, 4Dpt Surg, Nat Def Med Coll, 5Dpt Org Regen, Grad Sch Med, Shinshu Univ, 6Anim Genome Res Unit, Div Anim Sci, Nat Inst Agrobiol Sci)

Therecentdevelopmentof theCRISPR/Cas9systemhasenabledgenomeeditingofmammaliangenomes;however,itsapplicabilityandefficiencyinthepighavenotbeenstudied.Here,usingthissystem,weaimedtodestroythefunctionoftheporcineα-1,3-galactosyltrans-ferase(α-GalT)gene(GGTA1)whoseproductisresponsibleforthesynthesisoftheα-Galepitope,acausativeagentforhyperacuterejec-tionuponpig-to-humanxenotransplantation.Porcineembryonicfibro-blastsweretransfectedwithaCas9expressionvectorandguideRNAspecificallydesignedtotargetGGTA1.Aftertransfection,thecellswere incubatedwithIB4conjugatedwithsaporin (IB4SAP),whicheliminatesα-Galepitope-expressingcells.AlmostallsurvivingcolonieswerenegativeforstainingwithIB4.SequencingofthemutatedportionofGGTA1revealedaframeshiftoftheα-GalTprotein.Porcineblas-tocystsderivedfromthenucleartransferoftheseα-Galepitope-neg-ativecellsalsolackedtheα-Galepitopeontheirsurface.TheseresultsdemonstratedthattheCRISPR/Cas9systemcanefficientlyinducethebiallelicconversionofGGTA1intheresultingsomaticcells,andisthusapromisingtoolforthecreationofKOclonedpiglets.NoCOI.

Symposium 11The time in physiology: Arrow of time

and Biological rhythms

(March 16, 9:00–11:00, Room J)


1S11J-1Misregulation of transcriptional program controlling the cellular differentiation disrupts the circadian clock developmentYagita, Kazuhiro(Department of Physiology and Systems Bioscience, Kyoto Prefectural University of Medicine)

Inmammals,variousphysiologicalaspectsarecontrolledbyaninter-naltime-keepingsystemcalledcircadianclock.Invivo,thedevelopmentofthecircadianclockstartsduringembryonicstages. Invitro, thecircadianclockformationoccurscell-autonomouslyinculturedmouseEScellsunderdifferentiatingconditions.Conversely,reprogrammingclockoscillatingsomaticcellsresultsinthedisappearanceofcircadianmolecularrhythms.Thesefindingsraisethehypothesisthatthede-velopmentofthecellularcircadianclockcloselycorrelateswithcellu-lardifferentiationmechanisms.Toaddressthisissue,wehereinves-tigatedthecircadianclockinhumanNeuroblastomacells,derivedfromanembryonic tumorwithabnormalcellulardifferentiation,duetohigh-levelexpressionofN-MYC.Thecircadianclockreportermoni-toringassayrevealedabolishedcircadianclockoscillationsinindivid-ualneuroblastomacells.Next,weestablishedmouseEScelllinesex-hibiting abnormal cellular differentiation because of alteredtranscriptionalprogramthroughdoxycyclin-induciblec-mycexpression.Constitutiveexpressionofc-mycresultedinthedisruptionofcircadi-anclockdevelopmentevenafterinvitrodifferentiationculture.Inter-estingly,allessentialclockgenesexpressed inthesecellswithoutshowingcircadiancycle.Altogether,ourdatasuggestedthatthemisregulationoftranscriptionalprogramcontrollingcellulardifferen-tiation,ratherthandirecteffectofMYC,mightdisruptthecellularcircadianclockdevelopment.NoCOI.

1S11J-2Toward a comprehensive analysis of Ataxin-2 funtions for the understanding of a common mechanism under-lying neurodegenerationKawahara, Yukio(Laboratory of RNA Function, Graduate School of Medicine, Osaka University)

Spinocerebellarataxiatype2isanautosomaldominantneurodegen-erativedisease.ThedisorderiscausedbyabnormalCAGrepeatex-pansioninthecodingregionoftheAtaxin-2gene(ATXN2).Recently,moderateCAGrepeatexpansioninATXN2wasidentifiedasariskfactorforamyotrophiclateralsclerosis.ThisfindingsuggestsacommonpathogenicroleofAtaxin-2intheseneurodegenerativediseases.There-fore,acomprehensiveunderstandingofthephysiologicalfunctionsofAtaxin-2,especiallyrelatedtodiseasepathogenesis, isnecessarytoelucidatethemechanismunderlyingAtaxin-2-mediatedneurodegen-eration.Ataxin-2isknowntodirectlybindtopolyadenylate-bindingprotein1(PABPC1),whichsuggeststhatAtaxin-2islikelyinvolvedinRNAmetabolism.However,thefunctionsofAtaxin-2havenotbeencharacterizedindetail.WehaverecentlyfoundthatAtaxin-2bindstosomeclass ofRNAsdirectly in aPABPC1-independentmanner.Throughdeepsequencinganalysis,wehaveelucidatedthatAtaxin-2recognizessomedistinctmotifssituatedin3’untranslatedregionofmRNAs.Inaddition,wewouldliketoshowthatAtaxin-2promotesthetranslationofmRNAthroughtheincreaseofRNAstability.Inter-actionwithPABPC1partiallycontributestothepromotionoftransla-tion;however,weidentifiedapreviouslyuncharacterizeddomainthatisindispensableforthisfunction.Furthermore,abnormalexpansionofthepolyglutaminestretchinAtaxin-2significantlyreducedtheeffi-ciencyoftranslation,whichmaycontributetothepathogenesisofneurodegenerativediseases.NoCOI.

1S11J-3Hypothalamic Sirt1 and energy balance regulationSasaki, Tsutomu; Kitamura, Tadahiro(Laboratory of Metabolic Signals, Instituite for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan)

Obesityisassociatedwithagingandincreasedcaloricintake,whilecaloricrestrictionimproveshealthandlongevityinmultipleorganisms;theNAD+-dependentdeacetylaseSirt1isimplicatedinthisprocess.Proopiomelanocortin(POMC)andagouti-relatedpeptide(AgRP)neu-ronsinthearcuatenucleusofthehypothalamus(ARC)arecriticalforenergybalanceregulation,andSirt1proteinleveldecreaseswithageintheARC.ConditionalSirt1overexpressioninmousePOMCorAgRPneuronspreventedage-associatedweightgain;overexpression inPOMCneuronsstimulatedenergyexpenditureviaincreasedsympa-theticactivity inadiposetissue,whereasoverexpression inAgRPneuronssuppresses food intake.However, thesephenotypeswereabsentinmiceconsumingahigh-fat,high-sucrosedietduetodecreas-esinARCSirt1proteinandhypothalamicNAD+levels.Meanwhile,Sirt1inthehypothalamusisregulatedbynutrient/metabolicsignalsinawaydifferent fromtheperipheral tissues.HypothalamicSirt1proteinlevelisdecreaseduponenergydeficitandtheubiquitin/pro-teasomesystemsisinvolvedinthisprocess.Theregulationofhypo-thalamicSirt1 isdisturbedbydiet inducedobesity.Asanefforttoelucidatethemechanismsforthedynamicregulationofenergyho-meostasisandhowthedisturbanceofthesystemcontributestothepathophysiologyofobesity, IwilldiscusshowhypothalamicSirt1regulatesenergybalancethroughPOMCandAgRPneurons,andhowmetabolicsignalsreflectingenergybalancestatusofthebodyfeedbacktothehypothalamicSirt1andregulateit.NoCOI.

1S11J-4Regulatory mechanism of wakefulness/non-REM sleep/REM sleep by the hypothalamic neuronsYanamaka, Akihiro; Tsunematsu, Tomomi(Department of Neuroscience II, Research Institute of Environmental Medicine, Nagoya University)

Melanin-concentratinghormone(MCH)isaneuropeptideproducedinneuronssparselydistributedinthelateralhypothalamicarea.RecentstudieshavereportedthatMCHneuronsareactiveduringrapideyemovement(REM)sleep,buttheirphysiologicalroleintheregulationofsleep/wakefulnessisnotfullyunderstood.Todeterminethephys-iologicalroleofMCHneurons,newlydevelopedtransgenicmousestrainsthatenabledmanipulationof theactivityandfateofMCHneuronsinvivoweregeneratedusingtherecentlydevelopedKENGE-tetsystem.Theactivityofthesecellswascontrolledbyoptogeneticsbyexpressingchannelrhodopsin2(E123T/T159C)orArchaerhodopsinTinMCHneurons.AcuteoptogeneticactivationofMCHneuronsat10Hzinducedtransitionsfromnon-REM(NREM)toREMsleepandincreasedREMsleeptimeinconjunctionwithdecreasedNREMsleep.ActivationofMCHneuronswhilemicewereinNREMsleepinducedREMsleep,butactivationduringwakefulnesswasineffective.AcuteoptogeneticsilencingofMCHneuronsusingArchaerhodopsinThadnoeffectonanyvigilancestates.Temporally-controlledablationofMCHneuronsbycell-specificexpressionofdiphtheriatoxinAincreasedwakefulnessanddecreasedNREMsleepdurationwithoutaffectingREMsleep.Takentogether,theseresultsindicatethatacuteactivationofMCHneuronsissufficient,butnotnecessary,totriggerthetransi-tionfromNREMtoREMsleepandthatMCHneuronsalsoplayaroleintheinitiationandmaintenanceofNREMsleep.NoCOI.


1S11J-5The circadian timing regulation of food intakeNakamura, Wataru1(1Laboratory of Oral Chronobiology, Graduate School of Dentistry, Osaka University, Osaka, Japan, 2JST, PRESTO, Saitama, Japan)

Animportantaspectofdailyhomeostasisisthetimingofdailyrhythmsinrestandactivity,feedingbehavior,energyutilization,andenergystorageacrossthedaily24hcycles.Thetimingoffoodintakeshowspronounceddiurnalrhythm,mostofwhichoccurduringtheactivephaseofanimals.Theneuralcircadianclocklocatedwithinthehypo-thalamicsuprachiasmaticnucleus(SCN)orchestrates24hcycles.Be-causerestrictionof foodavailabilitytoacertaintimeofdayelicitsanticipatorybehaviorevenafterablationoftheSCN,suchbehaviorhasbeenassumedtobeunderthecontrolofanothercircadianoscil-lator. Inthepresentstudy,micewithSCNlesionsorwithmutantcircadianperiodswereexposedtorestrictedfeedingschedulesatperiodswithinandoutsidecircadianrange.PeriodicfeedingledtotheentrainmentofFAArhythmsonlywithinalimitedcircadianrange.Cry1-/-mice,whichareknowntobeashort-periodmutant,entrainedtoashorterperiodoffeedingcyclesthandidCry2-/-mice.ThisresultindicatedthattheintrinsicperiodsofFAArhythmsarealsoaffectedbyCrydeficiency.Bmal1-/-mice,deficientinanotheressentialelementofthemolecularclockmachinery,exhibitedapre-feedingincreaseofactivityfarfromcircadianrange,indicatingadeficitincircadianos-cillation.Weproposethatmicepossessafood-entrainablepacemakeroutsidetheSCNinwhichcanonicalclockgenessuchasCry1,Cry2andBmal1playessentialrolesinregulatingFAAinacircadianoscil-latorymanner.NoCOI.

Symposium 12Smooth muscle myosin;

novel regulatory mechanisms in smooth muscle contraction

(March 16, 8:40–10:20, Room K)

1S12K-1Regulation of thick and thin filaments organization by smooth muscle myosinWatanabe, Masaru1; Ishida, Yukisato2; Nakahara, Naoya3; Taguchi, Mika3; Kimura, Masako3,4; Takemori, Shigeru3(Graduate School of Human Health Sciences, Tokyo Metropolitan University, Tokyo, Japan, 2Bunkyo Gakuin University, 3The Jikei University School of Medicine, 4Kagawa Education Institute of Nutrition)

Thick (myosin)filamentsareknowntoexist inthe livingsmoothmuscleevenwhenregulatorylightchainofmyosin(MLC20)isdephos-phorylated.However,severalgroupsincludingushavepresentedthatorganizationof thickandthinfilaments inthevertebratesmoothmusclewasmorelabilethanthatintheskeletalmuscle.Wefoundthatblebbistatin,apotentinhibitorofmyosinII,disruptedorganizationofthethickfilaments intheskinned (cellmembranepermeabilized)smoothmusclefromguineapigtaeniacecum(Watanabeetal.,2010).X-raydiffractionstudiesontheskinnedtaeniacecumintherestingstate,showedthatblebbistatininduceddecreaseintheintensityofbothmeridionalreflection14.4nmarisenfromthearrangementofmyosinmoleculesinthethickfilamentsanddiffuseequatorialpeak1/11.4nm-1originatedfromlattice-likearrangementofthethinfila-ments.Theseresultsindicatethatfunctionalchangesinsmoothmus-clemyosinmoleculesregulatetheorganizationofthick-filaments,re-sultinginthealterationofthin-filamentsstructureand/ororganizationevenintheabsenceofcross-bridgeformation.NoCOI.

1S12K-2Smooth muscle myosin: Novel roles in smooth muscle contractionSATO, OSAMU; Ikebe, Mitsuo(Department of Cellular and Molecular Biology, University of Texas Health Science Center, Tyler, Texas, USA)

MyosinsareknownasmolecularmotorsthatconverttheenergyofATPhydrolysistothemechanicalforcetomovealongactinfilamentsanddiversifiedinmorethan20differentclasses,thusconstitutingasuperfamily.Itisknownthatsmoothmusclemyosinisclassifiedintotype2myosininitssuperfamily.WhilemyosinIIiscalled“conven-tionalmyosin”,thepropertiesofmyosinIIarequiteuniquetoproduceforce.MyosinIIisalowdutyratiomotor,whichiscriticalformyosinIItofunctionasaforceproducingmotorprotein. AnothercriticalissueofmyosinIIisthelargethickfilamentformation,whichenablesmyosinIItoresideinactinfilamentsduringthepowerstrokecycle.Thus,myosinIIplaysroles inmusclecontraction,cellmotility,cellformation,maintenanceofcellstructure,andsoon.Ithasbeenwide-lyacceptedthatsmoothmusclemyosinisregulatedbyphosphorylationof itsregulatory lightchain (RLC).However, it isstillcontroversialhowsmoothmusclemyosinmoleculeisregulatedbyphosphorylation.Whilesingle-headedsmoothmusclemyosinhasbeenconsideredtobefullyactiveregardlessofRLCphosphorylation,currentstudyproposedthatthesingle-headedmyosinisneitherfullyactivenorfullyinhibited.RecentstudieshavealsoshownhowRLCkinasecausesphosphoryla-tionofRLC,andmyosinphosphatasereversesthephosphorylationlevel.Italsoremainstobeestablishedwhethertheformationofsmoothmusclethickfilamentsisregulatedinsitu.Iwilladdressthoseissuesfrombiochemicalaspectsinthissymposium.NoCOI.


1S12K-3Myosin phosphorylation dependent and independent smooth muscle contractionTakeya, Kosuke; Takai, Akira(Dept. of Physiology, Asahikawa Med. Univ., Asahikawa, Japan)

Signaltransductionintheregulationofsmoothmusclecontractionhasbeenstudiedbyusingphysiological,pharmacologicalandbiochemicaltechniques.Inpharmacologicalstudy,specificandselectivedrugsarenotalwaysavailable,andcomplexresultsleadtocontroversialinter-pretation.Biochemicalanalysiscanpresentdirectevidenceatmolec-ularlevel(e.g.phosphorylation)tosupportthepharmacologicalinter-pretation.However,duetothelackofsensitivity,theapplicationofbiochemicaltechniqueshasbeen limitedtorelatively largesmoothmuscletissues.Wehaveovercomethislimitationbyintroducingphos-tagmethods,andmeasuredmyosinphosphorylationinsmallsmoothmuscles.Wehavefound,insometissues,signalpathwaysproposedbypharmacologicalstudieswerenotconsistentwiththebiochemicalchanges.Forexample,pharmacologicalstudyshowedPKCinhibitioninratcerebralartery(RCA)resultedinattenuationofmyogenicre-sponse,suggestingtheinvolvementofCPI-17phosphorylationinthemyogenicresponse.However,wecouldnotfindanychangeinCPI-17phosphorylationduringmyogenicresponse.Moreover,myosinphos-phorylationdidnotdecrease intheRCAdilatedbyPKCinhibitor.Theseresultssuggesttheexistenceofunknownphosphorylation-in-dependentpathwaysinmyogenicresponse.Anotherexampleofphos-phorylationindependenceisciliarymuscle.Ciliarymusclecontractionrequires increase in intracellularCa2+.However,wecouldnotfindsignificantchangeinmyosinphosphorylationduringcontraction,sug-gestingtheinvolvementofphosphorylation-independentmechanisms.NoCOI.

1S12K-4The role of direct phosphorylation of myosin light chain by Rho-kinase in Ca2+-sensitization of smooth muscle contractionKobayashi, Sei(Department of Molecular Physiology and Medical Bioregulation, Yamaguchi University Graduate School of Medicine, Ube, Japan)

Rho-kinase(ROK)playsacentralroleinCa2+-sensitizationofvascularsmoothmuscle(VSM)contractionleadingtoabnormalVSMcontractionsuchasvasospasm.TheROK-mediatedelevationofmyosinlightchain(MLC)phosphorylationhasbeenwelldocumented,andinvitrostudiesshowedthat1)ROKindirectlyincreasesMLCphosphorylationthroughtheinhibitionofMLCphosphatase,and2)ROKdirectlyphosphorylatesMLC.However,thelatterpossibilityhasbeenexcludedwithoutanystrongevidenceandtheformermechanismisregardedassolepathwayforCa2+-sensitization. Inthisstudywetestedthe latterpossibility,usinginvitromotilityassaywithhighlypurifiedcontractileproteinsofsmoothmuscle,but inthecompleteabsenceoftheMLCkinase(MLCK)andphosphatase,bothofwhichareessentialfortheformermechanism.SmoothmusclemyosinswerepurifiedasrecombinantproteinsinabaculovirusexpressionsystemandconfirmedbyMAL-DI-TOFMS.ThemyosinMLCwasphosphorylatedbyeitherROKorMLCK.TheinvitromotilityassayrevealedthataveragevelocityofactinslidingonROK-phosphorylatedmyosinwas30timesfasterthanthatonunphosphorylatedMLCandwascomparabletothemaximumvalueobtainedwithMLCK-phosphorylatedmyosin.Finally, inthepermeabilizedVSMconstitutivelyactiveROKinducedlargeCa2+-sen-sitizationandMLCphosphorylationintheabsenceofcytosolicCa2+andMLCKactivity.TheseresultssuggestthatdirectphosphorylationofMLCbyROKisalonesufficientfortheCa2+-sensitization.NoCOI.

Symposium 13 New aspects of the hierarchical study

on function and morphology of epithelial membrane

[CollaborationSymposiumwithTheMembraneSocietyofJapan]

(March 16, 10:25–12:05, Room K)

1S13K-1Pathophysiological roles of an actin-binding protein, ezrin in epithelial tissues.Asano, Shinji; Hatano, Ryo; Matsumoto, Yosuke; Yoshida, Saori (College of Pharmaceutical Sciences, Ritsumeikan University, Kusatsu, Japan)

Ezrinisanactin-bindingprotein,whichcross-linksmembraneproteinsandF-actindirectlyorindirectlythroughscaffoldproteins.Itiscon-centratedonapicalsurfaceofmanyepithelialcellsespeciallyinsmallintestines,stomachs,andkidneys.Tamuraet al.preparedezrinknock-down(Vil2kd/kd)miceinwhichexpressionlevelofezrinwasdecreasedto lessthan10%comparedwithwild-type.Here,we introducethephenotypesofthesemiceinepithelialtissues.Instomachs,ezrinisexpressedonapicalcanalicularmembraneofparietalcells.TheVil2kd/kdmiceshowedachlorhydriaduetoimpairmentofmembranefusionoftubulovesicleswithapicalmembrane.Theyshowedfoveolarhyper-plasiaandatrophyinthefundicglandswiththenumberofparietalandchiefcellsbeingdecreased,andthenumberofneckcellsbeingincreased.Theneckcellsexpressedspasmolyticpolypeptide (SP),representingSP-expressingmetaplasia,SPEM.Therefore,ezrinisin-volvednotonlyingastricacidsecretionbutalsoinarchitectureanddevelopmentofgastricglandularepithelia.Inthekidney,ezrinislo-catedatthebrushbordermembraneofproximaltubuleswhere itinteractswithaNa+/phosphatecotransporter,Npt2a,throughascaf-foldingprotein,NHERF1.TheVil2kd/kdmiceexhibitedhypophospha-temia,osteomalacia,andurinary lossofphosphate.TheNpt2aandNHERF1expressionsatbrushbordermembranewerereducedintheVil2kd/kdmice.Theseresultssuggestthatezrin is involved incellsurfaceexpressionofNpt2a,andrequiredfortheregulationofsys-temicPihomeostasis.NoCOI.


1S13K-2Mechanisms for ABCB4- and PEMT-mediated resis-tance of hepatocytes against bile saltsMorita, Shin-ya(Department of Pharmacy, Shiga University of Medical Science Hospital, Otsu City, Shiga, Japan)

Bilesaltshavepotentdetergentpropertiesanddamagehepatocytesbyaffectingtheintegrityofcellularmembranes,whichareresponsibleforcholestasisandhepatocellularnecrosis. However, it isnotfullyunderstoodhowthecanalicularmembraneofhepatocytesacquiretheresistanceagainstbilesalts.ABCB4,amemberoftheABCtransporterfamily,ispresentinthecanalicularmembranesofhepatocytes.Lipidraftsareplasmamem-branemicrodomainscontaininghighlevelsofsphingomyelin(SM)andcholesterol.WeshowedthatABCB4waslocalizedmainlytononraftmembranes,andthattaurocholatesignificantlystimulatedtheAB-CB4-mediatedeffluxofphosphatidylcholine(PC),phosphatidylethanol-amine(PE)andSM,whiletheeffluxofPEandSMwasmuchlessthanthatofPC.Thebiliaryphospholipidsecretionprotectsthecanalicu-larmembranesofhepatocytesagainstbilesalts.PEN-methyltransferase(PEMT)convertsPEtoPCintheliver.WedemonstratedthatPEMTexpressionelevatedthelevelsofPCspeciescontaininglongerpolyunsaturatedacylchains,increasedthemicrovil-lusdiameter,andresultedinincreasedresistancetoconjugatedbilesalts.ThesefindingssuggestthatABCB4andPEMTplayacrucialroleintheresistanceofhepatocytesagainstbilesalt-inducedcytotoxicityandincholestasisandhepatocellularinjury.NoCOI.Ref. [1]Moritaetal. (2007)Hepatology,46,188–199. [2]Moritaetal.(2010)Biochem.J.,432,387–398.[3]Moritaetal.(2011)FEBSJ.,278,4768–4781.[4]Moritaetal.(2013)J.LipidRes.,54,1221–1230.

1S13K-3Regulatory mechanism of Na,K-ATPase function in plasma membrane microdomainsSakai, Hideki; Fujii, Takuto; Shimizu, Takahiro(Department of Pharmaceutical Physiology, University of Toyama, Toyama, Japan)

Lipidraftsandcaveolaeareglycosphingolipid-andcholesterol-enrichedmicrodomainsandarethoughttobethefunctionaldomainsinvolvedinmembranetraffickingandsignaltransduction.Here,wewilltalkaboutourrecentfindingsaboutnovelfunctionsofNa,K-ATPaseintheplasmamembranemicrodomains.Ingastricparietalcells,wefoundthatNa,K-ATPaseandK,Cl-cotransporter3a(KCC3a)werecoimmu-noprecipitated,andthatbothofthemwerehighlylocalizedinalipidraftfraction.TheNa,K-ATPaseactivitywassignificantlyinhibitedbyaKCCinhibitor.ThestableexogenousexpressionofKCC3ainLLC-PK1cellsresultedinassociationofKCC3awithNa,K-ATPaseanditrecruitedNa,K-ATPase in lipidrafts,accompanyingan increase inNa,K-ATPaseactivity.Wesuggest thatKCC3aformsa functionalcomplexwithNa,K-ATPaseinlipidraftsofthecells.IncaveolaeofLLC-PK1cells,ithasbeenreportedthatnon-pumpingNa,K-ATPasebutnotpumpingATPaseispredominantlylocalized.Inhumancancercells,wefoundthatdisruptionofcaveolaebymethylβ-cyclodextrin(MβCD)significantlyinhibitedtheactivationofvolume-sensitiveout-wardlyrectitfyinganionchannels(VSOR)inducedbylowconcentrationofouabain(100nM).MβCDalsoinhibitedtheouabain-inducedmito-chondrialdysfunctionanddecreaseincellproliferation.Ourresultssuggestthatbindingofouabaintonon-pumpingNa,K-ATPaseinca-veolaeupregulatestheVSORactivityand inhibits thecancercellproliferation.NoCOI.

1S13K-4Molecular mechanism of regulation of epithelial Na+ channel (ENaC) activity at the plasma membrane - Roles of raft domainYokoyama, Noriko1; Marunaka, Yoshinori1,2,3(1Department of Molecular Cell Physiology, Kyoto Prefectural University of Medicine Graduate School of Medical Science, 2Department of Bio-Ionomics, Kyoto Prefectural University of Medicine Graduate School of Medical Science, 3Japan Inst for Food Education & Health, St. Agnes Univ.)

ENaCexpressedintheapicalmembraneofsodium-transportingepi-thelialcellsplaysacriticalrole inhomeostasisoffluidcontentandbloodpressure.TheamountofENaC-mediatedNa+transportdependsontheopenprobabilityofthechannelandthenumberofchannelexpressedontheapicalmembrane.ENaCiscleavedbyaldosterone-in-ducedproteases.AreleasinginhibitorytractfromtheextracellularloopofENaCresultedinanincreaseinopenprobability.ThenumberofENaClocatedattheapicalmembraneisregulatedbyubiquitinligase(Nedd4-2).InthisstudywequantifiedtheabundanceandlocalizationofENaCattheplasmamembranewith/withoutaldosteronestimulationinrenalepithelialcells.Aldosteronestimulatedexpressionofbothfull-lengthandcleavedENaCs;i.e.,thefull-lengthENaCwasexpressedinbothcytoplasmandplasmamembrane (raftdomain),whereasalargepartofthecleavedENaCwasobservedatthenon-raftplasmamembrane.DepletionofcholesterolfromtheraftdomaindiminishedthemovementofENaCbyaldosteronestimulation.AldosteronealsoincreasedexpressionofNedd4-2andSGK1inraftdomain,suggestingthattheubiquitination-mediatedregulationofENaClifetimewouldoccurinraftdomain.Basedonourfindings,weproposenovelrolesofthelipidraftdomainontheregulationofENaCactivity/trafficking.NoCOI.

Symposium 14Neurobiology of glutamate synaps

(March 16, 15:05–17:05, Room D1)


1S14D1-1Physiological importance of the rate of synaptic vesi-cle filling for maintaining fast synaptic transmission in the mammalian CNSHori, Tetsuya1,2; Takahashi, Tomoyuki1,2(1Cellular and Molecular Synaptic Function Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan, 2Laboratory of Molecular Synaptic Function, Graduate School of Brain Sciences, Doshisha University)

Atthepresynapticterminal,afterexocytosisofsynapticvesicles,fusedvesiclemembranesarere-internalizedbyendocytosis,andsynapticvesiclesarerefilledwithneurotransmitterandrecycledforreuse.Atglutamatergicpresynapticterminals,vesiclesarerefilledwithgluta-matebyvesicularglutamatetransportersbyusingaH+electrochem-icalgradientproducedbytheH+-ATPase.Aswehavepreviouslyreported,vesiclerefillingwithglutamatehasameantimeconstantof15s,andthistimeconstantvariesdependingupontemperature,de-velopmentalstageofanimals,andpresynapticCl-concentrations([Cl-]).Inisolatedvesicles,magnitudesofglutamateuptakedependonex-tra-vesicular[Cl-]concentrations,withlessuptakebothatlowandhighCl-concentrations(Naito&Ueda,1985).Likewise,atthecalyxofHeldglutamatergicpresynapticterminals,vesiclesareoptimallyrefilledat30mM[Cl-],butrefillingislessefficientatlowerorhigher[Cl-](Hori&Takahashi,2012).Here,atthecalyxofHeld-MNTBsynapse,weaskedwhethertheslowingoftheglutamaterefillingratebyCl-wouldaffectfastsynaptictransmissionduringhighfrequencyfiring.Mypresenta-tionhighlightstheimportanceofvesiclefillingrateformaintainingthefidelityofpostsynapticfiringinthemammalianCNS.NoCOI.

1S14D1-2Imaging of glutamate release at synapsesHirose, Kenzo(Department of Neurobiology, Graduate School of Medicine, the University of Tokyo, Tokyo, Japan)

Glutamatergicsynapsesplayessentialrolesinbrainfunctions.Tostudypropertiesofglutamatergicsynapses,wehavedevelopedfluorescentglutamateprobesnamedEOSprobes.EOSprobe isahybridtypeprobewhichconsistsofasyntheticfluorescentdyeandaglutamatebindingproteinderivedfromAMPAreceptorsubunit,GluA2.EOSwhenappliedinculturedhippocampalneuronsvisualizedglutamatereleasefromglutamatergicsynapsesatasinglesynapseresolution.Fromquantalanalysisfortheglutamatereleasedata,wefoundthateachsynapsehasmultiplereleasesites.Thenumberofreleasesites(N)wasfoundtobeheterogeneousamongsynapses.WeproposethattheheterogeneityinNprovidesuniqueweightsinsynapticcomputa-tion.NoCOI.

1S14D1-3Roles of postsynaptic density in spatiotemporal con-trol of glutamate receptorsSuzuki, Etsuko1,2; Kamiya, Haruyuki1(1Department of Neurobiology, Graduate School of Medicine, Hokkaido University, 2JSPS)

Spatialandtemporaldistributionofsynapticglutamatereceptorsarecontrolledbymultiplexmolecular interactionsbetweenglutamatereceptorsubunitsandpostsynapticscaffolds.ThereisaccumulatingevidenceshowingthatPSD95,oneofthemajorproteinsinamacro-molecularcomplexof thepostsynapticdensity,playakeyrole incontrollingsubcellularlocalizationofsynapticglutamatereceptors.Inthissymposium,wewillintroduceourrecentattemptstoidentifyingtherolesofPSD95insynapticAMPAreceptordynamicsinthehip-pocampalCA1synapses,aswellasinspecificlocalizationofsynaptickainatereceptorsatthemossyfiber-CA3synapses.Recentlywede-velopedaphotochemicalinactivationtechnique,usingaphotoreactiveAMPAreceptorblockerANQX,tomonitortemporaldynamicsofsynapticAMPAreceptors.WeappliedthismethodtohippocampalslicesobtainedfromknockoutmiceofPSD95.OurresultsupportsthenotionthatPSD95limitmobilityandtraffickingofAMPAreceptors.SincePSD95alsohaveshowntobinddirectlytokainatereceptorsubunits,wealsoexaminedchanges inkainatereceptor-mediatedEPSCsinPSD95knockoutmice.StimulationofhippocampalmossyfiberselicitedmixedEPSCscomposedofbothAMPAreceptor-medi-atedandmuchslowerkainatereceptor-mediatedcomponentsinCA3neurons.Preliminaryresultshavesuggestedthattherelativecontri-butionofthetwocomponentswasaffectedinPSD95knockout.WewilldiscussaboutourmodeloftherolesofPSD95 inthespatiallyrestricteddistributionofkainatereceptorsonthemossyfiber-CA3synapses.NoCOI.

1S14D1-4The C1q complement family complements glutama-tergic synapses on cerebellar Purkinje cells.Kakegawa, Wataru1,2; Yuzaki, Michisuke1,2(Department of Physiology, Keio University Schook of Medicine, Tokyo, Japan, 2JST-CREST, Saitama, Japan)

CerebellarPurkinjecellsreceivetwomajorglutamatergic inputs,parallelfibersfromgranulecells (PFsynapses)andclimbingfibersfrominferiorolive(CFsynapses).Cbln1,amemberofC1qcomplementfamilycharacterizedbyaconservedglobalC1qdomain,ishighlyex-pressedingranulecellsandreleasedfromPFterminalstoregulatePFsynapse formationand long-termdepression (LTD),asynapticplasticityunderlyingcerebellarmotorlearning(Hiraietal.,Nat Neu-rosci,’05;Matsudaetal.,Science,’10).Recently,weidentifiedanovelC1q-familyprotein,C1q-likeprotein1 (C1qL1),which isselectivelyexpressedininferiorolive(Iijimaetal.,Eur J Neurosci, ’10).Inthisstudy,toexaminewhetherC1qL1controlsCFsynapseformationandfunctions,weanalyzedamutantmouselackingC1qL1gene(C1qL1KOmouse). Immunohistochemistryusinganti-C1qL1antibodiesre-vealedthatC1qL1proteinswererestrictedonCFsynapsesinwild-type(WT)mice.Interestingly,CFsofC1qL1KOmicewereseverelyre-tractedcomparedtoWTandhadadecreasednumberofvesicularglutamatetransporterII,apresynapticmarkerofCFsynapses.C1qL1KOmicealsoshowedsignificantlysmallexcitatorypostsynapticcur-rentsatCFsynapseswithseveralstepsofamplitudes,reflectingamultipleCFinnervation,acharacteristicofimmaturetypesofCFsinWT.Furthermore,theseKOmiceimpairedLTDatPFsynapsesandcerebellarmotorlearning.Theseresultsindicatethat,likeCbln1atPFsynapses,C1qL1complementsCFsynapseintegrityandcerebellarmotorlearning.NoCOI.


Symposium 15New Aspects of Zinc Ion Biology

in Homeostasis, Diseases and Signal transduction

(March16, 15:05–17:05, Room D2)

1S15D2-1Zinc signaling: New regulatory system in physiology and diseasesFukada, Toshiyuki(RIKEN, IMS, Homeostatic networks)

Zinc isanessential traceelementthat isrequired fornumbersofcellularfunctions.Impairmentofitshomeostasisexertsvariousprob-lemsincludinggrowthretardation,abnormalboneformation,fragileskin,andimmunodeficiency,etc.Zinchomeostasisisregulatedbyzinctransportersandchannels,andrecentgeneticandmolecularapproach-esonthesemoleculesrevealedtheimportantrolesofzincionasasignalingmediator.Theemergingattentionshavebeendrawntoac-tionsofintracellularandextracellularzincions,whichhasbecomeafocusofitspotentialtoidentifynoveltargetsfortherapeuticinterven-tioninhumandisease.IFirstlywillintroducethesignificantcontribu-tionofzinctransporter-mediatedzincsignaling inourhealth,anddiscussupdatedinformationbyhighlightingzincsignalthatselective-lycontrolsmoleculesandthefollowingcellularevents,calledZn signal axiswhichmaintainthephysiologicalphenomena.NoCOI.Reference:Fukadaetal,J. Bone Miner. Meta.31:129–135,2013Review.

1S15D2-2Intracellular Zn2+ signal in the dentate gyrus is re-quired for object recognition memoryTakeda, Atsushi(Department of Bioorganic Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan)

Brainzinchomeostasisisstrictlycontrolledunderhealthycondition,indicatingtheimportanceofzinchomeostasisinphysiologicalfunctioninthebrain.Zincisrelativelyconcentratedinthehippocampus.Thehippocampusisrequiredformemoryretentionforalimitedperiodoftimeafterlearning.Hippocampalthreesynapses,i.e.,perforantpath-way-dentategranulecell,mossyfiber-CA3pyramidalcellandSchaffercollateral-CA1pyramidalcell,areglutamatergic(zincergic).Thethreesynapsescontainzincinthepresynapticvesicles,whichservesasaZn2+signal.Zn2+isco-releasedwithglutamateandmodulatesglutamatesignalingatzincergicsynapses.However,theroleofsynapticZn2+signalinlearningandmemoryispoorlyunderstood.Synapticplastici-tysuchas long-termpotentiation (LTP) isbelievedtobeacellularmodel formemory.Onthebasisof theevidencethatZn2+signalmulti-functionallymodulatesLTPinductioninhippocampalslices,invivoreal-timecorrelationbetweenLTPinductionandmemorywasexaminedusingyoungrats.Theroleofperforantpathway-dentategranulecellsynapsesincognitivebehaviorwasexaminedfocusingontheselectivelossofsynapticZn2+signalinthedentategyrus,whichwasinducedbyinjectionofzincchelatorsintothedentategyrus.ThepresentstudyindicatedthatintracellularZn2+signalinthedentategyrusisrequiredforobjectrecognitionmemory,probablyviadentategyrusLTPexpression.ItislikelythatdentategyrusLTPisreal-time-lylinkedtoobjectrecognitionmemory.NoCOI.

1S15D2-3The diabetes susceptible gene SLC30A8/ZnT8 regu-lates hepatic insulin clearanceFujitani, Yoshio(Department of Metabolism & Endocrinology, Juntendo University, Tokyo, Japan)

Recentgenome-wideassociationstudiesrevealedthatcommonvariantsofSLC30A8increasesusceptibilitytotype2diabetes.SLC30A8encodeszinctransporter-8(ZnT8),whichtransportszincionfromthecytoplasmintoinsulingranulesinpancreaticbetacells.Whileitiswellknownthat insulingranulecontainshighamountofzinc, itsphysiologicalmeaninghasnotbeenfullyelucidated.Hereweshowthatzincco-se-cretedwithinsulinregulateshepaticinsulinclearance.TodeterminetheroleofZnT8inglucosemetabolism,wecharacterizedbeta-cell-spe-cificSlc30a8-deficientmice.Lowperipheralbloodinsulinlevelswereobserved inmutantmiceafterglucosechallenge,although insulinsecretionfrompancreaticbetacellswasenhanced.Aseriesofexper-imentsassessinginvivozincfluxrevealedthatlossofzincsecretionfrompancreasfailedtosuppresshepaticinsulinclearanceinpostpran-dialstate.Consistentwiththesefindings,human individualswithrs13266634,amajorriskalleleofSLC30A8,exhibitedincreasedinsulinclearanceassessedbyc-peptide/insulinratio.Theresultsindicatethatzinc-mediatedpancreas-to-livercommunicationregulateshepaticinsu-linclearance,andthatdysregulationofthissystemcouldplayaroleinthepathogenesisoftype2diabetes.NoCOI.


1S15D2-4Zinc transporter ZnT2/Slc30a2 is involved in skin-wound healingNishida, Keigo1,2(Lab. for Homeostatic Network., Center for IMS., (IMS-RCAI) RIKEN, 2Immune system., Grad. Sch. Frontier Biosci., Osaka Univ.)

Zinc(Zn)isanessentialnutrient,anditsdeficiencycausesimmunode-ficiencyandskindisorders.ManymetalloenzymesandtranscriptionfactorsrequireZntoexerttheirfunctions.Znalsoactsasanintracel-lularsignalingmolecule.AchangeinZn’sintracellularstatusinre-sponsetoextracellularstimuliaffectsavarietyofsignalingpathways;thus,Zniscriticallyinvolvedinvariousphysiologicalfunctions,includ-ingimmuneresponsesandwoundhealing.Inadditiontotheseintra-cellularroles,Znappearstoactasaneurotransmitter.Mastcellgran-ulesarerichinZn,whichisreleasedwhenthecellisstimulated,asdoneuronsatsynapses.However,therolesoftheZnreleasedfrommastcellsareunknown.HerewereportthattheZntransporterSlc30a2/ZnT2islocalizedtothemembraneoftheZn-containinggran-ulesinmastcells.ZnT2-KOmicedonotcontainZnintheirmast-cellgranulesandshowdefectiveZnreleaseuponstimulation.Importantly,byusingthesemice,wefoundthatZnreleasedbymastcellsplaysamajorroleinskinwoundrepair,functioningasafirstmessenger.WeshowedthatZnpromotestheproductionofinflammatorycytokinessuchasIL-6inmacrophagesandfibroblastsviatheZnreceptorG-pro-tein-coupledreceptor39(GPR39).WeobtainedgeneticevidencethatnotonlyIL-6butalsoGPR39arerequiredforskin-woundhealing.Collectively,ourfindingsindicatethatZnreleasedbymastcellsisaninflammatory“firstmessenger”andthattheZn/GPR39/IL-6axisplaysamajorroleinwoundhealing,inwhichinflammationisakeyprocess.NoCOI.

1S15D2-5Zinc deficiency and dermatitis: From the standpoint of zinc transporter functionsKambe, Taiho(Graduate School of Biostudies, Kyoto University, Kyoto, Japan)

Severezincdeficiencycausesabroadrangeofdefectsincludingim-munesystemdysfunction,tastedysfunction,andskinlesionsofder-matitisasparticularsymptoms.Inthelastfourdecades,inheritedzincdeficiencydisorderhasbeeninvestigated,andtwozinctransportergeneshavebeenidentified,mutationsofwhichcauseseveredermati-tis.Oneistherareautosomalrecessivegeneticdisorderacrodermati-tisenteropathica,whichiscausedbymutationsofzinctransporterZIP4/SLC39A4.ZIP4functionsasanessentialcomponent forzincabsorptioninthesmallintestine.AnumberofstudieshaverevealedthatexpressionofZIP4isdynamicallyregulatedbymultiplepost-tran-scriptionalmechanisms,andwefoundZIP4proteinundergoespro-cessingbyremovalofthelongextracellularamino-terminalhalfviaproteolyticcleavageduringprolongedzincdeficiency.ThesimilarprocessinghasbeendetectedinotherZIPtransporterproteins,whichsuggestsitsimportancetoregulateactivityofZIPtransporters.Theotheristhetransientneonatalzincdeficiencywithseveredermatitis.Thistypeofzincdeficiencyiscausedbylowzincbreastmilkonlyduringbreast-feedinganddoesnotreoccurafterweaning.Recently,wefoundthatnovelcompoundheterozygousmutations inZnT2/SLC30A2causethelowzinclevelsinthebreastmilkoftheJapanesemother,whichcausedseverezincdeficiencyinherinfant.Aspointedoutabove,thestudyofzinctransportersisnowofgreatclinicalinter-est.Here,theinheritedzincdeficiencyandzinctransportersfunctionsarereviewedfromthemolecularstandpoint.NoCOI.

Symposium 16Late-breaking topics

in the ion transport system[CollaborationSymposiumwithTheJapanesePharmacologicalSociety]

(March 16, 15:05–17:05, Room E)

1S16E-1Pathophysiological roles of HCN channel family in the heartTakano, Makoto1; Ohshita, Kensuke1; Ito, Masayuki1; Ishihara, Keiko1; Kuwahara, Koichiro2(1Dept.Physiol., Sch. Med., Kurume University, Kurume, Japan, 2Dept.Cardiovasc.Med., Grad.Sch.Med., Kyoto Univ., Kyoto, Japan)

Amonghyperpolarization-activatedcyclicnucleotide-modulated(HCN1-4)channelfamily,HCN4ishighlyexpressedinthepacemakercellsofsino-atrialnode. Intheventricularmyocytes,HCN2andHCN4areexpressedinthefetalheart,butdown-regulatedduringdevelopment.Theyarere-expressed inthehypertrophiedheart,andthoughttoincreasethesusceptibilitytoarrhythmia.Recentstudiesusingtrans-genicmicehaverevealedunexpectedrolesofHCNchannels,bothinphysiological-andpathophysiologicalcondition.Inthepacemakercells,functionalconsequenceofHCN4knockoutappearedcontroversial;Baruscottietal.reportedHCN4knockoutcauseddeepbradycardiaandheartblock,whereasHermannetal.suggestedthatHCN4onlyprovideddepolarizationreserveandwasnotrequiredforheartrateacceleration.Inventricularmyocytes,overexpressionofHCN2channelreducedrepolarizationreserveandprolongedtheactionpotentialduration,butthiswasnotsufficientto induce lethalarrhythmia inphysiologicalcondition.Inthetransgenicmiceoverexpressingdominantnegativemutantofneuron-restrictedsilencingfactor(dnNRSF),mul-tiplefetaltypecardiacchannelsincludingHCN2wereup-regulated,anddiedsuddenlyfromtachyarrhythmia.Mostnotably,ivabradine,ablockerofHCNchannelswerereportedtoimprovethesurvivalofdnNRSFmice.TheseresultssuggestedthatHCNchannelsmaybeatherapeutictargetofventriculararrhythmiaincardiachypertrophy.COIproperlydeclared.


1S16E-2Selective modulation of TRPA1 channels by O2 and trans-nitrosylationKozai, Daisuke1; Kiyonaka, Shigeki1,2; Numata, Tomohiro1,2; Takahashi, Nobuaki1; Ohwada, Tomohiko3; Mori, Yasuo1,2(1Dept. Synth. Chem. and Biol. Chem., Grad. Sch. Engineer., Kyoto Univ., 2Hall of Global Environmental Studies, Kyoto Univ.)

Certainmembersoftransientreceptorpotential(TRP)cationchannelsactascellsensorsforchangesinredoxstatus.Oursystematicevalu-ationofTRPchannelsusingreactivedisulfideswithdifferentredoxpotentialsrevealedthecapabilityofTRPA1tosenseO2.Weprevious-lydemonstratedthatsensitivitytoactivationbysyntheticNO-releas-ingagentsviaS-nitrosylationisacommonfeatureofmembersofTRPchannels.However,strategiestoconfersubtypeselectivitytonitrosylat-ingagentstargetedtoTRPchannelsareyettobedeveloped.Here,weshowselectiveactivationofTRPA1channelsbynovelNOdonorsderivedfromthe7-azabenzobicyclo[2.2.1]heptane (ABBH)N-nitrosa-mines,whichexhibit trans-nitrosylationreactivitytothiolswithoutreleasingNO.TheN-nitrosamineelicitsS-nitrosylationofTRPA1proteins,anddose-dependentlyinducesrobustCa2+influxviarecom-binantTRPA1channels,butnotviaotherNO-activatedTRPchannels.TRPA1activationbytheN-nitrosamine issuppressedbyspecificcysteinemutationsbutnotbyNOscavenging.Interestingly,non-elec-trophilicderivativesofABBHalsoactivateTRPA1selectivelybutlesspotentlycomparedtotheN-nitrosamine.Thus,ABBHN-nitrosaminesconfersubtypeselectivityonS-nitrosylationinTRPchannelsthroughsynergeticeffectsoftwochemicalprocesses:cysteinetrans-nitrosyla-tionandmolecularrecognitionofthenon-electrophilicmoiety.NoCOI.

1S16E-3Involvement of Na+/Ca2+ exchanger in Ca2+ regulation of pancreatic β cellsKita, Satomi; Iwamoto, Takahiro(Department of Pharmacology, Faculty of Medicine, Fukuoka University)

Na+/Ca2+exchangertype1(NCX1)isaplasmamembranetransporterinvolvedinintracellularCa2+regulation.NCX1exchangesNa+andCa2+ineitherCa2+effluxorCa2+ influxmode,dependingonmembranepotentialandtransmembraneiongradients.Pancreaticislettransplan-tationisanattractivetherapyforthetreatmentofinsulin-dependentdiabetesmellitus,butthelowefficiencyofthisproceduremainlyduetotheearlylossoftransplantedisletsisamajorobstacle.Previousstudyshowedthatalargeamountofhigh-mobilitygroupbox1protein(HMGB1)releasedfromisletssoonaftertheirtransplantationinmicetriggersinnateimmunerejectionwithactivationofDC,NKTcellsandneutrophilstoproduceIFN-γ,ultimatelyleadingtotheearlylossoftransplantedislets.Thus,HMGB1releaseplaysaninitialpivotalroleinthisprocess,however, itsmechanismremainsunclear.HerewedemonstratethatreleaseofHMGB1fromtransplantedisletsisduetohypoxicdamageresultingfromCa2+influxintoβcellsthroughNCX1whichisoformmainlyexpressedinβcells.Moreover,thehypoxia-in-ducedβcelldamagewaspreventedbypretreatmentwithaspecificNCX1inhibitorpriortotransplantation,resulting inprotectionandlong-termsurvivalof transplantedmouseandhuman isletswhengraftedintomice.ThesefindingssuggestthatNCX1inhibitorcouldbeappliedtohumandonorisletstoimproveefficiencyofislettrans-plantation.NoCOI.

1S16E-4Inhibition of metastatic cell growth by MagExFunato, Yosuke; Yamazaki, Daisuke; Miki, Hiroaki(Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan)

PRLoverexpression isassociatedwithcancerprogression,butthemolecularmechanismofinducingmetastaticcellgrowthbyPRLhasremainedunknown.WehereperformacomprehensivesearchforinvivobindingproteinsforPRL,andidentifyanuncharacterizedmem-braneproteinMagnesium-Exportingprotein(MagEx).LiveimaginganalysesrevealthatMagExstimulatesMg2+-effluxbyNa+/Mg2+ex-changeanddecreasesintracellularMg2+levels.PRLdirectlybindstoMagExandinhibitsitsMg2+-effluxfunction.Overexpressionandknock-downanalysesofPRLand/orMagExrevealthattheMg2+ level iscorrelatedtothatofATP,andaugmentedMg2+andATPlevelsbyPRLoverexpressionconferscellresistancetoenergystressunderglucosestarvation.Also,theimportanceofMagExinmetastaticgrowthiscorroboratedbyexperimentalmetastasisanalyses inmice,anddownregulationofMagExexpressioniscorrelatedwithcancermalig-nancyinhumancoloncancers.TheseresultspresentanovelroleofMg2+incontrollingtheenergystatusandgrowthofcancercells.NoCOI.

Symposium 17Tissue homeostasis of the myocardium

and its disruption

(March 16, 15:05–17:05, Room F)


1S17F-1Inhibition of hypoxia-induced apoptosis of cardiomyo-cytes by synthetic peptide from ischemia-inducible activator of G-protein signaling 8 (AGS8)Sato, Motohiko; Suzuki, Hiroko; Sakima, Miho; Mamun, Abdullah Al; Hayashi, Hisaki; Iwase, Satoshi; Inukai, Yoko; Nishimura, Naoki; Sato, Maki; Shimizu, Yuuki(Department of Physiology, Aichi Medical University, Nagakute, Aichi, Japan)

IschemicinjuryoftheheartisassociatedwithactivationofmultiplesignalingpathwaysincludingthosemediatedbyheterotrimericG-pro-tein.Previously,weidentifiedanischemia-inducibleG-proteinactivator,activatorofG-proteinsignaling8(AGS8),whichinteractedwithGβγandtriggeredtheapoptoticcascadesofcardiomyocytesunderhypox-ia.Here,wereporttheprotectionofcardiomyocytesfromhypoxia-in-ducedapoptosisbyasyntheticpeptide(AGS8-peptide),basedonami-no acids of the Gβγ interacting domain of AGS8. AGS8-peptidesuccessfullyblockedtheinteractionofGβγwithAGS8.FITCconju-gatedAGS8-peptidewassuccessfullydeliveredintocardiomyocytesbychemicalreagent.AGS8-peptideeffectivelyblockedapoptosis incardiomyocytes,determinedbyDNAend-labeling(57.7±5.1%ofapop-toticgroup,p<0.05,mean±SEM)andbycleavedcaspase-3(57.9±3.9%ofapoptoticgroup,p<0.05,mean±SEM)followingexposureofculturedcardiomyocytestohypoxia(6h)/reoxygenation(18h).IncontrastwithgeneralinhibitionofGβγ-signalingbysmallcompound,AGS8-pepetidedidnotshowcytotoxicityinMTTassay(62.5±2.3%ofcontrol,p<0.05,mean±SEM),suggestinganadvantageofAGS8-peptideforthepro-tectionofcardiomyocytes.Thesedataindicatedanimportanceofac-cessoryproteins forheterotrimericG-protein inpatho-physiologicalchallengeandtheirpotentialasnoveltherapeutictarget.NoCOI.

1S17F-2The role of cardiac fibroblasts in inflammation after myocardial infarctionKuorse, Hitoshi; Nakaya, Michio(Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University)

Atmyocardialinfarction,manycellsaredeadbylimitationofoxygensupplefromthecoronaryartery.Thesedeadcellsshouldberemovedproperly.Otherwise,cellularcomponentsreleasedfromthedeadcellscauseinflammation.Ithasbeenrecognizedthatexcessinflammationleadstodetrimentaleffectsontheheart.However,inflammationshouldbestoppedbyamechanism,whichisnotwellestablished.Althoughinflammatorycellssuchasmacrophagesandneutrophilsarebelievedtoberesponsible forremovalofdeadcells, it isunknownwhethertheseinflammatorycellsareactuallyinvolvedinremovalofdeadcells.Afterremovalofdeadcells,spacethatwasdroppedoutatinfarctionareashouldbefilledupwithcollagen.Accumulationofcollagen iscalledasfibrosis.Collagen isproducedbymyofibroblaststhataredifferentiatedfromfibroblasts.Therefore,differentiationfromfibro-blaststomyofibroblastsshouldbetightlyregulatedtoavoidexcessfibrosis.However, thetriggerofdifferentiationoffibroblasts intomyofibroblastsisnotwellunderstood,andtherelationshipbetweendifferentiationoffibroblastsand inflammationat infarctarea isnotwelldefined.Inmypresentation,Iwillshowtheimportantroleoffi-broblastsininflammationatmyocardialinfarction.NoCOI.

1S17F-3Another pathophysiological aspect of stress-related cardiomyopathyKakinuma, Yoshihiko1; Tsuda, Masayuki2; Akiyama, Tsuyoshi3; Okazaki, Kayo4; Iketani, Mitsue1; Oikawa, Shino1; Sato, Takayuki4 (1Department of Physiology, Nippon Medical School Graduate School of Medicine, Tokyo, Japan, 2Institute for Laboratory Animal Research, Kochi Medical School, Nankoku, Japan, 3Department of Cardiac Physiology, National Cardiovascular Center Research Institute, Osaka, Japan , 4Department of Cardiovascular Control, Kochi Medical School, Nankoku, Japan, 5Department of Physiology, Nippon Medical School Graduate School of Medicine, Tokyo, Japan)

Thepathophysiologyofstress-relatedcardiomyopathy,initiallyreport-edinJapanasTako-tsubocardiomyopathy,remainstobeelucidated.Despitethecatecholaminesurgeasoneoftriggers,itremainstobeinvestigatedhowcatecholaminecoulddisturbcardiac function.Todisclosethepathophysiology,aFDG-PETstudyinhumanswasper-formedtorevealthattheprevalenceofhealthyhumanfemaleswithmaximalFDGuptakewasmorethanthatofmalesspecifically inpostmenopausalages.Estrogenreplacementinpatientswithoopho-rectomyalsoenhancedcardiacFDGuptake,suggestingthatthefemaleheartmoredependsonglucose.Thetransgenicmicewiththeheart-spe-cificoverexpressionofanon-neuronalcholinergicsystemwasdevel-oped,whoseheartshowedaglucosepreferencewithupregulatedtransporters.Moreover,estrogen itselfactivatedthenon-neuronalcholinergicsystem.Inaratmodelofstress-relatedcardiomyopathy,glucoseadministrationbluntedcatecholaminesurgeandattenuatedimpairmentofcardiacperformance.Theseresultssuggestthatfemalesexuality, thecardiaccholinergicsystem,whichregulatesglucoseutilization,isinvolvedinthecardiopmyopathy.NoCOI.

1S17F-4The role of TCTP, a novel endogenous inhibitor of p53, in the development of doxorubicin-induced heart failureFujita, Takayuki; Cai, Wenqian; Hidaka, yuko; Jin, Hui-Lin; Suita, Kenji; Ishikawa, Yoshihiro(Yokohama City University Graduate School of Medicine, Cardiovascular Research Institute)

Anthracyclineantibiotics, includingdoxorubicin (DOX),arewidelyusedchemotherapeuticagents forcancertherapy.However, theirclinicalusagehasbeenlimitedbytheirseriouscardiotoxicity.p53-in-ducedapoptosisandproductionofreactiveoxygenspecies(ROS)havebeenrecognizedasmechanismsofthecardiotoxicity.TCTP(transla-tionallycontrolledtumorprotein)isananti-apoptoticprotein.Recent-ly,wedemonstratedthatTCTPisap53inhibitor.TCTPbindsdirect-ly to the DNA binding domain of p53, thereby interfering theinteractionbetweenDNAandp53,atranscriptionfactor.Inaddition,itisreportedthatp53andTCTPdownregulateeachother’sexpression.Moreover,TCTPisreportedtoprotectcells fromoxidativestressinducedcelldeath.Basedonthesefindings,wehypothesizethatTCTPplaysimportantroleinthedevelopmentofDOX-inducedheartfailure.WefoundthatDOXtreatmentdownregulatescardiacTCTPexpres-sioninvitroandinvivo.DownregulationofTCTPbySiRNAresultsinincreasedsusceptibilitytoapoptosisincardiomyocytes.Inaddition,TCTPdownregulationenhancedDOXinducedproductionofROS,atleastinpart,throughp53dependentmechanism.Moreover,overex-pressionofTCTPattenuatedDOX-inducedapoptosisofcardiomyo-cytes.Thetransgenicmicewithcardiomyocyte-specificoverexpressionofTCTPareresistanttothedevelopmentofDOX-inducedheartfailure.ThesefindingsindicatethatTCTPmaybeapotenttherapeutictargetforDOX-inducedheartfailure.NoCOI.


1S17F-5Fusion of prion protein (PrP)-expressing cardiac pro-genitor cells forms atypically-shaped cardiomyocytes (ACMs), a new subpopulation of self-beating heart cells in mouseOmatsu-Kanbe, Mariko; Matsuura, Hiroshi(Department of Physiology, Shiga University of Medical Science, Otsu, Japan)

Wehaverecentlyidentifiedatypically-shapedcardiomyocytes(ACMs),anewsubpopulationofspontaneouslybeatingheartcellsobservedwithinacultureofcardiacmyocyte-depletedfractioncellsfromadultmouseheart.ACMsaremostlymultinucleatedandrespondβ-adren-ergicstimulationonthespontaneousCa2+transients.However,manyofthecharacteristicsarestillunclear.Ithasbeenrecentlyreportedthatprionprotein(PrP)servesasasurfacemarkerforisolatingcar-diomyogenicprogenitors frommurineembryonicstemcells.Thepresentstudyexaminedtheoriginsandthedevelopmentofmultinu-clearbeatingACMs.ACMswerefoundtoexpressPrPmostlyintheplasmamembraneevenintheveryearlystageofculture.WeobservedthatthesmallcellsexpressingPrPmigratedtowardsthebeatingACMsandfusedwiththemtobecomelargercells.Furthermore,someoftheACMsconnectedattheedgesoftheplasmamembraneoftenmigratedtowardsthedifferentdirectionsuntiltheyweretornintheindividuallybeatingcells.Immunohistochemicalanalysesrevealedthatthecellsco-expressingPrPandcardiactroponinT(cTnT)wereresi-dentintheinterstitialsmallspaceamongventricularmyocytes.TheresultssuggestthatthePrP(+)/cTnT(+)cellsidentifiedinthemouseheartarecardiacprogenitorcellsandtheirfusionresultsinthedevel-opmentofmultinuclearbeatingACMs,whiletherearesomepossibil-itiesthatthesecellsplayaharmfulrole,suchasacauseofarrhythmia,ininjuredheart.NoCOI.

Symposium 18Central circuitries for psychologically induced physiological behaviors and

responses[CollaborationSymposiumwithThe

JapanNeuroscienceSociety]

(March 16, 15:05–17:05, Room G)

1S18G-1A direct hypothalamo-medullary pathway for socio-psychological stress-induced brown fat thermogene-sis and hyperthermiaKataoka, Naoya1; Nakamura, Kazuhiro1,2(1Career-Path Prom. Uni. for Young Life Sci., Kyoto Univ., Kyoto, Japan, 2PRESTO, JST, Japan)

Psychologicalstress-inducedhyperthermiaisafundamentalautonom-icresponseinmammals.Wehavepreviouslyshownthatstressinduc-esheatproductioninbrownadiposetissue(BAT)byactivatingsym-patheticpremotorneurons in therostralmedullaryraphe (rMR)(Lkhagvasurenet al.,2011).Inthisstudy,wefurtherinvestigatedthecentralcircuitmechanismthatdrivesstress-inducedBATthermogen-esisandhyperthermia.Exposureofratstosocialdefeatstress,aso-ciopsychologicalstressmodel,exhibitedanimmediateincreaseinBATtemperatureandadelayedelevationofabdominaltemperature,asdetectedwithatelemetrysystem.ThisresponsewaseliminatedbyananoinjectionintotherMRwithamixtureofAP5andCNQX,gluta-matereceptorantagonists.Thestress-drivenglutamatergicinputtotherMRmightbeprovidedfromthedorsomedialhypothalamus(DMH),sincerMR-projectingneuronsintheDMHwereactivatedinresponsetothestress.Consistentwiththishypothesis,inhibitionofneuronsintheDMHwithmuscimolinjectionseliminatedthestress-inducedBATthermogenesisandhyperthermia.Supportingthefunctionalcontribu-tionoftheDMH-rMRmonosynapticpathwayinthestress-drivenBATthermogenesis,in vivooptogeneticstimulationofDMH-derivednerveendingsintherMRelicitedBATthermogenesis,whichwasmediatedbyglutamatereceptors intherMR.Theseresults indicatethataDMH-rMRglutamatergicmonosynapticpathwaylikelymediatesstresssignalingtodriveBATthermogenesis,contributingtostress-inducedhyperthermia.NoCOI.

1S18G-2Neuronal basis of pain-induced aversionMinami, Masabumi(Dept of Pharmacol, Grad Sch of Pharm Sci, Hokkaido Univ, Sapporo, Japan)

Theneuralbasisunderlyingthesensorycomponentofpainhavebeenstudiedextensively,butweareonlybeginningtounderstandthatunderlying itsemotionalcomponent.Thebednucleusof thestriaterminalis(BNST)hasbeenimplicatedinnegativeaffectivestates,suchasanxiety,fear,andaversion.Inthepresentstudy,wefirstexaminedtheroleoftheCRFergictransmissionwithinthedorsolateralpartoftheBNST(dlBNST) inpain-inducedaversion. Invivomicrodialysisdemonstratedthe increasedreleaseofCRFwithinthedlBNSTbynoxiousstimulation.Intra-dlBNSTinjectionofCRFantagonistsatten-uatedthepain-inducedconditionedplaceaversion(CPA).Intra-dlBNSTinjectionofCRFproducedCPAevenintheabsenceofnoxiousstim-ulation.Takentogether,theseresultsrevealthatenhancedCRFergictransmissionswithinthedlBNSTareimportantforpain-inducedaver-sion.Next,inordertoaddresscellularmechanismsforthiseffectofCRF,weexaminedtheeffectofCRFonneuronalactivityindlBNSTneuronsusingawhole-cellpatch-clamprecording.WefoundthatCRFmodulatedtherestingmembranepotential inaparticular typeofneurons,typeIIneurons,inthedlBNST.Toclarifytheneuronalcir-cuit(s)involvedinpain-inducedaversion,wecharacterizedVTA-pro-jectingBNSTneuronsusingcombinedneurotracingandhistochemicaltechniques. The major BNST-VTA projection originates fromGAD67-expressingGABAergicneuronsintheBNST,andpreferen-tiallytargetsGABAergic interneurons intheVTA,suggestingthedisinhibitorycontrolofVTAdopaminergicneuronsbyBNST-VTAprojection.Iwilldiscusstheneuronalcircuitsforpain-inducedaversion.NoCOI.


1S18G-3The septo-habenular pathway in anxiety- and fear-re-lated behaviorsYamaguchi, Takashi1; Danjo, teruko1; Ira, Pastan2; Hikida, Takatoshi1; Nakanishi, Shigetada1(1Department of Systems Biology, Osaka Bioscience Institute, Suita, Japan, 2Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, USA)

Theposteriorseptumconsistingofthetriangularseptum(TS)andthebednucleusoftheanteriorcommissure(BAC)ispredominantlylinkedwiththemedialhabenula(MHb)andhasbeenimplicatedinthecontrolofanxietyandfearresponses.However,itsanatomicalandfunctionallinkagehas largelyremainedelusive. WeestablishedatransgenicmodelmouseinwhichtheTSandBACprojectionneuronswerevi-sualizedbyGFPfluorescenceandselectivelyeliminatedbyimmuno-toxin-mediatedcelltargeting.ThelinkagebetweentheTS/BACandtheMHbconstitutes2parallelpathwayscomposedoftheTS-ventralMHb-thecorepartof the interpeduncularnucleus (IPN),andtheBAC-dorsalMHb-theperipheralpartoftheIPN.AblationoftheTSandBACprojectionneuronsselectivelyimpairedanxietyandenhancedfearresponsesandlearning,respectively.InputsfromtheTSandBACtotheMHbarethussegregatedby2parallelpathwaysandplayspecializedrolesincontrollingemotionalbehaviors.NoCOI.

1S18G-4Regulatory mechanism of basal ganglia circuit in re-ward and aversive behaviorHikida, Takatoshi(Medical Innovation Center, Kyoto University Graduate School of Medicine, Kyoto, Japan)

Thebasalganglia-thalamocorticalcircuitryplaysacriticalroleinre-wardandaversivelearninganddecision-making.Theinputsofthenucleusaccumbens inthiscircuitryaretransmittedthroughtwoparalleldirectand indirectpathwaysandcontrolledbydopaminetransmitter.Toexplorehowtheassociativelearningbehavioriscon-trolledinthebasalgangliacircuit,wedevelopedareversibleneuro-transmissionblocking(RNB)technique,inwhichtransmission-blockingtetanustoxinwasspecificallyexpressedinthedirectstriatonigralortheindirectstriatopallidalpathwayand,inturn,blockedeachpathwayinadoxycycline-dependentmanner.Wehaverevealedthedistinctroleofthetwostriatalpathways:thedirectpathwaycriticalforre-ward-basedlearningandtheindirectpathwayforaversivebehaviorandflexibilityoflearning.Wealsoaddressedtheregulatorymecha-nismsofthebasalgangliacircuit,suggestingthedynamicshiftofpathway-specificneuralplasticityviaselectivetransmitterreceptorsisessentialforrewardandaversivebehavior.NoCOI.

Symposium 19Regulatory logic of stress response in

blood vessels

(March 16, 15:05–17:05, Room H1)

1S19H1-1Stretch-induced responses of Human Umbilical Vein Endothelial CellsNaruse, Keiji(Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University)

Humanumbilicalveinendothelialcells (HUVEC)are locatedattheinnersurfaceofvesselwallandarecontinuouslysubjectedtomechan-icalstimulationsinvivo,suchasshearstress,hydrostaticpressureandstretch,anditiswidelyrecognizedthattheyareplayinganimportantphysiologicalroleincardiovascularsystem.Inresponsetostretch,wereportedthatintracellularCa2+increasedtransientlythroughtheac-tivationofmechanosensitive(MS)cationchannelinHUVEC.However,themolecularentitiesthatformtheMScationchannelinthecellremainunknown.Recentpapersreportsomemembersofthetransientrecep-torpotential(TRP)channelshavebeensuggestedasoneofcomponentsofMSchannels.SinceHUVECexpressesseveraltypesofmechanosen-sitiveTRPchannelsincludingTRPV2,TRPC1,andTRPM7,wesup-pressedtheexpressionofthesechannelsinHUVEC.IntheTRPV2-knockeddowncells,thestretchinducedCa2+increasemeasuredbyfluorescenceimagingusingfura2wascompletelyabolished.Afterthecellsweresubjectedto20%uniaxialcyclicstretchat1Hzfor1h,neitherastretch-enhancedstressfiberformationnorachange inthecellorientationperpendiculartothestraindirectioncouldnotbeobserved.Finally,TRPV2-knockeddownHUVECdidnotshowstretch-inducednitricoxide(NO)production.TheseobservationsstronglysuggestthatTRPV2isaresponsibleionchannelforstretch-inducedCa2+increase,which leadsthecytoskeletalreorganizationandNOproduction inHUVEC.Thus,TRPV2wouldbeakeycomponentofMSchannelcomplexinHUVEC.COIproperlydeclared.


1S19H1-2Molecular mechanism of elastic laminae developmentNakamura, Tomoyuki(Department of Pharmacology, Kansai Medical University)

Elasticlaminaecompriseasmuchas50%ofthedryweightinlargearteries,andarenecessaryforelasticrecoilofstretchedarterialwall.Aging-relateddeteriorationofelasticlaminaecausesincreaseinpulsepressure.Becausedegradationofelastic laminae isapathologicalfeatureofaneurysms,ithasbeenofinterestwhetherelasticlaminaedegradationisacauseofaneurysmalenlargementofarterialwall.Weandothersfoundthatthreesecretedproteins,fibulin-4,5,andLTBP-4arerequiredforelasticfiberdevelopment.Geneticallyengineeredmicedeficientinanyoftheseproteinsshowedseverelydisorganizedelasticlaminaeinaortae.However,onlyfibulin-4mutantmice,butnotfibulin-5orLTBP-4deficientmiceshowedaorticaneurysms.Mytalkwillfocusonthedifferencesoffunctionsoftheseelastogenicproteins.Thepossibilityofelasticfiberregenerationwillalsobediscussed.NoCOI.

1S19H1-3Significant roles of matricellular protein in vascular development and disease.Imanaka-Yoshida, Kyoko1,2(The Department of Pathology and Matrix Biology, Mie University Graduate School of Medicine, Tsu, Japan, 2Mie University Research Center for Matrix Biology)

Theextracellularmatrix(ECM)iswellrecognizedtoprovidenotonlystructuralsupportbutalsoimportantbiologicalsignalingwhichinflu-encesvariouscellularfunctioninembryonicdevelopmentandphysi-ological/pathologicalresponsetoextrinsicstimuli.Amongmatrixmolecules,increasedattentionhasbeenfocusedonmatricellularpro-teins.Matricellularproteinsareagroupofnon-structuralECMproteinshighlyup-regulatedatactivetissueofremodeling,servingasbiologicalmediatorsbyinteractingdirectlywithcellsorregulatingtheactivitiesofgrowthfactors,cytokines,proteases,andotherECMmolecules.Tenascin-C(TN-C)isatypicalmatricellularprotein,stronglyexpressedduringembryonicdevelopment,woundhealing, inflammationandcancerinvasion.Invascularsystem,strongexpressionisdetectedinmedialsmoothmusclecellsduringmaturationofvascularwall,andalsoinkedwithseveralpathologicalconditionsuchascerebralvaso-spasm,intimalhyperplasia,pulmonaryarteryhypertension,andaorticaneurysm/dissection.OurrecentresultssuggestthatTN-Csynthe-sizedinresponsetodevelopmentalandenvironmentalcueincludinggrowthfactorsandmechanostressmaybeadeterminantformechan-icalpropertyoftissueandmodulateinflammation,synthesisofothermatrixmolecules.ThediscussionwillbefocusedonseveralrolesandtheirregulatorypathwayofTN-Cinadaptationduringnormalvascu-lardevelopmentaswellastissueremodelinginpathologicalcondition.NoCOI.

1S19H1-4Hemodynamic stress on aortic walls: adaptation and maladaptationAoki, Hiroki(Cardiovascular Research Institute, Kurume University, Kurume, Japan)

Aortaneedstocopewiththehemodynamicstressthroughtheremod-elingofitswall.Thisisaccomplishedbythedynamicinteractionbe-tweentheresidentsmoothmusclecellsandtheinflammatorycellsthatinfiltrateintotheaorticwallundertheabnormalstress.Tobetterunderstandtheadaptiveandmaladaptiveresponsesoftheaorticwall,wecreatedamousemodelofaorticwallstiffeningandaugmentedhemodynamicstressbyperiaorticCaCl2applicationandangiotensinIIinfusion.Theaorticstresscausedaminorinjurywithmacrophageinfiltrationintheaorticwalls,whichhealedin6weeks.WhenJAK/STATsignalingwasaugmentedspecificallyinmacrophagesbydelet-ingSocs3,anendogenousinhibitorofJAK/STAT,theminoraorticinjuryprogressedtoaorticdissectionin6weeks.Theaorticdissectionwasassociatedwiththedecreaseinextracellularstructuralproteinsandcelladhesionmolecules,suggestingthedysregulationofthetissueintegrity.Thus,adequateregulationoftheproinflammatoryresponseisessentialfortheprotectionofaorticwallsunderthestress.NoCOI.

1S19H1-5Extracellular matrix remodeling is critical for closure of the ductus arteriolesMinamisawa, Susumu1; Yokoyama, Utako2(1Department of Cell Physiology, The Jikei University School of Medicine, Tokyo, Japan, 2Cardiovascular Research Institute, Yokohama City Univ. Yokohama, Japan)

Vascularcellsaredefinedbythewaysinwhichtheyregulatetheirextracellularmatrix(ECM),andtheECM,inturn,changesvascularcellphenotype,i.e.theabilitytodifferentiate,proliferate,andmigrate.Elastin,collagens,proteoglycansandstructuralglycoproteinsaresyn-thesizedduringfetaldevelopmentandtheirthreedimensionalorga-nization isoptimal forvascular functions.Thus, theECMisakeycomponent inthedevelopmentandremodelingofspecificvascularstructure.HerewedemonstratehowcellsignalingpathwayssuchasprostaglandinE(PGE)regulatetheECMintheductusarteriosus,anessentialbypassarterybetweentheaorticarchandthepulmonaryarteryduringfetaldevelopment.TheproperorganizationoftheECMiscrucialforclosureoftheductusarteriosusafterbirth.WealsowouldliketodiscussaboutapotentialtherapeuticstrategytoregulatetheECMforvasculardiseasessuchasthepersistentpatentductusarte-riosus.NoCOI.


Symposium 20Metabolic regulation of

renal physiology and pathophysiology[FAOPSJointSymposium-Japan/China]

(March 16, 15:05–17:05, Room H2)

1S20H2-1Farnesoid X receptor (FXR) gene deficiency impairs urine concentration in mice.Zhang Xiaoyan1,2; Huang, Shizheng1; Guan, Youfei1,2(1Peking University Health Science Center, 2Shenzhen University Health Science Center, China)

TheFarnesoidXreceptor (FXR) isa ligand-activatedtranscriptionfactorbelongingtothenuclearreceptorsuperfamily.FXRismainlyexpressedinliverandsmallintestine,whereitplaysanimportantroleinbileacid,lipidandglucosemetabolism.ThekidneyalsohasahighFXRexpressionlevelwithitsphysiologicalfunctionunknown.HerewedemonstratethatFXRisubiquitouslydistributedinrenaltubules.FXRagonisttreatmentsignificantly loweredurinevolumeand in-creasedurineosmolality,whileFXRknockoutmiceexhibitedanim-pairedurineconcentratingabilitywhichledtoapolyuriaphenotype.WefurtherfoundthattreatmentofC57Bl/6micewithchenodeoxy-cholicacid(CDCA),anFXRendogenousligand,significantlyupregu-latedrenalaquaporin2(AQP2)expression,whileFXRgenedeficiencymarkedlyreducedAQP2expression levels inthekidney. InvitrostudiesshowedthattheAQP2genepromotercontainedaputativeFXREsite,whichcanbeboundandactivatedbyFXRresultinginasignificantincreaseofAQP2transcriptioninculturedprimaryinnermedullarycollectingductcells.Inconclusion,thepresentstudydemon-stratesthatFXRplaysacriticalroleintheregulationofurinevolumeanditsactivationincreasesurinaryconcentratingcapacitymainlyviaupregulatingitstargetgeneAQP2expressioninthecollectingducts.NoCOI.

1S20H2-2Interaction between renal medullary (pro)renin recep-tor and PGE2 in AngII-induced hypertensionYang Tianxin1,2; Lu, Xiaohan1; Wang, Fei1(1Institute of Hypertension, Sun Yat-sen University, Guangzhou, China, 2Department of Internal Medicine, University of Utah and Veterans Affairs Medical Center, Salt Lake City, Utah, the United States)

(Pro)reninreceptor(PRR),anewlydiscoveredcomponentofthere-nin-angiotensinsystem(RAS), isviewedasapotentialregulatoroftissuesRASinlightofitscapabilityofbindingreninandprorenintoincreasetheircatalyticactivity.Withinthekidney,PRRexpressionispredominantlyexpressedintheintercalatedcellsoftheCDwhereitsexpressionisinducedbyAngII.Thegoalofourstudywastoin-vestigatethemechanismofhowAngIIactivatedPRRintheCDcellsandfurtherexplorethefunctionalimplicationofthisphenomenon.OurfocuswasplacedondefiningtheCOX-2/PGE2/EP4pathwayinregu-lationofPRRintheCDcellsbyAngII.InvitrostudiesdemonstratedthatAngIIstimulatedPRRproteinexpressioninprimaryratIMCDcells,whichwascompletelyabolishedbyCOX-2inhibitororEP4an-tagonismandto lessextentbyEP1butnotEP3antagonism.ThemediumreninactivityvariedinparallelwithPRRexpressionlevel.InvivostudiesshowedthatchronicAngII infusionelevatedratrenalmedullaryPRRexpressionandtheactiveandtotalreninlevels,allofwhichwereabolishedbyCOX-2inhibitionorEP4antagonism,accom-paniedbyasignificantattentionofhypertensiondevelopment.Overall,theseresultshaveestablishedacrucialroleofCOX-2/PGE2/EP4pathwayinmediatingtheupregulationofrenalmedullaryPRRex-pressionandreninactivityduringAngIIhypertension.NoCOI.

1S20H2-3Crosstalk between cholesterol homeostasis and TLR4/6 pathway ikidney Xiong Zhong Ruan1; Li, Andrew2(1Centre for Lipid Research, Chongqing Medical University, China, 2John Moorhead Renal Research Laboratory, Centre for Nephrology, University College London (UCL) Medical School, Royal Free Campus, London, UK)

Bothmacrophagesandrenalproximaltubularcellsplay importantroleintheprogressionofchronickidneydisease(CKD).Sterolregu-latoryelementbindingprotein (SREBP)cleavage-activatingprotein(SCAP)isacholesterolsensorthatregulatesLDLreceptor(LDLr)and3-hydroxy-3-methylglutarylcoenzymeAreductase(HMGCoAR)tran-scriptionandmaintainstheintracellularcholesterolhomeostasis.Theaimofthisstudy isto investigate ifSCAPregulates inflammatoryresponseinPMAactivatedTHP-1andHK2cells.Over-expressionofSCAPincreased,whileknockdownofSCAPdecreasedLDLrandHMGCoARexpressioninbothcells.Intracellularcholesterolcontentwassignificantlyincreasedafterover-expressionofSCAPandremark-ablyreducedafterknockingdownSCAP.Interestingly,over-expressionofSCAPalsoincreasedthepro-inflammatorycytokinesIL-6andTNF-α.LPS increasedIL-6andTNF-αexpressionasexpected.However,knocking-downSCAPabolishedtheup-regulatoryeffectsbyLPSonIL-6andTNF-α,accompanyingwithreductionofphosphorylationofIκB(ratherthanJNKphosphorylation)andnuclearp65levels,indicat-ingthatSCAPmediatesinflammatoryresponseinTHP-1andHK2cellsviaIκBphosphorylation.ThissuggeststhatSCAPisnotonlyacholesterolsensorbutalsoakeyregulatorforinflammatoryresponseinTHP-1andHK2cells.SCAPmayserveasanoveltargetforbothlipid-loweringandanti-inflammatorytherapies inrenaldiseases.NoCOI.


1S20H2-4Molecular Mechanisms of Regulation of ENaC Ex-pression and Intracellular Trafficking in Renal Epithe-liumMarunaka, Yoshinori; Niisato, Naomi; Yokoyama, Noriko; Sasamoto, Kouhei(Kyoto Prefectural University of Medicine)

EpithelialNa+channel (ENaC)-mediatedNa+reabsorption inthecorticalcollectingductplaysan importantrole inregulationofex-tracelullarfluidvolumeandbloodpressure.TheNa+entrystepviaENaClocatedattheapicalmembrane istherate-limitingstep fortransepithelialNa+reabsorption.TheamountofNa+entryviaENaCdependson:1)theactivityofindividualENaCintheapicalmembrane,and2)theamountofENaCsurfaceexpressionintheapicalmembrane.ToinvestigateregulationofENaCactivityandtrafficking,weestab-lishedamathematicalmodelofENaCtrafficking including4stepsinvolvedinENaCtranslocation:i.e.1)insertion,2)endocytosis,3)recy-cling,and4)degradation.Combingthismathematicalmodelwithelectrophysiologicalandbiochemicalobservations,weobtainedtheamountofrecycledENaCsdependingonqualitycontrolofENaCintheintracellularstoresite.Inourtalk,weprovideinformationon:1)regulationofENaCactivitybyphosphorylation;2)regulationofENaCsurfaceexpressionintheapicalmembraneandrolesoflipidrafts,and3)theestablishedmathematicalmodelofENaCtraffickingandregu-lationofENaCrecycling.SupportedbyJSPS(24590283,25670111)andSaltScienceResearchFoundation(1235).NoCOI

Symposium 21Circadian clock signaling for adaptation to environments

(March 16, 15:05–17:05, Room J)

1S21J-1Role of HIF1α in the Mammalian Circadian SystemIkeda, Masaaki1,3; Kumagai, Megumi1,3; Ueno, Munehisa2; Okabe, Takashi2,3(1Department of Physiology, Saitama Medical University, Moroyama, Saitama, Japan, 2Department of Uro-oncology, International Hospital, Saitama Medical University, 3Molecular Clock Project, Research Center for Genomic Medicine, Saitama Medical University, Hidaka, Saitama, Japan)

PASfactorsaresonamedafterthediscoveryofadomaincommontoPer,Arnt,andSimproteins. These factorssenseandbindsmallmolecules,suchasoxygen,dioxins,andcellularmetabolites.Theyareinvolved intheadaptationtoexternaland internalchanges intheenvironment,suchascircadian,hypoxic,andtoxicstatechangesinmammals.Inthecircadiansystem,thecorefeedbackloopthataccountsforthegenerationofanapproximately24-hrhythminvolvesuptofourPASfactors(BMAL1,CLOCK,NPAS2,andBMAL2)andthreePERproteins.Cross-talkbetweenthePASfactorsinvolvedinhypox-icresponsesandcircadiansystemshasbeenreported.Whilestudyingthecross-talkamongtheadaptationsystemsthatmakeuseofPASfactors,wediscoveredthetranscriptionalregulationofPer2genesbyHIF1α.WewilldiscussthemechanismofthetranscriptionalregulationofthePer2genebyHIF1αandtheroleofHIF1αinthePer2regula-tionoftumorcells.NoCOI.

1S21J-2ROS stress resets circadian clocks to coordinate pro-survival signalsTamaru, Teruya(Dept. of Physiol., Toho Univ. Sch. of Med.)

Asanadaptiveresponsetodailyenvironmentalchanges,thebiologicalclock (circadiansystem)conferstemporalorderasamulti-cellular/tissuesynchronizationstateevokedbyresettingcuesanddailyantic-ipatoryrhythmicphysiologicalprocessesdrivenbycircadiansystem.Dysfunctionofcircadianclocksexacerbatesvariousdiseases,inpartlikelyduetoimpairedstressresistance.Itisunclearhowmammaliancircadiansystemrespondstoreactiveoxygenspecies(ROS)-inducedstress.Toaddressthisquestion,wesoughttoidentifyanovelclock-re-latedadaptivesignalingsystemevokedatthelife-deathboundarytomediateprotectionfromthestress.WeidentifiedaROS-responsivecircadianpathway inmammals.Near-lethaldosesofROS-inducedcriticaloxidativestress (cOS)atthebranchpointof lifeanddeathresetscircadianclocks,synergisticallyevokingprotectiveresponsesforcellsurvival.ThecOS-triggeredclockresettingandpro-survivalresponsesaremediatedbytranscriptionfactor,centralclock-regula-toryBMAL1andheatshockstress-responsive (HSR)HSF1.CaseinkinaseII(CK2)-mediatedphosphorylationregulatesdimerizationandfunctionofBMAL1andHSF1tocontrolthecOS-evokedresponses.ThecorecOS-responsivetranscriptomeincludesCK2-regulatedcross-talkbetweenthecircadian,HSR,NFκB-mediatedanti-apoptotic,andNrf2-mediatedanti-oxidantpathways.Thisnovelcircadian-adaptivesignalingsystemlikelyplaysfundamentalprotectiverolesinvariousROS-inducibledisorders,diseasessuchascancerandneurodegenera-tivediseases,anddeath.NoCOI.


1S21J-3Phase shift of cancer clock by hypoxiaMasubuchi, Satoru1; Yagita, Kazuhiro2; Nakamura, Wataru3; Honma, Sato4; Honma, Ken-ichi4(Department of Physiology, Aichi Medical University, Nagakute, Aichi, Japan, 2Department of Physiology and Systems Bioscience, Kyoto Prefectural University of Medicine, Kyoto, Japan, 3Laboratory of Oral Chronobiology, Graduate School of Dentistry, Osaka University, Suita, Osaka, Japan, 4Department of Chronomedicine, Hokkaido University Graduate School of Medicine, Sapporo, Japan)

Mammaliancircadianphysiologicalandbehavioralfunctionsaredriv-enby intracellular transcriptionalnegative-positive feedback loopsinvolvingseveralclockgenes.Clockgenesareexpressedinacircadi-anfashionnotonlyinthemasterclock,thehypothalamicsuprachias-maticnucleus(SCN),andperipheralclocksinextra-SCNorgansbutalsoincancercells.Partialpressureofoxygen(pO2)inthesolidtumordeclinesdependingonthedistancefrombloodvessels.Sincehypoxiccancercellsareresistanttotherapies, informationaboutcircadianrhythmofthesehypoxiccancercellsiscriticalfordesigningchrono-therapy.PreviousstudyondrosophillaeclosionrhythmsuggeststhatlowpO2affectsthecircadianrhythmsofcancercells.Inthepresentstudy,synchronizedcoloncancercell line (HCT116:DBP-luc)wasexposedto6hhypoxiaofdifferentpO2.Hypoxiaphaseshiftedcancerclockdependingonexposuretimingandoxygenconcentration.Incontrast,thesametreatmenthadlittleeffectonfibroblastclock.Buff-eringofhypoxia-inducedpHdecreaseattenuatedphaseshiftsofcan-cerclock.Theseresultssuggestthatcancerspecificmetabolicresponsetohypoxiamodifiesclockfunctioninsolidtumor.NoCOI.

1S21J-4Dissection of circadian clock system in humansHida, Akiko(Department of Psychophysiology, National Institute of Mental Health, National Center of Neurology and Psychiatry, Tokyo, Japan)

Circadianrhythmsofbehaviorandphysiologyaredrivenbyasystemofself-sustainedclocksandareentrainedbyexternalcues.Themam-maliancentraloscillator,SCNincorporatesenvironmentalinformationandorchestratesslaveoscillators inperipheralcells.Thecircadianclocksystemiscomposedofahierarchyofoscillatorsthat involvetranscriptionandtranslationfeedbackloopsofmultipleclockgenes.Disorganizationofthecircadiansystemisknowntoberelatedtomanydiseasesincludingsleep,moodandmetabolicdisorders.Evaluationofcircadianphenotypesiscrucialforunderstandingthepathophysiologyofdiseasesassociatedwithdisturbedbiologicalrhythms.Wemeasuredclockgeneexpressioninfibroblastsderivedfromindividualsubjectsandobservedcircadianrhythmsinthecells(in vitrorhythms).Periodlengthofthein vitrorhythm(in vitroperiod)wascomparedwiththeintrinsiccircadianperiod,τ,measuredundera forceddesynchronyprotocol(in vivoperiod)andcircadian/sleepparameters.Althoughnosignificantcorrelationwasobservedbetweenthein vitroandin vivoperiods,thein vitroperiodwascorrelatedwithchronotype.Evaluatingrhythmicexpressionofclockgenesinisolatedfibroblastcellsmightthereforebeanappropriatemethodtoassessanindividual’scircadianclockphenotype.Here,wehaveexaminedin vitrorhythmsinpatientswithdisturbedsleep-wakerhythmsandwilldiscusscircadianpheno-typesofthepatients.NoCOI.

1S21J-5Stress and circadian clockTakumi, Toru(RIKEN Brain Science Institute, Wako, Japan)

Several linesof indirectevidencesuggestthatmooddisordersanddysfunctionsincircadiantimingarelinked.Learnedhelplessness(LH)rats,amodelofdepression,exhibitshorterperiodsofcircadianrhythmsandtheseshortenedperiodsarelengthenedbylithium,amoodstabi-lizerandinhibitorofglycogensynthesiskinase3β(GSK3β).CircadianexpressionofphosphorylatedGSK3β(pGSK3β)disappearsinthesu-prachiasmaticnucleusandprefrontalcortexofLHratsandinfibro-blasts.ThealteredpGSK3βlevelisalsoobservedindifferentdepres-sionmodels.WeidentifyaphosphorylationsiteonPERIOD2(PER2)byGSK3βandpresentevidencethatphosphorylationonPER2mod-ulatesbothcircadiananddepression-relatedphenotypes.ThealteredpGSK3βandanewsignalingpathwayspecifiedinthisstudyopenthedoortodevelopnewdiagnosticandtherapeutictoolsformooddisor-ders.NoCOI.

Symposium 22Presentation skills for researchers

[JapanYoungPhysiologistAssociationSymposium]

(March 16, 15:05–17:05, Room K)


1S22K-1Presentation skills for scientists, in a view of present situation and tasksKamikubo, Yuji(Department of Pharmacology, Faculty of Medicine, Juntendo University, Tokyo, Japan)

Thepresentationofscientificknowledgeandresearchisanimportantpartofaresearcher’scareer,yetscientistsarerarelyofferedprofes-sionalinstructiononhowtopresenttheirachievement.Inthispresen-tation,Italkaboutpresentationinstructionsforyoungscientists.

1S22K-2Presentation skills for researchersHayashi, Hidetoshi(Tokyo JTECT Bldg.,11-15, Ginza7, Chuo-ku Tokyo Japan)

HidetoshiHayashiRICOHHUMANCREATESCO.,LTDPresentationskillsforresearchersThefollowingrequiredsinceaneffectivepresen-tationisputonexplainsthreepoints.1.Persuasionbylogos“logic”language,scenario,andreason.2.Ethos“reliance”speaker’scharacterandpersonalcharacter,senseofethics,credibility.3.Feelingofpathos“passion”opinion,earnestness,thought.

1S22K-3Easy to Understand and Beautiful Visual Design for Research Presentations Tanaka, Sayoko(University of Tsukuba, Tsukuba, Japan)

Visualexpressionshavepoweroftransmittingcontentseasilyeventhoughdifficultbylanguage.Visualexpressionsforresearchpresen-tationsthathelpunderstandingsareoccupyingpositionsmoreandmoreimportantforresearchers.Basedonthissituation,Italkvisualdesignmethodsforresearchpresentationsthatareeasytounderstandandbeautiful.Themaincontentsareasfollows.1.Drawing:HowtocreatecomplicatedfigureseasilybyPowerPointorKeynote.2.Color:Simplyandcontrastycolorscheme,coloruniversaldesignfortrans-mittingtomorepeople.3.Typography:Recommendationandnon-rec-ommendationfontstylesofJapaneseandEnglish,suitablefontsize,suitablespacebetween lines,andsuitable lengthof lines.4.Layout:Naturaleyesflow,alignment,closingandseparating,whitespace,hi-erarchyofinformation.

Symposium 23Regulation of brain functions by meals

and systemic metabolism

(March17, 9:00–11:00, Room B)


2S23B-1Facilitation of spatial memory and hippocampal plas-ticity during food intake through satiety signalsOomura, Yutaka1; Katafuchi, Toshihiko1; Aou, Shuji2; Moriguchi, Shigeki3; Fukunaga, Koji3(1Dept Integr Physiol, Sch Med, Kyushu Univ, Fukuoka, 2Dept Brain Sci Engineer, Kyushu Inst Technol, Kitakyushu, 3Dept Pharmacol, Sch Pharmacy, Tohoku Univ, Sendai)

Duringfoodintake3mMglucoseconcentrationinCSFbecomestwice.Intrahippocampalinjectionof6mMglucosefacilitatsspatiallearningandmemory.InvitroCA1slicepreparationwith3mMglucose inRingersolution,EPSPsamplitudesgeneratedbytheSchaffercollater-al/commissuralstimulationmarkedly increased in6mMglucose.NMDAandAMPAevokedcurrentsweresignificantlyaugmentedby6mMglucose.Thepaired-pulse facilitationexperiments indicatedaugmentationofpresynaptictransmitterreleaseby6mMglucose.TheseaugmentationswereassociatedwithenhancedphosphorylationsofCAMKII,PKCandpresynapticsynapsin.HighfrequencystimulationoftheSchafferpathwayfailedtoinduceLTPintheCA1regionin3mMglucosebutfacilitatedby6mMglucose.TheLTPinductioninthe6mMglucosewasassociatedwithfurtherincreaseinCAMKIIandPKCautophosphorylations.Ontheabovementionedglucoseef-fectsthereisonequestionwhethertheseeffectsaredirectglucoseorglucoseconvertedATPeffects.Thenwearenowexperimentingusingpinacidilanddehydorepiandrosteroneontheglucoseeffects,sincethesearemakingopeningtheATPsensitiveKchannels.Iftheglucoseisstilleffectiveontheseconditions,wecansagelymentionthedirectglucoseeffects.Takentogetherfoodintakeisvaluableforthebrainplasticityenhancingspatialmemoryoffoodrichplaces.NoCOI.

2S23B-2Pancreatic hormones affect the brain and regulate food intake via vagal afferent neural pathwayIwasaki, Yusaku; Yada, Toshihiko(Department of Physiology, Jichi Medical University School of Medicine, Tochigi, Japan)

Peripheralhormonesarethoughttosignaltothebrainviapenetratingthroughtheblood-brainbarrierand/oractingonthevagalafferents.Pancreatichormonesinsulinandpancreaticpolypeptide(PP)arere-leasedinresponsetomealintakeandcontributetoformationofsatiety.ThisstudyaimedtodeterminewhetherinsulinandPPdirectlyactonthevagalafferents.Wemonitoredtheactivitiesofisolatedvagalaffer-entnodoseganglionneurons(NGNs)bymeasuringcytosolicCa2+([Ca2+]i)andmembranepotential.PPincreased[Ca2+]iinNGNs.Theanorexi-geniceffectofPP isreportedlyabolishedbyvagotomy.Hence,theactivationofvagalafferentsbyPPislinkedtoinhibitionoffeeding.Insulindepolarizedandincreased[Ca2+]iin8%ofNGNs.Weexaminedwhetherthevagalafferentsinnervatingthepancreassenseinsulin.Theneuralfibersinnervatingpancreaticisletsexpressedtheinsulinreceptor.TheNGNsinnervatingthepancreasrespondedtoinsulinat10-7M,theestimatedinsulinconcentrationinthepancreas,withhighresponse incidence (20%). Intraperitoneal injectionofglibenclamiderapidlyinducedinsulinsecretionandphosphorylatedAKTinNGNs.Moreover,theinsulinactionwasmarkedlyattenuatedinNGNsisolat-edfromIRS-2KOmicethatexhibithyperphagiaandobesity.Theseresultssuggestthatvagalafferentneuronsinnervatingthepancreassensethereleasedinsulinwithinthepancreastorapidlyinformthebrainofmetabolicchanges,therebycontrollingappetite,andpossiblyenergyandglucosemetabolism.NoCOI.

2S23B-3Regulation of longevity and cognitive function through neural insulin-like signalingTaguchi, Akiko1,2; Makinodan, Manabu3; Fukuokaya, Wataru1; Kurata, Eiko1; Nakazato, Masamitsu1; Corfas, Gabriel3; White, Morris2(1Div. of Neurology, Endocrinology, and Metabolism, Faculty of Medicine, Univ. of Miyazaki, Japan, 2Div. of Endocrinology, Childrens Hosp Boston, Harvard Med Sch, USA, 3Div. of Neurology, Childrens Hosp Boston, Harvard Med Sch, USA)

Diabetesandobesitymaycontributetocognitive impairmentandreducedadulthippocampalneurogenesisthatisassociatedwithmem-oryfunction,suggestingthatmetabolicdisturbancespromoteaging-likeeffects.PreviousstudiesshowthatlessneuralIRS2(InsulinReceptorSubstrates2)increaseslifespanandreversesprematuremortalitywithreducingaggregatedAβinAlzheimerdiseasemodelmice,whilein-creasingbloodinsulinlevel.However,itremainsunknownhowneuralIRS2deficiency influencescognitivedeclineandadulthippocampalneurogenesis.HereweshowthatIRS2isexpressedinbothmatureneuronsandneuralstem/progenitorcellsinthehippocampaldentategyrus(DG).Proliferationandneuronaldifferentiationremarkablyaug-mentedinagedmicelackingneuralIRS2(BIRS2ko).Intheprocessofneuronaldifferentiation,neural IRS2depletion increaseddendriticcomplexityanddecreasedmicroglialactivationintheDGofagedmice.Moreover,theWaterT-mazetestdemonstratedthatthetestaccura-cyratiosinagedBIrs2komiceweresignificantlygreaterthanthoseinage-matchedcontrols.ThesefindingssuggestthatlessneuralIRS2promotesadultneurogenesisandretainscognitivefunctionsinagedmicewithoutage-associatedreduction indendriticcomplexityandincreasedneuroinflammation,regardlessofhyperinsulinemia.NoCOI.

2S23B-4The role of hypothalamic brain-derived neurotrophic factor (BDNF) in energy metabolism: therapeutic po-tential for visceral obesityMaekawa, Fumihiko1,2; Fujiwara, Ken2,3; Toriya, Masako2; Nohara, Keiko1; Yada, Toshihiko2(Center for Environmental Health Sciences, National Institute for Environmental Studies, Tsukuba, Japan, 2Department of Physiology, Jichi Medical University, Shimotsuke, Japan, 3Department of Anatomy, Jichi Medical University, Shimotsuke, Japan)

BDNFisimplicatedinfeedingbehaviorandenergybalance.Inthisstudy,wefocusedonexamining1)howBDNFregulatesenergyme-tabolismand2)whetherimpairedexpressionofBDNFplaysaroleinthedisorderofmetabolism.Firstly,wefoundthatBDNFregulatedtheneuronsexpressingcorticotrophin-releasinghormone(CRH)intheparaventricularnucleusofhypothalamus.TheCRHneuronsexpressedTrkB,areceptorforBDNF,andintracerebroventricular(icv)injectionofBDNFincreasedCRHmRNA.Inaddition, the increasedenergyexpenditurebyBDNFtreatmentwasantagonizedbysimultaneoustreatmentwithaCRHreceptorantagonist.Theseresultsdemonstrat-edthepossibilitythattheeffectofBDNFismediatedviatheCRHneuralpathway.Secondly,weinvestigatedBDNFexpressionintype2diabeticGKratsexhibitingvisceralobesity.Inmiddle-agedGKrats,BDNFandglucosetransporter-2expressionsweresignificantlyde-creased intheventromedialhypothalamus (VMH),suggestingthatimpairedglucosemetabolismcausedBDNFreduction.SinceBDNFtreatmentbyicvinjectionamelioratedvisceralfataccumulationandhyperleptinemia,thevisceralobesityisatleastinpartduetothere-ductionofBDNFinVMH.TheBDNFsupplementationcouldprovideaneffectivetreatmentofvisceralobesityintype2diabetes.NoCOI.


Symposium 24Frontline of molecular physiology of

hair cells in the inner ear

(March 17, 10:05–12:05, Room C)

2S24C-1Introduction of hair cell functionHibino, Hiroshi(Department of Molecular Physiology, Niigata University School of Medicine, Niigata, Japan)

Haircellsinthecochleaofthemammalianinnereararesensoryepi-theliathatlieabovethebasilarmembraneandplaycentralrolesinhearingsystem. Sound-inducedvibrationofthebasilarmembranedeflectsthestereociliaofthehaircellsandopensthemechnoelectricaltransductionchannelsattheirtop.Substantiallycationintheextra-cellularsolutionentersintothehaircell,whichdepolarizesthem.Thisprocesstransformsthemechanicalenergyofsounds intoelectricalsignals.Excitationofhaircellssecretestheneurotransmitterglutamatebyuniquemoleculararchitectureintheirpresynapticregion.Fur-thermore,haircellsamplifyfaintsoundenergybydynamicphysiolog-icalphenomenathatemergeinthestereociliaandinthecellbodies.Inthistalk,Iwillexplainhowhaircellsworkinthecochleaastheintroductionofthissymposium.NoCOI.

2S24C-2Mechanoelectrical transduction channels in the inner ear: TMC1 and TMC2Kawashima, Yoshiyuki(Department of Otolaryngology, Tokyo Medical and Dental University, Tokyo, Japan)

Transmembranechannel-like1(TMC1)genewasidentifiedthroughpositionalcloningof thegeneunderlyingdominantandrecessivenonsyndromichearinglossattheDFNA36andDFNB7/B11loci,re-spectively.MouseTmc1mRNAisexpressedininnerearhaircellsandp.M412KpointmutationinTMC1causehearinglossinBeethoven(Tmc1Bth)mice.ThecloselyrelatedTmc2geneisalsoexpressedinhaircells.Tmc1andTmc2encodesix-passintegralmembraneproteinswithsequenceandtopologysimilartoeachother.ArecentreportsuggeststhatC. elegance tmc-1formsnon-selectivecationchannelswhenexpressedinheterologouscells.TheonsetofTmc2expressionintheinnerearcoincideswithdevelopmentofhaircelltransduction.MicewithatargetdeletionofTmc1aredeaf,whilethosewithade-letionofTmc2arephenotypicallynormal.Micethataredoublehomo-zygotesfortargeteddeletionsofTmc1andTmc2exhibitvestibulardysfunctionaswellasprofounddeafness.Doublehomozygousmutanthaircellsshowintactstereociliayethavenotransduction.Exogenous-lyexpressedTMCproteinscanbelocalizedneartipsofstereociliaandeitherTMC1orTMC2canrescuetransductioninthedoublehomo-zygousmutanthaircells.HaircellsthatexpressedTmc2alonehaslargesingle-channelcurrentsrelativetothoserecordedfromhaircellsthatexpressedTmc1.Importantly,haircellsthatexpressedTmc1Bthhassignificantlysmallsingle-channelcurrentsrelativetothoserecord-edfromhaircellsthatexpressedwild-typeTmc1.ThedataindicatethatTMC1andTMC2arecomponentsofmechanoelectricaltransduc-tionchannels.NoCOI.

2S24C-3Tip link is an asymmetric filament formed by hetero-philic interaction of cadherin 23 and protocadherin 15 Sakaguchi, Hirofumi(Department of Otolaryngology-Head and Neck Surgery, Kyoto Prefectural University of Medicine, Japan)

Haircellsarespecializedmechanosensorsthatconductsmechanoelec-tricaltransduction(MET).METinhaircellsoccursinhairbundles,comprisedofdozensofstereocilia.METchannelsarelocalizedclosetotiplinks,theextracellularfilamentsconnectingthetipofeachste-reociliumtoitsneighbor.Hairbundledeflectiongeneratestensionintiplinks,whichrapidlyincreaseintheopenprobabilityofMETchan-nels.Twomembersofthecadherinfamily,cadherin23(CDH23)andprotocadherin15(PCDH15),hadbeenidentifiedasintegralcomponentsoftiplinks,butitstrueentitywasstillunknown.Wefirstdemonstrat-edthatheterophilicinteractionofCDH23homodimersandPCDH15homodimersformsatiplinkfilamentusingstructuralandbiochemicalassays.Immuno-TEMstudyshowedthatbothCDH23andPCDH15extracellularcadherindomains(ECDs)localizeattheupperandlowerpartofatiplink,respectively.LabelingofN-terminiofCDH23andPCDH15colocalizedinthetiplink,suggestingthatthetwocadherinsinteractviatheirN-termini.Negative-stainingTEMshowedthatre-combinantECDofCDH23andPCDH15formcoiledhomodimersthatinteractattheirN-termini,resemblingtiplinkstructure.InteractionoftheCDH23andPCDH15ECDswasalsoconfirmedbybiochemicalassay.Ourdataidentifiedtheconstituentsofthetiplinkandrevealeditsasymmetricmolecularproperty,whichshednewlightonthemo-lecularbasisofMETandthemechanismscausinghereditarydeafness,noise-inducedhearinglossandpresbycusis.NoCOI.


2S24C-4Dissection of synaptic function of otoferlin at the mouse inner hair cell afferent synapseTakago, Hideki1,2,3; Moser, Tobias2,3(1Perceptual Funtions Section, Department of Rehabilitation for Sensory Functions, Research Institute, National Rehabilitation Center for Persons with Disabilites, Tokorozawa, Japan, 2InnerEarLab, Department of Otolaryngology, Univerisity Medical Center Göttingen, Göttingen, Germany, 3Bernstein Centor for Computational Neuroscience, Univeristy of Göttingen, Göttingen, Germany)

Ca2+signalsandsubsequentexocytosisatthe innerhaircell (IHC)afferentsynapseunderlietheencodingofsound.Themulti-C2domainproteinotoferlin,whosemutationscausetheautosomalrecessivedeafnessDFNB9,hasbeenbelievedtocontrolCa2+-triggeredexocyto-sisatthissynapsebymeansofneuronalSNAREproteins.However,arecentstudyhasarguedthatthehaircellafferentsynapseappar-entlyoperateswithoutneuronalSNAREproteins,requestingadetailedevaluationofthesynaptic functionofotoferlinatasinglesynapselevel.Therefore,weperformedpatch-clamprecordingsfrompostsyn-apticboutonsofspiralganglionneurons inwild-typeandotoferlinmutantmice,eachofwhichcontactsasingleIHCthroughasingleribbon-typesynapse.Wefoundthatdeletionofotoferlindecreasedfrequencyofbothspontaneousandhighpotassium-evokedrelease,confirmingtheroleofotoferlinforprimingand/orfusionofsynapticvesicles.Furthermore,wefoundthatdeletionofotoferlindecreasedEPSCamplitude,butnotabolished largeandvariedamplitudeofEPSCs,supportingthehypothesisthatuniquantalreleaseofsynapticvesiclesisafundamentalmechanismattheIHCafferentsynapse.NoCOI.

2S24C-5Contributions of two force-generating mechanisms of hair cell to nonlinear amplification in the mammalian cochleaNin, Fumiaki1; Reichenbach, Tobias2; Fisher, Jonathan A.N.2; Hudspeth, J. A.2(1Department of Molecular Physiology, Niigata University School of Medicine, Niigata, Japan, 2Rockefeller University, New York, US)

Thecochleartravelingwaves’highsensitivitystemsfromtheactiveprocessofouterhaircells.Theactiveprocesspossessestwoforce-gen-eratingmechanisms:activehair-bundlemotilityelicitedbyCa2+influxandsomaticmotilitymediatedbythevoltage-sensitiveproteinprestin.Althoughinterferencewithprestinhasdemonstratedaroleforsomat-icmotilityintheactiveprocess,itremainsunclearwhetherhair-bun-dlemotilitycontributes invivo.Weselectivelyperturbedthetwomechanismsbyinfusingsubstancesintotheendolymphorperilymphofthechinchilla’scochleaandthenusedscanninglaserinterferometrytomeasurevibrationsof thebasilarmembrane.Blockingsomaticmotility,damagingthetiplinksofhairbundles,ordepolarizinghaircellseliminatedamplification.Whilereducingamplificationtoalesserdegree,pharmacologicalperturbationofactivehair-bundlemotilitydiminishedoreliminatedthenonlinearcompressionunderlyingthebroaddynamicrangeassociatedwithnormalhearing.Theresultssuggest thatnotonlysomaticmotilitybutalsoactivehair-bundlemotilityplaysasignificantroleintheamplification,andactivehair-bun-dlemotilitycontributescompressivenonlinearityofthecochleartrav-elingwave.NoCOI.

Symposium 25Pathophysiological roles of

TRPM subfamily in stressed heart

(March 17, 10:05–12:05, Room D1)

2S25D1-19-Phenanthrol, a TRPM4 Inhibitor, protects rat hearts from ischemia-reperfusion injuryTakahashi, Ken1; Piao, Hulin1,2; Wang, Jing1,3; Naruse, Keiji1 (1Okayama University Graduate School of Medicine, Dentistry and Pharmacological Sciences, 2Department of Cardiovascular Surgery, The Second Affiliated Hospital of Jilin University, Changchun, China, 3Qingdao Municipal Hospital, Qingdao, China)

Ischemicheartdiseasenotonlyremainstheleadingcauseofmortali-ty,butalsoleavescomplicationssuchasanginapectorisorarrhythmias.TRPM4channelsareinvolvedinthepathogenesisofvariouscardio-vasculardiseases.Hereweexploredthepossible involvementofTRPM4channels inthedevelopmentof ischemia-reperfusion (I/R)injury.Pretreatmentwith9-phenanthrol(9-Phe),aspecificinhibitorofTRPM4,improvedthesurvivalrateofratsunderwentleftanteriordescendingcoronaryarteryocclusionfollowedbyreperfusionin vivo.Inisolatedrathearts,9-Phepretreatmentrecoveredcardiaccontrac-tilitydramatically,withreducedtissuedamageandarrhythmicevents.Surprisingly,simultaneousapplicationof5-HD,ablockerofthemito-chondrialKATPchannel,didnotabolishthecardioprotectiveeffectproducedby9-Phe,suggestingthatthecardioprotectionby9-PheisnotderivedfromKATPchannelnecessaryforclassiccardioprotectionpathway.Furthermore,aratcardiomyocytecelllineH9c2wasusedtotestwhethercardiomyocyteshaveprotectivefunctionagainstI/Rinjurybythemselves.9-PhepretreatmentkeptviabilityofH9c2cellsunderwentoxidativestressbyH2O2 (mimickingI/Rdamage)andanoxia.TRPM4channelsmayserveasaneffectivepharmacologicaltargetforcardioprotectivetreatmentstrategiesagainstI/Rinjury.NoCOI.


2S25D1-2Enhanced PIP2 sensitivity of human TRPM4 channel with an arrhythmic mutationHu, Yaopeng1; Kurahara, Rin1; Okamura, Yasushi2; Mori, Masayuki3; Inoue, Ryuji1(1Dept. Physi., Grad.Sch. Med. Sci., Fukuoka Univ., 2Lab. Integ. Physiol., Dept. Physiol., Grad. Sch. Med., Osaka Univ., 3Dept. Synth. Biol.Chem., Grad. Sch. Engineer., Kyoto Univ.)

TRPM4isaCa2+-activatedmonovalentcation-selectivechannelandknowntobeinvolvedinbothcongenitalandacquiredformsofcardi-acarrhythmias.Inthisstudy,weexploredhowthischannelisregu-latedbyendogenousPIP2levelwhichwouldfluctuateunderneuro-hormonalstresses,andalsothefunctional impactofanarrhythmicmutationE7Kthereon,byusingtheDanioreriovoltage-sensingphos-phatase(VSP).VSPwascoexpressedinHEK293cellswithhumanTRPM4[EKE5-7 (wild-type),EKK5-7 (E7Kmutant),orENE5-7mutant]andcurrents inducedby intracellularperfusionof1µMCa2+wererecordedbythepatchclamptechnique.WhentheCa2+-activatedcurrentswerestabilized,asetofdepolarizingpulses (from-60to+120mV;300–2000msinduration)wererepetitivelyappliedtoreduceendogenousPIP2contentviaVSPactivation.ProlongingthedurationofthepulsesprogressivelyinhibitedtheCa2+-activatedcurrents,andrecoveryfromitrequiredseveralseconds.TheapparentsensitivitytoPIP2reductioncausedbyVSPactivationwasstrongerintherankorderofENE5-7>wild-type>E7K,whereastherecoveryfromtheinhi-bitionwasmorerapid;E7K>wild-type>ENE5-7.TheseresultssuggestthatendogenousPIP2isessentialtomaintainTRPM4channelactivitypresumably through electrostatic interactions dependent on thechargedstatusofthe5thto7thN-terminalaminoacidresidues,whichmightbepathologicallyenhancedintheE7Kmutation.NoCOI.

2S25D1-3Cardiac TRPM2 aggravate ischemia reperfusion inju-ryNumata, Tomohiro1; Shimizu, Shunichi2; Mori, Yasuo1(1Department of Technology and Ecology, Hall of Global Environmental Research, Kyoto University, Kyoto, Japan, 2Department of Pathophysiology, Showa University School of Pharmacy, Hatanodai, Shinagawa-ku, Tokyo, Japan)

Transientreceptorpotentialmelastatin2(TRPM2)isaCa2+-permeablenonselectivecationchannelactivatedbyoxidatativestress.However,itremainsunknownwhetherTRPM2contributestoheartfunctionduring ischemia-reperfusion (I/R).HereweshowthattheTRPM2aggravateI/Rinjuryincardiacmyocyte.Inisolatedcardiaccellsfromadultmousehearts,wholecellrecordingsrevealedthattheADPri-bose-inducedmacroscopiccationiccurrentsexhibitTRPM2-likeprop-ertiessuchaslinearrectificationandsensitivitytoeconazol.ThewholecellcationcurrentwasalsoactivatedbyH2O2andoxygenglucosedeprivation (OGD)/reperfusion.RT-PCRandrealtimePCRdemon-stratedmolecularexpressionofTRPM2inisolatedcardiaccellsfromadultmousehearts. In isolatedcardiaccells fromTRPM2-deficientmice,H2O2-orOGD-inducedwhole-cellcurrentsandcelldeathweresuppressed.IntheLangendorffperfusionmodel,wefoundthat leftventricularpressure,heartrate,ratepressurewererecoveredearlierafterreperfusioninTRPM2-deficientmicethaninwildtypemice.ItisconcludedthatactivationofendogenousmyocardialTRPM2isin-volvedinmyocardialinjuryinducedbyI/Rex vivoandin vitro.Car-diacTRPM2mayserveasatargetaccessibleevenafter ischemicattackforpharmacotherapeuticinterventioninI/R-inducedmyocar-dialinfarction.NoCOI.

2S25D1-4Activation of TRPM2 channels in neutrophils enhanc-es myocardial ischemia/reperfusion injuryShimizu, Shunichi1; Mori, Yasuo2(1Division of Physiology and Pathology, Department of Pharmacology, Toxicology and Therapeutics, Showa University School of Pharmacy, 2Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University)

Transientreceptorpotentialmelastatin2(TRPM2)isaCa2+-permeablenonselectivecationchannelactivatedbyoxidativestress,andisex-pressed inneutrophilsandcardiomyocytes.WeexaminedwhetherTRPM2contributestomyocardialischemia-reperfusion(I/R)injury.Wild-type(WT)andTrpm2knockout(KO)micewereexposedtoI/Rbyligationoftheleftcoronaryartery.MyocardialinfarctionfollowingI/RbutnotischemiaalonewasreducedinTrpm2KOmice,andcar-diaccontractilefunctionwasalsoimprovedinTrpm2KOmice.More-over,neutrophilaccumulationinthereperfusedareawasloweredinTrpm2KOmice.WhenWTorTrpm2KOpolymorphonuclearleuko-cytes (PMNs)wereadministeredtotheTrpm2KOheartexvivothroughperfusateorinvivobyintravenousinjection,WTPMNsin-ducedmoreseverecardiacinjuryfollowingI/RcomparedwithTrpm2KOPMNs.InWTbutnotinTrpm2KOPMNs,thecombinationofH2O2andleukotrieneB4(LTB4)resultedinenhancementoftheincreasein intracellularCa2+andtheiradhesiontoendothelialcells.Thesefindings indicatethatTRPM2is implicated inthedevelopmentofmyocardialreperfusioninjury.AccumulationofneutrophilsinthehearttriggeredbyactivationofneutrophilTRPM2byH2O2andLTB4islikelytohaveacrucialroleinmyocardialI/Rinjury.NoCOI.

Symposium 26Modulation of cellular functions by

heat[CollaborationSymposiumwith

TheBiophysicalSocietyofJapan]

(March 17, 9:00–11:00, Room E)


2S26E-1Detection of temperature change in norepineph-rine-stimulated brown adipocytes with a bimaterial microcantileverSato, Masaaki; Ishijima, Akihiko(Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan)

Temperaturegloballyaffectsbiologicalactivitiesandchemicalreactionsin livingthings. Inmammals,brownadipocytesgenerateheat tomaintainbodytemperature.Althoughthemechanismofheatgener-ationhasbeenstudiedincellsuspensionscontaining105ormoreadi-pocytes,heatgenerationandtemperatureelevationinindividualcellshasnotbeenmeasured. Inaddition,measuringtherateofoxygenconsumptioninaclosedvesselwoulddisruptthecell’snaturalcondi-tion,bythedepletionofsubstancesrequiredforgeneratingheat,suchasoxygendissolvedinthesolution.Inviewoftheneedforamethodtoinvestigatethermogeniccapacitynon-invasivelyatthecellularlevelwithkeepingcell’snaturalcondition,wefabricatedabimaterialmicrocantileverthatcandetectmillikel-vin-ordertemperaturechangesinsolution.Weobservedbendinginthemicrocantileverinresponsetoatemperatureriseofabout0.1Kpercell,whenitwasplacedwith4–7norepinephrine-stimulatedbrownadipocytes.Ourexperimentalsystemrevealedthatwhenadequatedissolvedoxygenwasavailable,theamountofheatgeneratedbythebrownadipocytesgraduallyincreasedoveraperiodofseveralhours.Ourbimaterialmicrocantilevercanbeusedtoobservechanges incellulartemperatureinawiderangeofcelltypes,includingvirus-in-fectedandcancercells,andourstudyofbrownadipocytesatthesingle-cell levelmaycontributetomedicalapproachestotreatingmetabolicsyndromesandlifestyle-relateddiseases.NoCOI.

2S26E-2Microscopic heat pulses reveal novel tempera-ture-sensitivities of living cellsOyama, Kotaro1; Shintani, Seine A1; Itoh, Hideki1; Ishii, Shuya1; Fukuda, Norio2; Suzuki, Madoka3,4; Ishiwata, Shin'ichi1,3,4(1Department of Physics, Faculty of Science and Engineering, Waseda University, Tokyo, Japan, 2Department of Cell Physiology, The Jikei University School of Medicine, Tokyo, Japan, 3Organization for University Research Initiatives, Waseda University, Tokyo, Japan, 4Waseda Bioscience Research Institute in Singapore (WABIOS), Waseda University, Singapore)

Thechemicalreactionsoflivingcellscauselocalandtemporarytem-peraturechanges,whichhavethepotentialtomodulatetheactivityofboththecellsthemselvesandalsothesurroundingcells.Theview-pointof“Thermo-ChemicalSignaling”shouldbeessentialforunder-standinghowthe livingcellsefficientlyusethesurrounding localtemperaturechangesandthosewithinthecellitself.Wehaverevealednoveltemperature-sensitivitiesoflivingcellsusingthemicroheatingsystemsand localtemperaturemeasurements.Inthispresentation,wewouldliketointroducethefollowing3topics.(1)AmicroscopicheatpulseinducesCa2+burstinhumancancercells.(2)Ca2+-independentcontractionsofcardiomyocytesinducedbymi-croscopicheatpulses.(3) “Walkingnanothermometers”reveal temperature-sensitivityoftransportedacidicorganelleinlivingcells.NoCOI.

2S26E-3Thermometry in aqueous solution at single cell-scale using fluorescent nanothermometersSuzuki, Madoka1,2(1Waseda Bioscience Research Institute in Singapore (WABIOS), Waseda University, Singapore, 2Organization for University Research Initiatives, Waseda University, Tokyo, Japan)

Carriermaterialsusedindrugdeliverysystems(DDS)shouldtargetthecertainregionsinabodywhileavoidingundesirableentrapmentsbycellsandorgans.Inadditiontothetargetingability,thecontrolledreleaseofdrugs intherightplaceattherighttimesupportsthesystem’sefficiencyandversatility.Currentstimuli-responsiveDDSinclinicaltrialsrelyontheexternallyappliedtemperaturechange.Yetthedetailsofheatingsuchasthetemperaturedistributionanditstimecoursearenotfullyclarified,especiallyatthescaleofsinglecell.Iwillpresentournewlydevelopedmethodsforthetemperaturemeasure-mentinaqueousconditionunderthefluorescencemicroscope.Iwillshowthatourfluorescentthermometersarecompatiblewithlivingcells,anddiscussournewfindingsinHeLacellsobtainedbycombin-ingourthermometrywith[Ca2+]imaging.Weenvisagethesemethodsimprovingourunderstanding in local temperaturedynamics fromsub-cellularscaletothespheroidcultures,possiblytowardsliveanimalsinnearfuture.NoCOI.

2S26E-4In vivo gene manipulation in the targeted single cells using an infrared laserKimura, Eiji1; Kamei, Yasuhiro2(1Dept. of Anat., Iwate Med. Univ., Iwate, Japan, 2Spectrography and Bioimaging Facility, NIBB, Aichi, Japan)

In vivospatiotemporalgeneregulationinasinglecellisobligatorytoanalyzespecificgenefunctionsassociatedwithmorphogenesisinde-velopingembryos.Theinfraredlaser-evokedgeneoperator(IR-LEGO)systemisanewmicroscopeoptimizedtoheatcellsusinganinfrared(IR)laser,whichhasasuperiorabilitytoheatwater.Acombinationoftheheatshockprotein(hsp)promoterandIRlaserirradiationenablesthespatiotemporalgeneinductionintargetedcells.Wereportedthissystemwasefficientinregulatingspatiotemporalgeneexpression,andIRlaser-mediatedgeneinductioninsingletargetedcellsinnematodes(Caenorhabditis elegans),andthelocalgeneexpressioninvarioustis-sues,suchasthemuscle,notochord,andretinainsomelivingverte-brates,forexample,zebrafish(Danio rerio)andmedaka(Oryzias latipes).Morerecently,weappliedthissystemtothevascularbiologyinze-brafishandestablishedanexcellentmethodtoinducelaser-mediatedgeneexpressioninsingletargetedendothelialcellsofvertebrateor-ganismin vivo.Weoptimizedtheirradiationconditions,whichresult-edinraisingtheefficiencyoflaser-mediatedgeneinductionupto60%.Furthermore,weappliedthismethodtotheendothelialcellsofthefirstintersegmentalarteries(SeAs)toevaluatethesystem,andrevealedtheircontributioninconnectingthevascularsystemsofthebrainandspinalcord.Ourachievementinthisstudywillleadustotheprospectofuncoveringthemechanismsunderlyingmorphogenesisofvertebratemodelorganisms.NoCOI.


2S26E-5The physiological roles of thermosensitive channel TRPM2Uchida, Kunitoshi1,2; Tominaga, Makoto1,2(1Division of Cell Signaling, National Institute for Physiological Sciences (Okazaki Institute for Integrative Bioscience), Okazaki, Aichi, Japan, 2Department of physiological Science, The Graduate University for Advanced Studies, Okazaki, Aichi, Japan)

Thegeneencodingthecapsaicinreceptorasanoxiousheatsensor,whichisnowcalledTRPV1,wasisolatedfromarodentsensoryneuroncDNAlibraryin1997andwasconsideredtobeabreakthroughfortheresearchconcerningtemperaturesensing.Sincethen,severalTRPchannelshavingthermosensitiveabilityhavebeenidentifiedinmam-mals,withtenthermosensitiveTRPchannelsreportedinmammalstodate.WhilesearchingforanovelthermosensitiveTRPchannel,wefoundthatTRPM2hasthermosensitivity,anditstemperature-depen-dentactivationisdrasticallyenhancedbyco-applicationofligandssuchascyclicadenosine5-diphosphoribose(cADPR).Interestingly,TRPM2ismainlyexpressedinthetissuesnotexposedtothedrastictempera-turechanges.TRPM2wasoriginallyclonedasthetargetofADPRthatcausesanintracellularCa2+increase,andhasbeenreportedtobeactivatedbynicotinamideadeninedinucleotide,hydrogenperoxideandintracellularCa2+.Inaddition,TRPM2hashighCa2+-permeability,indicatingthat itsphysiologicalrolesmightdependon intracellularCa2+ increases. Inthissymposium, Iwould liketotalkaboutthephysiologicalroleofTRPM2inpancreasandimmunocytes.Especial-ly,IfocusontheinvolvementofTRPM2inthedevelopmentofdiabe-tesanddegranulationfrommastcells.NoCOI.

Symposium 27New developments of mathematical

physiology

(March 17, 9:00–11:00, Room F)

2S27F-1Modeling analysis of inositol 1,4,5-trisphosphate re-ceptors (IP3R)-mediated calcium mobilization from intracellular stores in pancreatic beta cells.Takeda, Yukari; Takeda, Yukari; Noma, Akinori(Ritsumeikan University, Faculty of Bioinformatics, Kusatsu, Japan)

Uponelevationofplasmaglucoseconcentration,pancreaticβ-cellsgenerateburstsofactionpotentialsandinducecyclicchangesin[Ca2+]iregulatingpulsatileinsulinrelease.GLP-1,anincretinhormone,syner-gisticallyenhancesglucose-dependentinsulinsecretionbymodulatingmultiple ionchannelsandexocytoticmachineryproteinsthroughPKA-andEPAC-dependentmechanisms.PKA-mediatedintracellularCa2+mobilizationviaIP3RswassuggestedtobepartofthemechanismbywhichcAMPamplifies insulinrelease. Increase in [Ca2+]iwouldmodulateactivitiesofmultipleCa2+-sensitiveionchannelsandtrans-portersaswellasmitochondrialandmembraneenzymeswhichreg-ulate[ATP]iand[cAMP]i,thusintricatelyaffectingcellularresponsive-ness.IntracellularCa2+relesechannelsinconventionalβ-cellmodels,however,havebeendescribedasasimpleleakchannelwhichwouldnotbeabletoreproducedynamiccareleasefromstores.Herewedevelopedamathematicalmodelof IP3RandreconstructedCa2+transientsandoscillationsthataregeneratedduringGLP-1stimulationinpancreaticβ-cells.SimulationstudiesindicatedthattheseeventsofCamobilizationweregeneratedbypositivefeedbackontheCa2+-de-pendentactivationofthechannel.SlowrateofCa2+-dependentinacti-vationwasalsosuggestedtodeterminethetimecourseofdecayofCa2+transients.Interestingly,mathematicalanalysisrevealedthattheslowrateofCa2+-dependentinactivationofthechannelisthekeytogeneratingCa2+oscillations.NoCOI.

2S27F-2An updated model of interstitial cells of Cajal repro-ducing intestinal pacemaker activityYoum, Jae Boum1; Kim, Hyoung Kyu1; Heo, Hye-Jin1; Kim, Nari1; Leem, Chae Hun2; Han, Jin1(1Department of Physiology, College of Medicine, Inje University, 2Department of Physiology, University of Ulsan College of Medicine, Seoul, Korea)

InterstitialcellsofCajalplayasapacemakeringastrointestinalsystem.Previously,weconstructedabiophysicallybasedmodelofinterstitialcellsofCajalinmousesmallintestine.Sinceionchannelscontributingtothepacemakeractivityhavebeensubstantiallyupdated,wetriedto improveourmathematicalmodel.We incorporated5more ionchannelsintoourpreviousmodel.Theyarevoltage-gatedNa+channel(Nav1.5),Ca2+-activatedCl-channel,ERGK+channel,Ca2+-activatedK+(BK)channels,andNa+-leakchannel(NALCN).TheIP3-mediatedCa2+releaseisakeyeventtodriveregeneratingpacemakerpotentialsandwasupdatedtoreproduceitsstochasticbehavior.Thestochasticcurrentswerereproducedbysimulatingtherandomopeningsandclosingofindividualionchannel.Theupdatedmodelwasabletore-producestochasticfeaturesofpacemakerpotentials.Theywerenotuniforminsize,duration,andfrequency,whichcorrespondtothoseofexperimentalrecordings.ThemodelsuggeststhattheNa+-leakchan-nelcontributestodepolarizationabout10mVinrestingmembranepotential.ThemodelalsosuggeststhatCa2+-activatedCl-channelismorelikelytostabilizemembranepotentialratherthantoexciteunderthephysiologicalcondition.Weconcludethatthis improvedmathe-maticalmodelcouldgiveaninsighthowionchannelsandstochasticIP3-mediatedCa2+releasedrivepacemakeractivityingastrointestinalsystem.NoCOI.


2S27F-3Simulation study of Ca2+ response in lymphocytesMatsuoka, Satoshi1; Bongju, Kim2; Ayako, Takeuchi1; Orie, Koga3; Masaki, Hikida3(1Integr. Physiol. Fac. Med. Sci. Univ. Fukui, 2Inst. Genome Res., Univ. Tokushima, 3Cent. Innov. Immunoreg. Tech. and Therap. Kyoto Univ.)

Toclarifythemolecularmechanismsunderlyingantigen-receptorin-ducedCa2+responseoflymphocytes,wecreatedamathematicalmod-elofthelymphocyteCa2+dynamics.Followingprocessesaremodeled.1)Antigen-receptormediatedproductionofinositol1,4,5-trisphosphate(IP3)byphospholipaseCandthedegradationbyinositolphosphate5-phosphatasesandIP33-kinase.2)Endoplasmicreticulum(ER)Ca2+uptakebyCa2+pump,Ca2+releasebyIP3receptor.3)MitochondrialCa2+ influxbyCa2+uniporterandCa2+effluxbyNa-Caexchange(NCXm).4)ERCa2+dependenttransitionofstromalinteractionmole-cule1anditsactivationofCRACchannel.5)PlasmamembraneCa2+extrusionbyCa2+pumpandNa-Caexchange,andcytoplasmicCa2+(Ca2+

i)bufferbycalmodulin.Ordinarydifferentialequationswereinte-gratedbyRunge-Kuttamethods.Themodelwellreproducedanti-gen-receptormediated2+

iriseofBlymphocytes.SystematicanalysisofthemodelpredictedthatNCXmactivityaffectsERCa2+contentandantigen-receptormediatedCa2+

irise.ThemodelpredictionwasvalidatedinDT40andA20Blymphocytes.PharmacologicalinhibitionofNCXmbyCGP-37157andatargetedknockoutorknockdownofNCXmgene(NCLX)markedlydecreasedERCa2+content,suppressingtheantigen-receptor inducedCa2+

irise.WeconcludedthatNCXmfunctionsasaCa2+providertoERandisimportantinantigen-receptorinducedCa2+responseoflymphocytes.NoCOI.

2S27F-4Model prediction and experimental validation of mechanisms regulating synaptic plasticity in the cer-ebellum Kawaguchi, Shin-ya(Graduate School of Brain Science, Doshisha University, Kyoto, Japan)

Synapticplasticity,neuronalactivity-dependentsustainedalterationoftheefficacyofsynaptictransmission,isacellularbasisforlearningandmemory.Manyformsofsynapticplasticityare inducedbyanincreaseinintracellularCa2+concentration([Ca2+]i)throughactivationofcomplicatedintracellularsignalingnetworkincludingseveralfeed-backloops.Tointuitivelyunderstandthedynamicbehaviorofsynap-ticplasticityinduction,quantitativesimulationofthemolecularnetworkisuseful.Wehaveconstructedakineticsimulationmodelofinhibitorysynapticplasticityinthecerebellum,andsystematicallyanalyzedthebehaviorofintricatemolecularnetworkscomposedofproteinkinases,phosphatases,etc.Simulationanalyseshaveprovidedseveralpredic-tions,suchasthemechanismsregulatingCa2+threshold fortheplasticityinduction,thedynamicplasticityregulationbythetemporalcontextof[Ca2+]iincrease,andthecooperativelyregulationofdiffer-entformsofplasticityatexcitatoryandinhibitorysynapses.Inthissymposium,Iwillshowdynamicregulationmechanismsofsynapticplasticityrevealedbycombinedapplicationofmodelingandexperi-mentalvalidation.NoCOI.

2S27F-5EAD and DAD mechanisms analyzed by developing a new ventricular cell model; quantitative analysis by applying mathematical analysesAsakura, Keiichi1,2; Asakura, Keiichi1,2; Cha, ChaeYoung3; Horikawa, Yuusuke2; Memida, Hiraku2; Yamaoka, Hiroyo2; Trevor, Powell4; Noma, Akinori2(1Pharmacokinetics and Safety Assessment Dept., Nippon Shinyaku Co., Ltd., Kyoto, Japan, 2Department of Bioinformatics, Ritsumeikan University, Japan, 3Oxford Centre for Diabetes, Oxford University, UK, 4Laboratory of Physiology, Oxford University,UK)

DrugsthatprolongtheQTintervalpresentamajorsafetyconcernforpharmaceuticalcompanies.TheQTprolongationhasbeenlinkedwiththepotentiallylethalventriculararrhythmiaTorsadesdePointes(TdP).However,hERG-channelblockandQTprolongationareneithernecessarynorsufficientconditionsforatorsadogenicrisk,despiteastrongassociation.BecauseaprecisemechanismsthatleadtoinitiationofTdPremainundetermined.Proarrhythmicvulnerabilitylinkedtoimpairmentofrepolarizationthatsupportsearly-(EAD)ordelayed-af-terdepolarizations (DAD).Here,wefocusedonthemembrane-andCa2+-mechanismsunderlyingbothEADandDADinaquantitativemannerbyconstructinganewventricularcellmodel.TheionchannelmodelsweremostlybasedonelectrophysiologicaldatafromhumanmyocytesandtheLCC-RyRmodelbasedonlocalcontroltheorywasadopted.EADsandDADswereevokedbyapplyingseveralcondition.Contributionsofionchannelsandtransporterswerequantifiedwiththeleadpotential(VL)analysis,whichrevealedthatIK1andIKrplayedtheprimaryroleofEADinitiation.ICaLandINCXamplifiedEAD.Webelievethatsuchapplicationofmathematicalanalysestoarealisticcellmodelisapowerfultooltoevaluateandpredictproarrhythmicpotential.NoCOI.

Symposium 28Leading edge of research on neuronal

circuitry underlying cognitive functions

(March 17, 9:00–11:00, Room G)


2S28G-1Computational principles of microcircuit operations for representation and memory retrieval of objects in macaque temporal cortexHirabayashi, Toshiyuki(Dept. of Physiol., The Univ. of Tokyo Sch. of Med., Tokyo, Japan)

Inferiortemporal(IT)cortexinmonkeyslocatesatthefinalstageofvisualobjectprocessing.Althoughneuronalresponsesinthiscorticalregionhavebeencharacterizedwithvarioustaskparadigms,itremainslargelyunknownhowindividualneuronsinteracttoformfunctionalcircuitsforimplementingcognitivedemands.Here,Iwilldiscussmi-crocircuitoperations inmacaqueITcortex inanobject-associationmemorytask,focusingontwodifferenttopics.Thefirsttopicisthecircuitoperationforobjectmemoryretrieval.Wefoundthedirectedsignalflowbetweenfunctionallydifferentclassesofmemoryneuronsthatgeneratesmemoryretrievalsignalofvisualobjects.Thesecondtopicishierarchicalobjectprocessingacrosscorticalareas.Therep-resentationofagivenfeatureofvisualobjectshasbeenbelievedtobeconstructedinthecorticalareawherethatrepresentationisprev-alent.However,anotherpossibleschemeisthatpreparatorycodesofagivenfeaturearecreatedinalower-orderareabeforetheirincreasetobecomepredominantinahigher-orderarea.Intheobjectassociationtaskparadigm,wefoundthemicrocircuitsthatgenerateandincreasetherepresentationsofobjectassociations insuccessive lower-andhigher-orderareasintheITcortex,respectively,consistentwiththelatterhypothesis.Together,theseresultsdemonstratethatexamina-tionsofmicrocircuitoperationsinmonkeysperforminganobjectas-sociationmemorytaskprovidedtheprinciplesofcorticalcomputationsforrepresentationandmemoryretrievalofvisualobjects.NoCOI.

2S28G-2Cognitive signals in midbrain dopamine neuronsMatsumoto, Masayuki(Laboratory of Cognitive and Behavioral Neuroscience, Division of Biomedical Science, Faculty of Medicine, University of Tsukuba)

Dopamineneuronsinthesubstantianigraparscompacta(SNc)andtheventral tegmentalarea (VTA)arewellknownfortheircrucialrolesinrewardprocessing.Aseparatelineofresearch,ontheotherhand,hasestablishedthatneurotransmitterdopamineisessentialtocognitivefunctionssuchasworkingmemoryandattention.However,despiteabundantstudiesdemonstratingdopamineneuronactivityrelatedtoreinforcementandmotivation,littleisknownaboutwhatsignalsdopamineneuronsconveytopromotecognitiveprocessing.Inordertoidentifycognitivesignalscarriedbydopamineneurons,werecentlyconductedsingle-unitrecordingfromdopamineneuronsintheSNcandVTAwhilemonkeyswereengagedinacognitivetaskthatrequiredworkingmemoryandvisualsearch.Wefoundthattheactivityofdopamineneuronsatdifferent locationsreflectedsignalssuitablefordistinctrolesincognitiveprocessing.Asubsetofdopamineneuronswereactivatedbyvisualstimuliifthemonkeyhadtostorethestimuliinworkingmemory.Theseneuronswerelocateddorsolat-erallyintheSNc,whereasventromedialdopamineneurons,someintheVTA,representedconventionalrewardpredictionsignals.Fur-thermore,dopamineneuronsmonitoredvisualsearchperformance,becomingactivewhenthemonkeymadeaninternaljudgmentthatthesearchwassuccessfullycompleted.Ourfindingssuggestanana-tomicalgradientofdopaminesignalsalongthedorsolateral-ventrome-dialaxisoftheventralmidbrain.NoCOI.

2S28G-3Clarifying the neural mechanisms of social action monitoring using macaquesIsoda, Masaki(Department of Physiology, Kansai Medical University School of Medicine, Osaka, Japan)

Thelastdecadehasseenasurgeofinterestinthestudyofsocialbrainfunctions.Researchinthisfield,calledsocialneuroscience,hasbeencarriedoutmostlyusinganeuroimagingtechniqueforhumansubjects.However,giventhelimitedspatiotemporalresolutioninherentinthemethodology,asystemsneuroscienceapproachusingmacaquemon-keysmayprovideausefulplatformforunderstandingsocialbrainfunctionsatthecellularlevel,therebycomplementingneuroimagingtechniques.Werecentlydevelopedanexperimentalparadigminwhichtwomonkeysactivelymonitoredeachother’sactionsfortheirownactionselection.Usingthismodel,werecordedsingle-unitactivityinthemedial frontalcortex (MFC),acriticalnode inthesocialbrainnetworks.ResultsrevealedthattheMFCencompassedaselectiveneuralcodefortheactionofthepartner, inadditiontoaselectiveneuralcodeforone’sownactionsandasharedneuralcodefortheselfandpartner’sactions.Further,asizablenumberofMFCneuronsse-lectivelyrespondedtopartner’serrors.Thesepartner-errorneuronsweredistributedintwosegregatedpopulations:thedorsomedialcon-vexity,whereother’serrorsweredetected,andcingulatesulcuspatch-es,whereone’scorrectbehaviorfollowingother’serrorswasencoded.ThesefindingssuggestthattheMFCplaysaroleinself-otherdiffer-entiationandmonitoringothers’behaviorforadaptivesocialdecisionmaking.Thecontinuingeffortinthisresearchdirectionwilluncovertheneuralbasiswherebyprimateshavebecomesuchasuccessfulsocialbeingintheanimalkingdom.NoCOI.

2S28G-4What’s a function of the pulvinar as a subcotical hub for the visual system?Komura, Yutaka1; Nikkuni, Akihiko1,2; Miyamoto, Aki1(1National Institute of Advanced Industrial and Science Technology (AIST), Tsukuba, Japan, 2Ibaraki Prefectural University of Health Sciences (IPU), Ami, Japan)

Thepulvinar,avisualthalamicnucleus,doesnotexistintherodentbrainandshowsamarkedevolutionaryexpansionintheprimatebrain.It interconnectswithmultiplevisualcortices.Despiteof itscentralpositioninvisualsystem,thefunctionalroleofthepulvinarinvisionremainselusive.Usingacombinationofpsychophysics,neuralrecord-ings,pharmacological inactivationandcomputationalmodeling,wehavefoundtheneuralcorrelatesandcausesofperceptualconfidenceinthepulvinar.Wetrainedthemonkeystoperformacategorizationtaskforthevisualstimuliwithcolor-motionpairing.Inacategorizationtask,thepulvinarresponsescorrelatedwithstimulusambiguity.Inanopt-outtask, thepulvinarresponsestothesamephysicalstimulusdecreasedwhenthemonkeyschoseescapeoptions, indicating lessconfidenceinvisualcategorization.Functionalsilencingoftheunilat-eralpulvinarcausedtoincreasefrequenciesoftheopt-outbehaviors,onlywhenthevisualtargetappearedinthecontralateralvisualfieldtotheinactivationsiteofthepulvinar.Theseresultsindicatethatthepulvinarplaysacrucialroleinasubject’scertaintyoftheperceivedworld,therebypotentiallyunderlyingvisualconsciousness.NoCOI.


2S28G-5Neural basis of temporal processing: a role of the cer-ebellumTanaka, Masaki(Department of Physiology, Hokkaido University School of Medicine)

Temporalprocessingoverashortperiodoftimeisimportantforbothmotorcontrolandperception.Forexample,weareabletodetectslightchanges inmusicalrhythm,whichrequirestemporalpredictionofregularbeat.Previousstudiessuggestthatthecerebellummayplayaroleinsuchprocesses.However,theunderlyingneuronalmechanismremainslargelyunknown.Toaddressthis,weperformedexperimentsinmonkeys.Whentheanimalswererequiredtodetectasuddenomissionof isochronousrepetitivestimuli (missingoddballparadigm),neurons inthedeepcerebellarnucleiexhibitedfiringmodulationthatgraduallyincreasedastherepetitionprogressed.Themagnitudeoffiringmodulationforeachstimuluswaspositivelycorrelatedwiththe lengthof the in-ter-stimulusintervalinagiventrial,indicatingthatthesensorygaindependedonthetimeelapsedfromtheprecedingstimulus.Becauseinactivationofrecordingsitesdelayedthedetectionofstimulusomis-sion,thetemporallyspecificsensorysignalsinthecerebellummightbeconvertedintothepredictionerrorsignalsforstimulusomissioninthedownstreamthalamocorticalnetworks.Takentogetherwiththedataobtainedfromtheventrolateralthalamus,Iwilldiscussthepos-sible informationprocessingthroughthecerebello-thalamocorticalpathwaysfortemporalprediction.NoCOI.

Symposium 29Relationship between cardiovascular

physiology and physical therapy[CollaborationSymposiumwithJapanese

PhysicalTherapyAssociation]

(March 17, 8:40–10:20, Room H1)

2S29H1-1Central regulation of skeletal muscle blood flow during exerciseIshii, Kei; Matsukawa, Kanji(Department of Integrative Physiology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan)

Whetheracentrally-inducedneuralmechanismplaysaroleinexercisehyperemia inskeletalmuscle iscontroversial.Someanimalstudieshavereportedcentrally-inducedsympatheticcholinergicvasodilatationinskeletalmuscleatstartofexercise,whichmayserveasafeedfor-wardregulatorysignal for increasingmusclebloodflow.However,suchneuralmechanismhasbeendeniedinhumans,becausecervicalsympathectomyandmuscarinicblockadehadnoimpactonexercisehyperemia.Ontheotherhand,thesympatheticnervoussystempre-dominantlyregulatesbloodflowtonon-contractingmuscle.Recentlywehavedemonstratedthatbloodflowtonon-contractingmusclein-creasedatstartofvoluntaryone-leggedcyclingandduringimageryoftheexercise,suggestingthatcentralcommandcontributestothemusclehyperemia.Sinceimageryoftheexerciseincreasedbloodflowinbilateralmusclestothesameextent,thecentrally-inducedvasodi-latorsignalappearstobetransmittedequallytothebilateralmuscles.Inthiscontext,wefoundthatbloodflowincontractingmuscleaswellasnon-contractingmuscleincreasedatstartofone-leggedcyclingandthatthehyperemicresponsesinbothmuscleswerebluntedbyatro-pine.Thusitisconcludedthatcentralcommandcausescholinergicvasodilatationequallyinbilateralmuscles,whichcontributestomusclehyperemiaatstartofvoluntaryexercise.Suchcentralvasodilatormechanismmaybeappliedtoanewrehabilitationapproachtoimprovemusclecirculation,disorderofwhichmayresultinexerciseintolerance.NoCOI.

2S29H1-2Effect of exercise intensities on endothelial function in chronic and acute phasesGoto, Chikara(Department of Rehabilitation, Hiroshima International University, Hiroshima, Japan)

Background:Itiswellknownthatendothelialdysfunctionisaninitialstepofatherosclerosisprocess,leadingtotheonsetofcardiovascularevent.Also,physicaltrainingandexercisehavebeenestablishedtoimproveendothelialfunctionnotonlyinpatientswithhypertension,coronaryarterydisease,andchronicheartfailure,butalsoinhealthyindividuals.However,thereisnoinformationoftherelationshipbe-tweenexerciseintensitiesandendothelialfunctionsofar.Methodandresult:Todeterminetheeffectofdifferentexerciseintensitiesonen-dothelialfunction,weevaluatedtheendothelialfunctionbymeasuringforearmbloodflow(FBF)responsebeforeandafterdifferentintensitiesfor12weeks(mild,25%V02max;moderate,50%V02max;andhigh,75%V02max;bicycleergometers)inhealthyyoungmen.FBFresponsewassignificantly increasedaftermoderate-intensityexercisefor12weeks,butnotaftermild-andhigh-intensity. Interestingly,moder-ate-intensityexercisetendedtodecreaseboth indicesofoxidativestress,whereashigh-intensityexerciseincreased.Next,wesoughttodeterminetheacuteeffectofdifferentexerciseintensitiesonendothe-lialfunction.Then,similarly,wealsofoundthattheexerciseofmod-erate-intensity,butnotofmild-andhigh-intensities,couldsubstantial-lyimproveFBFeveninanacutephase.Conclusions:Thesefindingssuggestthatmoderate-intensityexercisecould improveendothelialfunctioneveninhealthyhumansthroughadecreaseofoxidativestressandsubsequentincreaseofNOproductionnotonlyinchronic,butalsoacutephases.NoCOI.


2S29H1-3Effect of exercise for Left Ventricular Assist Device patientsYanase, Masanobu1; Nakatani, Takeshi1; Kumasaka, Reon2; Arakawa, Tetuso2; Nakanishi, Michio2; Noguchi, Teruo2; Oohara, Takahiro2; Fukui, Shigefumi2; Goto, Yoichi2(1Department of Transplantation, National Cerebral and Cardiovascular Center, 2Department of Cardiovascular Medicine, National Cerebral and Cardiovascular Center)

TheLeftventricularassistdevice(LVAD)isanalternativetherapyforthepatientwithadvancedheartfailurewhodoesnotrespondtoconventionalmedicaltreatments.Wehavepreviouslyreportedthatinadditionto improveexercisetolerance,exercisetherapyforthepatientwithparacorporealLVAD(p-LVAD)couldpotentiallyimprovesurvivalonLVAD.FromApril2011,implantableLVAD(i-LVAD)hasbeenwidelyusedasbridgetotransplantinJapan.However,periodofLVADsupportinourcountryislongerthanthatofEuropeandUnit-edStates.Therefore,exercisetrainingisimportantforLVADpatientstoimprovetheirphysicalactivityandqualityoflifeoftheirLVADsupportperiod.Inthepatientwithi-LVAD,pulsepressureoftenin-creaseafterexercisetherapy.Thisphenomenonispartiallyaccountedforthephysiologicalfeatherofcontinuous-flowpumpthatpumpflowischangedbybeattobeatalongwithnativecardiaccycletomakepulsepressureandexercisecontributestoincreaseinleftventricularcontractionthroughactivationofsympatheticactivityandsecretionofcatecholamine.Further,exercisealsocontributestoenhancepulsepressurethroughincreasingcardiacpreloadbydrivingmusclepump.Thereremainsmuchtobeelucidatedaboutthephysiologicalroleofexercisetherapyini-LVADpatients.But,weconsiderthatexercisestrainingmaybeusefultoimproveoutcomewithi-LVADpatients.NoCOI.

2S29H1-4Quadriceps isometric strength as a predictor of exer-cise capacity and mortality in patients with coronary artery diseaseKamiya, Kentaro(Department of Rehabilitation, Kitasato University Hospital, Sagamihara, Japan)

Background:Improvement inexercisecapacity isoneofthemaingoalsofcardiovascularphysiotherapy,resultinginbothareductionofmortalityriskandanincreasedlevelofeverydayhabitualactivities.Quadricepsstrengthisrelatedtoexercisecapacityinnormalsubjectsanddifferentpatientpopulations,buttherelationshipbetweenmaximalquadriceps isometricstrength (QIS)anddifferentexercisecapacitylevelsincoronaryarterydisease(CAD)patientshasnotbeensystem-aticallyevaluatedyet.Method:Westudied621patients (60.6±9.9years)whowereadmittedtothehospitalbecauseofCAD.TheQISwasexpressedaspercentageofbodyweight(%BW).Multivariatelo-gisticregressionanalysisdeterminedapredictorofexercisecapacity.Thecut-offvaluesofQISfortheattainmentofexercisesof5,7and10METswereanalyzedusingreceiver-operatingcharacteristics(ROC)curves.Results:ROCcurvesrevealedthat theQISwas46%BW,51%BWand59%BWaspredictivecut-offvaluesfortheattainmentofexercisesof5,7and10METs,respectively.Moreover,wefollowedthiscohortfortotally2,027person-years,andthepatientswhohadlowerQIShadsignificantlylowersurvival.Conclusion:Thepredictivecut-offvaluesofquadricepsstrengthwere30%BWforprognosis,46%,51%and59%BWin5,7and10METsexercisecapacities,respective-ly.Thesefindingscanbeusedincardiovascularphysiotherapyforthedefinitionoftraininggoalsaccordingtopatients’needsabouttheirphysicalactivitylevels.NoCOI.

Symposium 30Cardiovascular diseases

and autonomic nerve intervention: Go back from clinical to basic science

(March 17, 10:25–12:05, Room H1)

2S30H1-1Open-loop analysis of the arterial baroreflex in heart failure and hypertensionKawada, Toru; Shimizu, Shuji; Turner, James Michael; Sugimachi, Masaru(Department of Cardiovascular Dynamics, National Cerebral and Cardiovascular Center)

Thearterialbaroreflexsystemcanbedivided intotheneuralarcsubsystemfrompressureinputtoefferentsympatheticnerveactivity(SNA)andtheperipheralarcsubsystemfromSNAtoarterialpressure(AP).Thearterialbaroreflexfunction isreportedtobealtered indiseasedstatessuchasheartfailureandhypertension.However,be-causeanychangesinmeanAPassociatedwiththediseasecanreflex-lyalterthelevelofSNAevenifthebaroreflexfunctionisunaltered,itishardtodeterminewhetherthebaroreflexfunctionistrulyalteredindiseasedstates.Toresolvethisquestion,wehaveemployedanopen-loopsystemsanalysiswherethearterialbaroreflexfunctioniscomparedbetweennormalanddiseasedstatesbyexactlycontrollingthebaroreceptorinputpressuretopredefinedlevels.Thetotal-loopbaroreflexfunctionapproximatesaninversesigmoidalcurve.Inratswithchronicheartfailure,theresponserangeofAPissignificantlynarrowedandthebaroreflexmaximumgainisdecreasedcomparedwithnormalrats,indicatingthelossofAPbufferingfunctionaroundtheoperatingpoint.Inspontaneouslyhypertensiverats,thesigmoidcurveisshiftedtowardhigherinputandoutputpressureswiththebaroreflexmaximumgainunchanged,indicatingtheshiftoftheoper-atingpointtowardhigherpressure(resetting)withoutthelossofAPbufferingfunctionaroundtheoperatingpoint.Togaininsightsintothedevelopmentofdisease,theneuralandperipheralarccharacter-isticsaretobecomparedbetweennormalanddiseasedstates.NoCOI.


2S30H1-2Effects of dihydropyridine calcium channel blockers on baroreflex sympathetic regulationYamamoto, Hiromi(Division of Cardiology, Depertment of Medicine, Faculty of Medicine, Kinki University, Osaka, Japan)

Calciumchannelblockersarewidelyusedforthetreatmentofhyper-tension.Firstgenerationdihydropyridinecalciumblockers (DHPs)exertastrongandrapidatrialpressure(AP)-loweringeffectviarelax-ationofvascularsmoothmuscles.Thehypotensivestatemayevokebaroreflex-mediatedsympatheticexcitationandvagalwithdrawal,leadingtoincreasedheartrate(HR)knownas“reflextachycardia”.SecondgenerationDHPshaveslow-onsetandreduceAPwhileeithermaintainingoractuallydecreasingHR.ThirdgenerationDHPshavelong-actingpropertyinadditiontotheslow-onsetproperty.Althoughlackoftachycardiainresponsetothe2ndand3rdgenerationDHPsmayindicatepossiblesympathoinhibitoryeffects,itremainsunknownwhethersympatheticoutflowfromthecentralnervoussystem issuppressedbyDHPs.Weexaminedtheacuteeffectof intravenousnifedipine(1stgeneration),cilnidipine(2ndgeneration)andazelnidipine(3rdgeneration)onpostganglionicsplanchnicsympatheticnerveac-tivity(SNA)usingamethodofbaroreflexopen-loopanalysisinanes-thetizedWistarKyotoratswithvagotomy.Whenthebaroreceptorinputpressurewasexactlycontrolledatpredefinedpressurelevels,nifedipine,cilnidipineandazelnidipinedidnotsignificantlyaffecttheSNAresponseovertheentireoperatingrangeofthecarotidsinusbaroreflex.Theseresultssuggestthattheacutehypotensiveeffectsof intravenousDHPsmaybechieflyattributabletotheperipheralvasodilatoractionbutnottothedirectinhibitionofthesympatheticoutflowfromthecentralnervoussystem.NoCOI.

2S30H1-3Renal Denervation Alters Neural Arc in Spontaneous-ly Hypertensive RatsSata, Yusuke1; Kawada, Toru2; Shimizu, Shuji2; Schlaich, Markus1; Esler, Murray1; Sugimachi, Masaru2(1Baker IDI Heart and Diabetes Institute, Melbourne, Australia, 2National Cerebral and Cardiovascular Center, Department of Cardiovascular Dynamics)

Catheter-basedrenalsympatheticnerveablationhasdemonstratedasignificantbloodpressuredropinpatientswithresistanthypertension.Throughthemodulationofrenalafferentandefferentsympatheticnerveactivities,renaldenervationmayprovidecentrally-mediatedanti-hypertensiveeffects.Toelucidatehowneuralandperipheralcharacteristicsarealteredbyrenalsympatheticdenervation(RDN),weexaminedtheopen-loopstaticcharacteristicsofthecarotidsinusbaroreflexinrenal-denervatedspontaneouslyhypertensiverats(RD-SHR)andcomparedwithnormotensiveWistarKyotorats(WKY)andnon-denervatedSHR.Theoperating-pointarterialpressure(AP)wasdeterminedfromtheintersectionbetweenthebaroreflexneuralandperipheralarcinabaroreflexequilibriumdiagram.RD-SHRshowedtheoperating-pointAPof129.8±6.9mmHg,whichwasbetweenthatofWKY(111.3±4.4mmHg)andthatofSHR(145.1±5.7mmHg).Renaldenervationmodulatesthebaroreflexcontrolofsympatheticnerveactivity,anddecreasedtheoperating-pointAPinRD-SHRcomparedtoSHR.However,RDNdidnotcompletelynormalizeSNAorAPregulationcomparedtoWKY.NoCOI.

2S30H1-4Water drinking-related muscle contraction induces the pressor response via mechanoreceptors in con-scious rats.Abe, Chikara; Morita, Hironobu(Department of Physiology, Gifu University Graduate School of Medicine, Gifu, Japan)

Waterdrinkingisknowntoinducethepressorresponse.Theefferentpathway inthisresponse involvessympathoexcitation,becausethepressorresponsewascompletelyabolishedbyganglionicblockadeoranα(1)-adrenergicantagonist.However,theafferentpathwayinthisresponsehasnotbeenidentified.Inthepresentstudy,wehypothesizedthatwater itselfstimulatestheupperdigestivetractto inducethepressorresponse,and/ordrinking-relatedmusclecontractioninducesthepressorresponseviamechanoreceptors.Toexaminethis,weevaluatedthepressorresponse inducedbyspontaneousorpassivewaterdrinkinginconsciousrats.Sincethebaroreflexmodulatesandobscuresthepressorresponse,theexperimentswereconductedusingratswithsinoaorticdenervation.Thepressorresponsewasnotsup-pressedby1)transientoralsurfaceanesthesiausinglidocaine,2)bi-lateraldenervationoftheglossopharyngealnerveandsensorybranchofthesuperiorlaryngealnerve,or3)denervationofthetunicaadven-titiaintheesophagus.However,thepressorresponsewassignificant-lysuppressed(by-52%)byintravenousgadoliniumchlorideadminis-tration.Electricalstimulationofthehypoglossalnerve inducedthepressorresponse,whichwassignificantlysuppressed (by -57%)byintravenousgadoliniumchlorideadministrationandcompletelyabol-ishedbyseveringthedistalendofthisnerve.Theseresultsindicatethatafferentsignalsfrommechanoreceptorsindrinking-relatedmus-clesareinvolvedinthewaterdrinking-inducedpressorresponse.NoCOI.

Symposium 31Pathophysiology for immunity-related

gastrointestinal dysfunctions

(March 17, 8:40–10:20, Room J)


2S31J-1Significant contribution of TRPC6-mediated Ca2+ in-flux to the pathogenesis of Crohn's disease fibrotic stenosisKurahara Hai, Lin1; Sumiyoshi, Miho1; Hung, Hsichin1; Aoyagi, Kunihiko2; Inoue, Ryuji1(1Department of Physiology, Fukuoka University, Fukuoka, Japan , 2Department of Gastroenterology, Fukuoka University, Fukuoka, Japan)

Crohn’sdiseaseischaracterizedbyrepeatedcyclesofinflammationandhealingofthegutwhichultimatelyprogressintointestinalfibro-sis.WeexaminedthemRNAexpressionofbiopsysamplesfromnon-ste-noticorstenotic intestinalareasofCrohn’sdisease’spatients.ThemRNA levels of canonical transient receptorpotential protein 6(TRPC6),N-cadherin,collagentypes1A1and3A1,and interleukins(IL-1β,IL-6,IL-8,IL-10,IL-11andIL-17β)weresignificantlyincreasedinthefibroticstenoticarea.Amongstthem,themRNAlevelofIL-11,animportantanti-fibrosis,anti-inflammationcytokine,wasmostprom-inentlyincreased(218fold).WenextexploredapotentialroleofTRPC6channelsforintestinalinflammationbyusingahumancolonicmyofi-broblastcelllineInMyoFib.TGF-β1,themajorstimulatorofintestinalfibrosis,activatedTRPC6-mediatedCa2+influx,whichwasrequisiteforthedifferentiationandinterleukinsynthesisofthemyofibroblast.InTGF-β1treatedInMyoFibs,TRPC6stronglyinteractedwithα-SMAandN-cadherinandpromotedmechanically-inducedCa2+entry.Fur-thermore,TRPC6-mediatedCa2+entrysignificantlysuppressedIL-11expressionbydown-regulatingERKandp38-MAPKphosphorylation.TheseresultssuggestthatTRPC6-mediatedmyofibroblasticdifferen-tiationcouldbeanimportantprocesspromotingintestinalfibrogenesis,andthusapromisingtargetforfutureanti-fibroticandanti-inflamma-torytherapiesinthegut.NoCOI.

2S31J-2Up-regulation of Tenascin-C in subepithelial myofi-broblasts is critical for intestinal mucosal protection in miceIslam, Shafiqul Md1; Kusakabe, Moriaki2; Horiguchi, Kazuhide3; Iino, Satoshi3; Nakamura, Tatsuro1; Iwanaga, Koichi1; Hashimoto, Hisashi1,4; Matsumoto, Hisashi5; Murata, Takahisa1; Hori, Masatoshi1; Ozaki, Hiroshi1(1Dept. of Vet. Pharmacol., Grad. Sch. of Agri. and Life Sci., The Univ. of Tokyo, Tokyo, Japan, 2Develop. of Adv. Tech. Lab, Res. Center for Food Safety, The Univ. of Tokyo, Tokyo, Japan, 3Dept. of Anatomy, Fac. of Med. Sci., Univ. of Fukui, Fukui, Japan, 4Dept. of Anatomy, Jikei Univ. Sch. of Med., Tokyo, Japan, 5Yakult Central Institute)

Tenascin-C(TnC)isamulti-domainextracellularmatrixglycoprotein.DSS-inducedcolitismicegreatlyexpressedTnCinthedamagedmu-cosalareasandsignificantlyup-regulatedmRNAofTnC,pro-inflam-matorycytokinesandgrowthfactors incolitis tissues. Inaddition,TNBS-inducedcolitisandSAMP1/YitspontaneousCrohn’sdiseasemodelmicealsoexhibitedanup-regulationofmucosalTnCincolonandilea,respectively.PDGFRαpositiveISEMFwerefoundtobetheprimaryTnC-producingcellsinthecolontissues.Accordingly,ISEMFcollectedfromtheratcolonconstitutivelyexpressedbothTnCandPDGFRα.PDGF-BBandTGF-β1up-regulatedbothTnCmRNAandproteinlevelsinISEMF.KnockdownofTnCgenemademicemoresusceptibletoDSS-inducedcolitisandremarkablyshowedabrasionofintestinalmucosalbarrier.Moreover,TnCacceleratedbothtrans-wellmigrationandwoundhealing inepithelialcells.Conclusions:ThepharmacologicalprofilesofPDGF-BBandTGF-βincolitistissuesandISEMFsuggestthatincreasedTnCproductionduringinflammationcontributesepithelialcellmigration,remodelingandprotectionofin-testinalbarrier.NoCOI.

2S31J-3Leptin receptor signaling pathways is required for high-fat diet-induced atrophic gastritis in mice.Inagaki-Ohara, Kyoko1,2; Okamoto, Shiki2; Minokoshi, Yasuhiko2 (1Department of Gastroenterology, Research Center for Hepatitis and Immunology, Research Institute, National Center for Global Health and Medicine (NCGM), Japan, 2Division of Endocrinology and Metabolism, Department of Developmental Physiology, National Institute for Physiological Sciences (NIPS))

Obesity isassociatedwithgastrointestinaldiseases inadditiontocardiovasculardisease,diabetes,anddyslipidemia.Atrophicgastritisisoneofobesity-relateddiseases,however,themechanismsofitsonsethaveremaineduncertain.Wenowshowthatmicefedahigh-fatdiet(HFD)developedatrophicgastritiswithenhancingexpressionofgas-tricleptinanditsreceptorsignalingpathway.HFDinducedagastrichyperplasiawithincreasedleptinexpressionat1wk-feedingandtheprogressionofmucosalhyperplasiawasdevelopedwithahigherKi67-positiveproliferatingcell frequencyand ledtoatrophy inthepresenceofinflammationinanage-dependentmanner.ActivationofObR,STAT3,AktandERK,whicharemoleculesinleptinreceptorsignalingwasdetectedinthegastricmucosainHFDfedmice.Atrophyingastricmucosadisplayedpathologyandmolecularalterations,suchascdx2andmuc2,resemblinghumanintestinalmetaplasia,whichisconsideredtobemostprogressivefeatureofatrophicgastritis.UnlikeWTmice,leptin-deficientob/obmiceandleptinreceptormutateddb/dbmicedidnotshowsignificantincreaseincdx2expressionduetoHFD-feeding. Theseresultssuggestedthatenhancementof leptinsignalinginthestomachisrequiredtodevelopmentofobesity-associ-atedatrophicgastritis.NoCOI.

2S31J-4Gastrointestinal immune activation and esophageal motility disordersIhara, Eikichi; Tanaka, Yoshimasa; Muta, Kazumasa; Bai, Xiaopeng; Akiho, Hirotada; Nakamura, Kazuhiko; Takayanagi, Ryoichi(Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University)

Coordinatedregulationofgastrointestinal(GI)motilityisindispensableformaintaininggeneralhealthandwellness.GI inflammationandimmuneactivationareaccompaniedbyalterationofGImotilitywithimpairedfunctionofGIsmoothmuscle.ThedelicatebalancebetweenmicrobesandhostdefensiveresponsesatthemucosalbarrierhasapivotalroleinthepathogenesisofGIinflammation.AdaptiveimmuneresponseseemstoplayanimportantroleinGIsmoothmusclefunctionwhereTh1andTh2immuneresponseisassociatedwithhypocontrac-tilityandhypercontractilityofinflamedGIsmoothmuscle,respective-ly.Basedonbasicresearch,wewillreviewacurrentunderstandingofrelationshipsbetween immuneactivationandGIsmoothmusclefunction,focusingoncytokine-inducedalterationofGIsmoothmusclecontractility.Fromclinicalviewpoint,somemotilitydisordersareas-sociatedwiththe immuneactivation,suchas inflammatoryboweldisease,esophagealmotilitydisordersandfunctionalgastrointestinaldisorders.Itisveryimportanttotestifywhethertheevidenceledbybasicresearchcanbealsoappliedtoclinicalconditions.Inthisregardwewillpresentheresomeclinicaldatafrompatientswithesophagealmotilitydisordersincludingachalasia,esophagogastricjunctionoutflowobstruction(EGOO)andeosinophilicesophagitis,andwewilldiscussanypossibilityofcytokine-inducedalterationofesophagealmotilityintheclinicalsetting.NoCOI.


Symposium 32Gut motility: Understandings

from diverse viewpoints

(March 17, 10:25–12:05, Room J)

2S32J-1The role of interstitial cells in the regulation of gastro-intestinal motilityKito, Yoshihiko(Department of Pharmacology, Faculty of Medicine, Saga University, Saga, Japan)

Recentstudieshaveshownthattwotypesofinterstitialcellsarein-volvedintheregulationofgastrointestinal(GI)motility.ThefirsttypeisinterstitialcellsofCajal(ICC),whichareimmunopositivetoc-Kit.IthasbeendemonstratedthatanetworkofICCwhichliesinthemy-entericregion(ICC-MY)functionaspacemakercellsandICCattheplaneofthedeepmuscularplexus(ICC-DMP)asmediatorsofentericmotorneurotransmissioninthesmallintestine.ICC-MYgeneratelarge,rapidlyrising,potentialchanges(pacemakerpotentials),whichhadtwocomponents,arapidupstrokecomponentthatwasfollowedbyaplateaucomponent.Wefoundthattheupstrokecomponent is initiatedbynifedipine-insensitivevoltage-dependentCa2+currentsandtheplateaucomponentisproducedbyCa2+-activatedCl-currentsinthemousesmallintestine.Thesecondtypeofinterstitialcellsisfibroblast-likecells(FLCs),whichareimmunopositivetosmallconductanceCa2+-ac-tivatedK+(SK3)channelandplateletderivedgrowthfactorreceptorα(PDGFRα).SinceP2Y1receptorsareexpressedinFLCs,itisbelievedthatFLCsareresponsibleforpurinergicinhibitoryneurotransmission.Although,purinesactivatedapamin-sensitiveK+currentsthatwereblockedbyP2Y1antagonistinPDGFα+cellsisolatedfromthemousecolon,itisunclearhowPDGFRα+cellsin situcontributetotheregu-lationofsmoothmuscleexcitabilityinGItract.Inthissymposium,IwillshowsomeofthepropertiesofICC-MYandFLCsrecordedfromtherabbitsmallintestine.NoCOI.

2S32J-2Depolarization-induced inward current in the intersti-tial cells of CajalGoto, Kazunori(The Department of Psychiatry, Kyoto University Hospital, Kyoto, Japan)

ToexploretheelectrophysiologicalpropertiesoftheinterstitialcellsofCajal(ICCs),wedevelopedanewpreparationbytreatingthemurinesmallintestinewithcollagenase.Thisthinmusclelayerpreparationcontained interstitialcellswithandwithout intercellularcontactsaroundtheentericnervebundles,andtheclusterofsmoothmusclecellsdisplayedarhythmiccontraction.WemorphologicallyidentifiedICCsandconductedpatchclampexperimentsonthecells.Thesinglec-kit-positiveICCsshowedspontaneousandrhythmicpotentialfluc-tuations,andalargetransientinwardcurrentwasevokedbydepo-larizationundervoltageclumpconditions.Oncetheinwardcurrentwastriggered,ittookaregenerativetimecourseandlastedapproxi-mately500ms.Thecurrentwasinactivatedbycontinuousdepolar-ization,andbyremovalofexternalCa2+.Theapplicationofacetyl-cholineprolongedthedurationofspontaneousdepolarizationaswellasthedepolarization-induced inwardcurrent.This inwardcurrentshowedareversalpotentialofaround+3mVandwasconsideredtobeduetonon-selectivecationchannels.NoCOI.

2S32J-3Hyperpolarization-activated cyclic nucleotide chan-nels as a pacemaker channel in colonic interstitial cells of CajalJun Jae Yeoul(Department of Physiology, College of Medicine, Chosun University, Gwangju, Korea)

Hyperpolarization-activatedcyclicnucleotide(HCN)channelsarepace-makerchannelsthatregulateheartrateandneuronalrhythminspontaneousactivecardiacandneuronalcells.InterstitialcellsofCajal(ICCs)arealsospontaneousactivepacemakercells intheGItract.Here,weinvestigatedtheexistenceandtheroleofHCNchannelsinpacemakingactivityincolonicICCsbyusingwhole-cellpatchclamp,RT-PCR,andCa2+-imaginginculturedICCsfrommousecolon.SQ-22537ordedeoxyadenosine (adenylatecyclase inhibitors)decreasedthefrequencyofpacemakerpotentials,whereasrolipram(cAMP-specificphosphodiesteraseinhibitor)orcell-permeable8-bromocAMPincreasedthefrequencyofpacemakerpotentials.CsCl,ZD-7288,zetabradine,andclonidine(HCNchannelinhibitors)suppressedthepacemakeractivity.RT-PCRrevealedexpressionofHCN1channelandHCN3channelincolonicICCs.InrecordingofspontaneousintracellularCa2+[Ca2+]ios-cillation,rolipramand8-bromocAMPincreased [Ca2+]ioscillations,whereasSQ-22537,CsCl,ZD7288,andzetabradinedecreased [Ca2+]ioscillations.TheseresultssuggestthatHCNannelsincolonicICCsaretonicallyactivatedbybasalcAMPproductionandactaspacemakerchannelsforpacemakingactivity.NoCOI.


2S32J-4Acceleration of Ileal Pacemaker Activity in Mice Lacking Interleukin 10Shozib, Bari Habibul1; Suzuki, Haruhiko2; Iino, Satoshi3; Nakayama, Shinsuke2(1University of Dhaka,Dhaka,Bangladesh., 2Nagoya University, Nagoya ,Japan, 3Fukui University, Fukui, Japan)

InterstitialcellsofCajal(ICC)playapivotalroleingutmotilitybycoordinatingtheelectricactivityofcellularmembersaswellasgen-eratingpacemakerpotentials.PatientswithIrritableBowelDisease(IBD)sufferfromgutmotilitydisorders.Thus,ourstudywastoassesswhetherICCpacemakingandtheircoordinatedactivityispreservedintheileumofIL-10deficientmice,amodelanimalofIBD.Interest-ingly,inapplyingmicroelectrodearray(MEA)experimentwerevealedthatspontaneouspacemakerelectricactivityissynchronizedovertherecordingareainbothWTandIL-10deficientmice,butoscillationsaresignificantlyacceleratedinIL-10-deficientmice,despitenosignif-icanthistologicalalterationsobservedinICC,macrophagesandenter-icneurons.Theilealtissuesusedinthepresentstudydidnotdiffermacroscopically.NeitherulcersnorbleedingwereobservedinbothWTandIL-10deficientmice.Inordertoassesswhethertheacceler-atedelectricalactivityisassociatedwithanyhistologicalchangesand/orimmunecellscontainedtherein,wegothroughimmunohistochem-istrybutnosignificantchangeswereobservedinICC,macrophagesandentericneuronsintheileumofWTandIL-10deficientmice.ThusweconcludedthattheresearchprovidesevidenceforacceleratedpacemakeractivityintheileumofIL-10deficientmice,notaccompaniedbyanysignificanthistologicalchanges.Thiscouldbeaccounted,asanexample,byageneticcross-linkbetweenIBDandIBS.NoCOI.

Symposium 33New Approaches to Study Cell Volume/Body Fluid Balance Regulatory System

(March 17, 8:40–10:20, Room K)

2S33K-1Correlation between the mechanisms for cell volume regulation and for body fluid osmolarity controlOkada, Yasunobu; Sato-Numata, Kaori(National Institute for Physiological Sciences, Okazaki, Japan)

Bodyfluidosmolarityismaintainedataconstantlevelbyregulatingtheextentofexocytoticreleaseofargininevasopressin(AVP)fromosmosensorybrainAVPneuronswhichareconstantlyinteractingwithnearby-existingastrocytes.Ontheotherhand,alltypesofmammaliancellssensechangesinambientosmolaritythroughalteredactivitiesofcellvolume-sensingionchannelsand/ortransportersthatarethere-afterparticipating incellvolumeregulation (CVR).Therefore, it ishighlylikelythatthesevolume-sensingorvolume-regulatorychannels/transportersareinvolvedinosmosensorymechanismsinAVPneuronsandnearby-interactingastrocytes.Actually,ithasbeenhypothesizedthatenhancementofAVPsecretioniscausedbydepolarizationinducedbyactivationofstretch-inactivatedcationchannel (SICC) inAVPneurons,andthatsuppressionofAVPsecretionisbyhyperpolarizationinducedbyglycinereceptorsinresponsetotaurinereleasedviavol-ume-sensitiveoutwardlyrectifyinganionchannels(VSOR)fromastro-cytes.Thus,ourstudiesshouldfocusonthecorrelationbetweenthemechanismsforCVRandforregulationofbodyfluidosmolarity,in-cludingreexaminationofabove-mentionedhypothesesinvolvingneu-ronalSICCandglialtaurinerelease.NoCOI.

2S33K-2Visualization from osmosensitive areas in the brain to vasopressin-containing vesicles by fluorescent pro-teins in transgenic ratsUeta, Yoichi(Department of Physiology, School of Medicine, University of Occupational and Environmental Health, Japan)

Thecircumventricularorgans(CVOs)includingtheorganumvascu-losumofthelaminaterminalis(OVLT),thesubfornicalorgan(SFO)andtheareapostrema(AP)areknowntobeosmosensitiveareasinthecentralnervoussystem.Recently,wehavegeneratedtransgenicratsthatexpressthec-fos-monomericredfluorescentprotein1(mRFP1)fusiongeneafteradequatephysiologicalstimulisuchasacuteandchronicosmoticchallenge.Theexpressionofthec-fos-mRFP1fusiongeneintheOVLTandtheSFOwereobservedafterchronicdehydra-tion.However, it is interestingthatthefusiongene intheAPwasmarkedincreasedafterrehydrationfollowedbychronicdehydration.Wealsogeneratedanothertransgenicratsthatexpressthevasopres-sin-enhancedgreenfluorescentprotein(eGFP)transgeneinthesupra-optic (SON)andparaventricularnuclei (PVN).ThemagnocellularneurosecretorycellsintheSONandthePVNcontaineGFPfluorescentvesicles.Thefluorescentimagingtechniquesgiveusnewinsightstounderstandcentralosmosensitivecircuitsanddynamicsinvasopres-sin-containingvesicles.NoCOI.


2S33K-3Xenograft of vasopressin neuron derived from mouse ES cellNagasaki, Hiroshi1; Kodani, Yu1; Suga, Hidetaka2; Kaneko, Yoko1; Nakashima, Akira1; Ota, Akira1(1Department of Physiology, Fujita Health University, Toyoake, Japan, 2Department of Endocrinology and Diabetology, Nagoya Graduate School of Medicine, Nagoya, Japan)

Recently,mouseembryonicstemcell-derivedhypothalamus(mES-hy-po)reportedtodevelopvarietyofhypothalamicneuropeptidesinclud-ingvasopressin,neuropeptideY,AGRP,andtyrosinehydroxylase.ThefeederlessmESline,EB5,quicklyaggregatestoformembryoidbodyinserum-freemediuminthephospholipid-coatedwell(SFEBq).Then,itdifferentiatestohypothalamicprogenitorcellsinSFEBqwhencul-turedingrowthfactor-freechemicallydefinedmedium.Vasopressin-ergicneuronisoneofthemajorpopulationsinthemES-hypo,andbothhistologicallyandfunctionally, it isconsidered identical tothenativeone:Clustersofparvocellularormagunocellular-likeneuronsarefoundinES-hypo,andtheysecretevasopressintotheconditionedmediumaftervariousstimulationsincludingpotassium,sodium,man-nitolandangiotensinII.ToevaluateES-hypowasviableinthecentralnervesystem,thedispersedES-hypocellswereinjectedtothecortexregioninadultSpragueDawleyrats.Vasopressinergicneuronsurvivedmorethan60daysafterthegraft,andnotumorformationfoundinthestudiesmorethanthirtyxenograftssofar.Thematurityoftheneuroninthegraftdependsonthedaysaftertheinductioninvitro:whenimmaturecellswereinjected,theysurvivebutpoorlydifferen-tiated,andwhenmaturecellswereinjected,magunocellular-likeneu-ronarefoundinthegraft.ThesefindingssuggestES-hypomaybevaluabletodeconvolutethephysiologyofvasopressinsystem.NoCOI.

2S33K-4Exploring the role of anion channels in cell volume regulation and intercellular communications in the de-veloping brainAkita, Tenpei; Fukuda, Atsuo(Department of Neurophysiology, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan)

Cellvolumeregulationisanessentialfunctioninvolvedincellshapechanges,proliferation,migrationandprogrammedcelldeath, thusplayingimportantrolesindevelopingtissues.Theregulationisattainedbyregulatingthenetinfluxoreffluxofsolutesandwateracrossthecellmembrane,andmanydifferenttypesofionchannelandtransport-ermayparticipateintheregulation.Inthedevelopingbraininem-bryos,neuronalcellvolumeisdynamicallychangedandadjusted,buthowandwhattypesofvolume-regulatedionchannelandtransportercontrol theregulationhasyettobeexamined.Recentlywehavecheckedthat intheneurons inganglioniceminences, fromwhichcorticalGABAergicinterneuronsoriginate,thecurrentthroughthevolume-sensitiveoutwardlyrectifying(VSOR)anionchannel,themajorregulatorofanionfluxduringcellvolumeregulationinmanytissues,hastypicalcharacteristicsofmildoutwardrectification,inactivationandsensitivitytoitsinhibitorDCPIB.Wearenowinvestigatinghowthechannelregulatesneuronalcellvolumeduringbraindevelopment.Moreover,wearealsoexaminingthepossibilityofthechannelasapathwayforthereleaseofGABAandtaurine,themajorneurotrans-mittersintheembryonicbrain,sincesynapticstructuresdonotde-velopyetattheembryonicstage.Wediscusstheimportanceoffinecontrolofthemultimodalrolesofanionchannelsinresponsetoguid-ingcuestodeterminethepreciselocationanddifferentiationofmi-gratingneuronsinthedevelopingbrain.NoCOI.

Symposium 34Local regulation of Ca2+ signaling and

cellular response in excitable cells

(March 17, 10:25–12:05, Room K)

2S34K-1Regulation and molecular mechanisms of Cav1.2 channel by calmodulin and ATPMinobe, Etsuko1; Han, Dong-Yun1; Asmara, Hadhimulya1; Feng, Rui1,2; Xu, Jianjun1; Kameyama, Masaki1(1Dept. Physiol., Grad. Sch. Med. Dent. Sci., Kagoshima Univ. Kagoshima, Japan, 2Dept. Pharmac. Toxicol., Sch. Pharmac. Sci., China Med. Univ., Shenyang, China)

Ca2+ ions,awidespreadsignalmessenger,enter intothecytoplasmfromoutsidethecell throughvoltage-gatedCa2+ (Cav)channels, li-gand-activatedcationchannels,orarereleasedfromSR/ER.Intheheart,Cavchannelsplaykeyrolesinpace-makeractivity,ECcouplingand linkageofelectricalactivitytometabolicadaptationandgeneexpressions.Itsactivityismodifiedbyauxiliarysubunitsandseveralregulatoryfactors.Intheinside-outrecording,Cav1.2channelmain-tainedtheactivityinthepresenceofcalmodulin(CaM)andATP.CaMisaubiquitousCa2+bindingproteinthatmediatesCa2+-dependentregulationofvarioustargetproteins.ThechannelactivityincreasedandthendecreasedwithincreasinginCaMorCa2+,andincreasedinATPdependentmanner.Asemi-quantitativepull-downassaytoex-aminethebindingofCaMorATPtoCav1.2channelsuggestedthatbothCaMandATPboundtotheproximalregionof thechannelC-terminaltail.Basedonourexperiments,weproposeamodelfortheregulationofCav1.2channelsbyintracellularCa2+,CaMandATP.ThemodelconsistsoftwoCaM-bindingsitesinthechanneleachforCa2+-de-pendent facilitation (CDF)and inactivation (CDI),andoneormoreATP-bindingsite(s)forreprimingthechanneltobeavailable,indepen-dentoftheregulationbycAMP-PKAsignalingpathway.OurmodelexplainsthatthebalanceamongCa2+,CaMandATPnearthechanneliscriticalforitsactivity.NoCOI.


2S34K-2SR counter-ion channels mediated blood pressure regulationYamazaki, Daiju1,2; Tao, ShengChen2; Takeshima, Hiroshi1,2 (1Research Unit for Physiological Chemistry, Kyoto University, Kyoto, Japan , 2Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan)

TheTRIC (trimeric intracellularcation)channelsubtypes,namelyTRIC-AandTRIC-B,whichassembleintobullet-shapedhomo-trimerstoformmonovalentcation-selectivechannelsinthesarco/endoplasmicreticulum(SR/ER).Double-knockoutmicelackingboththeTric-aandTric-bgenesshowembryoniccardiacfailure,andthemutantcardio-myocytesdisplayweakryanodinereceptor(RyR)-mediatedCa2+release.Moreover,mutantskeletalmuscle fromTric-a-/-miceoccasionallyexhibitsalternancontractionresponses, likelyduetodestabilizedRyR-mediatedCa2+release.ThesedefectsobservedintheknockoutmicesupportourhypothesisthatTRICchannelsubtypespartlyme-diatecounter-ionmovementstofacilitateSR/ERCa2+release.Vascularsmoothmusclecells(VSMCs)containbothTRICsubtypesandtwoCa2+releasemechanisms;incidentalopeningofRyRsgenerateslocalCa2+sparkstoinducehyperpolarizationandrelaxation,whereasago-nist-inducedactivationofIP3RproducesglobalCa2+transientscausingcontraction.Tric-a-/-micedevelophypertensionduetoinsufficientCa2+sparks inVSMCs.Ontheotherhand, thesmoothmuscle-sepcifictransgenicmiceoverexpressingTRIC-Achannelsdevelopedcongen-italhypotensionduetofrequentCa2+sparksinVSMCs.Moreover,inourclinicalstudy,theTRIC-AgenepolymorphismsintheJapanesepopulationwereassociatedwithbothhypertensionsusceptibilityandsensitivitytoantihypertensivemedications.NoCOI.

2S34K-3Regulation of nuclear and cytoplasmic Ca2+ signaling and its function in cardiomyocytes: role of neuronal Ca2+ sensor-1 (NCS-1)Nakamura-Nishitani, Tomoe Y; Nakao, Shu; Wakabayashi, Shigeo (Dept. of Mol.Physiol.,Natl.Cer.Cardiovas.Ctr., Osaka, Japan)

Incardiomyocytes,cytoplasmicCa2+ transientsoccurduringexci-tation-contraction(E-C)couplingandhormonalstimulation,leadingtomusclecontractionandgeneexpression(excitation-transcription/E-Tcoupling),respectively. Interestingly,suchCa2+elevationsarealsodetectedinorganellessuchasthenucleus,whereE-Ccouplingisnotthoughtbeinvolved.However,whyandhownuclearCa2+transientsoccurisnotcompletelyunderstood.OurresultssuggestthatduringE-Ccoupling,nuclearCa2+ isderivedfromthecytoplasm,possiblythroughthenuclearporetobuffercytoplasmicCa2+,whereasuponreceptorstimulation,itisderivedfromthenuclearenvelopeCa2+store,atleastinpartthroughtheinositol1,4,5-trisphosphatereceptors(IP3Rs).SincewehadreportedthataCa2+-bindingproteinNCS-1interactswithIP3Rsintheheart(Circ.Res.2011),weexaminedwhetherNCS-1isinvolvedinthenuclearCa2+signalregulation.Wefoundthatstimula-tionofIP3RsignalbyIGF-1extensivelyincreasedNCS-1levelsandstrengthenedtheinteractionbetweenNCS-1andIP3Rsinallsubcel-lularfractions,includingthenucleus,incardiomyocytes.Furthermore,IGF-1-inducednuclearCa2+transientswassignificantlylesserinNcs1-/-myocytescomparedtowild-typemyocytes,withaconcomitantde-creaseinthelevelsofhypertrophy-relatedmoleculessuchasnuclearphospho-CaMKII.TheseobservationssuggestanovelmechanismofnuclearCa2+regulationmediatedbyNCS-1andIP3RsthatmaybeinvolvedinE-Tcoupling.NoCOI.

2S34K-4Mitochondrial Na-Ca exchanger NCLX-mediated mi-tochondria-sarcoplasmic reticulum Ca crosstalk and cardiomyocyte automaticityTakeuchi, Ayako; Matsuoka, Satoshi(Integr. Physiol. Fac. Med. Sci. Univ. Fukui)

NCLX,amitochondrialNa-Caexchanger,servesasaCaextrusionsysteminmitochondria(Paltyetal.,2012;Kimetal.,2012).Veryre-cently,wereportedthatNCLXregulatesautomaticityofHL-1,aspontaneouslybeatingcardiaccelllineoriginatingfrommouseatrialmyocytes(Takeuchietal.,2013).WeclarifiedthatNCLXfunctionsasaCaprovidertosarcoplasmicreticulum(SR),therebymodulatingtheautomaticitywhichisdrivenby“spontaneousCaleakfromSR”.How-everthereisfewinformationontheroleofNCLXintheintactcardi-acpacemakercells,SAnodecells.Therearetwocontroversialmech-anismsforexplainingtheautomaticityofSAnodecells,a“membraneclock”mechanisminwhichmembranechannelsdeterminestheauto-maticity,anda“Caclock”mechanisminwhicha“spontaneousCaleakfromSR”determinestheautomaticity.Inordertoclarifythecontri-butionofNCLXontheautomaticityofSAnodecells,weincorporatedmitochondrialCadynamicsintotwodifferentSAnodecellmodels,the“Caclock”-drivenmodel(MaltsevandLakatta,2009)andthe“membraneclock”-drivenmodel(Himenoetal.,2008).Inbothmodels,reductionofNCLXresultedinthemarkeddecreaseofSRCacontent.Interesting-ly,thebeatingratewasprolongedinthe“Caclock”-drivenmodel,butshortenedinthe“membraneclock”-drivenmodel,whichwasattribut-abletotheincreaseofsustainedinwardNacurrentIstvia[Na]idecrease.TheseresultssuggestthatcontributionofNCLXonthepacemakeractivitydependsoncompositionofionchannels.NoCOI.

Symposium 35Expanding frontiers in appetite

research explored by young investigators

(March17, 15:05–17:05, Room B)


2S35B-1Paraventricular nucleus oxytocin to arcuate nucleus POMC: a novel anorexigenic neuronal pathway in hy-pothalamusMaejima, Yuko; Sakuma, Kazuya; Shimomura, Kenju; Yada, Toshihiko(Jichi Medical University, School of Medicine)

RecentstudiesreportedthatOxytocin (Oxt)actsasaregulatorofenergymetabolism.AlthoughtheroleofOxtinreducingfoodintakeiswellestablished,theunderlyingmechanismsarelessdefined.Re-cently,wehaveshownthatOxtneuronsintheparaventricularnucle-us(PVN)regulatepro-opiomelanocortin(POMC)neuronsatleastinthebrainstemnucleustractussolitarius(NTS)andtherebyinducemelanocortindependentanorexia.SincePOMCneuronsarealsopres-entinthearcuatenucleus(ARC),weinvestigatedtheeffectofOxtonARCPOMCneurons.WefoundthattheinjectionofOxtintothe3rdventricleincreasedtheexpressionofc-FosinARCPOMCneurons.OxtreceptorshavebeenfoundintheARCPOMCneurons.ToassessthedirecteffectofOxt,theintracellularCa2+concentration([Ca2+]i)wasmeasuredinsingleARCPOMCneurons.TheapplicationofOxtin-creased[Ca2+]iwithreversibleinhibitionbyOxtreceptorblocker.Also,confocalimagesrevealedthatOxtterminalscontactedARCPOMCneurons,andintra-ARCretrogradetracerinjectionshowedthattheOxtfibersinARCwereoriginatedfromPVNOxtneurons.Moreover,intra-ARCinjectionofOxtdecreasedfoodintake.TheseresultsindicatethattheprojectionofPVNOxtneuronstoARCPOMCneuronsisanorexigenic.Physiologicalsignificanceofthisneuralpathwayremainsunclear.Howeverweshow,forthefirsttime,thatOxtactsasanac-tivatingfactoroftheARCPOMCneuronsandthatthePVNOxttoARCPOMCpathwaycanserveasanewmelanocortindependentpathway.NoCOI.

2S35B-2Dnmt3a in Sim1 cells is necessary for the normal con-trol of body weight and energy homeostasisKohno, Daisuke1,2,3; Lee, Syann3; Harper, Matthew J3; Kim, Ki Woo3; Sone, Hideyuki4; Fan, Guoping5; Elmquist, Joel K3 (1Advanced Scientific Research Leaders Development Unit, Gunma University, Gunma, Japan, 2Laboratory of Metabolic Signaling, Institute for Molecular and Cellular Regulation, Gunma University, Gunma, Japan, 3Division of Hypothalamic Research, University of Texas Southwestern Medical Center, Dallas, TX, USA, 4Faculty of Human Life Studies, University of Niigata Prefecture, Niigata, Japan, 5Department of Human Genetics, University of California Los Angeles, Los Angeles, CA, USA)

Globalobesityratesarerapidlyincreasing.Howenvironmentalfactorscauselong-termchangesinmetabolismremainsunclear.Theepigen-eticstatusofagenecanbealtered inresponsetoenvironmentalchanges.ToinvestigatetheroleofDNAmethylationintheregulationofenergybalance,wedeletedthedenovoDNAmethyltransferase,DNMT3a,inSim1neurons,whichisexpressedintheforebraininclud-ingthePVH.Sim1-specificDNMT3adeletionmicebecameobesefrom7-weeksonwards.Theamountoffatwasincreasedinthemice.Themicealsoshowedhyperphagiaandglucose-intolerance.Furthermore,themicedevelopedhyper-LDL-cholesterolemiawhenfedahigh-fatdiet.GeneexpressionprofilingrevealedthattheexpressionoftyrosinehydroxylasewashighlyupregulatedinthePVHofSim1-specificDN-MT3adeletionmice.ThiswasaccompaniedbydecreasedDNAmeth-ylationofthetyrosinehydroxylasepromoter,whichsuggeststhatthegeneisapotentialtargetofDNMT3a.TheseresultsdemonstratethatDNMT3a,akeymethylationenzyme,inthePVHneuronsisnecessaryfornormalregulationofbodyweightandenergyhomeostasis.NoCOI.

2S35B-3AMPK in the paraventricular hypothalamus regulates food selection behavior in miceOkamoto, Shiki; Minokoshi, Yasuhiko(Division of Endocrinology and Metabolism, National Institute for Physiological Sciences, Okazaki, Japan)

HypothalamicAMP-kinase(AMPK)regulatesfeedingbehaviorinre-sponsetohormonalandnutrientsignals.However,theeffectofAMPKonfoodpreferenceremainstobeestablished.Wefoundthatrefeedingafterovernightfasting,whichactivatesAMPKinthePVH,increasedthechoiceofhighcarbohydratediet(HCD)butdecreasedthatofhighfatdiet(HFD)inmice.Theeffectoffastingwassuppressedbyexpres-sionofshRNAforAMPKalpha1and2inthePVHwithlentivirus.Incontrast,expressionofconstitutively-activeAMPK(CA-AMPK)inPVHneuronsincreasedHCDselection.WeexaminedtheprincipleneuronsinthePVHfortheregulationoffoodselectionbehavior.MicroinjectionofCRHintothePVHwasfoundtoincreaseHCDselection,andex-pressionofshRNAforCRHinthePVHbluntedtheHCDselectioninresponseto fasting.PreferenctialexpressionofCA-AMPKinCRHneuronsalsoincreasedtheHCDselection.WefoundthatthechangeinfoodselectionwasdependentontheAMPK-inducedfattyacidox-idation(FAO)inthePVH.Furthermore,pharmacologicalactivationofAMPKincreasedcytosolic[Ca2+]inCRHneuronsisolatedfromthePVH,andtheeffectofAMPKwasabolishedwiththeexpressionofshRNAforAMPKorwiththesuppresionofFAO.Diet-inducedandgneticallyobesemicehavebeenshowntopreferHFD.WefoundthattheobesemicedecreasedAMPKandFAOactivityaswellasCRHmRNAexpressioninthePVH.Thus,ourresultssuggestthatAMPK-FAOsysteminCRHneruons inthePVHregulates foodselectionbehaviorforHCDandHFD.NoCOI.

2S35B-4Role of ghrelin signaling in the ventral midbrain in binge-like sugar overconsumption in miceYasoshima, Yasunobu; Yamaguchi, Erina; Nishioka, Haruna; Shimura, Tsuyoshi(Division of Behavioral Physiology, Graduate School of Human Sciences, Osaka University, Suita, Japan)

Binge-likesugaroverconsumptionissuggestedtobeduetometabol-icneedandhedonicdesirefortaste/postingestivereward.Ourprevi-ousstudyhasshowedthathedonic-drivensucroseconsumption isenhancedaftersucrosebingeinginmice.Otherstudiesshowedele-vateddopaminereleaseinthenucleusaccumbensduringsugarbinge-inginrats,suggestingthatactivatedbrainrewardsystemincludingtheventraltegmentalarea(VTA)isinvolvedinthebinge-likesugaroverconsumption.However,molecularregulationoftheVTAactivityinthebinge-likebehaviorremainstobesolved.Toexplorertheissue,weexaminedrolesofghrelin-relatedsignalingintheVTAinbinge-likesucroseoverconsumption.An intraperitoneal injectionofaghrelinreceptorantagonist,D-Lys3-GHRP-6(DG-6),andbilateralmicroinfusionsofDG-6 intotheVTAremarkablyreducedhedonic-drivensucroseconsumptionintrainedmice.Next,wecomparedpost-trainingchowintakeelicitedbyintra-VTAinfusionsofghrelinbetweenbingeingandnon-bingeingmousegroups.InfusionsofghrelinintothebilateralVTAproducedgreaterchowintakeinbingeinggroupbutnotincontrolgroups,suggestingthatghrelinreceptorsintheVTAaresensitizedduringorafterthebinge-likebehavior.Thesedatasuggestthatsen-sitizationofghrelinsignalingintheVTAplaysacrucialroleinhedon-ic-drivencomponentofbinge-likesugaroverconsumption.NoCOI.


2S35B-5The molecular mechanism of feeding regulation from the viewpoint of anorexia nervosaAmitani, Haruka; Asakawa, Akihiro; Amitani, Marie; Inui, Akio(Department of Psychosomatic Internal Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan)

Anorexianervosa(AN)isaseriousdisorderaffectingadolescentsandyoungadults,anditdecreasesthequalityoflifeofaffectedindividualsforprolongedperiods.Inspiteofmanytreatmentssuchasdrugther-apy,behavioraltherapy,cognitive-behavioraltherapyandfamilyther-apy,ANcontinuestobearefractorydisorderbecauseofitsunknownpathogenesis.Themechanismsunderlyingpersistentanorexiaarelargelyunknown,butwereportedtherelationbetweenANandfeed-ingregulatorypeptidessuchasPP,CCK,ghrelin,obestatinandnes-fatin.Adiponectinisaproteinhormoneproducedalmostexclusivelyinadiposetissueandexistsasmultipleisoformsinthebloodcirculation:alow-molecular-weight(LMW)form,amiddle-molecular-weight(MMW)form,andahigh-molecular-weight (HMW)form.Previousstudyre-vealedthatHMWhadaneffectoppositetoothersonbodyweightgain.Theklothogene,whichencodesasingle-passtransmembraneproteinexpressedprimarilyinrenaltubes,hasbeenidentifiedasasystemicanti-aginghormone.Klothoplaysaroleinadipocytematu-rationandsystemicglucosemetabolism,andalsoincreasesadipocytedifferentiationinvitro.Recently,wereportedtheassociationofANwithadiponectinandklotho.Inthissymposium,wereviewtherela-tionshipthesefeedingregulatorypeptidesandAN.NoCOI.

Symposium 36The functional roles of Parabrachial nucleus as an coordinator between

sensory and autonomic system

(March 17, 15:05–17:05, Room C)

2S36C-1The parabrachial nucleus as a bridge linking nocicep-tion and emotional memoryKato, Fusao(Dept Neurosci, Jikei Univ Sch Med, Tokyo, Japan)

Theexternallateralparabrachialnucleus(elPB)isthemostpredomi-nanttargetofthenociception-specificneuronsinthesuperficiallayerofthespinalcord.Theneurons intheelPB inturnprojecttothecapsularpartofthecentralamygdala(CeC),asubregionofcentralamygdalainvolvedinnociception-inducedemotionalresponses,regu-lationofspinalnociceptionandconditionedfear/threatlearning.Thispathwayisthuswellsituatedasanon-thalamocorticalrouteforsend-ingaversiveinformationtotheamygdala.Todirectlyexaminethis,wepharmacologicallyinactivatedthebilateralelPBduringfearacqui-sitionandfoundasignificantlydecreasedfreezingtimeinresponsetotheretrievingauditorycue,indicatingthattheelPBactivelypartici-patesinfearlearning(Sato,Watabeetal).AsCeCneuronsarecom-posedofdistincttypesofneuronsplayingdistinctrolesinfearlearning,weexaminedwhichtypereceivestheinputofelPBoriginbyselec-tivelystimulatingtheaxonsofelPBneuronsinthecentralamygdalawithoptogeneticsapproach.Surprisingly,almostall typesofCeCneurons,asclassifiedbyfiringpattern,exhibitedmonosynapticexcit-atoryresponsestolightstimulation,suggestingthatparabracialinputisapotentglobalactivatorofCeCneurons(Sugimura,Takahashietal).TogetherwithourpreviousfindingsofrobustsynapticpotentiationoftheelPB-CeCtransmissioninchronicpainmodels(Nakaoetal,2012)andfear-conditionedmice(Watabeetal,2013),weconcludethattheelPBrelaysnociceptiveinformationtotheamygdalarsiteofconver-gence/integration/plasticityforaversivememoryformation.NoCOI.

2S36C-2Roles of the lateral parabrachial nucleus in body tem-perature regulationNakamura, Kazuhiro1,2(1Career-Path Promotion Unit for Young Life Scientists, Kyoto University, Kyoto, Japan, 2PRESTO, JST, Japan)

Tomaintainbodytemperature,changesinenvironmentaltemperaturearesensedbythermoreceptors intheskinandthethermosensoryinformationistransmittedtothethermoregulatorycenter,preopticarea.Thisfeedforwardthermosensorysignalingisessentialtoelicitdefensivethermoregulatoryresponsesbeforethechangesinenviron-mentaltemperatureaffectbodycoretemperature.Ihavestudiedthisthermosensorypathwayandfoundthatthisascendingsignaling ismediatedbytwopopulationsofglutamatergicneuronsinthelateralparabrachialnucleus (LPB).Aneuronalpopulation intheexternallateralpartoftheLPB(LPBel)receivescutaneouscool-sensorysignalsfromthespinaldorsalhornandtransmitsthemtothepreopticareathroughtheirdirectaxonalprojection.Theotherpopulation,whichislocatedinthedorsalpartoftheLPB(LPBd),mediatesthetransmissionofcutaneouswarm-sensorysignalsfromthespinaldorsalhorntothepreopticarea.Thesetwopopulationsarelocatednearby,butformedsegregatedneuronalgroupsintheLPB.InhibitionoftheseLPBneuronseliminatesthefeedforwardthermoregulatoryresponsestochangesinskintemperature.Thespinal-LPB-preopticpathwayisessential forelicitingrapidandpreciseresponsesfortheregulationofbodytem-peratureandimportantly,constitutesanovelthermosensoryneuralpathwaythatisdistinctfromthespinothalamocorticalpathwaywell-knownforperceptionanddiscriminationofskintemperature.NoCOI.


2S36C-3Information processing in the brainstem neurons de-fined by genetic tracing of taste representationSugita, Makoto; Yamamoto, Kuniyo; Hirono, Chikara; Shiba, Yoshiki(Department of Physiology and Oral Physiology, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan)

BitterandsweettastestimuliaredetectedbythefamiliesofGpro-tein-coupledreceptors,T2RsandT1Rs,expressedindistinctpopula-tionsoftastereceptorcells.Werecentlyappliedageneticapproachtodelineatetheneuronalcircuitriesofbitterandsweettastesbyse-lectivelyexpressingthefluorescenttransneuronaltracer,tWGA-DsRed,ineitherbitter-orsweet-responsivetastereceptorcellsinmice,andthenvisualizingthespatialdistributionoftWGA-DsRed-labeledneuronsinthebrain.ThelocationsoftWGA-DsRed-labeledneuronssuggestedthetopographicsegregationoftasterepresentationinthesolitarytractnucleiandtheparabrachialnuclei,wherethegustatoryneuronsareorganizedwithsweet inputs locatedrostralandwithbitter inputslocatedcaudal,exceptforbitterinputsintotheexternal-lateralandexternal-medialsubdivisionsoftheparabrachialnuclei.Herewecom-binedgenetictracingwithelectrophysiologicalandimmunohistochem-icalapproachestopredicttherolesofthetracer-labeledneuronsinthesolitarytractnucleiandtheparabrachialnuclei.AnalysesoftheneurontypesandthemechanismsofsynaptictransmissionoftWGA-DsRed-labeledneuronsrevealedtheheterogeneitiesoftaste-relayingneurons,showingthearchitecturalsolution inthebrainstemthatprocessestaste information.Ourdatasuggesttheneuronalbasesunderlying interactionbetweentheprocessingoftaste informationandthehomeostaticcontroloffeeding.NoCOI.

2S36C-4Cardiovascular control by the cerebellum via parabra-chial nucleusNisimaru, Naoko1,2(1Brain Science Institute, RIKEN, Wako, Japan, 2Dept. of Physiol., Facult. of Med., Oita Univ., Yufu, Japan)

Thecerebellumisgenerallyregardedasamotorcenter,butitisin-volvedinautonomicfunctionsandalsocognitivefunctions.Thecere-bellarcortexconsistsofnumerousmicrozones,eachextending10mm2orso.Eachmicrozoneiscombinedwithsubcorticalstructuresconsti-tutingamicrocomplex,thatis,afunctionalunitofthecerebellum.Amicrcomplexisequippedwithinputsviamossy,climbingandbeadedfibersandanoutputviacerebellarorvestibularnuclearneurons.Duringpastnearly30years,fivecardiovascularcontrolmicrozonesinthecerebellarcortexhavebeenfound:(1)regionoflobulesI,IIandIIIofanteriorvermis,(2)medialregionoflobuleVIIandVIIIofpos-teriorvermis,(3)medialuvula,(4)lateralnodulus-uvulaand(5)foliump inflocculus.Recently, ithasbeenfoundthatthreeofthese (3–5)projectedtothelateralparabrachialnucleus(l-PBN)toconstitutemi-crocomplexs(cardiovascularmodules).Thesecerebellarareasreceiveclimbingfibersignalsfromaorticnerves,andmossyfibersfromvagusnerves,nucleustractussolitaliusorpedunculopontinenucleus.l-PBNreceivesinputsfromcardiovascularsystemandservesasoneofthemajorcardiovascularcontrolcenters inthebrainstem.Behavioralexperimentsshowedthatmedialuvularegulatebaroreceptorreflexduringexcise(Bradleyetal.,1990)andthatthelateralnodulus-uvulamaintainsbloodpressureduringpositionalchangesofthebody(Nisi-maruetal.,1998).Wenowrevealedthatthefoliumpregulatesbloodflowwhileanimalsperformdefensebehaviors(Nisimaruetal.,2013).NoCOI.

2S36C-5Nucleus Parabrachialis is the mode switch as an in-spiratory-expiratory system and as a sleep-arousal systemArata, Akiko(Div. of Physiome, Dept. of Physiology, Hyogo Collge of Medicine)

Thenucleusparabrachialiscomplex(NPB)oftheponsisknownasarespiratorymodulatingcenterandtheNPBplaysacrucialroleintheinspiratoryoff-switch.WeaimedtoexaminehowtheNPBparticipatesintheinspiratoryoff-switchusingpons-medulla-spinalcordpreparationsobtainedfrom0to4daysoldrats.First,theeffectsofNPBelectricalstimulationonC4ventralrootinspiratoryactivitywereexamined.TheelectricalstimulationofNPBinduced inspiratory-expiratoryphaseswitching.Herewedescribetherespiratoryneuronsinthedorsalponsandtheirinteractingtherespiratorycenter,centralchemoreceptionandadrenergicmodulation.Wefoundseveral typesofrespiratoryneurons;inspiratory,tonicinspiratory,expiratoryandinspiratory-ex-piratory(I-E)neuronsintheNPBusingwholecellpatchclampmeth-od.ThepopulationofI-Eneuronswasmorethan50%oftherecordedneurons.I-Eneuronsmightdeterminerespiratorycycleusinginspi-ratory-expiratoryphaseswitching.Orexinisthepeptideofmaintenanceofarousal,andfacilitatedI-Eneuronrhythm.Respiratorycyclesfast-erwhenOrexinisincreasedunderstresscondition.NPBissendingaxonstoamygdalaregardingemotion,andisreceivinginputsfromrespiratorycenterrelatedtorespiratoryrhythm.NPBmightbein-volvedinhyperventilationsyndromeandpanicdisorderbycontrollingrespirationusing inspiratoryterminationasanactivephase-switchunderstresscondition.Thepontinerespiratoryneuronswouldplayanimportantroleinthebalancebetweenstressandautonomicfunction.NoCOI.

Symposium 37Cutting Edge of

the TRP Channel Research

(March 17, 15:05–17:00, Room D1)


2S37D1-1Role of TRPC3 channels in mechano-chemo trans-duction in heartsNishida, Motohiro(Division of Cardiocirculatory Signaling, Okazaki Institute for Integrative Bioscience (National Institute for Physiological Science), National Institute of Natural Sciences, Aichi, Japan)

Mechanicalstretchduringdiastolicfillingoftheheartactivatesthemechanotransductionsignallingpathwaysthathavebroadimplicationsforadaptationandmaladaptationoftheheartagainstmechanicalload.Adiastolicstretchofventricularmyocytesreportedlyinduceslocalproductionofreactiveoxygenspecies(ROS)inaprocessdependentonmicrotubule-localizednicotineamideadeninedinucleotidephosphate(NADPH)oxidase2(Nox2).However,themolecularmechanismunder-lyingNox2activationbymechanicalstretchisstillunclear.Wedemon-stratedthatcanonicaltransientreceptorpotentialsubfamily3(TRPC3)contributetoNox2-mediatedROSproduction inrodenthearts.Me-chanicalstretchofratneonatalcardiomyocytesincreasedROSpro-ductionaccompanyingtheincreaseinfrequencyofCa2+oscillations.AlthougheitherinhibitionofTRPC3orTRPC6suppressedmechnicalstretch-inducedCa2+oscillation,onlyTRPC3proteinphysicallyinter-actswithNox2protein,and inhibitionofTRPC3,butnotTRPC6,abolishedmechanicalstretch-inducedROSproduction.Futhermore,pressureoverload-inducedcardiacfibrosis (butnothypertrophy)aswellasROSproductionweresignificantlysuppressedinTRPC3-defi-cientmousehearts. Theseresultsstronglysuggest thatTRPC3participatesinchronicmechano-chemotransductioninrodenthearts,andwillbeastrongtherapeutictargetofheartfailure.NoCOI.

2S37D1-2Roles of TRPM2 channel in neurological diseases based on neuroinflammatory responsesNakagawa, Takayuki1,2; Shirakawa, Hisashi1; Kaneko, Shuji1 (1Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, 2Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University)

Accumulatingevidencesuggeststhatneuroinflammationcontributestotheetiopathogenesisofneurologicaldiseases,suchasneurodegen-erativediseases,cerebrovasculardisorders,multiplesclerosisandchronicpain.NeuroinflammatoryresponsesintheCNSaremediatedbyglialcellsandCNS-infiltratedperipheral immunecells.TRPM2channelsareexpressedabundantlyinimmuneandglialcells,andactsasareactiveoxygenspeciessensor.RecentstudiesfocusontherolesofTRPM2inimmuneandinflammatoryresponses.Inthisstudy,weexaminedtheinvolvementofTRPM2insomeneurologicaldiseasesbyusingTRPM2-knockout (KO)mice. Intransient focalcerebralischemicmicebymiddlecerebralarteryocclusion,TRPM2-KOreducedcerebralinfarctvolume,neurologicalscoresandmigrationofactivatedmacrophages/microgliainischemicpenumbra.Inexperimentalauto-immuneencephalomyelitis (EAE)mice,TRPM2-KOreducedEAEclinicalsymptomsandmechanicalallodynia.Inperipheralnerveinju-ry-inducedneuropathicpainmodels,TRPM2-KOinhibitedmechanicalallodynia,whichwasaccompaniedwithreducedactivationofspinalresidentmicrogliaandinfiltrationofmacrophagesintothespinalcord.Inthissymposium,IwilldiscussabouttherolesofTRPM2anditsmechanismsinneuroinflammationintheseneurologicaldiseases.NoCOI.

2S37D1-3Functional coupling between Ca2+-sensing receptor and TRPC6 channel in pulmonary hypertensionYamamura, Aya1; Yamamura, Hisao2; Yuan, Jason X.-J.3(1School of Pharmacy, Kinjo Gakuin University, Nagoya, Japan, 2Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan, 3Department of Medicine, University of Illinois at Chicago, Chicago, USA)

Idiopathicpulmonaryarterialhypertension(IPAH)isarare,progres-sive,andfataldiseaseofunknownpathogenesis.Sustainedvasocon-strictionandvascularremodelingduetopulmonaryarterialsmoothmusclecell(PASMC)proliferationarekeypathogeniceventsthatleadtoearlymorbidityandmortality.TheseeventshavebeenlinkedtoCa2+mobilizationandsignalinginPASMCs.WehavepreviouslyshownthatCa2+-sensingreceptor (CaSR) isupregulated inPASMCsfromIPAHpatientsandcontributestoenhancedCa2+response.Inthisstudy,weexaminedwhetherTRPCchannel is involved inenhancedCa2+influxfollowedbyCaSRactivationinIPAH-PASMCs.ApplicationofLa3+inhibitedtheplateauphaseofextracellularCa2+-induced[Ca2+]cytincreasethroughCaSRactivationinIPAH-PASMCs.ThemRNAandproteinexpressionlevelsofTRPC6inIPAH-PASMCsweregreaterthan in normal PASMCs. Knockdown of TRPC6 channel in IP-AH-PASMCswithsiRNAattenuatedtheCaSR-mediated[Ca2+]cytin-crease.Incontrast,overexpressionofTRPC6channelwithCaSRinnormalPASMCsmimickedtheextracellularCa2+-induced[Ca2+]cytin-crease.TRPC6currentwasevokedbytheactivationofCaSRinIP-AH-PASMCs. Inconclusion, theextracellularCa2+-induced [Ca2+]cytincreaseduetofunctionalcouplingbetweenCaSRandTRPC6channelisanovelpathogenicmechanismcontributingtotheaugmentedCa2+influxandexcessivePASMCproliferationinIPAHpatients.NoCOI.

2S37D1-4The molecular mechanism of muscle hypertrophy; roles of TRPV1Takeda, Shinichi(National Center of Neurology and Psychiatry, Translational Medical Center, Tokyo, Japan)

Skeletalmuscleatrophyoccursinagingandpathologicalconditionsincludingcancer,diabetesandAIDS.Treatmentofatrophyisbasedoneitherpreventingproteindegradationpathways,oractivatingproteinsynthesispathways.Neuronalnitricoxidesynthase(nNOS),theenzymethatproducesnitricoxide,ismainlylocalizedatthesar-colemma,asaperipheralmemberof thedystrophin-glycoproteincomplex.WepreviouslydemonstratedthatnNOSwastranslocatedtobytoplasmandpromotedmuscleatrophybyactivatingFoxO3apathway(SuzukiNetal,.J.Clin.Invest.117:2468-76,2007).Here,weshowthattheoverload-inducedhypertrophywaspreventedinnNOS-nullmice.Moreover,nNOSwastransientlyactivatedwithinthreeminutesaftertheinitiationofoverload.Thisactivationpromotedfor-mationofperoxynitrite,areactionproductofnitricoxidewithsuper-oxide,whichwasderivedfromNADPHoxidase4(NOX4).NitricoxideandperoxynitritethenactivatedTRPV1,resultinginanincreaseofintracellularCa2+concentration([Ca2+]i)thatsubsequentlytriggeredactivationofmammaliantargetofrapamycin(mTOR).Notably,admin-istrationoftheTRPV1agonist,capsaicin,inducedhypertrophywithoutoverload,andalleviatedtheunload-ordenervation-inducedatrophy.Thesefindingsidentifynitricoxide,peroxynitriteand[Ca2+]iasthecriticalmediatorsthatconvertamechanicalloadintoanintracellularsignalingpathway,andsuggestedthatTRPV1couldbeanovelther-apeutictargetfortreatmentofmuscleatrophy(ItoNetal,.NatMed.19:101-106,2013).NoCOI.


2S37D1-5Regulation mechanisms of thermosensitive TRP chan-nelsTominaga, Makoto1(1Division of Cell Signaling, Okazai Institue for Integrative Bioscience (National Institute for Physiological Sciences), Okazaki, Japan, 2Department of Physiological Sciences, SOKENDAI)

TherearetenthermosensitiveTRPchannelshavingdistincttempera-ture thresholds for activation. Are the temperature thresholdschanged?It iswellknownthattemperaturethresholdsforTRPV1andTRPM8activationaredecreasedandincreased,respectively,inthepresenceofminimalamountsofchemicalactivators(capsaicinandmenthol).Inaddition,wepreviouslyreportedthattemperaturethresh-oldforTRPV1activationcanbedecreasedtothebodytemperaturerangeuponPKC-dependentphosphorylation,explainingtheacuteinflammatorypain. Wealso foundthattemperaturethresholdforTRPM8activationischangeddynamicallywithchangesinPIP2bind-ing,whichcouldexplainthewell-knownthree-bowlexperiment.Fur-thermore,wefoundthattemperaturethresholdforTRPM2activationwasdramaticallyreducedbyoxidationofthesinglemethioninewithhydrogenperoxide,whichcouldexplainthetemperature-dependentchangesinmacrophagefunctions.IntermsofmouseTRPA1,wefoundasplicevariantwhichregulatesTRPA1activityandisinvolvedintheinflammatoryandneuropathicpainalthoughtemperature-sensitivitywasnotchanged.Thus,functionsofthermosensitiveTRPchannelsareregulatedinamanydifferentways.NoCOI.

Symposium 38Mechanism and role of

inhibitory synaptic plasticity

(March 17, 15:05–17:05, Room E)

2S38E-1IP3/Ca2+ signaling-dependent regulation of GABAer-gic synapsesBannai, Hiroko1; Niwa, Fumihiro2; Mark W Sherwood2; Triller, Antoine3; Mikoshiba, Katsuhiko2(1Dept. Biol. Sci., Grad. Sch. Sci. Nagoya Univ., Nagoya, Japan, 2RIKEN BSI, Saitama, Japan, 3IBENS, Paris, France)

RegulationofthenumberofGABA-Areceptors(GABAAR)clusteringattheinhibitorysynapseisacrucialdeterminantofGABAergicsyn-aptictransmissionefficacy,andthusplayakeyrole for learning,memoryandtheirpatho-physiology.Ca2+influxfromtheextracellularspaceinducesthedispersalofGABAARs.Ca2+releasefromtheintra-cellularCa2+storealsoplaysvariousrolesinneurons,however,theimpactofCa2+releaseontheGABAergicsynapseremainstobeelu-cidated.Wereportherethatinositol1,4,5-trisphosphate(IP3)-inducedCa2+release(IICR)isresponsibleforthestabilizationofGABAergicsynapticstructuresinhippocampalneurons.LossofIICRactivitybygeneknockoutofintracellularCa2+releasingchannelIP3receptortype1(IP3R1)andbyspecificinhibitorsresultedinthedecreaseinGABAARandgephyrinclustersizeandGABAergicmIPSCamplitude,withoutreducingtheamountofGABAARonthecellsurface.Furthermore,wefoundthatIICRisrequiredforthereclusteringofGABAARsafterdispersionofclustersevokedbyCa2+-influx.Singleparticletrackingwithquantumdot(QD-SPT)revealedthatthelossofIP3R1andinhi-bitionofIICRenhancedthelateraldiffusionofGABAARs.TheincreaseofGABAARlateralmobilityresultingfromthelossofIICRwascom-pletelypreventedbyaninhibitionofdephosphorylation.OurresultssuggestthatIP3/Ca2+signalingcontributestothestabilizationofsyn-apticGABAARclustersthroughtheregulationofGABAARlateraldiffusion,possiblythroughphosphorylation.COIproperlydecleared.

2S38E-2Postsynaptic determination of the polarity of long-term modifications at inhibitory synapses in neocorti-cal pyramidal cellsKomatsu, Yukio(Dept. of Neurosci., Res. Inst. Env. Med., Nagoya Univ., Nagoya, Japan)

Inhibitorysynapsesinpyramidalcellscanundergolong-termpoten-tiation(LTP)ordepression(LTD).Becausetheroleofinhibitionistoregulatethe initiationofspikesproducedbyexcitatory inputs, thepolarityofinhibitorysynapticplasticitymaybeaffectedbyexcitatoryinputsarrivingatpyramidalcellssimultaneouslywithinhibitoryinputs.Inthisstudy,weinvestigatedthemechanismdeterminingthepolar-ity.Werecordedpostsynapticresponsesevokedinlayer2/3pyrami-dalcellsbyextracellularstimulationofpresynapticfibersinmousevisualcorticalslices. Inhibitorypostsynapticcurrents (IPSCs)wererecordedat-50mVinthepresenceoftheglutamatereceptorantag-onistkynurenate.High-frequencystimulation(HFS,50Hz,1s)wasappliedinthecurrentclampmode,whilekynurenatewastemporari-lywashedout.HFSinducedLTDofIPSCs.WhentheNMDAreceptor(NMDAR)antagonistMK801was loaded intopostsynapticcellsorwhenthemembranepotentialwasheldat-90mVduringHFS,HFSinducedLTPinsteadofLTD.PostsynapticloadingoftheCa2+chelatorBAPTApreventedbothLTDandLTP,indicatingthatbothmodifica-tionsrequirepostsynapticCa2+increasesfortheirinductionandthatLTDinductionrequirespostsynapticNMDAR-mediatedCa2+elevation.AdditionalpharmacologicalstudiesshowedthatLTPinductionre-quiredGABABreceptor-mediatedPLCactivationandCa2+releasefrompostsynapticIP3-sensitivestoreswithoutNMDARactivation.Theseresultssuggestthatthepolarityisdeterminedbythebalanceofex-citatoryandinhibitoryinputs.NoCOI.


2S38E-3Stress-induced (meta)plasticity at hypothalamic GABA synapse during stressInoue, Wataru; Baimoukhametova V Dinara; Fuzesi Tamas; Wamsteeker Cusulin I Jaclyn; Koblinger Kathrin; Whelan J Patrik; Pittman J Quentin; Bains S Jaideep1(Hotchkiss Brain Institute, University of Calgary)

Exposuretoastressorsensitizesbehavioralandhormonalresponsestofuturestressors.Stress-associatedreleaseofnoradrenalineenhanc-esthecapacityofcentralsynapsestoshowplasticity(metaplasticity).Iwillpresentnoradrenaline-dependentmetaplasticityatGABAsyn-apsesintheparaventricularnucleusofthehypothalamusthatcontrolsthehypothalamic-pituitary-adrenalaxis.TheseGABAsynapsesun-dergoactivity-dependentlong-termpotentiation(LTPGABA)inacutebrainslices,onlywhenslicesarepreparedimmediatelyafterinvivostressexposure:inslicesfromnaive,non-stressedanimals,LTPGABAdidnotoccur.Activationofβadrenergicreceptors (β-ARs)duringstress isnecessaryforthisstress-dependentgatingofLTPGABA.Mechanistically,β-ARscauseafunctionalupregulationofmetabotropicglutamatereceptor1(mGluR1).Thisallowsglutamatespillover,duringhighfrequencystimulation,toinducemGluR1-dependentLTPGABA.ThisLTPGABAwasexpressedpostsynapticallyandmanifestedastheemergenceofnewfunctionalsynapses.Ourfindingsprovidethefirstdemonstrationthatnoradrenalinereleaseduringaninvivochal-lengealterstheinformationstoragecapacityofGABAsynapses.Thesechangesmaybethebuildingblocksof learningandmemorythatcontributetoneuroendocrineadaptationstostress.NoCOI.

2S38E-4Regulation mechanism and functional role of inhibito-ry synaptic plasticity in a cerebellar Purkinje neuronHirano, Tomoo(Department of Biophysics, Graduate School of Science, Kyoto University)

AtinhibitorysynapsesonaPurkinjeneuroninthecerebellum,post-synapticdepolarizationinduceslong-termpotentiationofGABAergicsynaptictransmission,whichiscalledreboundpotentiation(RP).WehavebeenstudyingmolecularregulationmechanismsandfunctionalrolesofRP.TheincreaseinintracellularCa2+concentrationcausedbydepolarizationinducestheenhancedresponsivenessofGABAArecep-tor(GABAAR)inRP.BindingofGABAARassociatedprotein(GABAR-AP)toGABAARregulatedbyCa2+/calmodulin-dependentkinaseII(CaMKII)isnecessaryforRPinduction.WhetherRPisinducedornotisdeterminedbythebalanceofphosphorylationandde-phosphor-ylationactivitiesregulatedbyintracellularCa2+andbymetabotropicGABAandglutamatereceptors.Werecentlyfoundthatsubunitcom-positionofCaMKIIhassignificantimpactontheRPinduction.Whentherelativeamountofα-toβ-CaMKIIislarge,RPinductionissup-pressed.FunctionalsignificanceofRPhasalsobeenstudiedusingtransgenicmice,inwhichapeptidewhichinhibitsbindingofGABAR-APandGABAARisexpressedselectivelyinaPurkinjeneuron.ThetransgenicmiceshowedabrogatedRPandsubnormaladaptationofvestibulo-ocularreflex,atypeofmotorlearning.Thus,RPisimplicat-edinmotorlearning.NoCOI.

Symposium 39Current Status of Physiome & Systems

Biology: the Efforts in Japan

(March 17, 15:05–17:05, Room F)

2S39F-1Multi-scale modeling of the brain and the bodyDoya, Kenji; Yoshimoto, Junichiro(Neurobiology Research Unit, Okinawa Institute of Science and Technology Graduate University)

Itwasgenerallyregardedthatthemajorroleofskeletalmusclewastoproducemovementbycontracting.However,recentstudiesreport-edmultiplecasesinwhichmetabolismwasadjustedbycouplingtoskeletalmusclecontraction,whichindicatedthatskeletalmusclesmighthaveotherbiologicalrolesinadditiontotheproductionofmovement.Forexample,musclecontractionstronglystimulatesglucosetransportfrombloodintothemusclecells,theeffectofwhichisequivalenttothatofinsulin.Inaddition,ithasbeenrevealedthatskeletalmusclesecretesseveralhumoral factors (myokines) formetaboliccontrol.Skeletalmusclewasrarelyregardedasatargetformedicationinthepast,butrecentfindingshaveledtoitbeingfocusedonasapotentialdrugtarget.Thispresentationwillfocusonthemolecularmechanismofcouplingofmusclecontractiontometaboliccontrol.NoCOI.


2S39F-2Analysis of the toxicity of molecular target drugsSuzuki, Hiroshi; Honma, Masashi(Department of Pharmacy, The University of Tokyo Hospital, Faculty of Medicine, The University of Tokyo)

Systemsbiologyhasbeenmostsuccessfulinunderstandingandpre-dictingthedynamicswithinsinglecells, suchasgeneregulatorynetworksandmolecularcascades.Thefunctionofthebrain,ontheotherhand,dependscriticallyonspecificinteractionsofalargenum-berofneurons.Furthermore, thereal functionofthebraincanbetestedonlywhenitisexposedtocomplexsensory-motorenvironments.Thisnecessitatesmulti-scalemodeling,simulation,andanalysisoftheneural,musculoskeletal,andenvironmentaldynamics. Inordertounderstandhowthebrainrealizesreinforcementlearning,orlearningbyrewardandpunishment,wearemodelingthefunctionofthebas-algangliaandthedopaminergic functionatmultiple levels. InourKakenhiproject(ScientificResearchonInnovativeAreas:PredictionandDecisionMaking,www.decisions.jp),wecombinetheoreticalandexperimentalstudiesofhuman/animalbehaviors,neuralnetworkdynamics,andtheirmolecularcontrol.IntheStrategicResearchPro-gramforBrainScience(brainprogram.mext.go.jp/missionG),wesys-tematicallycollectconnectomeandproteomedataandconstructmulti-scalemodelsofsignalingcascade,cellularmorphology,andelectricactivities.UndertheStrategicProgramsforutilizingRIKEN’sKsu-percomputer(www.kobe.riken.jp/stpr1-life),inordertounderstandthemotorsymptomsinParkinson’sdisease,wearecombininglarge-scaleneuralnetworkmodelsofthethalamo-cortico-basalganglialoopwithmulti-scalemodelsofthemusculoskeletalsystem.Wediscusscurrentachievementsandfuturechallengesinmulti-scalemodelingofthebrainandthebody.NoCOI.

2S39F-3UT Heart, a multi-scale, multi-scale heart simulator to bridge the gap between bench and bedsideSugiura, Seiryo1; Cui , Xiaoke1; Okada, Jun-ichi1; Washio, Takumi1; Yamashita, Hiroshi1; Kariya, Taro1; Nagai, Ryozo2; Kadooka, Yoshimasa3; Watanabe, Masahiro3; Hirahara, Takao3; Yamazaki, Takashi3, Iwamura, Takashi3; Nakagawa, Machiko3; Hatanaka, Kohei3; Yoneda, Kazunori3; Hisada, Toshiaki1(13rd Department of Internal Medicine, University of Toyama, 2Jichi Medical University, 3Fujitsu Ltd)

Manyofthemoleculartargetdrugsareassociatedwiththeirsevereandselectivetoxicity.Oneofthemechanismsforthedevelopmentoftoxicityisrelatedtotheiroff-targeteffects.Inthispresentation,wewilldiscussonthemechanismoftoxicityofsunitinib,andwillfurtherdiscussonthemethodstopredictthetoxicityofmoleculartargetdrugs.Althoughsunitinibisusedforthetreatmentofrenalcarcinoma,itsadministrationoftenresultsinthedevelopmentofadverseeffectsinseveral tissues includingheart, liverandthyroid. Decrease inplateletcountisalsoaseversideeffect.Wecomparedthemagnitudeofinhibitionoftyrosinekinasesinthebodybetweensunitinibandlesstoxicsorafenibattheirtherapeuticconcentrations.Itwasfoundthatphosphorylasekinasegammasubunits1and2(PHKG1/2)areexten-sivelyinhibitedbysunitinib,butnotbysorafenib.Systems-biologicalanalysisrevealedthattheinhibitionofPHKG1/2resultsinthedecreaseinthecellularglutathione(GSH)levels.InvitrostudiesdemonstratedthatthereductionofthecellularGSHpotentiatestheintrinsictoxicityofsunitinib.InvivostudiesinmicedemonstratedthattherecoveryofGSHbytheadministrationofanti-oxidantsresultsinthedecreasedtoxicityofsunitinib,withoutaffecting itsanti-tumoreffects. Theseresultssuggestthatthepharmacodynamics/systems-biologicalanaly-sisisusefulinthepredictionofmechanism-basedtoxicityofmoleculartargetdrugs.COIproperlydeclared.

2S39F-4Reproducing neuronal images of visual circuits in-duced by natural scenes in silicoYagi, Tetsuya1; Okuno, Hirotsugu; Kameda, Seiji; Sanada, Tadashi; Hayashida, Yuki; Hasegawa, Jun; Kawasetsu, Takumi(Graduate school of Engineering, Osaka University)

Wehavedevelopedamulti-scale,multi-physicsheartsimulatoraimingatawiderangeofapplications.Thissimulatorisbasedonthefiniteelementmethodandthemolecularmodelsofexcitation-contractioncouplingprocessareimplementedineachelement(20millionelementsfortheelectrophysiologicalanalysis,onemillionelementsformechan-icalanalysis).Excitationinitiatedatthepace-makersitepropagatesfromatriatoventricleandinducessynchronizedcontractionandre-laxationtocausephysiologicalbloodflowintheheartchamber.Wehavealsocreatedtheconductionsystem,valvesandcoronarycircu-lation.Suchmulti-scale,multi-physicsnatureofthemodelenablesustoexaminetheimpactofmolecularorcellularabnormalitiesonthemacroscopicfindingsobservedatthebedside.Examplesofapplicationsinbasicandclinicalscienceswillbepresented.COIproperlydeclared.

Symposium 40Recent advancements in our understanding of central pattern generators for rhythmic movements: a phylogenetically preserved circuit from non-vertebrates to mammals

(March 17, 15:05–17:05, Room G)


2S40G-1Structural and physiological analysis for a central pat-tern generator controlling swimming locomotion of the ascidian larvaHorie, Takeo1,2; Ohkura, Masamichi3; Kusakabe, Takehiro G4; Nakai, Junichi3; Nakagawa, Masashi5(1Shimoda Marine Research Center, University of Tsukuba, 2JST, PREST, 3Saitama University, Brain Science Institute, 4Department of Biology, Konan University, 5Graduate School of life Science, University of Hyogo)

Itisessentialinvisionresearchtorevealtherepresentationofthenaturalimagesinneuronalcircuitsandunderstandunderlyingcircuitproperties.However,thenaturalscenesarefarmorecomplexanddynamiccomparedwithimagesusedinphysiologicalexperiments,e.g.,orientedslitsandmov-inggratings.Furthermore,theimagesprojectedontheretinaarecontinu-ouslyinfluencedbyeyemovements.Inthisregards,animalexperimentsareconfrontedwithunsurmountabletechnicaldifficultiestostudynaturalvisioninrealisticenvironments.Onepossibleapproachtoovercomethisproblemisvirtuallyreproducingtheneuronalactivities,namelyneuronal images,withphysiologicalmodelsinsilicoundernaturalvisualenvironments.Thisapproachallowsonetovisualizehownaturalscenesaremappedontheneuronalcircuitsandelucidatetherelationbetweenthenaturalscenesandtheirneuronalrepresentations.Forthispurpose,asimulationplatformwiththefollowingfeaturesisrequired:real-timereproductionofneuronalimagesfornaturalscenes,aconfigurablemodelstructurethatperformsparallelprocessingsimilartothevisualcircuits,andcompacthardwaretobeinstalledonamockupsystemthatcanmimiceyemovements.Here,wedevelopedahigh-speed,compactandconfigurablesimulationplatformtostudyearlyvisioninnaturalenvironments.Theplatformconsistsofbuilt-inanalogcircuitsmimickingtheessentialcircuitstructureofouterretinaandreconfigurablecircuitsthatsimulatetheneuronalimagesofinnerretinaandprimaryvisu-alcortex.Computationsthatemployedimagedatasampledatahighframerate(200frames/s)achievedreal-timereproductionofneuronalimagesinresponsetonaturalscenesunderrealisticcondition.NoCOI.

2S40G-2Normal locomotion speed requires pre-motor inhibito-ry interneurons in Drosophila larvaKohsaka, Hiroshi1; Takasu, Etsuko2; Nose, Akinao1,2(1Dept. of Complexity Science and Engineering, Grad. Sch. of Frontier Science, the Univ. of Tokyo, Chiba, Japan, 2Dept. of Physics, Grad. Sch. of Science, the Univ. of Tokyo, Tokyo, Japan)

Speedoflocomotionissignificantinanimallife.Whilespatio-temporalactivitypatternofmotorneuronsiscontrolledbythecentralnervoussystem,theidentityofinterneurons(INs)andthemechanismsofthespeedcontrolremainunclear.WeuseDrosophila larvaasamodelanimaltodissectneuralcircuits for locomotion.Larvaeprogress inspacebypropagationofmuscularcontractionfromtheposteriortotheanteriorsegments.ToidentifyINsregulatingthelarvallocomotion,weconductedageneticscreeningwithCaimagingandidentifiedaclassofINs,termedPMSIs.Byanatomicalandoptogeneticanalyses,wefoundthatPMSIsareglutamatergic,inhibitory,premotor,localandsegmentalINs.WhentheactivityofPMSIsissilenced,thespeedoflocomotion isreduced, indicatingthatPMSIsplaycrucialroles ingeneratinginnatespeedoflocomotion.Bydual-colorCaimaging,wefoundPMSIsandmotorneuronsinthesamesegmentbecomeactiveatasimilartiming,suggestingthatPMSIsshapethetemporalpatternofmotorneuronalfiringthroughlocalinhibition.Totestthispossibil-ity,weacutelyblockedtheactivityofPMSIsbyoptogenetics,whileextracellularlyrecordingmotornerveburstactivity.WefoundthatblockingPMSIsactivityprolongsthedurationofburstofmotornerveactivity.TheseresultssuggestthatPMSIscontrolthespeedofloco-motionbyregulating,throughdirectinhibition,thedurationofmotorneuronalburstineachsegment.NoCOI.

2S40G-3Functional analysis of locomotor circuits in the spinal cord and brainstem in zebrafishHigashijima, Shin-ichi; Kimura, Yukiko; Satou, Chie(National Institutes of Natural Sciences, Okazaki Institute for Integrative Bioscience)

Neuronalcircuitsinthespinalcordandbrainstemplayanimportantroleforproducinglocomotioninvertebrates.Investigationoflocomo-torcircuitsinamniotes,however,isnottrivialduetoenormouscom-plexityoftheirneuronalcircuits.Zebrafishlocomotorcircuitsaremuchsimplerwithlessnumberofdistinctclassesofneurons,makingitmorefeasibletoaddressthisissue.Wehavesettodefinethemorphologyandfunctionalpropertiesofneuronsthatexpressaparticulartran-scriptionfactor.Inthissymposium,wefocusonourstudiesinchx10positiveneuronsinthebrainstem.WefirstanalyzedthefunctionofChx10neuronsinlocomotionbyusingofchannelrhodopsin(ChR).ChRwasexpressedinChx10neuronsusingaGal4-UASsystem.Photo-stim-ulationofthehindbrainofChx10:Gal4;UAS:ChRtransgeniczebrafishreliablyelicitedswimming,indicatingthatactivationofChx10neuronsaresufficienttoevokeswimming.Then,weperformedelectrophysio-logicalrecordingstoaskwhetherhindbrainChx10neuronswereactiveduringfictiveswimming.WefoundthatsomeoftheChx10neuronswereactiveduringfictiveswimming.Finally,toconfirmnecessityoftheactivityofChx10neuronsforswimming,weusedhalorhodopsin(Halo)andarchaerhodopsin-3(Arch)foroptogeneticinhibition.BothChx10:Gal4/UAS:HaloandChx10:Gal4/UAS:Archfishstoppedsponta-neousswimmingupongreenlight-applicationtothehindbrain.Theseresults indicateChx10neurons inthehindbrainplay indispensablerolestogenerateswimming.NoCOI.

2S40G-4The role of excitatory neurons in the locomotor circuit in mammals: What we can learn from the αchimaerin knockout mouseNishimaru, Hiroshi(Fac. Medicine, Univ. Tsukuba, Tsukuba, Japan)

Neuronalcircuitsinthespinalcord(SC)generatethebasiccoordinat-edrhythmiclimbmovementsforwalkinginmammals.Thesecircuitsarecontrolledbysupraspinalinputssuchasthemotorcortex.Duringthedevelopmentofthesemotorcircuits,thereceptortyrosinekinaseEphA4isexpressedinthecorticospinaltract(CST)axonsandinasubpopulationof ipsilaterally-projectingspinalneurons. Ithasbeenshownthata-chimaerin(Chn1)playsacrucialroleinthedownstreamsignalingofEphA4andglobalChn1knockout(Chn1-KO)mouseshowsabnormalhoppinggaitaswellasaberrantmidline-crossingofbothoftheseaxons intheSC (Iwasatoetal.2007).SincetheseaxonsaresuggestedtoberesponsiblefortheabnormalgaitweexaminedthelocomotorcircuitinmicewhichlackChn1intheexcitatoryneurons(ENs)inthetelencephalonincludingtheCSTneurons(Emx1-Chn1-KOmouse)andinmicewhichlackChn1invesicularglutamatetransport-ertype2(VGLUT2)-positiveENsincludingthemajorityoftheexcit-atoryspinalneurons(VGLUT2-Chn1-KOmouse).Interestingly,Emx1-Chn1-KOmiceshowednormalalternatinggaitwhileVGLUT2-Chn1-KOmiceshowedbothhoppingandalternatinggaits.ElectrophysiologicalstudiesusingisolatedSCpreparationsin vitrorevealedthatthespinalnetworkofthesemice iscapableofgenerating locomotorpatternssimilartothoseobservedin vivo.Theseresultsindicatethataberrant-ly-crossedaxonsofVGLUT2-positiveENsarepartlyresponsibleforthegenerationofthehoppinggaitwhilethoseofCSTneuronsarelesslikelytobeinvolvedinsuchabnormalphenotype.NoCOI.


Symposium 41Recent progress in cardiovascular

research-morphological and physiological approach

[CollaborationSymposiumwithTheJapaneseAssociationofAnatomists]

(March 17, 15:05–17:05, Room H1)

2S41H1-1PGE-EP4 signaling is a key regulator of cardiovascu-lar stiffness and elasticityYokoyama, Utako(Cardiovascular Research Inst., Yokohama City Univ., Yokohama, Japan)

Collagenandelasticfiberformationareresponsibleforstructuralin-tegrityofcardiovascularsystem.SinceprostaglandinE(PGE)receptorEP4isabundantlyexpressedinthedevelopmentalanddiseasedar-teriesandintheheart,ourstudyhasbeenfocusingontheroleofPGE-EP4signaling intheregulationofcardiovascularextracellularmatrices.Duringfetalperiod,placenta-derivedPGE2activatesEP4andEP4-c-Src-PLC-signalingpathwayinhibitselastogenesisthroughdegradinglysyloxidaseproteinintheductusarteriosuswhichisafetalbypassarterybetweentheaortaandpulmonaryartery.Inarte-rialpathologicalcondition, i.e.aorticaneurysmandatherosclerosis,PGE2productionislocallyincreasedandenhancesinterleukin-6pro-ductionandmatrixmetalloprotease2activity,resultingindegradationofcollagenandelasticfibers.Inaddition,werecentlyfoundthatEP4isaprimarilyPGEreceptorincardiacfibroblastsandthatendogenousPGE2-EP4signalinghasaprotectiveroleagainstcardiacfibrosispos-siblythroughinhibitingconnectivetissuegrowthfactor(CTGF)-me-diatedactivationofmyofibroblasts.StimulationofEP4decreasedα-smoothmuscleactin,amyofibroblastmarker,andCTGFproteinexpressioninratcardiacfibroblasts.Incontrast,incardiacfibroblastsofEP4+/-mice,CTGFexpressionwassignificantlyhighercomparedtothoseofwild-typemice.SystemicadministrationofangiotensinII-inducedcardiacfibrosiswasgreaterinEP4+/-thaninwild-typemice.RegulationofEP4signalingmaybeanewtherapeuticstrategytopreventexcessivecardiacfibrosiswhichleadstoheartfailure.COIproperlydeclared.

2S41H1-2Endothelial class II PI3K-C2α controls vasucular bar-rier integrity through regulating membrane trafficking.Yoshioka, Kazuaki1; Takuwa, Noriko1,2; Okamoto, Yasuo1; Takuwa, Yoh1(1Department of Physiology, Kanazawa University School of Medicine, Ishikawa, Japan, 2Department of Health and Medical Sciences, Ishikawa Prefectural Nursing University, Ishikawa, Japan)

Vascularbarrierfunction,whichisstructurallysupportedmainlybytheadherens junction (AJ)comprisingVE-cadherin,maintains lowvascularpermeabilityinhealthyvasculatures.TheassemblyoftheAJistightlycontrolledbyintracellularsignalingmoleculesincludingRhoGTPases.Phosphatidylinositol (PI)3-kinase (PI3K) familyregulatesdiversecellularfunctions;whileclassIPI3KsandclassIIIVps34arewell-characterized, thephysiologicalrolesofPI3Kclass II,whichcomprisesC2α,C2βandC2γandproducesPI(3)P,remainlargelyun-known.WegeneratedC2αKOmice,whichwereembryoniclethalduetoseveredefectsinangiogenesis.C2α+/-miceandinducibleendotheli-alcell(EC)-specificC2α-deletedmiceexhibitedvascularbarrierdys-function:C2α-KOmiceweremuchmoresensitivetochallengewithananaphylaxismediatorplatelet-activatingfactorwithincreasedle-thalityandchronic infusionofangiotensinIIwiththeformationofdissectinganeurysms.InEC,siRNA-mediatedC2αknockdowninduceddecreasedPI(3)P+-endosomes, impairedendosomal trafficking,anddefectivedeliveryofVE-cadherintoAJ.C2αknockdownalsoimped-edcellsignalingincludingVEGFreceptor-2andS1P1receptorinter-nalizationandRhoactivationontheendosomes.Thus,ourdatadisclosethenovelcrucialfunctionsofPI3K-C2αinbarrierintegrityandvascu-larformationandrepresentsanewtherapeutictargetforvasculardiseases.NoCOI.

2S41H1-3Visualization of secretory dynamics of tissue-type plasminogen activator and cell surfase-associated fi-brinolytic activity on vascular endothelial cellsSuzuki, Yuko; Sano, Hideto; Brzoska, Tomasz; Urano, Tetsumei(Department of Medical Physiology, Hamamatsu University School of Medicine, Hamamatsu, Japan)

Vascularendothelialcells(VECs)contributetokeepthepatencyofvasculaturethroughanti-coagulatoryandpro-fibrinolyticactivities.Tissue-typeplasminogenactivator(tPA)secretedfromVECsasanactiveform,directlyenhancesfibrinolyticactivity.Recently,wesuc-ceededtovisualizeitssecretorydynamicsinGFP-taggedtPA(tPA-GFP)expressingVECsusingtotal internalreflectionfluorescencemicroscopy.tPA-GFPappearedtohaveauniquesecretorydynamicsandtoremainonthecellsurfaceafterexocytosisfromitssecretorygranules.StudiesusingmutantsoftPA-GFPsuggestedthatthebind-ingtothecellsurfacecouldbealteredeitherbymodifyingitsmolec-ularstructureviacatalyticallyinactivation,fibrin-associateddomaindeletion,replacementinglycosylationsites,orbymodifyingtheinter-actionwithitsspecificinhibitor.TheretainedactivetPAwasshowntoenhancethecellsurfacefibrinolyticpotentialtoactivateplasminogenintoplasminasfollows.Fluorescent-labeledplasminogenappearedtoaccumulateoncellsurfaceattPA-GFPretainedspotsaswellasperi-cellular/matrixadhesiveareainlysine-bindingsites-andtPA/plasminactivity-dependentmanner.FibrinnetworkformedonVECswasef-fectivelydissolvedwhentPA-GFP,butnotcatalyticallyinactivetPA-GFP,wasexpressed.Ourresultsprovidenewinsightsintothemech-anismmaintaininghighfibrinolyticactivityontheVECsurface.NoCOI.


2S41H1-4Origin of cardiovascular cells and heart development controlled by VEGF-Flk1 signaling.Ema, Masatsugu1; Otsu, Ayaka2; Azami, Takuya2; Hirashima, Masanori3; Koshiba-Takeuchi, Kazuko4; Takeuchi, Jun4; Shibuya, Masabumi5; Rossant, Janet6; Nishie, Tomomi2(1Shiga University of Medical Science, RCALS, 2Tsukuba Univ., Fac. of Med., Dept. of Anat. and Embryol., 3Kobe Univ., Grad. Sch. of Med., Dept. of Vascular Biology, 4Tokyo Univ., Inst. of Mol. and Cell. Biosci., Div., of Cardiovascular Regeneration, 5Jobu University)

Duringthedevelopment,vasculardevelopmentreliesonVEGFanditsreceptorssuchasFlk1andFlt1.Previousdataindicatethathema-to-cardiovascularcellssuchashematopoieticcells,endothelialcells,cardiomyocytesandsmoothmusclecellsaregeneratedfromFlk1-pos-itivecellsduringmouseandhumanES(EmbryonicStem)celldiffer-entiation invitro.However,developmentalprocess invivoremainsunclear.Ourlineagetracingexperimentindicatesthatmostofcardio-myocytesandendothelialcells,ifnotall,originatefromFlk1positivecells,whilesmoothmusclecellsatE11.5originatefromFlk1negativepopulation,insharpcontrastwiththatofinvitrodata.Furthermore,wefindthattheVEGF-Flk1-Flt1signalingisinvolvedinheartdevel-opment.PreviousstudiesrevealedthathematopoieticandendothelialcellsarenotgeneratedintheabsenceofFlk1gene,whilehematopoi-eticandendothelialcellsincreaseintheabsenceofFlt1gene.Inter-estingly,wefindthatcardiomyocytemarkerssuchasNkx2.5,Tbx5,Isl1andMlc2aareup-regulatedintheabsenceofFlk1.Ontheotherhand,cardiomyocytemarkerssuchasNkx2.5,Tbx5,Isl1andMlc2aaredown-regulated intheabsenceofFlt1.Thus,VEGF-Flk1-Flt1pathwayisinvolvedinnormalheartdevelopment.NoCOI.

2S41H1-5Functional mechanisms of cardiomyocyte plasticity during heart regenerationTakeuchi, Jun K.(1Div. of Cardiovas. Res., IMCS, the University of Tokyo, 2Grad. Sch. of Science., the Univ. of Tokyo, 3JST PRESTO)

Transcriptionfactorshavespecificexpressiontoproduceheartcellsfrompluripotentstemcells,andtokeeptheirfunctionsintheadult.However,thesefactorsareinsufficientasmasterregulatorsofcardiaclineagesformpluripotentstemcells,andmaintenanceforahealthyheart.Toaddressthesequestions,wegeneratedtissue-specificgeneprofilestostimulatecardiacfate.Fortunately,wehavereportedthatBaf60c,amemberofSWI/SNF-BAFfamilyactsasakeymoleculetopromotecardiaccellsfrommesodermalcellswithcardiacTanscriptionFactors;TFs,Tbx5andGata4invivo(Takeuchi&Bruneau,Nature2009;Takeuchietal.,Nat.Commun.2011). Inaddition, this factorstronglyregulateschromatinconformationofBAFcomplexesandHistonemodificationontheseTFspromotersbyChIPanalysis.BAFcomplexesactaskeyfactorstokeepcardiomyocytesurvivalinadultheartaswellasitsinductioninembryonicstage.BAFoverexpressedheartprotectheartfailureaftermyocardialinfarctionviapromotingseveral-geneexpressionsuchascell-growthfactors,cardiactranscrip-tionfactors,angiogenesisaswellasrepressionofinflammationsignals.Theseresultsindicatethatstagespecificfactorswithchromatinre-modelerspromotecardiaccellfateandkeepitslife.NoCOI.

Symposium 42The Challenge of the Center for Promotion of Gender Equality

[SymposiumforComitteeforGenderEquality]

(March 17, 15:05–17:05, Room J)

2S42J-1The Challenge of the Center for Promotion of Gender Equality, Kagoshima UniversityMasuda, Mina(Department of Anesthesiology and Critical Care Medicine Kagoshima University Graduate School of Medical and Dental Sciences)

2S42J-2Personalized work-life balance—You can put bread on your table—Uchiyama, Asako(Shin Nippon Biomedical Laboratories, Ltd.)

2S42J-3「単身赴任＋子育て＋親の援助を年数回」での研究生活Nakasako Izumi, Hiroko(Dept. Pharmacol., Fclt. Med., Toho Univ., Tokyo, Japan)

2S42J-4子育て夫婦ポスドク生活と、単身赴任二重生活（親の援助有り）を振り返ってMiyakawa, Naohisa(Department of Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry)

2S42J-5大規模アンケート調査（科学技術系専門職の男女共同参画実態調査）から見る安定した研究者ライフとはSekino, Yuko1; Sugiura, Midori2(1National Institute of Health Sciences, 2Aichi Gakusen University)


Symposium 43ATP as a regulatory factor:

New biological function regulated via interaction with membrane proteins

(March 17, 15:05–17:05, Room K)

2S43K-1The Na+/H+ exchanger NHE1 as an ATP-binding pro-tein: regulation via lipid-interacting domainWakabayashi, Shigeo; Shimada-Shimizu, Naoko; Hisamitsu, Takashi; Nakamura-Nishitani, Tomoe Y(Dept. of Mol. Physiol.,Natl. Cer. Cardiovas. Ctr., Osaka, Japan)

Whilemanybiologicalreactionssuchasion-pumpingrequiretheen-ergyfromhydrolysisofATP,littleisknownabouttheroleofATPasadirectregulatoryfactorformembrane iontransporters.WeherereportthatATPdirectlyinteractswiththeplasmamembraneNa+/H+exchangerNHE1.NHE1doesnotrequireenergyinputfromATPandhasnoconsensusATP-bindingmotif,butcuriouslythephysiolog-icalNHE1activityisabolishedbycellularATPdepletion.UsingthepurifiedcomplexproteinofNHE1and itssubunitCHP1fromSF9insectcells,weexaminedaphoto-affinitylabelingreactionwith8-azi-do-ATP[γ]-biotin.UVirradiationpromotedtheincorporationof8-azi-do-ATPintoNHE1,butnotintoCHP1,whichwasinhibitedbynon-la-beled ATP at IC50 of 2.2 mM, close to the ATP concentrationactivatingNHE1.ATPwasfoundtobindtothemembrane-proximalcytoplasmicregion(G542-P598)calledthelipid-interactingdomain(LID)thathadalsopreviouslybeenidentifiedasanATP-sensitiveregionforNHE1regulation.ThesefindingssuggestthatNHE1isanATP-bind-ingtransporter.Interestingly,staurosporinederivativeswerefoundtobindtotheLIDincompetitionwithATPandstronglyinhibitedtheNHE1activationinresponsetovariousstimuli.TheseresultssuggestthattheLIDfunctionsasamolecularswitchthatdictatestheactiva-tionstateofNHE1inresponsetovariousstimuli.Thus,weproposethatATPisaprimarilyimportantmoleculeinNHE1regulation.NoCOI.

2S43K-2KATP channel mutation that causes hyperinsulinism in early life and progresses to glucose intolerance in adults.Shimomrua, Kenju1; Maejima, Yuko1; Ashcroft, Frances2; Yada, Toshihiko1(1Department of Physiology, Division of Integrative Physiology, 2Department of Physiology Anatomy and Genetics, Oxford University, Oxford, England)

Loss-of-functionmutationsintheKATPchannelgenesKCNJ11(Kir6.2)andABCC8(SUR1)causeneonatalhyperinsulinisminhumans.Dom-inantly inheritedmutationscause lessseveredisease,whichmayprogresstoglucoseintoleranceanddiabetesinlaterlife.OneofthesemutationsisE1506KmutationinSUR1subunit.Themouseexpress-ingSUR1-E1506KinSUR1wasgenerated.Inthesemutantmice,KATPchannelinhibitionbyMgATPwasenhanced,duetoimpairedchannelactivationbyMgADP.Mutantbeta-cellsshoweddecreasedKATPchan-nelactivityandfiringofactionpotential inglucose-freesolution.E1506Kmutatedmiceexhibitedenhancedinsulinsecretionandloweredfastingbloodglucosewithin8weeksofbirth.At6monthsofage,incontrast,thesemiceshowedreducedinsulinsecretionandimpairedglucosetolerance.Thereducedinsulinsecretioncorrelatedwithlow-erinsulincontent;insulincontentincreasedwithageinwild-typemicebutnotinE1506Kmice.Therewasnodifferenceinthenumberandsizeofisletsorbeta-cells.TheseresultsinthemousemodelsuggestthatthegradualdevelopmentofglucoseintoleranceinpatientswiththeSUR1-E1506Kmutationmightresultfromimpairedinsulinsecre-tionprimarilyduetothefailureofinsulincontenttoincreasewithage.NoCOI.

2S43K-3Regulation of L-type Cav1.2 Ca2+ channels by ATPKameyama, Masaki1; Feng, Rui1,2; Liu, Shunyuan1,2; Minobe, Etsuko1; Xu, Jianjun1; Kameyama, Asako1; Hao, Liying2 (1Dept. Physiol., Grad. Sch. Med. Dent. Sci., Kagoshima Univ., 2Dept. Pharm. Toxicol., Sch. Pharm., China Med. Univ.)

L-typeCav1.2classCa2+channels(LTCC)isregulatedbynumberofcytoplasmicsignalingsystemsandfactors,suchasproteinphosphor-ylation,Ca2+/calmodulin(CaM)andoxidation/reduction.Itisknownsince1980sthatactivityofLTCCisalsodependentonATP.However,theunderlyingmechanismhasnotbeenestablishedtodate.AlthoughATP-dependentchangeinthebalanceofphosphorylation/dephosphor-ylationstateofLTCCisimplied,amechanismindependentofphos-phorylationhasalsobeensuggested.Thisideahasbeensupportedbyinside-outpatchexperiments,inwhichactivityofLTCCrequiresATP(EC50~0.5mM)togetherwithCaM.SinceAMP-PNP,anon-hydrolyz-ableanalogofATP,waspartlysubstitutableforATP,wehypothesizedthatATPmightbinddirectlywiththeLTCCproteinsandregulateitsactivity.2’/3’-ο-(2-aminoethyl-carmamoyl)-ATP-biotin([EDA-ATP]-bi-otin;biotinconjugatedatribose),butnotγ-[(6-aminohexyl)]-ATP-biotin([6AH-ATP]-biotin,biotinconjugatedatγ-phosphate),mimickedtheATPeffect,implyingthatthephosphategroupofATPmightbein-volvedinthebindingofATP.Photo-affinitylabelingofLTCCproteinwith8-azido-[EDA-ATP]-biotinin vitrorevealedthatthereagentlabeledN-terminalandproximalC-terminaltailoftheα1-subunitofLTCC.TheseresultssupporttheideathatATPbindsdirectlywithLTCCproteinandregulateschannelactivity.Thismechanismmayberele-vanttothesuppressionofLTCCactivityduringhypoxiaofcells.NoCOI.


2S43K-4Mechanism of ligand recognition and activation of ATP-gated cation channelsHattori, Motoyuki1,2(1Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo Japan, 2JST PRESTO, Tokyo, JAPAN)

ATPisknownasthevitalenergysourceinvolvedinenergymetabo-lism,biosyntheticreactionsandactivetransport.ItisalsoanessentialextracellularsignalingmoleculethatactivatestwodistinctfamiliesofATPreceptors:ionotropicP2XandG-protein-coupledP2Yreceptors.P2Xreceptorsaretrimericnon-selectivecationchannelsinvolvedinvariousphysiologicalprocessesincludingsynaptictransmission,taste,neuronalpainandinflammation.Arecentcrystalstructureofzebraf-ishP2X4receptorinanapo,closedstate,revealedthechalice-shapedtrimericarchitectureofP2Xreceptors (Kawateet.al.Nature2009).However,duetothelackofacrystalstructureincomplexwithATP,theagonistbindingsiteandthemechanismofchannelactivationre-mainedunclear.Iwillpresenttheagonist-boundstructureofzebrafishP2X4receptoranddiscussthemechanismofligandbindingandacti-vationofP2Xreceptors(Hattori&GouauxNature2012).NoCOI.

Symposium 45Disorders of the autonomic nervous

system in the menopause and underlying mechanisms

[CollaborationSymposiumwithJapanSocietyofNeurovegetativeResearch]

(March 18, 10:05–12:05, Room C)

3S45C-1Involvement of sex steroids in the regulation of the central autonomic nervous systemUeyama, Takashi(Department of Anatomy and Cell Biology, Wakayama Medical University School of Medicine, Wakayama, Japan)

Receptorsforsexsteroidssuchasestrogenandandrogen(ERα,ERβandAR)arewidelyexpressedinthebrain,wheresexsteroidsmod-ulatecentralnervous function. Limbicsystems (amygdala, lateralseptum,infralimbic,insular,ventromedialtemporalcorticalregions),andseveralhypothalamicandbrainstemnucleihavebeenidentifiedasthecentralsitesthatregulatestress-inducedsympatheticnervousactivation.Allamygdaloidsubnucleireceivepsychologicalinformationfromotherlimbicregions,whilethelateralandcentralsubnucleire-ceivesensoryandimmuneinformationfromparabrachialnucleusandmedicalgeniculatenucleus.Outputtothehypothalamusmainlyorig-inatesfromthemedialamygdala,whileoutputtothebednucleusofthestriaterminalisoriginates fromthecentralamygdalaandthemedialamygdala.AsERα,ERβandARarehighlyexpressedinthemedialamygdala,sexsteroidsmodulatetheautonomicnervousactiv-ities.Estrogensupplementationattenuatedstress-inducedcardiovas-cularchanges.Italsoattenuatedthestress-inducedincreaseofc-Fosinthe lateralseptum,medialamygdaloidnucleus,paraventricularhypothalamicnucleus,dorsomedialhypothalamicnucleus,laterodorsaltegmentalnucleusandlocuscoeruleus.Italsodown-regulatedc-fosmRNAexpressionintheadrenalglandandtheheart,suggestinganincreaseofestrogenattenuatedthestress-inducedhypothalamo-sym-pathoadrenaloutflowfromthecentralnervoussystemtothetargetorgans.NoCOI.

3S45C-2The role of estrogen in thermoregulation: assessment from animal to human studies.NAGASHIMA, KEI(1Body Tem and Fluid Lab, Fac Human Sci, Tokorozawa, Japan, 2IABS, Waseda Univ., Tokorozawa, Japan)

Estrogenhasmanyrolesbesidesthoseforpubertyandreproduction.Inthepresentsymposium,Iintroduceourstudiesinvestigatingtheroleofestrogeninthermoregulation.Human StudyIn8women(age,20–22y),metabolicrate,bodycore(Tcore)andskintemperature(Tskin,skinbloodflow,andthermalsensationwereassessedduringcoolingofroomtemperaturefrom29°Cto23.5°C.Themeasurementswererepeatedtwiceinthefollicularandluteralphases.Tcorewashigherintheluteralphase.Tskinandbloodflowdecreasedandmetabolicrateincreasedwiththereductionofroomtemperature,buttherewerenodifferencesbetweenthephases.Thermalsensationofcoldwasstron-gerintheluteralphase.Theresultssuggestthatsexhormonesmayaffectthermalsensationinhumans,althoughitsinfluenceonautonom-icresponsesissmall.Animal StudyToassessthedifferenceinthermalsensationbetweengenders,and itsroleofestrogen,weestimatedthermalpreference.Normalmeleandfemalemice,andovariectomizedmicewithandwithoutestrogenreplacementwereused(5ea).Thermalpreferencewasassessedbyplacingthemiceinabehavioralbox,ofwhichbottomwascoveredwith5Pertierboardsandthetemperaturewasrandomlychanged(28°C,31°C,34°C,37°C,and40°C).Femalemicelikedhottertemperaturecomparedtomalemice (40°Cand34°C);however,therewasnodifferenceamongnormalfemaleandovariec-tomizedmice.Gendermayhasaninfluenceonthermalpreference;however,estrogenisnotinvolvedinthemechanism.NoCOI.


3S45C-3Estrogen-induced anorexia is dependent on light en-vironmentTakamata, Akira; Mabuchi, Kaori; Nishimura, Yuri; Morimoto, Keiko (Department of Environmental Health, Nara Women's University, Nara, Japan)

Estrogenhasanti-obesityandanorexigeniceffects.Theprevalenceofobesityinwomenincreasesaftermenopause,whichelevatesariskformetabolicandcardiovasculardiseases.Inanimals,ovariectomyincreas-esfoodintakeandbodyweightgain,andestrogenreplacementcanreversethesechanges,stronglysuggestingthatestrogenplaysacriticalroleincontrollingfoodintakeandbodyweightinfemales.Wefoundthatestrogenreplacementattenuatedfoodintakeandincreasedc-Fosexpression inthesuprachiasmaticnucleus (SCN)specificallyduringthelightphaseinovariectomizedrats.Wealsofoundthatre-movaloflightexposureduringthelightphaseincreasedfoodintakeanddecreasedc-Fosexpression intheSCNspecificallyduringthesubjectiveday inestradiol-replacedovariectomisedratsbutnot inestrogendeficitrats,indicatingthattheanorexigeniceffectanden-hancedneuronalactivityoftheSCNinducedbyestradiolreplacementareabolishedbyremovaloflightstimulationduringthelightphase.Thesesuggeststhatthedisruptedcircadianrhythmofingestivebe-haviorispossiblyinvolvedinthemechanismfortheestrogendeficien-cy-inducedhyperphagia,andthatlightstimulationduringthesubjec-tivedayisnecessarytoelicittheanorexigeniceffectofestrogen.NoCOI.

3S45C-4Estrogen replacement suppresses hypertensive re-sponses to psychological stress and intermittent hy-poxia in ovariectomized ratsMorimoto, Keiko1; Tazumi, Shoko1; Omoto, Sayo1; Takamata, Akira1; Kudo, Risa2; Hatake, Katsuhiko2; Nagai, Hisashi3; Yoshida, Ken-ichi3(1Dept. Environmental Health, Facult. Human Life and Environmental Sci., Nara Women's Univ., Nara, Japan, 2Dept. Forensic Med., Nara Medical Univ., Nara, Japan, 3Dept. Forensic Med. Grad. Sch. Med. Tokyo Univ., Tokyo, Japan)

Hypertensionisamajorcardiovascularriskfactor.Theprevalenceofhypertensioninwomenincreasesaftertheaverageageofmenopause.Estrogenhasbeenpostulatedtoinvolveinbloodpressure(BP)regu-lation.WeexaminedwhetherestrogenreplacementinovariectomizedratsaffectstheBPelevationinducedbyacutepsychologicalstressorchronicintermittenthypoxia(IH).FemaleWistarratsaged9wkwereovariectomizedandimplantedwithradiotelemetrydevicesforBPandheartratemeasurements.After4wk,theratswereassignedeithertoaplacebo-treated(Pla)grouporagrouptreatedwith17β-estradiol(E2).Ratswereexposedtocage-switchstressfor60min.Thestress-in-ducedpressorresponsewasattenuated intheE2groupcomparedwiththePlagroup.AnothersetofPlaandE2groupsunderwentIHexposurefor2wk(90sat4%O2every3min,8hday-1duringthelightphase).EstrogenreplacementcouldnotpreventtheelevationofBPinIH-phase,butinhibitedtheincreaseofBPinnon-IH-phaseat1-wkIHexposure.Further,weinvestigatedthemechanismsaccount-ingforthesuppressingeffectofestrogenonpressorresponsetothecage-switchstressorhypertensioninducedbyIH.Ourfindingssuggestthatrenin-angiotensinsystemorendothelium-dependentvasodilationwasinvolvedintheeffectofestrogen.NoCOI.

Symposium 46Risk stratification of lethal ventricular

arrhythmias: from bench to bedside

(March 17, 9:00–11:00, Room D1)

3S46D1-1Underlying mechanisms in acquired long QT syn-dromeItoh, Hideki1; Crotti, Lia2; Schwartz, Peter J2; Hayashi, Kenshi3; Nakajima, Tadashi4; Ohno, Seiko1; Makiyama, Takeru5; Yamagishi, Masakazu3; Imoto, Keiji6; Pascale, Guicheney7; Hoire, Minoru1(1Department of Cardiovascular and Respiratory Medicine, Shiga University of Medical Science, Shiga, Japan, 2Department of Molecular Medicine, University of Pavia, Pavia, Italy, 3Division of Cardiovascular Medicine, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan, 4Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, Maebashi, Japan, 5The Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan, 6Department of Information Physiology, National Institute for Physiological Sciences, Okazaki, Japan, 7Faculte de Medecine Pierre et Marie Curie, Paris, France)

WhileabouthalfofcongenitallongQTsyndrome(cLQTS)casesareassociatedwithcardiacionchannelsgenemutations,thegeneticback-groundoflongQTsyndrome(aLQTS)manifestedbyacquiredfactorsremainstobeclarified.From6centersinJapan,ItalyandFrance,thisstudyconsistedof168aLQTSprobands.InaLQTS,themeanQTcintervalwas450±41msatbaseline,significantlylongerthaninnoncar-riers (402±44ms,p<0.0001)andshorterthan incLQTS(475±48ms,p<0.0001).Geneticanalysesrevealed51mutationsinaLQTSandthesemutationsweredetectedregardlesstheracesandgender.WhenwesimulatedQTinterval inthepseudo-ECGwiththedynamicORdmodel,thesimulatedQTintervalinaLQTSwasmilderthancLQTS.aLQTShascollapsedreporalizationreserveand30%havemutationsassociatedwithaformefrusteofcLQTS.NoCOI.


3S46D1-2Is Ca2+/calmodulin-dependent protein kinase II in-volved in ventricular fibrillation degeneration? Tsuji, Yukiomi; Makita, Naomasa(Department of Molecular Physiology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan)

Ventricularfibrillation (VF) is themostcommoncauseofsuddencardiacdeath.Thetriggerlikeaprematureventricularcontractioninitiatesventriculartachyarrhythmia,whichdegenerates intoVF.However,molecularbasisunderlyingtheprogressiveprocessleadingtoVFispoorlyunderstood.Ca2+/calmodulin-dependentproteinkinaseII (CaMKII)hasemergedasapro-arrhythmicsignalingmolecule.CaMKIIactivationinitiatedbyCa2+/calmodulinissustainedbyauto-phosphorylationandoxidation.CaMKIIcanparticipateinarrhythmiasignalingbyeffectsonionchannelproteins,intracellularCa2+uptakeandrelease,regulationofcelldeath,andbyactivationofhypertrophicsignalingpathways.TheactionsofCaMKIIonvoltage-gatedCa2+andNa+channelsandCa2+releasingryanodinereceptorchannelsareim-portantcomponents forarrhythmia-initiatingafterdepolarizations.CaMKIIalsopromotestheventricularremodelingprogressionbyactivatingtranscription factors forhypertrophy, inflammationandapoptosis,producingasubstrateforreentry.StudiesusingavarietyofmodelsofcardiacdiseasesconsistentlyshowthatCaMKII-inhibitionprovidesantiarrhythmicbenefits.WehaverecentlycreatedarabbitmodelofVFstormthatfeaturesrepetitivedefibrillatorfiringforre-currentVFanddemonstratedtheconnectionbetweenVFstormandCaMKIIinthismodel.Here,wefocusonpotentialrolesofCaMKIIontransitionfromprematureventricularcontractionintoVF,basedonrecently-emergingexperimentalfindingsbyusandothers.NoCOI.

3S46D1-3A simulation study of ventricular arrhythmias generat-ed from the Purkinje network with gap junction muta-tionInada, Shin1; Harrel, Daniel Toshio2; Haraguchi, Ryo1; Ashihara, Takashi3; Makita, Naomasa2; Nakazawa, Kazuo1,3 (1National Cerebral and Cardiovascular Center, Suita, Osaka, Japan, 2Department of Molecular Physiology, Nagasaki University, Nagasaki, Japan, 3Department of Cardiovascular Medicine, Shiga University of Medical Science, Otsu, Shiga, Japan)

ThePurkinjefibernetwork is thepartof thecardiacconductionsystemsthatensurescoordinatedcontractionoftheventricles,butcanalsobeanoriginofventriculararrhythmiassuchasventricularfibrillation.Recently,amutationinconnexin40(Cx40),agapjunctionisoformpredominantlyexpressedinatriumandHis-Purkinjesystem,wasidentifiedinpatientswithprogressivecardiacconductiondefectassociatedwithlethalventriculararrhythmias.However,itremainsunknownhowthisgapjunctiondysfunctionofthePurkinjefiberresultsingeneratingarrhythmias.Toresolvethisissue,weinvestigatedtherelationshipbetweentheexcitationconductionpropertyofthePur-kinjefiberandtheinitiationofreentrantbeatsusingcomputersimu-lations.WeconstructedananatomicalmodelsoftherabbitPurkinjenetworkwithmultipleconductionpathwaysbetweentheHisbundleandventricle.TosimulateCx40mutation,thegapjunctionconductancewasreduceddownto5%.Underconditionsofreducedgapjunctionconductance,conductionblocksoccurredattheintersectionsbetweenPurkinjefibersthatresultinestablishmentofreentrantcircuits.OursimulationresultssuggestthatreductioningapjunctionconductanceinthePurkinjenetworkmaybesufficienttogeneratereentrantven-triculararrhythmiaswithoutstructuralabnormalities.NoCOI.

3S46D1-4The application of induced pluripotent stem cells-de-rived cardiomyocytes in drug safety testing and dis-ease modelingMakiyama, Takeru(Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan)

Humaninducedpluripotentstemcell-derivedcardiomyocytes(hiPS-CMs)areapromisingnewtoolindrugsafetytestingbecausewecanstudythe‘humancardiomyocytes’.Studiesusingmulti-electrodearraysystemwithbeatinghips-CMsorCatransientrecordingwithcellsheetshavebeenreportedtobeusefultoinvestigatetheantiarrhyth-micdrugresponseofiPS-CMs.Inaddition,diseasemodelingbyiPS-CMsfrompatientswithaninheritedcardiacdiseasesareexpectedtoelu-cidatethediseasecausingmechanismsandbeahelpfulinvitrosystemtoinvestigatethenewpharmacotherapies.However,therearesever-allimitationsiniPS-CMs.Theyhavespontaneousactivityandanom-alousbehaviorofactionpotentialparametersresultingfrommainlyincreasedIfanddecreasedIK1.Recently,wereportedtheultrastruc-turalmaturationprocessofiPS-CMsduringalongtimeculture.Wealsodiscussabouttheelectrophysiologicalmaturationprocessinthispresentation.Inaddition,wegenerateddisease-specificiPScellsfromapatientwithcatecholaminergicpolymorphicventriculartachycardia(CPVT).Thedifferentiatedcardiomyocyteshadanincreasedsuscep-tibilitytocatecholamineinduceddiastoliccalciumwavesandryanodinesuppressedthem.It issuggestedthatthis iPScell-basedmodelofCPVTisusefulforinvestigatingnewtherapeuticapproach.Wealsopresentourrecentprogressinothercardiacdiseasemodeling(long-QTsyndromeandcardiomyopathy).NoCOI.

Symposium 47Attractiveness of exercise physiology

based on molecular biological research

(March 17, 9:00–11:00, Room E)


3S47E-1Molecular exercise physiology of skeletal muscle—Basic research and its practical applications—Takemasa, Tohru(Faculty of Health and Sport Sciences, University of Tsukuba)

“Exercisephysiology”isstudyforelucidatinghowabodyrespondsandischangedbyexercisestimulation.Skeletalmuscleisextremelyplastictissuethatchangesitsquantitativeandqualitativecharacter-isticsbyexercise.Skeletalmuscle increases itsmassbyresistancetrainingand increases itsaerobiccapacitybyenduranceexercise,whereasdecreasesitsmassbyunloadingoraging.Inourlaboratory,inordertoelucidatethemolecularmechanismforabove-mentionedphenomena,weappliedoverload/unloadinmiceskeletalmuscleandanalyzeitbymolecularbiologicalmethods.Iwouldliketointroduceoneoftheresultsandplanforfuturestudiesinthissymposium.Genetherapyisbecominganimportanttechnologyformedicaltreat-ment,andfurtherprogressisanticipatedwithfurtherimprovementsinbiotechnology.Ontheotherhand,inthefieldofsports,anti-dopingauthoritiesfearthat“genedoping”,whichisabuseofgenetherapy,willbecomeameansbywhichathletescanimprovetheirperformance.Toinvestigatethefeasibilityofdevelopingamethodfordetectionofgenedoping inpower-athletes,wedevisedanexperimentalmodelsystem.Wetriedtodetectgenedopingwiththismodel,andIwilldiscussthelimitofgenedopingdetectionatthispoint.NoCOI.

3S47E-2Epigenetics for the preservation of characteristics in skeletal muscleKawano, Fuminori(Graduate School of Medicine, Osaka University)

Histonemodifications,suchasmethylationandacetylation,playacriticalroleforthetranscriptionalonsetofmostgenesviatheconfor-mationalchangeofchromatins.Themainpurposeofthepresentstudywastoidentifythemajorhistonemodificationinvolvedintheinductionofskeletalmusclecharacteristics.Infirst,fourfast-specificgeneswithminorexpressioninratsoleusandfourslow-specificgeneswithminorexpressioninratplantariswereselectedfromalltranscriptsasthetargetsbymicroarrayanalysis.Responsesoftheexpressionofthesegenestofunctionaloverloadingbytransectionoftendonsofsynergistsweredeterminedinplantarisofadultrats.Overloadingcausedtheincreaseinthenumberofslowfibers,hypertrophyinslowfibersandtheenhancedexpressionoffourslow-specificgenes.Nochangeswereobservedinfastgenes.Further,theenhancedexpressionoffastgenesinplantariswasnotedinneonatalratsat2-dayafterbirth,althoughtheexpressionofslowgenesstartedincreasingafterthebeginningofvoluntarylocomotioninthelatestageoflactationperiod.Thesedataindicatedthedifferentmannerinthegainofmuscletype-specificgenes.Next,ModificationofhistonewascheckedusingChIP-qPCR.Tran-scriptionallyactivemarks,H3K4me3andH3acetylation,werenotedinthetranscriptionstartsiteoffastgenesinplantaris,whereastheexpressionofslowgeneswasnotrelatedtotheepigeneticmarksinsoleus.Thesefindingssuggestedthatgeneswithdifferentepigeneticmarksshowedthedifferentresponsestothephysiologicalstimuli.NoCOI.

3S47E-3Development of biological strategy for preventing skeletal muscle wasting with exercise.Miyazaki, Mitsunori(Health Sciences University of Hokkaido, Graduate School of Rehabilitation Sciences)

Themaintenanceofskeletalmusclemassiscriticalforlong-termhealthandqualityoflife,becausemusclemasswastinginducedbydisuse,agingorcatabolicdiseasesishighlyassociatedwithfunctionalimpair-mentanddisability,whichthenleadstothelossofindependenceandincreasedriskofmorbidity/mortality.Despitethesignificanceofskel-etalmusclelossandweaknessasinevitableconcomitantswithcata-bolicconditions,thecoordinatedstrategyforpreventingmusclewast-inghasbeenunderdevelopment.At this time,onlyprovenandeffective interventiontocounteracttomusclemasswasting isthephysicalexercise(particularlyresistanceexerciseincombinationwithadequatenutrientssupplementation).Ithasbeengenerallyacceptedthatthenetbalancebetweenproteinsynthesisanddegradationisacriticaldeterminantoftheregulationofskeletalmusclemass.Recentstudiesindicatethattheskeletalmuscleproteinmetabolism(proteinsynthesisandproteindegradation)isregulatedlargelythroughenvi-ronmentalcuesincludingphysicalexercise.Theaimofthepresentsessionistosummarizeanddiscuss,1)recentprogressintheunder-standingofcellularmechanisms inanabolic (proteinsynthesis)andcatabolic (proteindegradation)signalingpathwaysthatgoverntheregulationofskeletalmusclemass,2)alteredcapacityintheproteinmetabolisminresponsetophysicalexercise.Itisquitepossiblethatpreciseunderstandingofcellularmechanismsthatgovernproteinmetabolisminskeletalmusclewilldevelopmoreeffectivetherapeuticinterventionstopreventthelossofmusclewithaginganddisease.NoCOI.

3S47E-4Coupling of contraction to metabolic control in skele-tal muscleFujii, Nobuharu(Dept. Health Promotion Sciences, Grad. Sch. Human Health Sciences, Tokyo Metropolitan University)

Itwasgenerallyregardedthatthemajorroleofskeletalmusclewastoproducemovementbycontracting.However,recentstudiesreport-edmultiplecasesinwhichmetabolismwasadjustedbycouplingtoskeletalmusclecontraction,whichindicatedthatskeletalmusclesmighthaveotherbiologicalrolesinadditiontotheproductionofmovement.Forexample,musclecontractionstronglystimulatesglucosetransportfrombloodintothemusclecells,theeffectofwhichisequivalenttothatofinsulin.Inaddition,ithasbeenrevealedthatskeletalmusclesecretesseveralhumoral factors (myokines) formetaboliccontrol.Skeletalmusclewasrarelyregardedasatargetformedicationinthepast,butrecentfindingshaveledtoitbeingfocusedonasapotentialdrugtarget.Thispresentationwillfocusonthemolecularmechanismofcouplingofmusclecontractiontometaboliccontrol.NoCOI.


Symposium 48Cutting-edge researches of membrane

proteins—Towards molecular mechanisms and physiological functions

(March 18, 10:05–12:05, Room F)

3S48F-1Activation-signal transmission in the trimeric P2X2 re-ceptor channel upon voltage- and [ATP]- dependent gatingKubo, Yoshihiro1; Keceli, Batu(Div Biophys and Neurobiol, Natl Inst Physiol Sci, Okazaki, Japan)

ATPreceptorchannelP2X2isatrimercomposedofthreesubunits.WepreviouslyreportedthattheactivationofP2X2 isvoltageand[ATP]dependentinspiteoftheabsenceofacanonicalvoltagesensor.IthasbeenknownthatP2X2canbeactivatedbythebindingoftwoATPmoleculestothetrimer.TheaimofthisstudyistoknowhowtheactivationsignalbythebindingoftwoATPtothetrimericP2X2istransmittedtotheporeopening.Towardsthisaim,weintroducedmutationsatdifferentlevels,i.e.ATPbindingsite(K308A),linkerregiononβ-14(D315A)andporedomain(T339S),bycontrollingthenumberofmutationsinthetandemtrimericconstructs(TTC).Weobservedthefollowings.(1)Twoorthree,butnotone,K308AmutationinthetrimeraffectedtheactivationofP2X2.(2)Twoorthree,butnotone,D315Amutationinducedtwodifferentgatingmodes.(3)T339Smuta-tion,whichinducedconstitutiveactivityatallmembranepotentials,showedgradualchangesfromWTwiththeincreaseinthenumberofT339S,suggestingindependentcontributionofthreesubunitsatthepore level. (4)WeintroducedoneK308AmutationandoneD315Amutationonthesame(cis)ordifferent(trans)subunit.Theirpheno-typeswereclearlydifferentoneanother.(5)InthecaseofT339SandK308A(orD315A), thephenotypeofcisandtransmutantsweresimilar.Takentogether,itwassuggestedthattheATPbindingsignalisdirectlytransmittedtothecorrespondingβ-14stranddowntothelevelofD315andthenitspreadstoallthreesubunitsequallyattheporelevel.NoCOI.

3S48F-2Molecular mechanisms of voltage-gated proton chan-nel, VSOP/Hv1Okamura, Yasushi1; Fujiwara, Yuichiro1; Kawanabe, Akira1; Sakata, Souhei2; Kurokawa, Tatsuki1(1Laboratory of Integrative Physiology, Osaka University, Suita, Japan, 2Inst. Academic Initiatives, Suita, Japan)

Protonplayskeyroles inhomeostasisandnumerousphysiologicalfunctionsincludingmetabolism,respiration,digestion,boneremodeling,andrenalexcretionandisalsoforcriticalformicroenvironmentforpathogeninfection,inflammationandcancerprogression.VSOP/Hv1istheproton-selective ionchannelthat isconservedfromalgaetohuman. It isdimericandeachprotomerhasfourtransmembranesegmentswithhomologytothevoltagesensordomainofvoltage-gat-ed ionchannels. Thefourthtransmembranesegment,S4,containsmultipleargininesasthesignaturepatternofvoltagesensor.VSOP/Hv1alsocontainsthecytoplasmicstretchofthecoiled-coilregionthatisessentialfordimerization.ChannelactivitiesareinnatetoprotomersofVSOP/Hv1,anddimerchannelsexhibitcooperativegatingwithsteepervoltagesensitivitythanprotomerchannel. Giventhattheminimumunitofprotonchannelisascribedtotheportionconsistingonlyofabout150aminoacids,VSOP/Hv1isthesmallestcationchan-nelamongsofarknowninmammals.Despiterecentintensivestudies,noatomicstructuralinformationhasbeenavailable,anditremainsyetunclearhowmolecularmechanismsofVSOP/Hv1arerelatedtothoseofothervoltage-sensor-regulatedchannelsandphosphatase.Inthissymposium,wewillintroducerecentfindingsofstructureandfunctionsofVSOP/Hv1anddiscussonhowsophisticatedpropertiessuchasvoltagesensing,protonpermeationandpHsensingcanbeachievedinthissimpleproteinarchitecture.NoCOI.

3S48F-3TCA suppress the transduction channel activity in the olfactory transduction cascadeTakeuchi, Hiroko; Kurahashi, Takashi(Graduate School of Frontier Biosciences, Osaka University)

Weexaminedtheeffectofoff-flavorsgeneratedinfoods/beveragesontheolfactoryreceptorcells.Theoff-flavorsubstancesinduceexogenousunpleasantsmellsevenataverylowconcentration(pptlevel).Althoughnotyetscientificallydemonstrated,ithasalsobeenpointedoutthatoff-flavorsreducethepleasantflavorscontainedinfoods/beverages.Oneofthemostpowerfuloff-flavorsis2,4,6-trichloroanisole(TCA),thatisespeciallyknownforinducingthecorktaintinwines.Inthepresentstudy,weshowwithhumanpsychophysicalteststhatTCAactuallyreducesflavorsoffood(banana)andbeverage(wine)withverylowconcentration.Inparallel,itwasshownthatTCAsuppressedcyclicnucleotide-gated (CNG)channelspotently,whenexaminedonnewtolfactorysensorycilia.TCAsuppressedCNGchannelsevenwithat-to-molar(aM)level.Toexplainsuchsuper-efficiency,theTCAeffectshowedthetime-integrationandslowrecoveryfromthecurrentsuppression,presumablyrepresentingtheintegrationofthesubstancewiththehydrophobicsiteof themembrane. Thefindingswillbeusefulforthebasicsciencesandtheindustryoffoodsandbeverages.NoCOI.


3S48F-4Calcium Homeostasis Modulator (CALHM): A novel ion channel family encoding voltage-gated ATP re-lease ion channels involved in non-synaptic neuro-transmission from taste cellsTaruno, Akiyuki1; Marunaka, Yoshinori1; Foskett, J Kevin2(1Department of Molecular Cell Physiology, Kyoto Prefectural University of Medicine, Kyoto, Japan, 2Department of Physiology, University of Pennsylvania, Philadelphia, PA, USA)

Ithasbeenalong-standingenigmahowtastecellsdevoidofsynapsestransmittasteinformationtothenervoussystem.Recognitionofsweet,bitterandumamitastesrequiresthenon-vesicularreleasefromtastecellsofATP,whichhasbeensuggestedasaneurotransmittertoac-tivate afferentneuralpathways.We recentlydemonstrated thatCALHM1,whichhasawideion-conductingpore(~1.4nmindiameter),wasanovelvoltage-gatedATP-permeableionchannelandservedasabona fideconduitforATPreleasefromsweet-,umami-andbitter-sens-ingtypeIItastecells.Calhm1isexpressedintastebudsexclusivelyintypeIIcellsanditsproducthasstructuralandfunctionalsimilaritieswithconnexinsandpannexins,twofamiliesofchannelproteincandi-datesforATPreleasebytypeIIcells.Calhm1knockoutinmiceleadstolossofperceptionofsweet,umamiandbittercompoundsandtoimpairedgustatorynerveresponsestothesetastants.ThesenewstudiesvalidatetheconceptofATPastheprimaryneurotransmitterfromtypeIIcellstogustatoryneurons.Furthermore,theyidentifyvoltage-gatedATPreleasethroughCALHM1ionchannelasanessen-tialmolecularmechanismofnon-vesicularATPreleaseintastebuds.Wediscussthesenewfindings,aswellasunresolvedissuesinperiph-eraltasteATPsignalingthatmayinvolveyet-unknownsubunitsintheATPreleasemachinery.NoCOI.

Symposium 49Mechanisms on distributive shock and other blood pressure control failure[CollaborationSymposiumwithThe

JapanShockSociety]

(March 18, 9:00–11:00, Room G)

3S49G-1Central blood pressure regulation in salt-sensitive hy-pertensionTandai-Hiruma, Megumi; Kemuriyama, Takehito; Nishida, Yasuhiro(The Department of Physiology, National Defense Medical College, Saitama, Japan)

Bloodpressureisthedrivingforceofthecirculationandintegratesfunctionallyorgansthroughoutthebody.Besidesthelocalregulationofperipheralorgans,centralsympatheticnervoussystemalsoinvolvesinlong-termcontrolofbloodpressureandthepathogenesisofhyper-tension. Inthepresentstudy,wewill focusonsympathoinhibitoryfunctionsofcentralneuronalnitricoxidesynthase(nNOS)neuronsinsalt-sensitivehypertension.InhibitionofcentralnNOSbyacuteintra-cerebroventricular(i.c.v.)infusionofS-methyl-L-thiocitrulline(SMTC)significantlyenhancesperipheralsympatheticactivity inbothDahlsalt-sensitive(DSS)normotensiveandhypertensiverats.TheriseofactivitywasmoreprominentandbrainstemnNOSactivityissignifi-cantlygreater inhypertensiveDSSrats.Next,Hypertensionwasnormalizedbynifedipine inDSSratsonhigh-saltdiet,resulting inrestoringthenumberofnNOSneuronsinthebrainstem.Furthermore,centralnNOSwasinhibitedforalong-termbychronici.c.v.infusionofSMTCandthemeanarterialpressurewasassessedinDSSratsusingaradiotelemetrysystem,resultinginworsenhypertension.OurdatasuggestthatcentralnNOSneuronsmaybeoneofsuppressorsofthesympatheticsystemeveninnormotension,andbeenhancedbyhypertensionratherthanhigh-saltdietinsalt-sensitivehypertensiontorelievehighbloodpressure.However,thiscompensatoryresponsemaybe insufficienttoovercomesympathetichyperactivityduetoincreasedbrainrennin-angiotensinsystemandabnormalitiesassociat-edwithperipheralorgans.NoCOI.

3S49G-2Sphingosine-1-phosphate receptor-2 protects against anaphylactic shock through inhibiting eNOSOkamoto, Yasuo1; Cui, Hong1; Yoshioka, Kazuaki1; Takuwa, Noriko1,2; Zhao, Juanjuan1; Takuwa, Yoh1(1Dept. of Physiol., Kanazawa Univ. Sch. Med. Ishikawa, Japan, 2Dept. of Health & Med. Sci., Ishikawa Pref. Nursing Univ. Ishikawa, Japan)

Sphingosine-1-phosphate(S1P)playsanimportantroleofvascularandimmunesystemsviaafamilyofGprotein-coupledreceptors(S1P1-S1P5).S1P1contributestomaintenanceofvascularbarrierintegrity.Howev-er,theroleofS1P2inbarrierintegrityisunknown.Inmouseanaphy-laxismodelsofantigenchallengeandplatelet-activatingfactor(PAF)injection,S1P2deletionaugmentedvascularleakduetobarrierdisrup-tion,hypotensionandlethality.Nitricoxide(NO)isimplicatedinbar-rierdisruption.InS1P2-nullmice,PAF-inducedactivationofendothe-lialNOsynthase (eNOS)andAkt,anactivatingkinaseofeNOS, inaortaand lungwasenhancedcomparedwithwild-type (WT)mice.Consistently,PAF-inducedincreaseinthecyclicGMPlevelinaortawasenhancedinS1P2-nullmice.EitherpharmacologicaleNOSblockadeorgeneticeNOSdeletionprotectedS1P2-nullmicefromaggravationofanaphylaxisafterantigenchallengeandPAFinjection.Endothelialcells (EC) isolated fromS1P2-nullmice (KO-EC)exhibitedgreaterstimulationofAktandeNOSwithenhancedNOproductionbyeitherS1PorPAF,comparedwithWT-EC.Moreover,KO-ECshowedmoreseveredisassemblyofadherensjunctionswithaugmentedS-nitrosyla-tionofβ-cateninbyPAF,whichwasrestoredbypharmacologicaleNOSblockade.TheseresultsindicatethatS1P2inhibitseNOSstim-ulationandtherebyimprovesanaphylaxis,suggestingpotentialuse-fulnessofS1P2agonistsasanoveltherapeuticdrugforanaphylaxis.NoCOI.


3S49G-3The Role of Autophagy in Septic ShockWatanabe, Eizo1,2; Hatano, Masahiko2; Takahashi, Waka1; Hotchkiss, Richard S3; Hirasawa, Hiroyuki1(1Department of Emergency and Critical Care Medicine, Graduate School of Medicine,Chiba University, 2Biomedical Research Center, Chiba University)

Itisnotwellunderstoodwhethertheprocessofautophagyisaccel-eratedorblocked,andwhetheritisbeneficialorharmfultotheimmunedefensemechanismoveratimecourseduringsepticshock.Theaimofourstudywastodetermineboththekineticsandtheroleofauto-phagyinsepticshock.Electronmicroscopy(EM)wasperformedonliversamplesobtainedfrombothanobservationalclinicalcohortofseverelysepticpatientsandcontrolpatientswithoutanyincidentsofsepsis.EMdemonstratedincreasedautophagicvacuolesinsepticpa-tientscomparedtonon-septicpatients.Membranealterations(mem-branevacuoles,invaginationintoadjacentorganellesandmyelinfig-ure-likechanges)occurinasubpopulationofmitochondriainseveresepsis,butotherhepatocyteorganellesshowednoconsistentultra-structuralinjury.Weexaminedautophagyfluxinmicewithsurgicalsepsis(viacecalligationandpuncture(CLP)withC57BL/6NmiceandGFP-LC3transgenicmice),usingwesternblotting,immunofluorescence,andEM.Autophagyisinducedinseveralorgansintheearlyphaseofsepsis,andthattheentireprocessofautophagy,fromearlyenvel-opmentofdamagedcytosolicelementstofusionofautophagosomeswithlysosomes,isactivated.Also,inhibitionofautophagyprocessbychloroquineadministrationafterCLPresulted in liver injuryandhighermortality,indicatingthatautophagymayplayaprotectiveroleinsepticanimals.Thedevelopmentsofbothrealtimeauthophagymonitoringmethodandthespecificmodulatorsofauthophagywillbecriticaltothesuccessfulintroductionofpro-autophagictherapiestosepticshock.NoCOI.

3S49G-4Interferon 15 reverses age-related T cell inactivation by enhancing interferon-gamma production and STAT 5 phosphorylation in aged mice.Inoue, Shigeaki1; Komori, Yukako2; Inokuchi, Sadaki1; Hozumi, Katsuto3; Sato, Takehito3(1Tokai University,School of Medicine, Department of Emergency and Critical Care Medicine, 2 Tokai University, Institute of Innovative Science and Technology)

Background:Agingisassociatedwithimpairedimmuneresponsestopathogensandvaccines.Interleukin15(IL-15)isapluripotentantia-poptoticcytokinethatpromoteslymphocytesactivationandprolifer-ation.WeexaminedwhetherIL-15increasesT-cellactivationbymod-ulatingcytokineproductionandsignaltransductionofTcells.Method:Splenocytesfromyoung(6to8weeks)andaged(20to22months)C57B6micewerestimulatedovernightusingananti-CD3antibodywithorwithoutrecombinantmouseIL-15.Weperformedflowcyto-metricanalysisforT-cellactivationidentifiedbyCD25expressionandtheintracellularexpressionofphosphorylatedSTAT5,whichisoneofkeymoleculesofIL-15signaltransduction.Wealsomeasuredinter-feron-gammalevelsinthesupernatants.Results:Invitrostimulationofsplenocytesshowedthatcomparedtoyoungmice,agedmiceshowedimpairedT-cellactivation(68%reductioninbothCD4+andCD8+Tcells,p<0.01).IL-15reversedthisimpairedactivationwithincreasingSTAT5phosphorylationinCD4+andCD8+Tcellsfromagedmice.IL-15reversedthisimpairedCD8+Tcellsactivationinagedmiceandincreasedinterferon-gammalevelsdosedependently.Conclusion:IL-15reversesage-relatedTcellinactivationbyenhancinginterferon-gam-maproductionandSTAT5phosphorylationinagedmice.NoCOI.

3S49G-5Neutrophil Extracellular Traps under critical conditionHamaguchi, Shigeto1; Hirose, Tomoya2; Masafumi, Seki1; Naoya, Matsumoto2; Yukihiro, Akeda3; Norihisa, Yamamoto1; Osamu, Tasaki4; Takeshi, Shimazu2; Kazunori, Tomono1(1Division of Infection Control and Prevention, Osaka University Graduate School of Medicine, Osaka, Japan, 2Department of Traumatology and Acute Critical Medicine, Osaka University Graduate School of Medicine, Osaka, Japan)

Neutrophilextracellulartraps(NETs)arestructurescomposedofDNAandgranularproteins.NETsarethoughttobeapartofinnateimmu-nityanditrapidlytrapsandkillspathogen.AlthoughtheformationofNETshasbeenobservedduringinfection,theirroleinvivoisstillunclear.Inourpreviousstudies,weevaluatedNETsinthebloodofpatientswithsystemicinflammatoryresponsesyndrome(SIRS)andvariousclinicalconditionsbyimmunohistochemicalstainingofbloodsmearscollectedfrompatientsintheintensivecareunit.Wealsore-portedthedynamicalterationoftheexpressionofNETsinsputumcollected frompatientswithacuterespiratory infection.NETsarethoughttoreflectsomepartofdiseaseprogressionundercriticalconditions.QuantificationofNETsmighthaveapotentialasanovelinflammatorybiomarker.WewilldiscussaboutNETsfunctionundervariousdiseaseconditionsincludingliteraturereview.NoCOI.

Symposium 50Beyond the front line of neural optical

imaging

(March 18, 10:05–12:05, Room H1)


3S50H1-1Voltage-sensitive dye imaging of neural activities in the embryonic central nervous systemSato, Katsushige1; Sato, Yoko2(1Dept Hlth & Nutr Sci, Fac Human Hlth, Komazawa Women's Univ, Tokyo, Japan, 2Dept Hlth & Nutr, Coll Human Enviro Studies, Kanto-Gakuin Univ, Yokohama, Japan)

Investigatingthedevelopmentalorganizationoftheembryonicnervoussystemhasbeenoneofthemajorchallengesinneuroscience.Despitetheirsignificance,functionalstudiesofthevertebrateembryonicCNShavebeenhampered,sinceconventionalelectrophysiologicalmeanshavesometechnicallimitations.First,earlyembryonicneuronsaresmallandfragile,andtheapplicationofmicroelectrodesisoftendifficult.Second,thesimultaneousrecordingofelectricalactivityfrommultiplesites is limited,andasaconsequence,spatio-temporalpatternsofneuralnetworkresponsescannotbeassessed.Theadventofopticaltechniquesusingvoltage-sensitivedyeshasenabledthenon-invasivemonitoringofelectricalactivityinlivingcellsandalsofacilitatedthesimultaneousrecordingofneuralresponses frommultipleregions.Usingopticalrecordingtechniques, it isnowpossibleto followthefunctionalorganizationoftheembryonicnervoussystemandtoimagethespatio-temporaldynamicsoftheneuralnetwork’sformation.Here,wepresentrecentprogressinopticalstudiesontheembryonicnervoussystemwithspecialemphasisonthespontaneousdepolarizationwave,whichdemonstratestheutilityoffastvoltage-sensitivedyeimagingasapowerfultoolforelucidatingthefunctionalorganizationoftheembryonicCNS.NoCOI.

3S50H1-2Functional organization of the auditory cortex re-vealed by application of in vivo voltage-sensitive dye imagingSong, Wen-Jie(Dept of Sensory and Cognitive Physiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan)

Optical imagingusingvoltage-sensitivefluorescentdyescanrevealcorticalactivityinanareaoftensofsquaremillimeters,withaspatialresolutioninthetensofmicrometersandatimeresolutionlessthanonemillisecond.Inthistalk,Iwillpresenttwoexamplesshowinghowthehighspatialandtemporalresolutionofthistechniquecanbeex-ploitedtoadvanceourunderstandingoftheauditorycortexandtheauditorythalamus.Oneexampleshowsthepowerof the imagingtechniqueinrevealingafundamentalrulegoverningspatialrepresen-tationofsoundfrequencyintheprimaryauditorycortex.Theotherwillshowhowthetechniquecanbeusedfordeterminingcorticalpositionswithhighprecision, forthepurposeofguidingmultipletracerinjections.Retrogradelabelingofcellsinthemedialgeniculatebodyrevealednewprinciplesoftheauditorythalamocorticalorgani-zation.NoCOI.

3S50H1-3Surround modulation in cortical orientation map re-vealed by optical imaging based on intrinsic signalsWang, Gang; Okamura, Jun-ya(Dept. of Information Science and Biomedical Engineering, Kagoshima University)

Weappliedopticalimagingbasedonintrinsicsignalstoinvestigatethemodulationofthesurroundstimuluspresenceanditsdependenceonthereceptivefieldeccentricityincatvisualcortex.Presentationofcenter?surroundstimuliatthecenterofgazeresulted innoclearsurroundmodulation,nosignificantmodulationontheorientationmapwasobservedintheresponsemagnitudeandspatialpatternofre-sponse.However,at10degeccentricityinmoreperipheralpartsofthevisualfield,significantmodulationwasobservedinitscorrespond-ingcorticalareawhenthecenter-surroundstimuliwerepresented.Modulationwasobservedbothintheresponsemagnitudeandinthespatialpatternoftheresponse.Surroundorientationperpendiculartotheorientationofacenterpatchgratingshowedthelargestmodulationintheresponsemagnitude.Themodulationbecameweakerastheorientationdifferencebetweenthecenterandsurroundgratingsbe-camesmaller.Theseresultsdemonstratethedifferenceininformationprocessingbetweenthecorticalareacorrespondingtothecenterofgazeandthatcorrespondingtotheperipheralpartofvisualfield,andsuggestarelativespecializationforvisualinformationprocessinginperipheralrepresentationsofcorticalareas.NoCOI.

3S50H1-4Higher visual functios revealed by flavoprotein fluo-rescence imaging in miceShibuki, Katsuei(Department of Neurophysiology, Brain Research Institute, Niigata University, Niigata, Japan)

Neuralactivities,coupledwithenergymetabolism,canbeimagedasactivity-dependentchangesinendogenousgreenfluorescencederivedfrommitochondrialflavoproteins.Thismethodisespeciallyusefulforvisualizinghighercorticalfunctionsintheintactmousebrainthroughthetransparentskull.Micenavigatenearbyspaceusingtheirvisionandwhiskers.Therefore,youngmicemustlearntointegratetheseheterogeneousinputsinperceptualspace.Wefoundthatcorticalre-sponsesweredepressedintheprimaryvisualcortexofyoungmiceafterwearingamonocularprismgoggle.Thisdepressionwasuniform-lyobserved intheprimaryvisualcortex,andwaseliminatedbywhiskertrimmingorlesionsintheposteriorparietalcortex.However,oculardominanceplasticitywasnotaffectedbythesemanipulations.Compensatoryvisualmapshiftsofresponseselicitedviatheeyethathadworntheprismwerealsoobserved.Asaresult,corticalrespons-eselicitedviaeacheyewereclearlyseparatedwhenavisualstimuluswasplacedinfrontofmice,andocular-dominancecolumn-likestruc-tureswereformed.Comparisonofresponseareasbeforeandafterprismwearingindicatedthatthemapshiftswereproducedbyde-pressionwithspatialeccentricity.Thecorticaldepressionandtheresultingvisualmapshiftsbasedonwhisker-guidedcuesmayserveasasimplemodeltoinvestigatecellularandmolecularmechanismsunderlyinghighervisualfunctionsinthemammalianbrain,andtran-scranialflavoproteinfluorescenceimagingisausefultoolforinvesti-gatinghighervisualfunctionsinmice.NoCOI.


Symposium 51Morphological analysis of

3D reconstructed image using serial block-face electron microscope

(March 18, 9:00–11:00, Room J)

3S51J-1Serial neuronal EM images using TEM, FIB/SEM or DiK-SEMKubota, Yoshiyuki(Div. Cerebral Circuitry, NIPS, Okazaki, Japan)

Theserialelectronmicroscopic(EM)neuronalimagescapturingmeth-odsusingconventionalTEM,FocusIonBeam(FIB)/SEM,ordiamondknifecuttingtype(DiK)-SEMareintroduced.WecaptureimagesfromserialultrathinsectionsusingconventionalTEMandreconstructneuronalprofiles,suchasdendrites,spinesandsomawith3Drecon-structionsoftware.Itisatimeconsuminglaborwork,andcanbedoneonlybyskilledworker.ItwasdesiredforlongyearstodevelopEMsystemtocapturetheserialEMimagesautomatically.RecentlythenewEMsystemsareavailable:FIB/SEMorDiK-SEM.BothEMsarebasedonSEMthatcapturesimagesonblocksurface.FIB/SEMusesveryfinefocusionbeamtomilltheblocksurfaceasthinas2nminz-step.DiK-SEMusesdiamondknifeonultramicrotome inSEMchambertocuttheentireblocksurfaceserialy.Thecuttingsectionthicknessisusually40-50nm.TheadvantagesoftheDiK-SEMmeth-odaretoobtainlargersizeimageandfasterimagingtimethantheFIB/SEM.ThemaximagesizeofDiK-SEMis100µm×100µm,andthatofFIB/SEMis20µm×20µm.TheDiK-SEMcanobtain120serialimages/hour,andFIB/SEM15-20serialimages/hour.TheDiK-SEMrequireshighlymetalstainedtissue,ontheotherhand, theconventionalstainedtissueforTEMcanbeusedforFIB/SEM,becauseofdifferentSEMdetectors,EsBorBSE.Thedifferentnatureoftheblocksmakestheimagequalitydifferent.ImageoftheDiK-SEMiswithhighercontrastduetolackofmid-toneelectrondensitythanthatofFIB/SEM.Asintroducedabove,theeachmethodshassuperiorityandinferiority.Ideallyweusethemdependingontheresearchprojectpurpose.NoCOI.

3S51J-23D entire membrane organization of the mitochondria revealed by high resolution FIB/SEM tomography methodOhta, Keisuke; Nakamura, Keiichiro(Department Anatomy, Kurume University school of Medicine)

Chemicallyfixedrathepatocytewasexaminedusinghigh-resolutionfocusedionbeam/scanningelectronmicroscope(FIB/SEM)tomogra-phythatrevealedtheentiremembraneorganizationofmitochondriaincludingitscristaestructure.UnderstandingofcristaeorganizationinthemitochondriawasadifficultsubjectforevenintheserialsectionsTEM(ssTEM)methodbecausethedistancebetweencristae(30nm)isusuallysmallerthanthethicknessofultrathinsection.Electronto-mographymethodhasahighenoughspatialresolutiontoobserveeachcristae,buttheobservationareaistoosmalltoobservetheentiremitochondria.TheFIB/SEMtomographyweusedisarecentadvancedpowerful3DreconstructionmethodthathassmallerspatialresolutionthanthessTEM,andisabletoobservelargervolumethantheelectrontomographymethod.Inthismethod,3Dvolumeisreconstructedfromserialimagesobtainedbyfullyautomatedcyclesofsurfacemillingofspecimenbygalliumion-beamandimagingbySEMfromthenewlycreatedflatsurfaceofthespecimenwithintheFIB/SEMmachinery.Thiscyclewasrepeated650timesata10nmmillingpitch.Thefinalreconstruction (216µm3)containeddozensofmitochondriaandhadhighenoughresolutiontoanalyzeeachcristainamitochondrion.Thisreconstructionshowsthattheinnermembraneofmitochondriainvag-inatedtoformcristaethroughnarrowbridgesandtubularopenings.Thetubularopeningthenextendstosheet-likecristae.Mostofthecristaehavemultipletubularjunctionstotheinnermembranethrough-outthewholeofthemitochondrion.NoCOI.

3S51J-3SBF-SEM for ultrastructural analyses of physiology and pathology of the nervous systemOhno, Nobuhiko1,2; Lu, Haiyan3; Ransohoff, Richard M3; Trapp, Bruce D3(1Department of Anatomy and Molecular Histology, University of Yamanashi, Chuo, Japan, 2National Institute for Physiological Sciences, Okazaki, Japan, 3Department of Neurosciences, Cleveland Clinic, Cleveland, USA)

SerialBlockFace-ScanningElectronMicroscopy(SBF-SEM)facilitatesrelativelyrapidacquisitionofserialelectronmicroscopicimagesfromareasaslargeastenstohundredsmicrometersquare.Thisacquisitionisachievedbyrepeatedmillingoftissueblocksbybuilt-inultramicro-tomeandsubsequentobservationoftheblocksurfacewithSEM.Thisapproachmakesprecise3-dimentionalultrastructuralanalysesofcellsandorganelleseasierandfastercomparedwithconventionalobserva-tionofserialultrathinsectionswithtransmissionelectronmicroscopy.Suchultrastructuralanalyseshaveprovidedevidencesthatmitochon-drialbehaviorinmyelinatedaxonswasmodulatedbyaxonalelectricalactivityandCa2+tosupporttheenergydemandofsaltatorynerveconduction.Furthermore,SBF-SEMwascombinedwithpre-embed-dingimmunohistochemistrytoexaminemorphologyandfunctionsofmonocyte-derivedandmicroglia-derivedmacrophagesininflammato-rydemyelinatinglesionsofspinalcord.Theresultssuggestedthatthetwopopulationsofmacrophagescoexistinthelesionsbuthavedistinctnuclearmorphology,andmonocyte-derivedmacrophagesplaypivotalrolesindemyelinationatdiseaseonset.Byintroducingtheseapplica-tions,thispresentationwilldiscussthecharacteristicsofthe3-dimen-tionalultrastructuralanalysesusingSBF-SEM.NoCOI.


3S51J-4Correlative three-dimensional reconstruction for con-focal laser-scanning microcopy and FIB/SEM in the Double immunohistochemical stainingSonomura, Takahiro1; Furuta, Takahiro2; Nakatani, Ikuko3; Yamamoto, Yo3; Honma, Satoru1; Kaneko, Takeshi2(1Anatomy II, Kanazawa Medical University, Uchinada, Japan, 2Dep. of Morph. Brain Sci.,Grad. Sch. of Med., Kyoto Univ., Kyoto, Japan, 3Hitachi High-Tech Science Corporation, Chiba, Japan)

Serialblock-facescanningelectronmicroscopy(SFB-SEM)hasmadeeasytoacquireimagestacksofthetransmissionelectronmicroscopy(TEM)inthemesoscale,whichistakenwiththeconfocallaser-scanningmicrocopy (CF-LSM).Wetried immunocytochemistry forFIB-SEMandcorrelatedthisimmunoreactivitywiththatinCF-LSM.Dendritesofneuronsintheratneostriatumwerevisualizedusingarecombinantviralvector.Moreover, thethalamostriatalafferentterminalswereimmunolabeledwithCy5fluorescenceforvesicularglutamatetrans-porter2(VGluT2).AfterdetectionofthesitesofterminalsapposedtothedendritesbyusingCF-LSM,GFPandVGluT2immunoreactivitieswerefurtherdevelopedforEMbyusingimmunogold/silverenhance-mentand immunoperoxidase/diaminobenzidine (DAB)methods,re-spectively.Weshowedthatconventionalimmuno-cytochemicalstain-ing forTEMwas applicable to FIB-SEM. Furthermore, severalsynapticcontacts,whichwerethoughttoexistonthebasisofCF-LSMfindings,wereconfirmedwithFIB-SEM,revealingtheusefulnessofthecombinedmethodofCF-LSMandFIB-SEM.COIproperlydeclared.

3S51J-5Analysis of Neural Circuit using Automated Tape Ul-tra Microtome;ATUMIwasaki, Hirohide; Okabe, Shigeo(Department of Cellular Neurobiology, Graduate School of Medicine, The University of Tokyo)

Thebrainconsistsofahugenumberofneuronsandglialcells,andneuronsconnecteachotherviasynapsestogenerateneuronalcircuits.Inordertounderstandhowthebrainworksatthecircuitlevel,itisimportanttofigureoutthecompleteconnectionsofneuronsinneuro-nalcircuits.However,therearesomedifficultiestodrawthecompleteneuronalmapandoneofthemajorproblemsisthatsynapsesaresotinythatitisessentialtouseelectronmicroscopytoobserve.Forthethree-dimensionalreconstructionofneuronalcircuitsatthesynapselevel,itisnecessarytocollectcontinuousseriesofultrathinsectionssuccessfully.ATUM(AutomatedTapeUltraMicrotome)istheequip-mentattachedtotheulramicrotometocollectultrathinsectionsontheplastictape.Bycomparingtootherequipmentsforthesimilarpurpose,suchasFIB-SEMorSBF-SEM,oneoftheadvantagestouseATUMisthatsectionsremainonthetapeafterobservationandwecanobservethesamesectionsformanytimesbyusingseveralmeth-ods.Inthissymposium,wewillintroducetheATUMandwillshowsomedatafromsectionscollectedbyATUMbyusingbothelectronmicroscopyandlightmicroscopy.COIproperlydeclared.

Symposium 52Essential Brain and

Physiological Function[FAOPSJointSymposium]

(March18, 10:15–12:05, Room K)

3S52K-1Sympathetic-respiratory coupling and the develop-ment of hypertensionAllen, Andrew M; Menuet, Clement(Department of Physiology, University of Melbourne, Parkville, Australia)

Thatbreathingpatternsregulatecardiovascular functionhasbeenunderstoodforaverylongtimeandthisknowledgeformsabasisformanymeditativepractices.Severalyearsagowedemonstratedthatanalteredinteractionbetweenthecentralrespiratorygeneratorandcircuitsgeneratingsympatheticvasomotoractivitytothecardiovas-cularsystemoccursfromveryearlypostnatalperiodsintheSponta-neouslyHypertensive(SH)rat?manyweeksbeforethedevelopmentofalteredbloodpressure.This increasedrespiratory-sympatheticcouplingwasresponsiblefortheelevatedsympatheticactivityandTraube-Herringwavesobserved intheSHrat.Usingavarietyoflentiviraltransductionapproachestoenablecell-specificactivationorinhibitionofneuronswithparticularchemicalphenotypesinadults,wehaveshownthatthisalteredrespiratory-sympatheticcouplingintheSHratinvolvesthecatecholaminergicC1neuronsoftherostralventrolateralmedulla.Activationofthesecellsenhancessympathet-ic-respiratorycoupling,whilstinhibitionselectivelyreducestheinspi-ratory-relatedpeakofsympatheticactivity.Weproposethatthesedatashedlightonamechanismbywhichalteredbreathingpatternsaffectcardiovascularfunctionandsuggestapotentialapproachforamelioratingtheelevatedsympatheticnerveactivitythatoccursinsomepre-hypertensivehumans.NoCOI.


3S52K-2Odor-induced analgesia in mouseKashiwadani, Hideki1; Tashiro, Shogo1,2; Kanmura, Yuichi2; Kuwaki, Tomoyuki1(1Department of Physiology, Graduate School of Medical and Dental Sciences, Kagoshima University, 2Department of Anesthesiology, Graduate School of Medical and Dental Sciences, Kagoshima University)

Painisessentialforanimalsincludinghumantodetecttheactualorpotentialtissuedamages.Howevertheexcessivepainofteninducestheimmobilizationtoanimalsandsometimespreventstheappropriatebehaviors.Thereforethegaincontrolofthepainisoneoftheessentialfunctionsinourbrain.Forthegaincontrol,animalshavedevelopedtheintrinsicneuronalcircuitsforanalgesia.Infolkremedy,odorouscompoundswereoftenprescribedtodrivethe intrinsicanalgesiccircuits.However, ithasnotyetknownwhether“theodor”,orthesenseofsmell,couldreallyinducetheanalgesia.Toaddresstheques-tion,weperformedthehotplatetestandtheformalintestunderodorexposure.Amongseveralodormoleculesexamined,theexposureofanodormolecule(“OdorantX”)showedsignificantanalgesiceffectsinwildtypemice.Theanalgesiceffectswerenotobservedinolfacto-ry-deprivedmice, indicatingthattheolfactoryinputevokedbytheodorantXinducedtheanalgesia.Furthermore,theodor-inducedan-algesiawasdisappearedinmicelackingorexinergicneuronsinhypo-thalamus,suggestingthatthe intrinsicanalgesiccircuits includinghypothalamicorexinergicneuronsmediatetheodor-inducedanalgesia.Inconclusion,wefoundtheodor-inducedanalgesiamediatedbyhy-pothalamicorexinergicneuronsinmouse.Ourfindingssuggestthatodorinputcouldmodifythepainprocessingbydrivingtheintrinsicanalgesiccircuitsincludinghypothalamicorexinergicneurons.NoCOI.

3S52K-3Hypothalamic orexin-synthesizing neurons have a physiological role in emotional hyperthermiaMohammed, Mazher1; Ootsuka, Youichirou1; Yanagisawa, Masashi2; Blessing, William W1(1Centre for Neuroscience, School of Medicine, Flinders University, Adelaide SA, Australia, 2Department of Molecular Genetics and HHMI, University of Texas Southwestern Medical Center, Dallas, USA.)

Whenanimalsencountersalient,potentiallythreateningenvironments,bodytemperatureincreasesandthermoregulatorycutaneousbloodflowdecreases,aresponsethatcanbedescribedasemotionalhyper-thermia.Thehypothalamicorexin-synthesizingneurons influenceanumberofphysiologicalandbehavioralprocesses, includingbodytemperatureandbrownadiposetissue (BAT)thermogenesis [1,2].Restraint-inducedhyperthermiaisreducedinorexin-deficientmice[3].Wehypothesizedthatorexin-synthesizingneuronscontributetoemo-tionalhyperthermiabyinductionofBATthermogenesisandcutaneousvasoconstriction.Weusedtheorexin-ataxin3transgenicratsinwhichorexinneuronsarepostnatallydestroyed.AcagedintruderratwasintroducedintothehomecageofaresidentmaleSprague-Dawleyrat.Intruder-evokedincreasesinBATthermogenesisandtheassociatedtailarteryvasoconstrictionwerereducedintransgenicrats.Theseresultsaddtothegrowingbodyofevidencethatorexinneuronsparticipateinthecoordinationoftheanimal’sresponsetoenvironmen-talthreats.[1]Inutsukaetal.,2013;[2]Tuponeetal.,JNeurosci2011;[3]Zhangetal.,JPhysiol2010.NoCOI.

3S52K-4Preoptic-raphé connections for thermoregulatory and febrile cutaneous vasoconstrictionTanaka, Mutsumi1,2; McKinley, Michael J.1; McAllen, Robin M.1(1Florey Institute of Neuroscience and Mental Health, University of Melbourne, Australia, 2Japan Automobile Research Institute)

Bodytemperatureismaintainedbythebalancebetweenheatproduc-tionandheatdissipation.Inrats,thetailcirculationisamajororganofheatdissipation,andiscontrolledbysympatheticvasoconstrictornervesthatareactivatedduringcoldexposureandexperimentalfeverinducedbyprostaglandinE2 (PGE2).Thepreopticarea isthekeystructureforbodytemperatureregulationandthefebrileactionofPGE2.Ithasbeenconsideredthatunderwarmconditionspreopticneuronsdirectlyinhibittailsympatheticpremotorneuronslocatedinthemedullaryraph?[1],andthatcoldsignalsandPGE2inhibitthosepreopticneurons,causingtailvasoconstrictionbydisinhibition[1].Thisinhibitoryrestraintoriginatesfromtwodistinctpreopticregions;arostromedialregion(RMPO)surroundingtheorganumvasculosumofthelaminaterminalisandthemedianpreopticnucleus,andasecondregioncentred~1mmcaudolaterally(CLPO)[2].Werecentlyshowedthat,inparallelwiththeinhibitorypathway,anexcitatorypathwayfromtheRMPOtothemedullaryraphémediatestailvasoconstrictorresponsestocoldskin[3]andtoexperimentalfever[4].Ourfindingssuggestthatbothinhibitoryandexcitatorypreoptic-raph?descendingdrivesregulatetailvasoconstrictioninconcert.NoCOI.

Symposium 53Mind-body interaction: How does brain

know visceral condition?

(March 18, 14:00–16:00, Room B)


3S53B-1A cerebral cortical region that responds to amino acid injection into the hepatic portal veinSekiguchi, Masayuki1; Koppensteiner, Peter1; Odagiri, Saori1; Hatanaka, Yusuke1; Yamada, Daisuke1; Yamada, Tetsuya2; Katagiri, Higeki2; Wada, Keiji1(1Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan, 2Department of Diabetes and Metabolism, Tohoku University Hospital, Sendai, Japan)

Informationaboutvisceralactivities(gastrointestinal,respiratory,andcardiovascular)islocalizedintheinsularcortexviavagusnerveaffer-ents.However,itisunknownwhetherthereisacorticalregionthatrespondstothehepaticbranchofthevagusnerve.Thisbranchinner-vatesthehepaticportalvein,bileduct,etc.Thespontaneousactivityofthisbranchischangedbyinjectionofaminoacidsintotheportalvein.Inthepresentstudy,activitymappingwascarriedoutinarc(activity-regulatedcytoskeleton-associatedprotein)-Venusmice.Arcisanimmediate-earlygeneandexpressedinresponsetopotentneu-ronalactivity. Inarc-Venusmice, thefluorescentproteinVenus isexpressedmainly incorticalregionsunderthecontrolof thearcpromoter.Portal-veininjectionofaminoacidsknowntoenhancevagusactivityincreasedthenumberofVenus-positivecellsspecificallyinthesecondarysomatosensorycortex(S2)(saline-injectionascontrol).Correspondingly,injectionofaminoacidsthatreducevagusactivityledtoadecreaseofVenus-positivecellsinS2.VagotomyabolishedtheactionofaminoacidsuponVenusexpression inS2.TheseresultssuggestthatS2includesneuronsthatpotentlyrespondtotheinjectionofaminoacidsintotheportalveinviathevagusnerve.NoCOI.

3S53B-2Inter-organ neural network mediate body weight regu-lationYamada, Tetsuya; Tsukita, Sohei; Katagiri, Hideki(Division of Metabolism and Diabetes, Tohoku University Graduate School of Medicine, Sendai, Japan)

Despiteremarkableadvancementsinobesityresearchoverthepastdecade,themechanismsunderlyingobesityarestillnotfullyunder-stood.Inrecentyears,ithasbeenrevealedthatnumerousmetabolicinteractionsbetweenorgans,whichareorganizedbythebrain,func-tionasanegative-feedbackmechanismandareinvolvedinmaintain-ingbodyweighthomeostasisagainstexcessenergy intake.Ontheotherhand,werecentlydiscoveredanewinter-organneuralnetwork,fromtheliver,possiblyrepresentingapositive-feedbackmechanism.Underconditionsofexcessiveenergyintake,changesinglucoseme-tabolismoccurintheliverwithincreasedexpressionofhepaticglu-cokinaseandthe inductionofneuronalsignal transmissionviatheafferentvagusnerve.Thesesignals,receivedbythemedulla,resultintheinactivationofsympatheticinnervationofbrownadiposetissue(BAT).ThisleadstosuppressionofthermogenesisinBATandthere-byfavorsobesitydevelopment.Furthermore,theefficacyoftheliver-to-BATinteractiondiffersamongmousestrainsandthesedifferencesmaycontributetodeterminingtheobesitypredispositionsofvariousstrains. Inconclusion, thisnovel inter-organneuronalrelaysystemfunctionstosuppressenergyexpenditurewhenenergyintakeisin-creased.Duringperiodswhensufficientfoodwasnotalwaysavailable,thissystemworkedinfavorofsurvival.However,inthecurrentageofplenty,itisassumedtoworkasamechanismflippingametabolicswitchtowardobesity.NoCOI.

3S53B-3Clinical neuromodulation by vagal stimulation: vagus nerve stimulation for epilepsyKawai, Kensuke1; Kawai, Kensuke1; Takahashi, Hirokazu2; Usami, Kenichi3(1Department of Neurosurgery, NTT Medical Center Tokyo, Tokyo, Japan, 2Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan, 3Department of Neurosurgery, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan)

Vagusnervestimulation(VNS)isanestablishedtreatmentofepilepsyanddepression.Itchronicallystimulatestheleftcervicalvagusnerveusinganimplanteddevice,anddecreasesfrequencyandseverityofepilepticseizure.WeintroduceclinicallyutilizedVNSforepilepsyandpresentourdataonthemechanismsofactionwithreviewofliterature.ClinicalVNSinJapanwasapprovedandreimbursedin2010.385pa-tientswereenrolledfor3yearsintheJapanesepostmarketingsurvey.PreliminarydatademonstratedtheefficacyandsafetysimilartothepreviousreportsfromtheUnitedStates.Patientrateswithaseizurereductiongreaterthan50%after3,6and12monthswere37,47and60%,respectively.OurelectrophysiologicalstudydemonstratedthattheupwardneuraltransmissionofthevagusnerveactuallyoccursinhumanclinicalVNS.OurexperimentalstudydemonstratedthatVNSmodulatesthecorticesinthewaythatitincreasesthephaselockinginanonepilepticstatebutdecreasesitinanepilepticstate,thusex-ertingthehomeostaticinfluenceonthebrain.AlthoughbiologicaleffectandmechanismsofclinicalactionofVNSarestilllargelyunknown,accumulatingevidencesuggeststhattheupwardneuralsignalsmod-ulatethebroadareasofcerebralcortexviavariouspathwaysinclud-ingnoradrenergic,serotonergicandcholinergicsystems.NoCOI.

3S53B-4Energetics of the synaptic transmission in the nucleus of the solitary tractNagase, Masashi; Watabe, Ayako M; Kato, Fusao(Dept. Neurosci., Jikei Univ. Sch. Med., Tokyo, Japan)

Thesynaptictransmissionbetweentheprimaryafferentsandthesecond-orderneuronsinthenucleusofthesolitarytract(NTS)isthefirstsiteinthebrainwheretheCNSobtainsthedirectinformationregardingtheon-goingstateoftheinternalenvironment.Suchinfor-mation,especiallythatontheenergeticandmetabolicstateofthebody,iscrucialnotonlyformaintaininginternalorganfunctionsbutalsoforassuringenergysupplytothebrain.ItisthereforeimperativethattheintegrityofthisNTSsynapseshouldberobustlymaintainedeveninthesituationswithreducedenergysupply,suchashypoxiaandhypoglycemia.Aslactatesuppliedbyastrocytes,whicharerichinglycogenunlikeneurons,cansupportthehighrequirementforATPoftheneurons,weexaminedtheroleofastrocyte-to-neuronlactatetransportintheNTSsynaptictransmission.Wefoundthat1)inhibitionofmonocarboxylate transporters (MCTs),keymolecules inastro-cyte-to-neuronlactatetransport,inhibitedexcitatorysynaptictrans-mission,2)thiseffectofMCTinhibitionwasmorepotentatreducedintracellularATP,whichwascounteractedbyintracellularadditionoflactateand3)extracellularadditionoflactaterescuedaglycemia-in-ducedsuppressionofsynaptic transmission inaMCT-dependentmanner.Interestingly,MCTdidnotaffectbasalmembranepotentialinNTSneurons,unlikeinthecerebellum.Weconcludethattheinteg-rityoftheNTSsynapseismaintainedlargelybylactatetransferfromastrocytes,thusallowingrobusttransferofthevisceralinformationtothebrain.NoCOI.


Symposium 54Involvement of TRP channels in

respiratory regulation

(March 18, 14:00–16:00, Room C)

3S54C-1Transient receptor potential channels and synaptic activitiesKumamoto, Eiichi; Fujita, Tsugumi; Jiang, Chang-Yu; Xu, Zhi-Hao; Ohtsubo, Sena; Matsushita, Akitomo(Dept. Physiol., Saga Med. Sch., Saga, Japan)

Transientreceptorpotential(TRP)channelslocatedintheperipheralterminalsofprimary-afferentneuronsreceivevariouschemicalandthermalstimuligiventotheperiphery;theinformationistransmittedtotheCNSasanactionpotential.TRPsarealsoexpressedinthecentralterminalsoftheneuronsandinvolvedinmodulatingthetrans-missiontotheCNS.ManyofthepropertiesoftheTRPshavebeeninvestigatedinthecellbodyoftheprimary-afferentneuronwhilethecentralTRPshavenotyetbeenexaminedthoroughly.WeexaminedtheactionsofvariousTRPagonistsonsynaptictransmissioninlami-naIIneuronsofadultratspinalcordslicesbyusingtheblindwhole-cellpatch-clamptechnique.TheagonistsincreasedthefrequencyofspontaneousEPSCwithaminimal increase intheamplitude inamannerresistanttoaNa+-channelblockertetrodotoxin;thiswasac-companiedbyaninwardoroutwardcurrentat-70mV.Thefacilita-toryactionswereinhibitedbycapsazepineorHC-030031.Primary-af-ferentevokedEPSCswere inhibited inpeakamplitudebyTRPagonists. Ontheotherhand,spontaneous inhibitorytransmissionswerepotentiatedbysomeTRPA1butnotTRPV1agonistsinaman-nersensitivetotetrodotoxin.TheseresultsindicatethatTRPagonistsactivateTRPV1orTRPA1to facilitatethespontaneousreleaseofL-glutamateontolaminaIIneurons.Theseactivationsweredifferentinpharmacologyfromthoseinthecellbodyofprimary-afferentneuron.CentralTRPsweresuggestedtobedifferentinpropertyfromperiph-eralones.NoCOI.

3S54C-2TRPA1 contributes to arousal and respiratory activa-tion to mild hypoxiaKuwaki, Tomoyuki(Department of Physiology, Kagoshima University Graduate School of Medical and Dental Sciences)

TRPA1channel,amemberofthetransientreceptorpotentialsuperfamily,isexpressedinasubsetofsensoryneuronsinthetrigeminal,nodose,anddorsalrootganglia.Atthevagalafferentnerveterminalsinthelowerairway,TRPA1detectsoxygenconcentrationofthein-spiredgasandtriggersrespiratoryaccelerationorslowingdependingonhypoxiaorhyperoxia.WehypothesizedthatTRPA1wouldalsocontributetoarousalfromsleepandrespiratoryactivation,asadefensemechanismtohypoxia.Totestourhypothesis,wemeasuredarousaltimeandrespiratoryvolumeandfrequencyinTRPA1knockoutmice(TRPA1-KO)andwild-typemice(WT)withindwellingEEGandEMGelectrodes.Duringnaturalsleeping,thecontinuousflushingairintothebodyplethymographicchamberwasswitchedtoahypoxicgasmixture.Oxygenconcentrationinthechamberwasgraduallydeclinedto10%by~40sandkeptforanother2minat10%.Thetimetoarous-alfromnaturalsleepinresponsetohypoxiainTRPA1-KOwasex-tremelylongerthanthatinWTmice.Notably,WTmiceawokebeforeoxygenconcentrationreached10%whereasTRPA1-KOdidnot.Theincreaseofrespiratoryminuteventilation,justafterarousalfromsleep,wasattenuated inTRPA1-KOthan inWTmice.WeconcludethatTRPA1isimportantforarousalandrespiratoryactivationinresponsetomildhypoxia.TRPA1,probablylocatedinthenasalcavity,seemstobeafrontlinesensorforasubtlechangeintheinspiredgasoxygen.NoCOI.

3S54C-3Putative mechanisms underlying pH sensitivity of the brainstem astrocytesMarina, Nephtali(Neuroscience, Physiology & Pharmacology, University College London, London, United Kingdom)

Ionchannelsofthetransientreceptorpotential(TRP)familyarekeychemosensoryionchannelsintherespiratorysystem.However,theroleofTRPchannelsinthecontrolofrespiratorychemosensitivityhasneverbeendescribed.AstrocytesresidingintheventralaspectofthemedullaoblongatarespondtoacidificationwithelevationsinintracellularCa2+andATPreleasewhichpropagatesCa2+excitationamongneighbouringastrocytesandactivatesneuronesoftheventralrespiratorycolumntriggeringadaptiveincreasesinbreathing.Here,weperformedapharmacologicalsurveyaimingtodeterminethemechanismsunderlyingpHsensitivityofastrocyteswhichresideneartheventralsurfaceofthemedullaoblongata(VMS).Acidification-evoked[Ca2+]iresponsesinVMSastrocyteswereunaffectedbyblockadeofTRPA1orTRPVchannels (HC030031,AMG9810,rutheniumred).However,significantreductionsin[Ca2+]iresponsestriggeredbyadecreaseinextracellularpHfrom7.4to7.2wereobservedafterappli-cationofNa+/Ca2+-exchangeinhibitorSN-6,Na+/HCO3-cotransportinhibitorS0859andanionexchangeinhibitorDIDS.Similarly,hyper-capnicacidosis-evoked [Ca2+]iresponses inVMSastrocyteswerereducedinthepresenceofeitherS0859orDIDS.ApplicationofS0859totheVMSinanaesthetized,vagotomizedandmechanicallyventilat-edrats,reducedrespiratoryresponsetriggeredbysystemichyper-capnia(10%inspiredCO2)by~50%.NoCOI.


3S54C-4Role of Transient Receptor Potential (TRP) channels in respiratory motor pattern generationKoizumi, Hidehiko(NINDS, National Institutes of Health (NIH), Bethesda, USA)

Thepre-Botzingercomplex(pBC)isakeybrainstemmicrocircuitthatgeneratesandtransmitsrhythmicrespiratoryactivity.TRPMchannelshavebeenproposedtocontributetorespiratoryrhythmgenerationinpBCnetworkbymediatingcalcium-activatednon-selectivecationiccurrents (ICAN).TRPCchannelshavealsobeensuggestedtobefunctionally important.However,TRPchannel-basedmechanisms,plausiblealternativetopersistentsodiumchannel-basedmechanisms,arenotcompletelyunderstood,andthereiscontinuingcontroversyabouttherolesofTRPchannelsinrespiratoryrhythm/patterngen-eration.WefirstexaminedexpressionofTRPM/CchannelsinpBCinspiratoryneuronsidentifiedinrhythmicallyactiveinvitrobrainstemslicepreparationsbycombiningpatch-clamprecordingandsingle-cellmultiplexRT-PCR.WedetectedTRPM4andTRPC3mRNAexpressionprimarilyinexcitatoryneurons.OnlyasmallpercentageofinhibitoryneuronsexpressedTRPC3mRNA.TofurtherdefinethecontributionsofTRPM/CchannelstothefunctionalbehaviorofpBCnetwork,wetestedperturbationsofthefrequencyandamplitudeofhypoglossalinspiratoryactivityfollowingbathapplicationofpharmacologicalblock-ersofTRPM4,TRPC3andICANinbrainstemslicepreparations.Inallcaseswefoundsignificantreductionsintheamplitudeofinspirato-ryactivitywithoutanysignificantperturbationsof the frequency.TheseresultssuggestthatwhileTRPM4/TRPC3/ICANcontributestotheformationofinspiratorydrive,thisfunctionisindependentofrhythmgenerationmechanismsatleastinvitro.NoCOI.

3S54C-5Neuronal mechanisms of effects of TRP channel relat-ed substances on respiratory center neuronsOnimaru, Hiroshi(Department of Physiology, Showa University School of Medicine, Tokyo, Japan)

Itisnotwellunderstoodwhetherchemicalsthatareknownastransientreceptorpotential (TRP)channelagonistsorantagonistsexertanyeffectsonthemedullaryrespiratorycenter.Recently,wehaveexam-inedeffectsoftransientreceptorpotential(TRP)channel-relatedsub-stancesonrespiratoryrhythmgenerationinthebrainstem-spinalcordpreparationfromnewbornrats.Thesesubstancesaredivided intoroughlytwogroupsthatinducedexcitatoryeffects(e.g.TRPV1agonist,capsaicin; TRPA1 agonist, cinnamaldehyde) or inhibitory effects(TRPM8agonist,menthol;TRPV3?agonist,carvacrolandeugenol).Oneofcharacteristicsofcapsaicineffectswasaninductionofstrongdesensitizationthatmightbeproducedbyextracellularcalcium-inde-pendentmechanisms.WepresumedthatcapsaicinmightinducethereleaseofintracellularstoredCa2+fromtheendoplasmicreticulum.Cinnamaldehydeinducedlong-lastingfacilitationofrespiratoryrhythmformorethan2hrsafterwashedout.Incontrast,carvacrolandeu-genolinducedinhibitionofrespiratoryrhythmfollowedbypronouncedshorteningofpre-inspiratoryandinspiratoryburstduration,suggest-ingthateugenolorcarvacrolinhibitedcellular(and/ornetwork)mech-anismsthatareessentialformaintenanceofburstdurationofrespi-ratoryneurons.Effectsofthesecompoundsmayilluminatenewaspectsofcellularmechanismsofrespiratoryrhythmgeneration.Inthispre-sentation,IwillshowrecentresultsregardingeffectsoftheseTRPchannel-relatedsubstancesanddiscussthecellularmechanisms.NoCOI.

Symposium 55What lies between physiology and

pathophysiology[CollaborationSymposiumwithJapanese

SocietyofPathophysiology]

(March 18, 14:00–16:00, Room D1)

3S55D1-1Prostaglandin E2 Receptor EP4 Signaling in smooth muscle cells regulates arterial elasticityIchikawa, Yasuhiro; Yokoyama, Utako; Ishikawa, Yoshihiro (Cardiovascular Research Institute, Yokohama City University, Yokohama, Japan)

Chronicinflammationisknowntocontributeadvancingaorticaneu-rysm(AA)inwhicharterialelasticitywasimpaired.Inadditiontotheroleofinflammatorycells,usinghumantissues,wehavedemonstrat-edthatthePGE2receptorEP4maybeinvolvedinchronicinflamma-tioninAA.Therefore,wefurtherexaminedwhetherPGE2-EP4sig-nalinginSMCsdecreaseselasticityintheaorta.Wegeneratedmicewithvascularsmoothmuscle-specificoverexpressionofhumanEP4(hEP4)usingtheCre-loxPsystem(EP4TG).hEP4mRNAwasover-ex-pressedintheaortaofEP4TG(n=8,respectively).VasodilativeeffectofEP4agonist (10µM)wasgreater in theaortaofEP4TGthannon-transgenicmice(non-TG)bywiremyograph(25.0±2.9vs.9.6±2.0%ofphenylephrine-inducedcontraction,p<0.01,n=4–6).Pressure-induceddilation(20to220mmHg)wasdecreasedinEP4TGthaninnon-TG(0.84±0.04-fold,n=5–6,p<0.05),althoughunderbasalcondition,aorticdiameterandelasticitywerenotdifferentbetweenEP4TGandnon-TG.Furthermore,afterangiotensinII(ATII)infusion(3.0µg/kg/minfor28days),theaortaeofEP4TGweredeformedandenlargedtoagreaterdegreethanthatofnon-TG(1.1±0.02-fold,n=5-6,p<0.05).MMP-2activ-ityofEP4TGishigherthanthatofnon-TGbygelatinzymography(1.7±0.07-fold,n=5,p<0.05)underbasalconditions.AfterATIItreat-ment,enhancementofMMP-9activationwasgreaterinEP4TGthaninnon-TG(1.6±0.19-fold,n=5,p<0.05).ThesedatasuggestthatEP4signalinginSMCsdecreasesaorticelasticitybyMMPactivation.NoCOI.


3S55D1-2Regulation and roles of TRPM7 kinase activity in pathophysiologyMatsushita, Masayuki(Department of Molecular and Cellular Physiology, University of the Ryukyus, Okinawa, Japan)

Themammaliantransientreceptorpotential (TRP)superfamilyofcationchannelsisdividedintosixsubfamiliesbasedonsequenceho-mologyTRPC,TRPV,TRPM,TRPA,TRPPandTRPML.Theseionchannelscanbeactivatedbyadiverserangeofchemicalandphysicalstimuli.Physicalstimuli includetemperature,membranepotentialchangesandosmoticstress,andsomeofthemorewellknownchem-icalstimuliincludecapsaicin(TRPV1),menthol(TRPM8)andacrolein(TRPA1).AmongTRPfamilies,TRPM7isuniquemoleculecomposedofthedomainwiththeionchannelstructureandthekinaseactivity.Asfortheroleofthekinaseactivity,itisstillunclearthoughthein-tensiveworkhasbeenperformed.ToclarifythephysiologyfunctionofTRPM7andtheroleofthekinaseactivity,wemadeTRPM7genet-icallymodifiedmousebyoneaminoacidsubstitutioninATPbindingsiteinkinasedomain.Thismutantmousegrewnormally,whilekinasedomaindeletionmousewasembryoniclethal.Interestingly,TRPM7kinasedeadmutantmouseshowmetabolicabnormality.ThenovelmetabolicroleofTRPM7kinaseactivitywillbeusefulforestablishmentofphysiologicalroleofTRPM7channel.NoCOI.

3S55D1-3Sleep hygiene and problems in healthFujiki, Nobuhiro(Department of Ergonomics, Institute of Industrial health, Ecological Science University of Occupational and Environmental Health, JAPAN)

Itisimportantforustomaintainahighqualityofsleepsothatwecouldobtainourbestmentalandphysicalperformanceinourdailylife.Insufficientsleepand/ordyssynchronybetweeninternalclockandexternallight/darkcycleacutelycausedaytimesleepinessandchron-icallyinducehealthproblems.Thedaytimesleepinesscouldresultinareductionoftheworkingefficiencyanditmightcauseseconomicloss.Moreover,italsocouldbeafatalrisksuchastrafficaccidentduetotheirlowlevelofvigilance.Recentreportalsosuggestedthatlackofsleepmightbeariskfactorforchronichealthproblemssuchasmetabolicsyndromeanddepression. It is thusreally importanttounderstandthe importanceofthesleephygieneandto investigatehowtheinadequatesleepand/orinappropriaterhythmofsleepcausethechronichealthproblems.Forthisinvestigation,someofcommonsleepdisorderssuchasanobstructivesleepapneasyndromeandacircadianrhythmsleepdisorder,shiftworktypewouldbethetargetsasthesediseasesareknowntobetherisks forthechronichealthproblems.NoCOI.

Symposium 56Mechanism of acupuncture for

musculoskeletal disorders[CollaborationSymposiumwithTheJapanSocietyofAcupunctureandMoxibustion]

(March 18, 14:00–16:00, Room E)

3S56E-1Effect of acupuncture treatment for skeletal muscle disorders in the field of sportsMiyamoto, Toshikazu(1Doctoral Program in Sports Medicine, Comprehensive Human Sciences Department, University of Tsukuba, Tsukuba, Japan, 2Acupuncture and Physical Therapy Teacher Training School, Tokyo, Japan)

Acupunctureisatreatmentthatyouwanttopenetrateinvivousingacupunctureneedlesuchasstainlesssteel,expectingabiologicalre-action.Acupunctureisthatitcanstimulatethetissueskin,subcuta-neoustissues,muscles,andnerves.Weareperformingacupuncturetotheathletesforthepurposeofthetreatmentandpreventionofsports injuries.Wehavealsoexaminedtheeffectofacupuncturestimulationontherepairofmusclestrainanddisusemuscleatrophytargetingmice.1.EffectofacupuncturetreatmentforsportsinjuriesanddisabilityManyofathleteswhomwetreatedarelumbagoandmusclestrain.Inourtreatment,wegivetheinsertionofacupunctureonmusclepainpart,andaredoingalow-frequencyelectroacupuncture.2.EffectoftackneedlesformusclepainandmusclefatigueTackneedlesareabout0.6mminlengthandcanbeappliedtoathleteswhiletheyareplaying.Wewillshowyoutheeffectofthetackneedlesbyadouble-blindrandomizedtrials.Oneisaboutmusclepainsandmusclefatigueafterthemarathon,andtheotherisaboutmusclefatigueandmusclehardnessoflongdistancerunnersinthelong-termtrainingcamp.3.Effectofelectroacupunctureformuscledamage,muscleatrophyWe introducethehistologicalstudyof themuscledamagemousemodel,andalsoshowtheeffectofelectroacupunctureondisusemus-cleatrophyofusingmicehindlimbsuspension.NoCOI.


3S56E-2Molecular and cellular mechanisms underlying mus-cle plasticityOno, Yusuke(Department of Stem Cell Biology, Nagasaki University Graduate School of Biomedical Sciences)

Musclestemcells,calledsatellitecells,arelocatedbetweenthebasallaminaandthesarcolemmaofmyofibresandplayimportantrolesinmuscleplasticitysuchashypertrophyandregeneration. Inadultmuscle,satellitecellsexistasmitoticallyquiescentstate.Satellitecellsareactivatedrapidlyinresponsetostimulationsuchasmuscleinjuryandproliferateextensivelytogiverisetotheirprogeny.Followingproliferation,themajorityofsatellitecellsundergomyogenicdifferen-tiationtoproducenewmyonuclei,whereasaminoritypopulationre-turnstoaquiescentstatetoself-renewformaintenanceofastemcellpool.Understandinghowthefatedecisionssuchasself-renewal,pro-liferationanddifferentiationareregulatedinsatellitecellsiscentraltounderstandinghowskeletalmuscleregulatestheplasticity.Byusingsatellite-cellspecificconditionalknockoutmousemodels,weareundertakingaseriesofexperimentstounderstandsignallingnetworksthatregulatesatellitecellfatedecisionswithaparticularemphasisonplanarcellpolarity(PCP)andNotchpathways.Wearealsoinvestigat-ingonhowsatellitecellsareafunctionallyheterogeneouspopulationbothbetweenmuscles,andwithinasinglemyofibre,throughoutbody.Here,wewilldiscussemergingfindingsofthemolecularandcellularbasisunderlyingmuscleplasticity.NoCOI.

3S56E-3Molecular mechanism: Electroacupuncture induces satellite cell activationTakaoka, Yutaka1,2(1Division of Medical Informatics and Bioinformatics, Kobe University Hospital, Kobe, Japan, 2Life Science Research Center, Kobe Tokiwa University, kobe, Japan)

WehavebeeninvestigatingthemolecularmechanismofElectroacu-puncture(EA)andhaveobtaineditsmolecularevidenceinskeletalmuscle[1-2].WefoundthemolecularevidencethatEAsuppressedtheexpressionofthemyostatingene,whichisanendogenousinhibitorofmusclegrowthandareducerofAKT/mTOR/p70S6Ksignaling,andEAinducedaproliferativereactionofmusclesatellitecells(stemcells).Theseresults ledustothepossibilitythattheEApreventmuscleatrophy.WethenelucidatedtheeffectofEAondisusemuscleatrophybyusinghindlimb-suspended(HS)micewhichweretreatedbyEA[3-4].WefoundthatEA/HSmicemaintainedasoleusmusclemassthatwasnotsignificantlydifferentfromthatofcontrolmice.Also,thediametersofmyofibersinmicehadthesametendency.RepeatedEAtreatmentsuppressedgeneexpressionofmyostatinandthreeubiquitinligasegenesinEA/HSmicebutinducedexpressionofthesegenesinHSmice.ThesefindingssuggestthemolecularmechanismofinhibitingthedisusemuscleatrophybyEAthroughtheAKT/mTOR/p70S6Ksignalingpathway.NoCOI.[1]TakaokaY,etal.PhysiolGenomics2007;30:102–110.[2]OhtaM,etal.eCAM2009;doi:10.1093/ecam/nep121[3]IkemuneS,etal.JJapSocofAcupandMoxi2010;60(4):707–715.[4]IkemuneS,etal.JJapSocofBalneo,ClimatoandPhysMed2011;74(2):103–111.

3S56E-4Recent advancements in controversial topics of skel-etal muscle research viewed with a special interest in fatigueTakemori, Shigeru(Department of Molecular Physiology, Jikei University School of Medicine, Tokyo, Japan)

Skeletalmuscle,buildwithabeautifullyaligned liquidcrystallinestructureofsarcomere, isessential forusanimals torealize forcegenerationandmotion.Withtheseuniquefeatures,skeletalmusclehasbeenattractingscientific interest intensively fora longtime.Symbolizedbyan1:1neuro-musculartransmissionandastraightforwardexcitation-contractioncouplingmechanism,skeletalmuscleisseeminglyasimpleandobedienttissueunderthecontrolofcentralnervoussystem.However,itisstillelicitingseveralconfusingargu-mentsinthefieldofmuscleresearch.Amongthearguments,fatiguehasbeenoneof themostcontroversial issues.Therecertainly isvariousaspectsoffatigue;musclefatigue,fatigueinneuro-musclulartransmission,fatigueintaskperformance,mentallyinducedsubjectivesenseoffatigue,andsoon.However,thesedifferentaspectshasbeenoftenconfusinglydiscussedasanidenticalconcept.Thisisprobablybecausewesomehowprojectoursenseoffatigueontoourskeletalmuscle.Forinstance,wefeelsomedullsenseinourproximalmuscleswhengotdeadlytiredwithmentalstress.Thissuggeststhatthereisanintimate(directorindirect)cross-talksbetweenourskeletalmuscleandcentralnervoussystem,andskeletalmuscleisnotamereobedi-entmotor.Recently,skeletalmuscleisrealizedasasignificantsourceofvarioussignalsaffectingtheactivityofwholebody.Withaspecialinterestinfatigue,recentadvancementsinseveralcontroversialtopicsofskeletalmuscleresearchwillbereviewedanddiscussed.NoCOI.

Symposium 57Mechanosensitive regulation of

biological function: update[FAOPSJointSymposium—Japan/Korea]

(March 18, 14:00–16:00, Room G)


3S57G-1Reno-pathogenic mutations and PKG-mediated phos-phorylation alter TRPC6 mechanosensitivity.Inoue, Ryuji; Ichikawa, Jun; Nakagawa, Midori; Kurahara, Lin; Hu, Yaopeng(Department of Physiology, Fukuoka University School of Medicine)

TRPC6 isamajorcardiovascularTRP isoformandsusceptible tomechanicalstressesinbothdirectandindirectways.Inrenalphysi-ology,mutationsinthisgenearepartlycausativeforfocalsegmentalglomerulosclerois(FSGS),culminatinginanexcessiveCa2+influxintopodocytesthroughreceptor-mediatedpathways. Inthisstudy,wefocusedonthreeFSGSmutationsidentifiedneartheankyrinrepeatsofTRPC6N-terminus(P111Q,M131T,N142S,)andinvestigatedhowthesemutationsaffectreceptoraswellasmechanicalresponsesofthechannel.Throughfunctionalevaluationofthesemutants,itturnedout;(1)allmutantsshowedenhancedreceptorresponses; (2)mechanicalresponsesalmostcompletelydiminishedexceptforexaggeration inM131Tmutation,whichwasabolishedbyactindepolymerizationwithcytochalasinD;(3)physicalinteractionofthemutantswithactinwassignificantlychanged; (4)coexpressionofaslitdiaphragmproteinpodocinenhanced,whereasPKG-mediatedphosphorylationonN-ter-minalThr69attenuatedbothreceptorandmechanicalresponsesofTRPC6channelanditsinteractionwithactin.TheseresultssuggestthatphysicalinteractionwithactincytoskeletonisessentialforthemechanosensitivityofTRPC6channelwhichissignificantlyaffectedbyFSGSmutationsorphosphorylatedstatusofThr69neartheN-ter-minalankyrindomains.Thismechanismmaynotonlyexplainabnor-mallyenhancedCa2+mobilizationinFSGSpatients’podocytesbutalsohintsanewtherapeuticstrategythatmayhelppreventtheprogressionofthedisease.NoCOI

3S57G-2A mechanosensitive ion channel, TRPV2 expressed in gastric myenteric plexus contributes to gastric adaptive relaxation and emptying in miceMihara, Hiroshi1; Tominaga, Makoto2; Sugiyama, Toshiro1(13rd internal medicine University of Toyama, 2Cell Signaling OIIB)

Aim:Gastricadaptiverelaxation(GAR)reducesmeal-inducedintra-gastricpressure.NOreleasedfrominhibitorymotorneuronshasanimportantroleintheprocess.Gastricemptying(GE)isincreasedwhenGARisimpairedorenhanced.However,themolecularmechanismispoorlyunderstood.TRPV2detectsmechanicalstimuliandchemicalsincludingprobenecid.WeshowedTRPV2expression inthemouseintestininalinhibitorymotorneuronsanditsinvolvementinintestinalrelaxation.ThisstudyevaluatedTRPV2distributioninmousegastricmyentericplexusandthephysiologicalroleinGARandGE.Methods:C57BL/6,TRPV2KO(V2KO)micewereused.RT-PCRandimmuno-histochemistrydetectedTRPV2mRNAandprotein,respectively.GARwasdeterminedwithanisolatedstomachusingapressuretransduc-er.GEinvivowasdeterminedusingphenolredmealwith/withoutTRPV2activatorsand/oraTRPV2inhibitor,tranilast.Results:TRPV2mRNAwasdetectedinWTstomach,butnotinV2KO.TRPV2proteinwasdetected in86.5%of inhibitorymotorneuronsthroughoutthestomach,butnotinV2KO.GARwassignificantlyenhancedbypre-treatmentwithprobenecidandtheenhancementwasinhibitedwithtranilast,TTXorL-NAME.GEwassignificantlyenhancedwithTRPV2agonistsandtheprobenecid-inducedenhancementwasinhibitedwithtranilast.GEwithtranilastonlyor inV2KOwasalsosignificantlyenhanced.Conclusion:TRPV2isexpressedinmousegastricinhibitorymotorneuronsandmayplayrolesinGARandGE.NoCOI.

3S57G-3Mechano-electric interaction and arrhythmiaIribe, Gentaro(Cardiovascular Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences)

Mechanicalstimulusmodulateselectrophysiologicalpropertiesoftheheart(mechanoelectricfeedback:MEF).Forinstance,abluntprecordialimpactontheheartmaycausefatalarrhythmiawhichisknownascommotiocordis.Mechanicallyinducedectopicbeatsarebelievedtobecausedby inwardcurrentviacation-selectivestretch-activatedchannels(SACs).Ontheotherhand,sarcolemmalstretch-activatedK+channel,forinstance,stretch-activatedBKCa(SAKCA)channelcausesoutwardcurrent,whichmaybalanceoutthearrhythmogeniceffectsofSACs.Mechanicalstimuliaretransmittedtowholecellareainclud-ingionchannelsinanyintracellularmembranestructureviacytoskel-eton.Therefore,intracellularorganellesmayshowmechanosensitiveresponsestoplayaroleinMEFaswellassarcolemmalmechanosen-sitivechannels.Wehaverecentlyrevealedthatmyocardialstretchincreasesmitochondrialreactiveoxygenspeciesproduction,whichcausesstretch-induced increase inCa2+spark,spontaneousCa2+releasefromryanodinereceptorsonsarcoplasmicreticulum(SR).SRcalciumreleasecausesCa2+extrusionviaNa+/Ca2+exchangerwhichcausesinwardcurrentandmaycontributetoinitiateectopicactionpotential.Mechanicallyinducedectopicbeatsaretheintegratedresultof theseresponses fromvariousmechanosensitivecomponents.NoCOI.

3S57G-4Mechanosensitive regulation of vascular tone; com-parison between systemic and pulmonary arteriesKim, Sung Joon; Yoo, Hae Young ; Kim, Hae Jin(Department of Biomedical Sciences, Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul, KOREA)

Vasculartone isdirectlyor indirectlyaffectedbyvarietyofmechanicalstimulisuchasshearstressandwall tension.Smallarteries invivoarepartiallycontracted,theextentofwhich isaffectedby luminalpressure(Plum)ofthevessel.Suchcontractioninsystemicarteryiscalledmyogen-icresponse(MR)thatiscriticalformaintainingrelativelyconstantregionalbloodflowdespitefluctuationofperfusionpressure.Mechanosensitivenonselectivecationchannels(NSCms),proteinkinaseC(PKC),andRhokinase(ROCK)aresuggestedasunderlyingmechanismsforMR.InPartI,wecomparethevariabilityofMRsaccordingtothearterialtypes (cerebralartery (CA),deepfemoralartery (DFA)andmesentericartery (MA)) intermsoftheirpressure-sensitivityandunderlyingmechanismsthatarerecruitedaccordingtoPlum.InPartII,mechanosensitiveregulationofpulmonaryartery(PA)wouldbedemonstratedregardingtheNSCmsinPAmyocytesandmechanosensitiverecruitmentofNOsynthase (NOS)putativelyexpressedinPAsmoothmusclelayer.AlbeittheexpressionofNSCmsinPAmyocytes,theisolatedPAsdonotshowmyogenicresponsesbutshoweddecreasedtonebyrepetitivepressureincrease.Withpartialcontractionbyapplying3nMU46619(TXA2agonist)inendotheliumde-nudedPAs,additionalcontractionbyNOSinhibitor[nitro-L-argininemeth-ylester(L-NAME)]wasfeeble.However,withcombinedincreaseinwalltensionequivalentto10to30mmHgofPlum,L-NAMEinducedrobustcontractionofPAs.Apartialdepolarizingcondition(20mMKCl)combinedwith3nMU46619 inducedsimilarsensitivitytoL-NAME.RT-PCRandimmunohistochemistryanalysisshowfaintexpressionofeNOSinPAsmoothmusclecells.ThesupposedactivationofeNOSbyTXA2andincreasedwalltensionmightcontributetotherelatively lowperipheralresistanceofpulmonarycirculationandpulmonaryarterialpressureinvivo.NoCOI.


Symposium 58Optical approaches toward the

understanding of physiological functions

(March 18, 14:00–16:00, Room H1)

3S58H1-1Developing high-performance indicators for live cell imagingHorikawa, Kazuki(Institute of Health Biosciences, The University of Tokushima Graduate School, Japan)

Inthephysiologicalanalysis,thereisanincreasingdemandforreliableandquantitative imaging.Here,fluorescentandchemiluminescentindicatorsthatvisualizethedynamicsofmolecules,cellsandtissuesareessential.Althoughavarietyoffunctionalindicatorshavebeendeveloped,analysisunderthe“purelyphysiologicalcondition”isstillchallenging.Thisoftencomesfromthenon-negligibledifferencebe-tweenexperimentalandphysiologicalconditionwearefocusingon.Forexample,liveCa2+imagingisperformedbydeliveringverystrongstimuliatthelevelcellswouldneverexperienceunderthenaturalcodition.Insuchacase,cellsshowthe“death-cry”likeresponsethatcanbeeasilydetectedduetotheirexceptionallylargeamplitudeofCa2+concentrationchange.Ontheotherhand,cellsinvivoaccomplishthesignaltransductionwithvanishinglysmallamplitudeforwhichultrasensitiveindicatorsareneededforitsdetection.Oneofourgoalsistodevelopaseriesofhigh-performanceindicatorswhichcanbeusedforimagingevenunderthe“purelyphysiologicalcondition”.Inthistalk,Iwill introduceoursimpleandsystematicapproachtoobtainhigh-performanceindicatorswithimproveddynamicrangeandsensi-tivity,beingthemostimportantparameterforsuccessfuldetectionofsubtlephysiologicalresponses.Withproof-of-conceptdemonstrationforCa2+,cAMPandcGMPindicators,Iwouldliketoshowtheeffec-tivenessofourstrategyindevelopingtheindicatorforotherphysio-logicalphenomenasuchastheinflammatoryresponseintheimmunesystem.NoCOI.

3S58H1-2Large-scale imaging of circadian rhythm in the mam-malian master clockEnoki, Ryosuke1,2,3,7; Mieda, Michihiro4; Ono, Daisuke1; Kuroda, Shigeru5; Mazahir, Hasan6; Honma, Sato2; Honma, Ken-ichi2(1Hokkaido Univ, Grad Sch of Med, Photonic Bioimaging, Sapporo, Japan, 2Hokkaido Univ, Grad Sch of Med, Dep of Chronomed, Sapporo, Japan, 3Hokkaido Univ, Grad Sch of Med, Dep of Cell Physiol, Sapporo, Japan, 4Kanazawa Univ, Faculty of Medicine, Dep of Mol Neurosci & Integrative Physiol, Kanazawa, Japan, 5Hokkaido Univ, RIES, Sapporo, Japan, 6Charite-Universitatsmedizin, NeuroCure Cluster of Excellence, Berlin, Germany, 7JST PREST, Chiyoda, Japan)

Thecircadianpacemakerinthehypothalamicsuprachiasmaticnucle-us (SCN)isahierarchicalmulti-oscillatorsysteminwhichneuronalnetworksplaycrucialrolesinexpressingcoherentrhythmsinphysi-ologyandbehavior.Usingalarge-scaleimagingmethodwithgeneti-cally-encodedcalciumsensors,wevisualizedintracellularcalciumfromtheentireSCNneuronalnetworkinculture(J.NeurosciMethods,2012).Wefoundcircadiancalciumrhythmsatasingle-celllevelintheSCN,whichweretopologicallyspecificpatternsintheventralregionthanthedorsal(PNAS,2012).Therobustnessoftherhythmwasreducedbutpersistedevenafterblockingtheneuronalfiringwithtetrodotox-in(TTX).TTXdissociatedthecircadiancalciumrhythmsbetweenthedorsalandventralSCN.Inaddition,wedevelopedadual-colorfluores-cenceimagingmethodandsuccessfullyvisualizedexpressionpatternsofPer1:EGFPandR-GECO(calcium)intheSCNatsinglecellresolution.Ourrhythmanalysisshowedthedistinctspatio-temporalpatternsofPer1andcalciumintheSCN.NoCOI.

3S58H1-3non-invasive in vivo NIR fluorescence imaging using iRFP Transgenic mice and iRFP-flox miceMiwa, Yoshihiro; Sakaguchi, Shota; Sugiyama, Yuka; Tanaka, Junko(Molecular Pharmacology, Faculty of medicine, University of Tsukuba, Tsukuba, Japan)

Threedimensional(3D)fluorescenceopticaltomographyoflivingmiceisanecessarytechniquefornoninvasivemonitoringofthetimecourseofdiseaseconditionsorchangesofbiologicalstatespreciselyavoidingsurgicaleffects.ItiswellknownthatNear-Infrared(NIR)lightof650-900nmcanpropagatebymultiplescatteringthroughseveralcenti-metersoftissues,becauseofthelowabsorbanceoftissuechromophoressuchasoxy-anddeoxy-hemoglobin,melaninandfat.However, thedirectionalityof lightpropagation israndomized intissues, imagerenderingrequirestomographicreconstruction.Becauseregulardietcontainsmanykindsoffluorescentcompounds,thefluorescenceoffoodisseriousimpedimentofopticaltomography.Non-alfalfadietandlow-fluorescencedietarecommerciallyavailable,however,non-alfalfadietstillfluoresces inNIR.Thefeedingof low-fluorescentdiet ledrapidlossofweightofmiceandfinallyresulteddeathofallmicebythreeweeks.Therefore,weanalyzedthefluorescencespectrumofallcomponentsofformulafood,andbasedonthesedata,wedevelopednewlow-fluorescentdiet.In2011,Filonovetal.reportedthenovelNIRfluorescentprotein, iRFPthatcanautonomouslymaturatewithoutadditionofexogenousbiliverdin.ThusiRFPwouldbesuitableforinvivoimagingasaNIRfluorescentprobe.Therefore,weattemptedtodetectthefluorescencesfromiRFPandICGseparately.Forfurtherbiologicalapplication,wehavetriedtoestablishiRFP-transgenicmiceandiRFP-floxknockinmice.NoCOI.


3S58H1-4Visualization of signaling pathways by FRET and its clinical applicationOhba, Yusuke(Dept. Cell Physiol., Hokkaido Univ. Grad. Sch. Med., Sapporo, Japan)

Thedevelopmentbiosensorsutilizingfluorescentproteins (FPs), in-cludingthosebaseontheprincipleofFörsterresonanceenergytrans-fer(FRET),hasrevealedspatiotemporaldynamicofcellsignaling.Herewe introduceourcurrenttrialofclinicalapplicationofbioimagingtechniqueswithchronicmyeloidleukemia(CML)asamodel.CMLisamyeloproliferativediseasecharacterizedbytheemergenceofPhil-adelphiachromosome,whichresultsintheexpressionofthecausativeproteinBCR-ABL.ThespecifictyrosinekinaseinhibitorofBCR-ABLimatinibmesylate(IM)hasradicallyinnovatedthetreatmentofthisdisease,witha5-yearsurvivalrateofapproximately80%.Nowadays,CMLhasthusbecomeadiseasecontrollablebyoralmedication;how-ever,continuousmedicationisrequiredtosuppressthediseasepro-gression.Furthermore,thereremainconcernsthatsubstantialpatientsdisplayIMresistancebothbeforeandduringthetreatment.Toover-comethese issues,wehavedevelopedtheFRET-basedbiosensorPickles(namedafterphosphorylationindicatorofCrkLensubstrate)usingtheBCR-ABLsubstrateCrkL.Pickleswasabletoevaluatedrugefficacywithhighersensitivityandidentifysmallerpopulation(<1%)ofdrug-resistantcells,comparedtoanypreviouslyestablishedmethods.Furthermore,itwaspossibletonotonlydeterminethecurrenteffec-tivenessofIM,butalsopredictfutureemergenceofdrugresistance.Inthissymposium,wewilloverviewthecurrentprogressinthede-velopmentofthismethodandwouldliketodiscussitspotentialtoovercomedrugresistance.NoCOI.

3S58H1-5Spatial and temporal dynamics of ensemble and sin-gle-neuron activity in the mouse motor cortex during a voluntary movementMatsuzaki, Masanori1(1Division of Brain Circuits, National Institite for Basic Biology, Okazaki, Japan, 2SOKENDAI)

Two-photonimagingisapowerfultoolusedtoexaminemolecularandcellularfunctionsinlivingtissues.Inparticular,calciumimagingcanquantitativelymeasureneuronalactivityi.e.actionpotentialfiring.Weconductedtwo-photoncalciumimagingofmousemotorcortexduringaself-initiatedlever-pulltask.Inthistask,head-restrainedmicehadtopullaleverfor~600mstoreceiveawaterdrop,andthenhadtowaitformorethan3stopullitagain.Intheimagingsessionaftertraining,wefoundseveral typesoftask-relatedcells inthemousemotorcorticalareas.Forexample,cellswhosepeakactivitiesoccurredduringleverpullsandcellswhosepeakactivitiesoccurredaftertheendof leverpulls.Bypredicting levermovementtrajectories fromensembleandindividualactivities,wefoundspatiotemporaldynamicsofthemotorcorticalactivity.Wealsoconductedtwo-photoncalciumimagingintheprimarymotorcortexduring14trainingsessionsoftheself-initiated lever-pull task.Motorcorticalactivitydynamicallychangedatbothensembleandindividual-neuronlevelsoversessions.NoCOI.

Symposium 59Rehabilitation from the aspect of nutrition and visceral function

(March 18, 14:00–16:00, Room K)

3S59K-1Responses of glucose output from the liver to somatic afferent stimulationKurosawa, Mieko1; Shimoju, Rie1,2(1Center Med. Sci., Intl. Univ. Health & Welfare, Otawara, Japan, 2Dept. Physical Ther., Intl. Univ. Health & Welfare, Otawara, Japan)

Somaticafferentexcitationbyphysicaltherapytreatmentcanproducevariouseffectsonmotorandautonomicfunctions.Wehaveinvestigat-edthereflexresponsesofvariousautonomic functionstosomatic(muscleorskin)afferentstimulationinanimalswhoseemotionalinflu-encesareeliminatedwithuseofanesthesia.InthepresentsymposiumIwillshowourstudiesontheresponsesofglucoseoutputfromthelivertomusclestimulationinanesthetizedrats.Electricalstimulation(10mA,20Hz)wasdeliveredtotheunilateralanteriortibialmusclefor10min.Themusclestimulation increasedthehepaticglucoseoutput(HGO),leadingincreasesintheplasmaglucose.TheHGOre-sponsewasdisappearedaftersectionofthesomaticnervesinnervat-ingtheanteriortibialmuscle,andaftertreatmentwithphenoxyben-zamineandpropranolol,analphaandabetaadrenoceptorblockades.Ontheotherhand,theresponsewasaugmentedaftertreatmentwithatropine,amuscarinicreceptorblockade.TheseresultsindicatethattheresponseofHGOtomusclestimulationismediatedviathesomat-icnervesasanafferentlimb,andthesympatheticnervesasaneffer-entlimb,andthatatthesametimetheresponseisreflexlyinhibitedviatheparasympatheticnerves.Finally,IwillalsoshowthatthesamemusclestimulationincreasestheinsulinsensitivityofbloodglucoseintheanimalmodeloftypeIIdiabetesviaexcitationofthesomaticaf-ferentnerves.NoCOI.


3S59K-2Resistance exercise-induced endocrine activation and it mechanismIshii, Naokata(Department of Life Science, Graduate School of Arts and Sciences, University of Tokyo, Tokyo, Japan)

EExercisesstimulatesecretionsofavarietyofhormonesinanexerciseintensity-dependentfashion.Inparticular,anumberofstudieshaveshownthatresistanceexercisecausesmarked increases inbloodconcentrationsofanabolichormonessuchasgrowthhormone(GH)andtestosterone.However, thiseffect isnotsimplydependentonexerciseintensity,butstronglydependsontheotherexercisevariables,e.g.,exercisevolumeandrestperiodbetweensets(Kraemer,1995),suggestingthatmultipleprocessesareinvolvedinitsmechanism.Wehaveshownthatresistanceexercisewithrestrictedmuscularbloodflow(RRB)causesdramaticincreasesinbloodGHandnoradrenalin,evenatlowexerciseintensity(Takaradaetal.,2000).Inaddition,alow-intensityresistanceexercisewithslowmovementandtonicforcegeneration(LST;Tanimoto&Ishii,2006)causeslargerincreasesinbloodGH,freetestosteroneandnoradrenalin(Tanimotoetal.,2005;Gotoetal.,2008)thanafternormalhighintensityexercise.BothRRBandLSTarecharacterizedbysustaineddeclineofmuscleoxygenationlevelandregionalincreasesinmetabolicsub-productssuchaslactate.ThuswehaveinvestigatedtheeffectsofdirectelectricstimulationofmusclewithandwithoutrestrictionofmuscularbloodflowonthesecretionofGH.Electricstimulationofmusclecausedsignificantin-creasesinbloodGHandlactateonlywhencombinedwithrestrictionofmuscularbloodflow,suggestingthatchemoreceptionofmetaboliteswithinmuscleplaysanimportantpartinthestimulationofGHsecre-tion(Inagakietal.,2011).NoCOI.

3S59K-3Combined effects of interval walking training with nu-trient supplement intake on physical fitness and im-provement of life-style related diseases in middle aged and older subjects.Nose, Hiroshi1,2; Masuki, Shizue1; Morita, Atsumi1; Okazaki, Kazunobu1; Kamijo, Yoshi-ichiro1(1Dept. of Sports Med. Sci., Shinshu Univ. Grad. Sch. of Med., 2Jukunen Tai-ikudaigaku Research Center)

Wehavedevelopedahealthpromotionprogramformiddleagedandelderpeople,JukunenTaiikudaiikuProgram,toexaminethephysicalandmentaleffectsofhigh-intensityintervalwalkingtraining(IWT)on5,400subjectsforthese10years.SinceaprescriptionofIWTcanbeconductedbyusinganITnetworksystem,theparticipants intheprogramwereabletoreceivetheprescriptioneveniftheylivedremotefromtrainers,enablingthemtoperformIWTattheirfavoredplacesandtimes,andalsoatlowcost.WefoundthatIWTfor4monthsin-creasedphysicalfitnessby10–20%anddecreasedtheindicesoflife-stylerelateddiseasesby10–20%.Inaddition,weexaminedtheeffectsofamixtureofproteinandcarbohydrate intake immediatelyafterdailyaerobicexercisetraining includingIWTandfoundthatthesupplementsenhancedtheincreasesinplasmaalbumincontentandthighmusclestrength.Further,weexaminedtheeffectsof5-amino-levulinicacidintakeduringIWTandfoundthatitimprovedtheworkefficiencyduringexerciseandincreasedanIWTachievement.Theacidisknowntobecontainedinmanykindsoffoodsandthesoleinitialmaterialofthehemebiosynthesis.Theseresultssuggestthatnutritionalsupplementintakeacceleratestheeffectsofaerobictrainingonphysicalfitnessandtheimprovementoflife-stylerelateddiseases.NoCOI.*Currentaffiliation:UrbanHealthandSports,OsakaCityUniversity.

3S59K-4Nutrition and rehabilitationWakabayashi, Hidetaka(Department of Rehabilitation Medicine, Yokohama City University Medical Center, Yokohama, Japan)

Malnutritionoftenoccursinpatientswithdisability.Theprevalenceofmalnutrition ingeriatricrehabilitationwashigherthanhospital(50.5%vs.38.7%)accordingtoMNAclassification.Asnutritionalstatusisassociatedwithrehabilitationoutcome,acombinationofbothreha-bilitationandnutritioncaremanagementmaybeassociatedwithabetteroutcome.Thisconceptisdefinedrehabilitationnutrition.Reha-bilitationnutritionistoassesswiththeInternationalClassificationofFunctioning,DisabilityandHealthincludingnutritionstatusandtopracticenutritioncaremanegementtodemonstratefunctions,activities,andparticipationtothefullest.Itisnotenoughforpatientswithdis-abilitytocoordinateonlyrehabilitationorclinicalnutrition.Sarcopeniaisasyndromecharacterizedbyprogressiveandgeneralizedlossofskeletalmusclemassandstrength.Primarysarcopeniaisconsideredtobeage-relatedwhennoothercauseisevident,otherthanageingitself.Secondarysarcopeniashouldbeconsideredwhenothercausesareevident,suchasactivity-relatedsarcopenia,disease-relatedsarco-penia,ornutrition-relatedsarcopenia.Activity-relatedsarcopeniacanresultfrombedrest,deconditioning,orzero-gravityconditions.Dis-ease-relatedsarcopeniaisassociatedwithinvasion(acuteinflammato-rydiseases),cachexia(cancer,advancedorganfailure,collagendiseas-es,etc.),andneuromusculardisease.Nutrition-relatedsarcopeniaresultsfrominadequatedietaryintakeofenergyand/orprotein.Treatmentincludingrehabilitationandnutritioncaremanagement isdifferentaccordingtothecausesofsarcopenia.NoCOI.

Oral Presentations


Oral Presentation 01Ionic Channel, Receptor (1)

(March 16, 9:00–10:00, Room H2)

1O1H2-1Inhibitory effects of monoterpenes on human TRPA1 and the structural basis of their activityTakaishi, Masayuki1; Fujita, Fumitaka1; Shimizu, Mayumi1; Shimada, Tadashi1; Urabe, Shun1; Matsui, Hiroshi1; Uchida, Kunitoshi2; Tominaga, Makoto2(1Technical Development center, Mandom corp., Osaka, Japan, 2Section of Cell Signaling, National Institute for Physiological Sciences, Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences)

Monoterpenes,suchasmenthol,camphor,and1,8-cineole,compriseagroupofnaturallyoccurringorganiccompoundsthathavebeenusedforanesthetic,analgesicandanti-inflammatorypurposes.Amongmono-terpens,camphorand1,8-cineole,botharewell-knowncomponentsofessentialoils,arereportedtoexertanalgesiceffectsthroughtheinhi-bitionofTRPA1.Inthisstudy,wescreenedseveralmonoterpeneanalogsofcamphortoidentifymoreeffectivenaturallyoccurringTRPA1antagonists.Wefoundthatborneol,2-methylisoborneol,andfenchylalcoholexhibitedhigher inhibitoryeffectsonhumanTRPA1(hTRPA1)activitythaneithercamphoror1,8-cineole.Furthermore,sensoryirritationtestsinvivoshowedthatborneolconferredananalgesiceffectonthesensoryirritationproducedbymenthol.Thesedatasuggest thatborneol,2-methylisoborneolandfenchylalcoholhavethepotentialtobeeffectiveanalgesiccompounds.Inaddition,wefoundthattheS873,T874,andY812residuesofhTR-PA1wereinvolvedintheinhibitoryeffects,suggestingthatthehy-droxylgroupinthesix-memberedringoftheinhibitorsmayinteractwiththeseaminoacids.Furtherresearchonborneol,2-methylisoborneolandfenchylalcoholcouldleadtothedevelopmentofanti-nociceptiveagentsthroughTRPA1inhibition.NoCOI.

1O1H2-2ATP-mediated odontoblast-odontoblast communica-tion following TRP channel activationSato, M; Tsumura, M; Soya, M; Kawaguchi, A; Nishiyama, A; Ogura, K; Mochizuki, H; Kodama, S; Shibukawa, Y; Tazaki, M(Tokyo Dental Collge)

Odontoblastsproducethedentinmatrixduringtoothformationandcontrolitsmineralizationunderphysiologicalandpathologicalcondi-tions.Thesecellslocalizeattheinterfacebetweenthedentalpulpanddentinandareorganizedasapalisadelayer.Althoughodontoblastsformdentincooperatively,howthesecellscommunicateremainsun-clear.Inthisstudy,weinvestigatedthechemicalsignalingthatmedi-atesintercellularcommunicationbetweenodontoblastsfollowingme-chanicalstimulation.Singlemouseodontoblast lineagecells (OLCs)werestimulatedusingaglasspipettefilledwithstandardextracellularsolution.Wemeasuredtheintracellularfree-Ca2+concentration([Ca2+]i)byusingfura-2instimulatedOLCsandintheOLCslocatednearthem.DirectmechanicalstimulationofsingleOLCsincreased[Ca2+]ibyac-tivatingTRPV1,V2,andV4channels.Moreover,weobservedincreas-esin[Ca2+]inotonlyinthestimulatedOLCs,butalsoinnearbyOLCs.Thisincreasein[Ca2+]iinthenearbyOLCs,butnotinthestimulatedOLC,wasblockedbypannexin-1 inhibitor inaconcentration-anddistance-dependentmanner.Moreover,inthepresenceofPLCinhib-itor,theincreasein[Ca2+]iproducedinthenearbyOLCsaftermechan-icallystimulatingsingleOLCswasabolished.OurresultsindicatethatATPisreleasedfrommechanicallystimulatedodontoblastsviapan-nexin-1becauseofTRPchannelactivationandthatATPsignalstonearbyodontoblastsbyactivatingP2Yreceptors.Theresultsalsostronglysuggestthatodontoblastscommunicatetodrivetheircellularfunctions.NoCOI.

1O1H2-3Molecular mechanisms for developmental acquisition of single-spiking property of Mauthner cell among ho-mologous reticulospinal neurons in zebrafish Shimazaki, Takashi; Watanabe, Takaki; Oda, Yoichi(Graduate School of Science, Nagoya University, Nagoya, Japan)

TheMauthner(M)cells,whicharebilaterallypairedgiantreticulospi-nalneurons(RSNs)locatedatthefourthsegment(r4)inthehindbrainofteleostandknowntotriggerfastescape.M-cellsdeliverasinglespikeat theonsetofdepolarizing input,whereastheirhomologs,MiD2cmandMiD3cm,whicharelocatedinadjacentsegment(r5andr6)firerepetitively.TheuniquefiringpropertyofzebrafishM-cellisacquiredduringlarvaldevelopment:immatureM-cellsexhibitburstfiringastheirhomologs.Here,weshowtheexpressionoftwodifferentKvsubunitsandtheirmodificationareakeyfortheacquisitionoftheM-cellexcitabilityin larvalzebrafish.First,pharmacologicalstudiesrevealthattwotypesofKvchannel,Kv1andKv7,arenecessaryforsingle-spikingofM-cell.Second,in situhybridizationshowedthattwodifferentKvα-subunits,Kv1.1andKv7.4,areexpressedinM-cells.Bothchannels,however,arealreadyexpressedbeforeM-cellsexhibitsinglespikingandKv1.1isalsoexpressedinMiD2/3cm.Nevertheless,thecell-surfaceexpressionofKv1.1α-subunitsisuniquelyenhancedbyanauxiliarysubunitKvβ2andthegatingofKv.7.4ismodifiedbyproteinkinaseCspecificallyintheM-cellswhentheyacquirethesingle-spik-ingproperty.Thus,theseresultsshowcriticalstepsfordevelopmentalacquisitionoftheuniquefiringpropertiesof theM-cellamongthesegmentallyhomologousneurons.NoCOI.


1O1H2-4The interaction of new IK1 blocker (PA-6) with intracel-lular spermine and magnesium.Takanari, Hiroki1,2; Houtman, Marien2; Stary-Weinzinger, Anna3; Ono, Katsushige1; van der Heyden, Marcel2(1Department of Pathophysiology, Oita University School of Medicine, Yufu, Japan, 2Department of Medical Physiology, University Medical Center Utrecht, Utrecht, The Netherlands, 3Department of Pharmacology and Toxicology, University of Vienna, Vienna, Austria)

Background:The inwardrectifierK+current (IK1)contributestoastablerestingmembranepotentialandactionpotentialrepolarizationincardiaccells.WedevelopedanewspecificIK1blocker,pentamidineanalogue(PA-6).NowweinvestigatedtheinterplayofPA-6,intracel-lularspermineandMg2+onchannelinhibition.Methods:HEK293TcellsweretransfectedwithhumanKIR2.1bylipo-fectamine,andusedforinside-outpatchclampexperimentwitharampprotocolfrom-100mVto100mVin5s.Bothwildtypeandmutant(E224A)channelsweretested.Results:PA-6 (200nM)aloneblockedIK1completely.Sperminede-creasedtheoutwardcomponentofIK1by70%at0.1µM,andtheinwardcomponentby5%.Inthepresenceof0.1µMspermine,PA-6provided41%and39%ofadditionalblockonIK1ininwardandoutwardcompo-nents,respectively.Mg2+(1mM)hadasmallblockingeffectonIK1by7%,anddidnotinterferewithIK1blockofPA-6.BothPA-6andsper-minehadlesseffectonE224A-KIR2.1current,andnoreciprocalinter-ferencewasobserved,suggestingthatthenegativeresidueE224isessentialforPA-6andsperminetointeractwiththeKIR2.1channel.Conclusion:Spermine,butnotMg2+,partiallyrelievedIK1blockofPA-6inadose-dependentmanner.PA-6mayhaveanoverlappingbindingsiteonKIR2.1channelswithspermine,butnotwithMg2+.NoCOI.

Oral Presentation 02Motor Function / Sensory Function (1)

(March 16, 11:05–12:05, Room J)

1O2J-1Hierarchical connectivity among morphologically ho-mologous and repeated reticulospinal neurons in the segmented hindbrain for escape behavior in goldfish and zebrafishNeki, Daisuke; Oda, Yoichi(Graduate School of Science, Nagoya University, Nagoya, Japan)

Segmentationalongtheneuraxisisaprominentfeatureofthecentralnervoussysteminvertebrates. Inwiderangeoffishes,hindbrainsegmentscontainorderlyarrangedreticulospinalneurons (RSNs).IndividualRSNsingoldfishandzebrafishhindbrainaremorphologi-cally identified,andsimilarRSNsarecalledsegmentalhomologs.Segmentalhomologsarerepeatedinadjacentsegmentsofhindbrainandthoughttobe functionallyrelated.Here,we investigatedthefunctionalrelationshipsof thembyexaminingelectrophysiologicalconnectivitybetweentheMauthnercell(M-cell),pairedgiantRSNsinsegment4(r4)andknowntotriggerfastescapeorpreycaptureoffish,anddifferentseriesofhomologousRSNsinr4tor6.Pairedintra-cellularrecordingsinadultgoldfishrevealedunidirectionalconnectionsfromtheM-celltoRSNs.M-cellconnectivitywascloselycorrelatedwiththemorphologicalhomologyofrepeatedRSNsinr4tor6:SinglespikeoftheM-cellproducedIPSPsindorsalRSNsinr4tor6ontheipsilateralsideandexcitatorydepolarizationonthecontralateralside,whereasstrongdepolarizationsequallyinventralRSNsinr4tor6onthebothsides.TheseresultssuggestthateachfunctionalconnectionworksasafunctionalunitduringtheM-cell-initiatedescapeorpreycapture.NoCOI.

1O2J-2Tectal commissural connections and their functional roles in saccades in relation to the VOR and Listing’s lawTakahashi, Mayu; Sugiuchi, Yuriko; Shinoda, Yoshikazu(Dept of Neurophysiology, Tokyo Medical and Dental Univ, Tokyo, Japan)

Thecommissuralconnectionbetweenthesuperiorcolliculi(SCs)wasknowntobeinhibitory,butwerecentlyfoundthatstrongexcitatorycommissuralconnectionsalsoexist.Electrophysiologicalstudyshowedthatcaudal tectoreticularneurons (TRNs)receivedonly inhibition,whereasrostralTRNsreceivedexcitationand inhibitionfromtheoppositerostralSC.TRNsinthemedialandlateralSCsreceivedex-citationfromthecontra-medialandlateralSC,respectively,andre-ceivedinhibitionfromthecontra-lateralandmedialSC,respectively.Thesefindingssuggestthatinhibitorycommissuralconnectionsexistbetweenthemedial (lateral)SCrepresentingupward (downward)obliquesaccadesononesideandthelateral(medial)SCrepresentingdownward(upward)obliquesaccadesontheother.Thispatternofreciprocalinhibitionbetweentheup-obliqueanddown-obliquesaccadesinthetwoSCsisverysimilartothatseenintheanteriorandposte-riorcanal-relatedvestibularneurons.ThissimilarityimpliesthattheSCsaccadesystemmayusethesamecoordinatesastheVOR.Incontrast,mirror-symmetricexcitatoryconnectionsbetweenmedial-me-dialandlateral-lateralSCsplayaroleinconjugateupwardanddown-wardverticalsaccades,respectively,becausecoactivationofTRNsinsymmetricsitesofthetwoSCsmayoccurthroughthecommissuralexcitations,andtorsionalcomponentsofindividualeyesseemtocanceleachother,leavingmainlyverticalcomponents.Therefore,thesetec-talexcitatorycommissuralconnectionsmaycontributetoListing’slaw.NoCOI.


1O2J-3Cytoplasmic Regulation of Channel Opening of Elec-trical Synapses between Retinal Ganglion Cells.Hidaka, Soh(Dept. Physiol., Fujita Health Univ. Sch. of Med., Aichi, Japan)

Electricalsynapsesarepresentinretinalneuronsexpressingchannelsubunit,connexin36(JNeurosci,2004,2009).RecentstudiesrevealedchannelopeningofgapjunctionsbetweenamacrinecellsisregulatedbyintracellularcyclicAMPaswellasintracellularCa2+concentration(BrainRes,2012). Inthepresentstudy,I investigatedregulationofchannelopeningofelectricalsynapsesbyapplicationofligandsunderdualwhole-cellpatchclamprecordingsbetweenretinalganglioncells.Imeasuredpassagecurrentsthroughelectricalsynapsesbyapplicationofantibodiesagainstconnexin36andbychangeofintracellularCa2+concentration.Ialsostudiedmorphologicalchangeofgapjunctionsundertheapplicationbyelectronmicroscopy.ChelatingintracellularCa2+ledustoobservelargepassagecurrentsbetweentheretinalcellsandtransjunctionalconductance(Gj)betweenthecells (2.45nS).Gjsuppressedto0.23nSby intracellularapplicationofcyclicAMPinpipettewith5mMconcentration,comparedwiththatofcontrolcon-dition.Electronmicroscopyrevealedformationofannulargapjunctions.Intracellularapplicationofanantibodyagainstthecytoplasmicdomainofconnexin36(JNeurosci,2004)reducedGj(0.98nS).TheinhibitionofGjbythecytoplasmicantibodywasdose-dependentmanner.CocktailoftheantibodyandcyclicAMPleavesGjasinthelevelbysingleinvolvementoftheantibody.Theannulargapjunctionswerenotfoundunderapplicationof thecocktail.Theseresultsdemonstratethatchannelopeningofelectricalsynapsesisassociatedwithcytoplasmicdevelopmentofgapjunctions.NoCOI.

1O2J-4Functional analysis of Na+,K+,2Cl--cotransporters on the lateral cochlear wall contributing to the endolym-phatic potentialYoshida, Takamasa1,2; Nin, Fumiaki1; Ogata, Genki1; Uetsuka, Satoru1,3; Kurachi, Yoshihisa4; Hibino, Hiroshi1(1Dept Mol Physiol, Niigata Univ Med Sch. Niigata, Japan, 2Dept Otolaryngol, Grad Sch Med, Kyushu Univ, Fukuoka, Japan, 3Dept Otolaryngol, Grad Sch Med, Osaka Univ, Osaka, Japan, 4Dept Pharmacol, Grad Sch Med, Osaka Univ, Osaka, Japan)

Thelateralcochlearwallcomprisestwoepitheliallayers;strialmar-ginalcells (MCs) facingendolymph,andthesyncytiumfacingperi-lymph.Theselayerssandwichawell-vascularizedextracellularcom-partment, the intrastrial space (IS). K+-gradients established byunidirectionalK+-transportacrossthelateralcochlearwallelicitK+-dif-fusionpotentialsacrossK+-channels,andasaconsequence,endolymphhastheendocochlearpotential(EP)of+80mV.SincespecificinhibitorsgreatlyreducetheEP,basolateralNa+,K+,2Cl--cotransporters(NKCCs)expressedoneachlayeraregenerallythoughttocontributetotheunidirectionalK+-transport. Inthisstudy,weexaminedthe lateralcochlearwallofguineapigsinordertoanalyzetheindividualfunctionofNKCCsoneach layer.Whenbumetanide,aspecific inhibitorofNKCC,wasappliedperilymphatically,electrochemicalpropertiesofthesyncytiumwasbarelyaffected,whereas[K+]oftheISwaselevat-edjustlikewhenbumetanidewasvascularlyapplied.Theseresultssuggestedthatperilymphaticallyappliedbumetanideseemedtopen-etratethroughthesyncytiumintotheISandinhibittheMC’sNKCCs.Thesyncytium’sNKCCs,whichwasunlikelytobeaffected,mightbedispensablefortheunidirectionalK+-transportandtheEP.NoCOI.

Oral Presentation 03CNS Function

(March 17, 11:05–12:05, Room G)

2O3G-1Plasmalogens diet enhance hippocampal-dependent memory by enhancing neurogenesis in dentate gyrusHossain, Shamim Md1; Ifuku, Masataka1; Ahmed, Youssef Md.Saleh1; Miake, Kiyotaka2; Fuchuu, Hidetaka2; Katafuchi, Toshihiko1(1Department of Integrative Physiology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan, 2Center Research Institute, Marudai Food Company Limited, Osaka,Japan)

Theetherphospholipids,Plasmalogens(Pls),arecharacterizedbythepresenceofavinyletherlinkageatthesn-1position.IntheAlzheimer’sdisease(AD)patients,Plswerefoundtobereducedinthebraintissues.HumanbraincontainsalargeamountofPls,mainlyethanolaminePls(EthPls),inthehippocampus.HippocampuscontrolsthememoryandusuallyitishighlydamagedintheADpatients.Wetherefore,hypoth-esizedthatthereductionofPlsinthehippocampusofbrainmightbeassociatedwiththeneuronaldamagesandmemoryloss.Inourprevi-ousstudy,wehavefoundthatPlscaninhibithippocampalneuronalcelldeath.Inthepresentstudy,wehavefoundthatthemicehavingPls foodperformedbetterthancontrolmice inMorriswatertasksuggestingthatPlsenhancememory. In invitroexperimentstheculturedhippocampalneuronsshowedasignificant increase inthespineformationaftertreatmentwithEthPls(P<0.01).Inaddition,Plsdietsignificantly increasedneurogenesismarkedbyan increaseofdoublecortinpositiveneuronsintheDG(P<0.01).Moreinterestingly,wehavefoundthatPlsdietenhancemRNAexpressionofBDNFinthehippocampus.Wetherefore,proposethatPlsdietincreaseneuro-genesisandspineformationprobablythroughtheexpressionofBDNFinthehippocampusresultinginthememoryformation.NoCOI.


2O3G-2Subtypes of nicotinic acetylcholine receptors in nico-tine-induced vasodilation in the rat hippocampusWatanabe, Saori1,2; Misawa, Hidemi2; Uchida, Sae1(1Department of Autonomic Neuroscience, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan, 2Department of Pharmacology, Faculty of Pharmacy, Keio University, Tokyo, Japan)

Previousstudyinourlaboratoryhasshownthatstimulationofcholin-ergicneuronsinthemedialseptalnucleusincreasesbloodflowinthehippocampus (Hpc-BF)viaactivationof thenicotinicacetylcholinereceptors(nAChRs).Oftheseveralsubtypesofnicotinicreceptors,theα4β2andtheα7subtypesarethemostabundantinthehippocampus.Thepresentstudyaimedtodeterminewhethereitherorboththeα4β2ortheα7subtypeisinvolvedinthecholinergicvasodilationinthehippocampus.Inurethane-anesthetizedrats,Hpc-BFwasmeasuredbylaserDopplerflowmetry.WeexaminedtheresponseofHpc-BFinducedbyadministrationofnicotine(10–300µg/kg,i.v.)asanAChRagonist.Hpc-BFincreaseddose-dependentlyfollowingtheadministra-tionof30–300µg/kgnicotine.Meanarterialpressurewasnotinfluencedat10–30µg/kgnicotine,butwasincreasedat100–300µg/kgnicotine.TheincreaseinHpc-BFbynicotineat30µg/kg,withoutchangingmeanarterialpressure,wascompletelyabolishedbytheα4β2nAChRantagonistdihydro-β-erythroidine(DHβE,5mg/kg,i.v.),whileitwasnotinfluencedbytheα7selectivenAChRantagonistmethyllycaconi-tine(MLA,5mg/kg,i.v.).Thesefindingssuggestthatcholinergicva-sodilationinthehippocampusismediatedviaα4β2nicotinicreceptorswhileα7nicotinicreceptorsarenottobeinvolved.NoCOI.

2O3G-3Rat thalamic neurons encode complex combinations of facing and movement directions and trajectory route during translocation with sensory conflictNyamdavaa, Enkhjargal(System Emotional Science, Grad Sch of Med and Pharmaceu Sci, Univ of Toyama,Toyama, Japan)

Previousstudieshavereportedthatsomethalamicneuronsencodetheanimal’sdirectionalheading,andthesearereferredtoasheaddirectioncells.Thepresentstudyinvestigatedeffectsofsensorymis-matchamongideotheticcuesonthalamicneurons.Ratswereplacedonatreadmillstagethatmovedinafigure-8-shapedpathway.Theanterodorsalandlaterodorsalthalamicneuronswererecordedunder2conditions:1)controlsessions,inwhichboththestageandthetread-millmovedforward,and2)backwardsessions,inwhichthestagewasmovedbackwardwhiletheratsranforwardonthetreadmill.Ofthe222thalamicneuronsrecorded,60showeddifferentialresponsestothesouthandnorthdirections,alongwhichtheanimalsweretranslocatedinthelongaxisofthetrajectory.Ofthese60,19showedfacingdirec-tion-dependentresponsesregardlessofmovementdirection.Twentyneuronsdisplayedfacingandmovementdirection-dependentrespons-es;activityof10andtheremaining10neuronsincreasedduringfor-wardandbackwardmovement,respectively.Twentyoneneuronsshowedmovementdirection-relatedresponsesregardlessof facingdirection.Furthermore,theactivityofsomedirection-relatedneuronsincreasedonlyinaspecifictrajectory.Theresultssuggestedthattheactivityoftheseneuronsreflectscomplexcombinationsoffacingdi-rection,movementdirection,motor/proprioceptive information,andthepasthistoryofmovements.NoCOI.

2O3G-4Classical conditioning reinforced by visual stimuli af-ter V1 lesionTakakuwa, Norihiro1,2; Kato, Rikako1; Redgrave, Peter3; Isa, Tadashi1,2(1Dept Dev. Physiol, Nat'l Inst. Physiol. Sci., Okazaki, Japan, 2The Graduate Univ. for Advanced Studies, Hayama, Japan, 3Dept Psychol, Univ. of Sheffield, Sheffield, U.K.)

Classicalconditioningiswhenanimalslearnanassociationbetweenapredictingconditionedstimulus(CS)andareward.Duringconditioning,theCSacquiresthepropertiesofasecondaryreinforcersothat,sub-sequently,itcanreinforcetheacquisitionofnovelinstrumentalbehav-ior.Wehypothesisedthatthedirectprojectionfromthesuperiorcolliculus(SC)todopamine(DA)neuronsinthesubstantianigraparscompacta(SNc)playsakeyroleinassociativelearningwithconditionedvisualstimuli.TotestthishypothesisweclassicallyconditionedtwomonkeyswithunilateralV1lesions(ananimalmodelof“blindsight”)whenvisualCSswerepresentedinthelesion-affectedandintactvisu-alfields.AftertrainingwithtwoCSs,onepredictinghigh(large/imme-diate)andtheotherlow(small/delayed)reward,themonkeysexhib-itedappropriatelytimed,quantitativelymodulatedanticipatorylicking.TheseclassicallyconditionedresponseswereelicitedwhenthevisualCSswerepresentedineitherthelesion-affectedandintactvisualfield.Afterconditioning,werecordedtheresponsesofSNcdopamineneu-ronswhentheCSswerepresentedagaininthelesion-affectedvisualfield.WeshowedclearphasicresponseswhenCSswerepresentedinthelesion-affectedvisualfield.Togethertheseresultsshowthatvisu-alinputviatheSChasaccesstobasicassociativelearningcircuitryinthebasalganglia.NoCOI.

Oral Presentation 04Heart, Circulation (1)

(March 17, 11:05–12:05, Room G)


3O4G-1Alteration of the arterial baroreflex sensitivity by elec-trical stimulation of the mesencephalic ventral teg-mental area in decerebrate ratsLiang, Nan; Matsukawa, Kanji; Endo, Kana; Ishii, Kei; Idesako, Mitsuhiro(Dept. Integrative Physiology, Graduate School of Biomedical and Health Sciences, Hiroshima Univ. Hiroshima, Japan)

Wehaveprovidedevidencethatarterialbaroreceptor-heartrate(HR)reflexisbluntedattheonsetofspontaneousmotoractivityinparalyzed,decerebratecats(Matsukawaetal.AmJPhysiolHeartCircPhysiol2012).Giventhatthemesencephalicventral tegmentalarea (VTA)playsanimportantroleinthegenerationofthecardiovascularresponseduringexercise,wehypothesizedherethatarterialbaroreflexsensi-tivity (ABS) isalteredbyneuronalactivitieswithintheVTA.Ratsweredecerebratedatthepremammillaryandprecollicularlevelandthenparalyzed.ActivationoftheVTAwasevokedbyanelectricalstimulation(40–80µA)lastingfor30s.Abriefocclusionoftheabdom-inalaortawasusedtonaturallyactivatearterialbaroreceptors.ABSforHRwasestimatedfromthebaroreflexratiobetweenthepressorandbradycardiaresponsesduringaorticocclusionandfromtheslopeofthebaroreflexcurvebetweenchangesinthemeanarterialpressure(MAP)andHR.AscomparedtothatwithoutVTAstimulation,bothbaroreflexratioandslopeoftheδMAP-δHRcurvewerebluntedwhenaorticocclusionwasgivenattheonsetofVTAstimulation(0–3s).At10–25sfromthestartofVTAstimulation,surprisingly,thebaroreflexratioandtheslopeofthecurverevertedtothepre-stimulationlevelandeventurnedtoincrease.Thepresentfindingssuggestthatacti-vationofVTAleadstodeceaseoftheABSattheonsetof,butnotduring,thefictivemotoractivity.NoCOI.

3O4G-2β-adrenergic vasodilatation does not contribute to centrally-induced muscle hyperemia at start of volun-tary one-legged cycling and during motor imageryIshii, Kei1; Matsukawa, Kanji1; Liang, Nan1; Endo, Kana1; Idesako, Mitsuhiro1; Hamada, Hironobu2; Kataoka, Tsuyoshi3; Ueno, Kazumi3; Watanabe, Tae3(1Department of Integrative Physiology, Graduate School of Biomedical and Health Sciences, Hiroshima University, 2Department of Physical Analysis and Therapeutic Sciences, Graduate School of Biomedical and Health Sciences, Hiroshima University, 3Department of Health Care for Adults, Graduate School of Biomedical and Health Sciences, Hiroshima University)

We hypothesized that central command induces sympatheticβ-adrenergicvasodilatationatstartofexercise,which inturnmaycontributetoincreasingmusclebloodflow.Totestthehypothesis,theeffectsofpropranololonthebloodflowresponsestovoluntaryone-leggedcyclingandimageryoftheexercisewereexaminedin9sub-jects.Therelativeconcentrationsofoxygenated-hemoglobin(Oxy-Hb)inbilateralvastus lateralismusclesweremeasuredasan indexofmuscletissuebloodflowwithnear-infraredspectroscopy.IncreasesinOxy-Hbofthebilateralmusclesobservedatstartofone-leggedcyclingandduringmotorimagerywerenotbluntedbypropranololbutwereabolishedbyadditionalatropine.Whenfemoralbloodflowtothenon-exercisinglimbwasmeasuredwithultrasoundDopplerflowmetryin5subjects,bothone-leggedcyclingandmotorimageryevokedtheincreasesinfemoralbloodflowandvascularconductance.Thevaso-dilatationwasatropine-sensitiveandwasnotaffectedbypropranolol.Thusweconcludedthatcentralcommandevokescholinergic,butnotβ-adrenergic,vasodilatationinskeletalmusclesatstartofvoluntaryexerciseinhumans.NoCOI.

3O4G-3Drinking-induced bradyarrhythmias and cerebral inju-ry in Dahl salt sensitive rats with sinoaortic denerva-tionAbe, Chikara; Morita, Hironobu(Department of Physiology, Gifu University Graduate School of Medicine, Gifu, Japan)

Wehavedemonstratedthatadrinking-inducedpressorresponsewaslargerifthebaroreflexdidnotoperate,andthemeanarterialpressurereached163mmHginconsciousratswithsinoaorticdenervation(SAD).Thus,wehypothesizedthatadrinkingbehaviorbecameacardiovas-cularriskfactor,ifabasalarterialpressurewashigh.Toclarifythis,weanalyzedtheoccurrenceofarrhythmiasandtheaccumulationofmicrogliainDahlsaltsensitiverats(DahlS)withSAD.WemaintainedDahlSandDahlsaltresistant(DahlR)withhigh-sodiumdietfor5weeks.AfterSADsurgery,wemeasuredAPandelectrocardiogram(ECG)duringwaterdrinkingbehavior inallrats.Furthermore,wemeasuredTNF-αconcentrationinthecerebrospinalfluid(CSF)andmicroglialaccumulationsaroundthe3rdand4thventriclesinratswithprogrammeddrinkingatarapidorslowratefor7days.Incidenceofdrinking-inducedbradyarrhythmiasandprematureventricularcon-tractions(PVCs)weresignificantlylargerinDahlSthanDahlRrats.BothbradyarrhythmiasandPVCswerecompletelyabolishedbyat-ropineadministration.Accumulationsofmicrogliaaroundthe3rdventricleandincreaseinTNF-αintheCSFwereobservedinratsthatdrankwateratarapidrate;thesewerenotseeninratsthatdrankwaterslowly.Inconclusion,bothcardiovasculareventsandcerebralinjurymaybeincreasedbydrinkinginDahlSratswithSAD.Theserisksarereducedbymodifyingdrinkingbehaviorsuchasslowingthedrinkingrate.NoCOI.

3O4G-4The Analysis of the polyunsaturated fatty acids and the incidences of supraventricular arrhythmias of the elderly in Group HomeTodoroki, Kikue1,2; Ikeya, Yoshimori1; Tanaka, Etsuro2; Fukuyama, Naoto1; Mori, Hidezo1(1Dept Physiology, Tokai Univ School of Medicine, 2Dept Nutritional Science, Tokyo Univ of Agriculture)

Wereportedthattheplasmaeicosapentaenoicacid(EPA)concentrationin ischemicstrokepatientsespecially incardioembolismaresignifi-cantlylowerthanthecontrol(Ikeyaetal,2013).Atrialfibrillationin-creasestheriskofcardioembolism.However, thepolyunsaturatedfattyacidsandtheincidencesofsupraventriculararrhythmiaswithreferencetocardioembolismoftheelderlyinGroupHome(GH)hasnotbeenexamined.ToanalyzeEPAandotherpolyunsaturatedfattyacidsandtheincidencesofsupraventriculararrhythmiasbetweenthe30elderlyinGHand30age-matchedcontrolsubjects,wecomparedthebloodsampledata(EPA,arachidonicacid(AA),docosahexiaenoicacidandtriglyceride,LDL-cholesterol,HDL-cholesterolandHbA1c)andquantitatedtheincidencesofsupraventriculararrhythmiasusinganon-parametrictestandamultiplelogisticregressionanalysis.Wealsoinvestigatedlifestylehabitoffishconsumptionusingaquestion-naire.ThemultiplelogisticregressionanalysisrevealedthatlowerEPAandHbA1candhigherAAwerespecificintheelderlyinGroupHome.Theincidencesofsupraventriculararrhythmiaswerenotsignificant-lydifferentbetweenthetwogroups.Thiscross-sectionalstudyoftheelderlyinGHshowedthatsignificantlowerplasmaEPAconcentrationandfishconsumptionandhigherAAandthatnosignificantdifferenceintheincidencesofsupraventriculararrhythmias.TheseresultsmightleadtoEPAreplacementtherapyfortheelderlyinGH.NoCOI.


Oral Presentation 05Cell Physiology, Molecular Physiology

(1)

(March 18, 11:05–12:05, Room J)

3O5J-1Basic fibroblast growth factor-induced neuronal differ-entiation of canine bone marrow stromal cells: In-volvement of FGFR2/PI3K/Akt pathwayNakano, Rei1; Edamura, Kazuya1; Nakayama, Tomohiro2; Narita, Takanori3; Sugiya, Hiroshi3(1Lab. of Vet. Surg., Dep. of Vet. Med., Nihon Univ. Grad. school, Fujisawa, Japan, 2Lab. of Vet. Radiol., Dep. of Vet. Med., Nihon Univ. Grad. school, Fujisawa, Japan, 3Lab. of Vet. Biochem., Dep. of Vet. Med., Nihon Univ. Grad. school, Fujisawa, Japan)

Thedomesticdog isanoptimalmodel forthepre-humantrials inspinalcordinjury.However,thecapacityandmechanismofneuronaldifferentiationofcaninebonemarrowstromalcells(BMSCs)havenotwellbeenelucidated.Wehereinvestigatedtheeffectofbasicfibroblastgrowthfactor(bFGF)onneuronaldifferentiationofcanineBMSCsandthesignallingpathway.IncanineBMSCs,bFGF(100ng/ml)inducedanincreaseinmRNAexpressionofneuronmarkers,MAP2,NEFLandENO2,andadecreaseofthatofneuralstemcellandgliamarkers,NESandGFAP.Immunocytochemicalstudyshowedthatthecellsexpressedneuronmarkers,NF-LandNSE.InthebFGF-treatedcellsloadedwiththeCa2+indicatorFluo3,ahighconcentrationofKClandL-glutamateinducedanincreaseinintracellularCa2+levels.IncanineBMSCs,cross-linkingandimmunoprecipitationanalysisrevealedthatbFGFboundtotheFGFR2.InthepresenceoftheFGFRinhibitorSU5402,thePI3KinhibitorLY294002andtheAktinhibitorMK2206,bFGFfailedtoinducemRNAexpressionofMAP2.InbFGF-treatedcells,Aktwasphosphorylated,andthephosphorylationwasinhibitedbySU5402,LY294002andMK2206.Takentogether,itismostlikelythatFGFR2/PI3K/AktpathwayisinvolvedinbFGF-inducedneuronaldifferentiationofcanineBMSCs.NoCOI.

3O5J-2Aloin differentiates 3T3 fibroblasts to osteoblasts through MAPK mediated Wnt dependent BMP signal-ing cascadePengjam, Yutthana; Kumar, Harish; Madhyastha, Radha; Omura, Sayuri; Nakajima, Yuichi; Maruyama, Masugi(The Department of Applied Physiology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan)

Bonemassismaintainedbybalancebetweenboneformingosteoblastandosteoclastcells.Inthisstudy,weinvestigatedtheeffectsofaloin,ananthraquinoneglycoside,on3T3fibroblastcellsandstudiedtheproliferation,differentiationandboneformationparameterslikemin-eralizationandassociatedsignalingcascade.Initiallyweconfirmedtheactionofaloinontheproliferationpatternsof3T3cellsandresultsconfirmedthedosedependentactivity.Resultsshowed0.05µMofaloinincreasedALPactivitysignificantly.ThemineralizationoftheextracellularmatrixwasdeterminedbyAlizarinredSstainingandshowedaloin0.05µMconcentrationssignificantlydepositedcalcium.Westudieddifferentconcentrationofaloinonfluctuationsofosteoblastmarkergenes.Resultsrevealedthedosedependentfluctuationsofmarkergenes.Wealsoinvestigatedtheosteogenesissignalingpath-wayslikeMAPK,Wntdependentbonemorphogeneticprotein(BMP)signalingpathway.Furthermore, theBMPandRunx2antagonistnogginandMAPKinhibitors,includingp38inhibitor,SAPK/JNKin-hibitorandAktinhibitorattenuatedaloin-promotedproteinsignalingactivity.Takentogether,theseresultsindicatethataloindifferentiates3T3fibroblastcells intoosteoblastthroughMAPK,WntdependentBMPsignalingpathway.Furthermore,aloinhasanaboliceffectsofboneformationandmaybeuseful forthetreatmentofosteogenicrelateddiseases.NoCOI.

3O5J-3SCF/beta-TRCP Negatively Regulates Cdh1 Stability to Ensure Proper Cell-Cycle ProgressionFukushima, Hidefumi1; Inuzuka, Hiroyuki2; Okamoto, Fujio1; Kajiya, Hiroshi1; Okabe, Koji1(1Section of Cellular Physiology, Department of Physiological Sciences & Molecular Biology, Fukuoka Dental Collge, Fukuoka, Japan, 2Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, United States of America)

Cell-cycle,theseriesofeventsthattakeplaceinacellleadingtoitsdivisionandduplication,isthefundamentalmechanismofdevelopment,differentiationandaging.Mutationsofthecellcyclecomponentsfre-quentlyfounded inseveralcancers.Propercell-cycletransitions isprogressedby“KinaseEngine”containingCDK/Cyclincomplexesactivityandtunedbyubiquitinsystemcontrollingproteinamountofkinases.Skp1/Cullin/F-boxprotein(SCF)complexesandanaphase-pro-motingcomplexes(APC)representtwomajorclassesofubiquitinli-gaseswhoseactivitiesarethoughttoregulateprimarilytheG1/Sandmetaphase/anaphasecell-cycletransitions,respectively.However,transitionmechanismbetweenSCFandAPCcomplexactivitiesarestillunclear.WepreviouslyreportedthatAPC/Cdh1regulatestheSCFcomponentSkp2fordegradation.Here,wecontinuetoreportthatSCF/beta-TRCPreciprocallycontrolsAPC/Cdh1activitybygoverningCdh1ubiquitinationandsubsequentdegradation.Dis-func-tionofthismechanismcauseprotractionofG1/Stransitionandlossofstemness inmousestemcells.Thus,ourworkrevealsatimelycrosstalkbetweenAPCandSCFdefinesanorderedcascadeofAPCandSCFactivityduringcell-cycletransitions.NoCOI.


3O5J-4Calcineurin B homologous protein 3 (CHP3) modu-lates Akt-GSK3beta signaling in rat cardiomyocytesKobayashi, Soushi; Wakabayashi, Shigeo(Dept. of Mol. Physiol., Natl. Cer.)

CalcineurinBhomologousprotein3(CHP3)isa25-kDaEF-handcalci-um-bindingproteinmainlyexpressedinheart,butitsfunctionremainslargelyunknown.TounderstandtheroleofCHP3,weknockeddownthegeneinratneonatalventricularcardiomyocytesbyusingadeno-virus-mediatedRNAinterferencetechnique.CHP3knockdownsignifi-cantlyenlargedthesizeofcardiomyocytesandincreasedtheproteinexpressionlevelofthehypertrophymarkerANP.Inaddition,CHP3knockdownincreasedthephosphorylationlevelsofAktandGSK3be-ta,keyregulatorsofcardiachypertrophy.Ontheotherhand,whenCHP3wasoverexpressedinlung-derivedfibroblastcellswhichdonotexpresstheprotein,theinsulin-inducedphosphorylationlevelsofAktandGSK3betawasdecreased.Furthermore,co-immunoprecipitationexperimentsdemonstratedtheinteractionofCHP3withGSK3beta.TheseresultssuggestthatCHP3maybeanovelregulatorforcardiachypertrophythroughmodulatingAkt-GSK3betasignaling.NoCOI.

Oral Presentation 06Ionic Channel, Receptor (2) /

Neuron, Synapse

(March 18, 14:00–16:00, Room F)

3O6F-1Compromised GABAergic maturation causes abnor-mal network activity in the hippocampus of epileptic Ca2+ channel mutant mice, totteringNakao, Akito1; Miki, Takafumi1; Shimono, Ken2; Numata, Tomohiro1; Wakamori, Minoru3; Mori, Yasuo1(1Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan, 2Bioscience Technology Development Office, Panasonic Corporation, Kyoto, Japan, 3Department of Oral Biology, Graduate School of Dentistry, Tohoku University, Sendai, Japan)

Cognitiveimpairmentsaredevastatingco-morbiditiesofepilepsy,buttheirunderlyingmechanismsarestillelusive. Inthisstudy,using64-electrodearray(MED64system),weinvestigatednetworkactivityinthehippocampusinP/Q-typeCa2+channelmutanttottering(tg)mice,awell-establishedmodelofspontaneousabsenceepilepsy.Hippocampalslicesoftgmicedisplayedmuscarinicacetylcholinereceptor-inducedepileptiformdischargeswithabnormalhigh-frequencypatternsorigi-natingfromtheCA3region.Thesewereattributabletoadevelopmen-talretardationofGABAergicinhibitioncausedbyimmatureintracel-lularCl- regulationviaabnormality indevelopmentallyregulatedexpressionofCl-transportersandbyGABAAreceptorcompositionsinhippocampalneurons.OurstudysuggeststhatcompromisedGAB-Aergicmaturationcausingabnormalnetworkactivityinthehippo-campuspresumablycontributestocognitiveco-morbiditiesinepilepsyduetoaberrantP/Q-typechannelfunctions.NoCOI.

3O6F-2Genetic and functional analysis of HCN channel mu-tation in familial febrile seizure patientsNakamura, Yuki1; Shi, Xiuyu2; Numata, Tomohiro3; Mori, Yasuo3; Inoue, Ryuji4; Hirose, Shinichi1,5(1Department of Pediatrics, Faculty of Medicine, Fukuoka Univ., Fukuoka, Japan, 2Chinese PLA General Hospital, Beijing, China, 3Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto Univ., Kyoto, Japan, 4Department of Physiology, Faculty of Medicine, Fukuoka Univ., Fukuoka, Japan, 5The Research Institute for the Molecular Pathomechanisms of Epilepsy, Fukuoka Univ., Fukuoka, Japan)

Hyperpolarization-activatedcyclicnucleotide-gated(HCN)currents(Ih)areidentifiedinneuron.Ihmaintainrestingmembranepotentialsandcontrolneuronalrhythmicactivity.HCNchannelsinthehumanbrainareformedbyHCN1andHCN2subunits.RecentstudiesshowthatmutationsinsodiumchannelsandGABAwerefoundinFebrileseizure(FS)patients.However,thesewererareanddonotexplainthecommoncauseofFS.WeexaminedtheHCN2in160FSpatientsandfoundthesamemutation(S126L)in2ofthem.Weperformedawhole-cellpatchclampexperimentforwildtype(WT),homo-mutant(MT)andhetero-mericchannels.Toreplicatephysiologicalhumantemperature, thebathsolution’stemperaturewasset from35to38°C.At35°Candagainat38°C,therewasnosignificantdifferenceinvoltagedependencebetweeneachchannels.However,whenthetemperaturewassetfrom25to38°C,theshiftofthehalfmaximalactivationvoltage(V1/2)forMTchannelssignificantlyincreased.NosignificantdifferenceswerefoundintheresponseofcyclicAMP.TheseresultsindicatethattheS126Lmutationincreasestemperaturesensitivity,suggestingthatthismutationmaytriggertheonsetofFS.NoCOI.


3O6F-3Local temperature changes of epileptogenic zone re-late to disease progression through enhancement of TRPV4 activityShibasaki, Koji1; Tanaka, Kenji F2; Ishizaki, Yasuki1(1Dep Mol Cellular Neurobiology, Gunma Univ Grad Sch Medicine, Maebashi, Japan, 2Dept. Neuropsychiatry, Sch Medicine, Keio Univ, Tokyo, Japan)

Physiologicalbraintemperatureisanimportantdeterminantforneu-ronalfunctions,anditiswellestablishedthatchangesintemperaturehavedynamic influencesonbrainneuronalexcitabilities.Wehaveclearlyrevealedthatathermo-sensorTRPV4(activatedabove34°C)isactivatedbyphysiologicaltemperatureinhippocampalneuronsandtherebycontrolstheirexcitability.Therefore,iflocalbraintemperaturecandynamicallyelevatedependingontheneuronalactivities,ather-mo-sensorTRPV4canenhanceelectricalexcitabilityinneurons,andmightleadtohyperexcitability.Inthisstudy,wefocusedonepilepsy,sinceitwascausedbyhyperexcitabilityofneurons.Wegeneratedamodelofpartialepilepsybyutilizingkindlingstimuliinventralhippo-campusofwildtype(WT)orTRPV4KOmice,andmeasuredelectro-encephalogram(EEG).ThefrequenciesofepilepticEEGinWTmiceweresignificantlylargerthanthoseinTRPV4KOmice.TheseresultsstronglyindicatethatTRPV4activationisinvolvedindiseaseprogres-sionofepilepsy.Weexpectedthatthediseaseprogressionenhancedhyperexcitability,andleadtohyperthermiaintheepileptogeniczones.Toconfirmit,wedevelopedanewdevicetomeasureexactbraintemperatureonlyinrestrictedlocalarea.Fromtherecordingresultsbythenewdevice,werevealedthatthebraintemperaturesinepilep-togeniczonesweredramaticallyelevatedcomparedwithnormalre-gions.NoCOI.

3O6F-4Endocannabinoids shorten seizures presumably by suppressing excitatory synaptic inputs around the in-ner molecular layer of the dentate gyrusSugaya, Yuki1; Yamazaki, Maya2; Sakimura, Kenji2; Kano, Masanobu1(1Dep. of Neurophysiol., Grad. Sch. of Med., Univ. of Tokyo, Tokyo, Japan , 2Dep. of Cell. Neurobiol., Brain Res. Inst., Niigata Univ., Niigata, Japan)

Intemporal lobeepilepsy,expressionofcannabinoidCB1receptorsaroundtheinnermolecularlayer(IML)ofthedentategyrusismark-edlydecreased.However,itisunclearhowdeficientendocannabinoid(eCB)signalingaroundtheIMLaffectsseizures.ThepresentstudyaimedatelucidatingrolesofeCBsignalingaroundtheIMLofthedentategyrusduringseizures.Stimulusandrecordingelectrodeswereimplantedintotheangularbundleandthedentategyri,respectively,ofadultC57/bl6Jmice.Afterdischargeswereevoked30minafterintraperitoneal injectionofaCB1antagonist,AM251 (10mg/kg),orvehicle.Currentsourcedensitywascalculatedfromlocalfieldpoten-tials.WefoundthatseizuresinthedentategyrusweresignificantlylongerinAM251-treatedmicethanvehicle-treatedmice.SeizuresinAM251-treatedmiceconsistedofrepeatedburstdischargestriggeredbyexcitatoryinputsaroundtheIMLofthedentategyrus.Furthermore,thedurationof eachburstdischargewas significantly longer inAM251-treatedmiceduetothesequentialactivationofthetri-synap-ticcircuit inthehippocampus.OptogeneticstimulationoftheIMLprojectionsduringseizuressignificantly increasedthenumberofrepetition,butnotthedurationofeachburstdischargeinwild-typemice.TheseresultssuggestthateCBsignalingaroundtheIMLofthedentategyrusspecificallysuppressesrepetitionofburstdischargesandshortensseizures.NoCOI.

3O6F-5On-demand biosynthesis by diacylglycerol lipaseα is the major source of 2-arachidonoylglycerol, the endo-cannabinoid that mediates retrograde signaling.Hashimotodani, Yuki1; Ohno-Shosaku, Takako2; Kano, Masanobu3(1Dominick P. Purpura Dept Neurosci, Albert Einstein Col. Med., NY, 2Dept. Impair. Study, Grad. Sch. Med. Sci., Kanazawa Univ., Kanazawa, 3Dept. Neurophysiol, Grad. Sch. Med, Univ. Tokyo, Tokyo)

Theendocannabinoid (eCB)2-arachidonoylglycerol (2-AG)producedbydiacylglycerol lipaseα(DGLα) isoneof thebest-characterizedretrogrademessengersatcentralsynapses.2-AGisthoughttobeproduced‘ondemand’uponneuronalactivity.However,recentstudiesproposedthat2-AGispresynthesizedbyDGLαandstoredinneurons,andthat2-AGisreleasedfromsuch‘pre-formedpools’withoutcontri-butionofDGLα.Toaddresswhetherthe2-AGsourceforretrogradesignalingistheon-demandbiosynthesisbyDGLαorthemobilizationfrompreformedpools,weexaminedtheeffectsofacutepharmacolog-icalinhibitionofDGLbyanovelpotentDGLinhibitor,OMDM-188,onseveralformsofeCBsignaling.Wefoundthatbothdepolarization-in-ducedsuppressionof inhibition (DSI),apurelyCa2+-dependenteCBsignaling,andgroupImetabotropicglutamatereceptor(I-mGluR)-in-ducedeCBsignalingwereblockedbyOMDM-188inculturedhippo-campalneurons.WealsofoundthatOMDM-188blockedDSIinacuteslices fromthehippocampus,striatumandcerebellum.Moreover,OMDM-188blockedsynapticsuppressionofPurkinjecellsinducedbyburststimulationofparallelfibers,whichismediatedby2-AGreleasedbycombinationofCa2+influxandmGluR1activation.Ourresultsdonotsupportpre-formed2-AGhypothesisbutstronglysuggestthaton-demand2-AGbiosynthesisbyDGLα;initiatesretrogradesignaling.NoCOI.

3O6F-6BDNF derived from Purkinje cell regulates synapse elimination in the developing cerebellumChoo, Myeong Jeong1; Miyazaki, Taisuke2; Yamasaki, Maya3; Tanimura, Asami1; Uesaka, Naofumi1; Watanabe, Masahiko2; Sakimura, Kenji3; Kano, Masanobu1(1Department of Neurophysiology, Graduate School of Medicine, the University of Tokyo, Tokyo, Japan, 2Department of Anatomy, Hokkaido University Graduate School of Medicine, Sapporo, Japan, 3Department of Cellular Neurobiology, Brain Research Institute, Niigata University, Niigata, Japan)

Eliminationofredundantsynapsesformedatearlierdevelopmentalstageisknownas“synapseelimination”andisconsideredtobeanessentialprocessinneuralcircuitformation.Intheneonatalmousecerebellum,eachPCisinnervatedbymultipleCFs.Duringpostnataldevelopment,early-formedsurplusCFsaregraduallyeliminatedandmostPCseventuallybecomeinnervatedbysingleCFsinmaturemice.However,molecularmechanismsofCFsynapseeliminationstillremainlargelyunknown.Inthisstudy,weexaminedwhetherBrain-derivedneurotrophic factor (BDNF) inpostsynapticPCs is involved inCFsynapseeliminationusingconditionalknockoutmiceinwhichBDNFisdeletedinPurkinjecells(BDNF-PC-KOmice).Inmutantmice,about40%ofPCswereinnervatedbymultiple(twoorthree)CFswhileonly20%ofPCswereinnervatedbytwoCFsincontrolmicefrompostna-talday21(P21).Furthermore,excitatoryinputsfromparallelfibers(PFs)andinhibitoryinputsfrommolecularlayerinterneuronswerealsoalteredinmutantmice.TheseresultssuggestthatBDNFsignal-ingfrompostsynapticPurkinjecellregulatesdevelopmentalsynapseeliminationinthecerebellumeitherbydirectlyinteractingwithpre-synapticCFsor indirectlyaffectingPFsor inhibitorysynapses.NoCOI.


3O6F-7The role of primary somatosensory cortex in causing mirror-image painIshikawa, Tatsuya1; Ishibashi, Hitoshi1,2; Nabekura, Junichi1,2(1Dept Develop Physiol, Nat Inst Physiol Sci, Okazaki, Japan, 2Dept Physiol Sch Life Sci, SOKENDAI, Hayama, Japan)

Ithasrecentlybeenreportedthatplasticchangesofneuronalcircuitsplay importantroles inchronicpain.Wepreviouslyreportedthatchronichindpawpainincreasedtheneuronalactivityandspineturn-overrateincontralateralsomatosensorycortex(S1).Amongthesymp-tomsofchronicpainsyndrome,patientswithperipheralnerveinjurycansufferfromamirror-imagepainthatpersistsatuninjuredsitescontralateraltotheperipheralnerveinjury.However,thereisnoin-formationabouttheactivityofneuronalandglialcellpopulationofipsilateralS1(ipsi-S1)withsinglecellresolution.Inthepresentstudy,therefore,weinvestigatedtheactivityofneuronalandglialcellsinipsi-S1usingin vivo2-photonCa2+imagingunderchronicpaincondi-tions.Followingperipheralnerveligation(PSL),weobservedincreasedCa2+transientcurrentsintheipsi-S1forlayer1inhibitoryneuronsandastrocytes,butthespineturnoverofpyramidalneuronsremainedunchanged.Interestingly,chronicapplicationoftheGABAAreceptorantagonistgabazine (SR95531) tothe ipsi-S1ofPSLmice increasedspineturnoverrate inthe ipsi-S1,anddecreasedthethresholdtomechanicalstimuliintheintacthindpawcontralateraltothePSLsite.Synapticchangesintheipsi-S1inducedadditionalimpairment;thus,anexcitation-inhibitionbalancecouldbeanunderlyingmechanismformirror-imagepain.NoCOI.

3O6F-8Formation of synapses in somatosensory cortex of de-veloping mice: using in vivo two photon imagingMiyamoto, Akiko1,2; Eto, Kei2; Murakoshi, Hideji3; Shibata, Keisuke4; Koizumi, Schuichi4; Nabekura, Junichi1,2(1Dept Physiol, Sch Life Sci, SOKENDAI, 2Dept Homeostatic Develop, Natl Inst Physiol Sci, Okazaki, 3Supportive Center for Brain Research, Natl Inst Physiol Sci, Okazaki, 4Dept Pharmacol, Grad Sch Med and Eng, Yamanashi Univ, Yamanashi)

Ithasbeenrecentlyreportedthatrestingmicroglia,whichareimmunecellsinthecentralnervoussystems,activelyinteractwithsynapses.Forexample,restingmicrogliaselectivelycontactontosynapsesinintactbrainandarealsoinvolvedinsynapseeliminationatcorticalischemia,whichmaycontributetoneuralcircuitreorganization. Inaddition,activatedmicrogliaareknowntoreleaseseveralmoleculesrelatedtosynapseformation.Inthisstudy,wefocusedonwhethermicrogliaareinvolvedinsynapseformationofneocortexatpostnatalday8–10miceduringwhichexcitatorysynapsesarerapidlyincreasinginnumber.Usinganinvivotwophotonimagingtechnique,weob-servedthatfilopodiawasformedatmicroglialcontacteddendriteofL2/3pyramidalcell,whichoccurredagespecifically.Itisknownthatmicrogliainimmaturebrainaremorelikelytobeactiveinmorphol-ogy.Injectionofminocycline,whichlessenstheactivationofmicroglia,andtheirablationinducedbygeneticmanipulationdecreasedcorticalspinesindensity.Since,microglia-inducedfilopodiacouldeventuallybecometoexcitatorysynapses,weexaminedminiatureEPSC(mEPSC)frequency.mEPSCfrequencywassignificantlyreducedinmicrogliaablatedmice.Thesedata indicatesthatmicroglia iscontributedtofunctionalsynapseformationindevelopingmousecortex.NoCOI.

Oral Presentation 07 Kidney, Urination / Cell Physiology /

Sensory Function (2) / Nutrition, Moleculara Physiology (2), Metabolism,

Thermoregulation / Absorption / Heart, Circulation (2)

(March 18, 14:00–16:00, Room J)

3O7J-1Overexpression of Leukocyte Kv1.3-Channels Pro-motes Renal Fibrosis in Rats with Advanced Chronic Renal FailureKazama, Itsuro; Maruyama, Yoshio; Murata, Yoshimichi; Baba, Asuka(1Department of Physiology I, Tohoku University Graduate School of Medicine, Sendai, Japan)

Leukocytes,suchas lymphocytesandmacrophages,predominantlyexpressdelayedrectifierK+-channels(Kv1.3),andthechannelsplaycrucialrolesintheactivationandproliferationofthecells.Sincelym-phocytesareactivatedinpatientswithend-stagerenaldisease(ESRD),thechannelsexpressedinthosecellswouldcontributetotheprogres-sionofrenalfibrosisinadvancedstagechronicrenalfailure(CRF).Inthepresentstudy,usingaratmodelwithadvancedCRFthatunder-went5/6nephrectomyfollowedbya14-weekrecoveryperiod,weexaminedthehistopathologicalfeaturesofthekidneysandtheleuko-cyteexpressionofKv1.3-channelsandcellcyclemarkers.Age-matchedsham-operatedratswereusedascontrols.InthecorticalinterstitiumofadvancedCRFratkidneys, leukocytesproliferated insituandoverexpressedKv1.3-channelproteinintheircytoplasm.Treatmentwithmargatoxin,aselectiveKv1.3-channelinhibitor,significantlysup-pressedthenumberofleukocytesandtheprogressionofrenalfibrosiswithasignificantdecreaseinthecorticalcellcyclemarkerexpression.Thisstudydemonstratedforthefirsttimethatthenumberofleuko-cyteswasdramaticallyincreasedinratkidneyswithadvancedCRF.TheoverexpressionofKv1.3-channelsintheleukocyteswasthoughttocontributetotheprogressionofrenalfibrosisbystimulatingcellcyclingandpromotingcellularproliferation.NoCOI.


3O7J-2ERp57 up-regulates gastric H+,K+-ATPase activity apart from its chaperoning functionFujii, Takuto1; Awaka, Shun-ya1; Shimizu, Takahiro1; Tsukada, Kazuhiro2; Sakai, Hideki1(1Dept. Pharm. Physiol., Grad. Sch. Med. Pharm. Sci., Univ. Toyama, Toyama, Japan., 2Dept. Surg. II, Grad. Sch. Med. Pharm. Sci., Univ. Toyama, Toyama, Japan.)

WefoundthatERp57,which isanERchaperonehavingdisulfideisomeraseactivity,washighlyexpressed inhumangastricparietalcells.ToclarifydistributionofERp57ingastricparietalcells,twotypesofgastricvesicles,intracellulartubulovesicles(TV)andstimulation-as-sociatedvesicles(SAV)containingapicalmembrane,werepreparedfromhoggastricmucosa.ERp57waspredominantlyexpressedinSAVbutnotinTV.GastricH+,K+-ATPasewasexpressedbothinTVandSAV.ERp57wasco-immunoprecipitatedwithH+,K+-ATPase inthelysateofSAV.OverexpressionofERp57intheHEK293cellsstablyexpressingH+,K+-ATPaseα-andβ-subunits (HEK-αβ)significantlyincreasedtheATPaseactivity. Interestingly,overexpressionofacatalyticallyinactivemutantofERp57intheHEK-αβcellsalsostim-ulatedtheATPaseactivity.Incontrast,knockdownofendogenousERp57intheHEK-αβcellssignificantlydecreasedtheATPaseactiv-ity.ItisnotedthatoverexprssionandknockdownofERp57hadnoeffectsontheexpressionlevelofH+,K+-ATPaseα-andβ-subunits.Ontheotherhand,expressionandfunctionofendogenousNa+,K+-ATPasewerenotsignificantlychangedbyoverexpressionandknockdownofERp57inthecells.TheseresultssuggestthatERp57positivelyregu-latesgastricH+,K+-ATPaseactivityapartfromitschaperoningfunction.ItmayregulatetheH+,K+-ATPaseactivityinbasalgastricacidsecre-tion.NoCOI.

3O7J-3Contribution of developmental changes in ionic sys-tems to myocardial excitation-contraction coupling: a simulation studySano, Hitomi I1,2; Toki, Tamami1,3; Aoki, Riko1,2; Naito, Yasuhiro1,2; Tomita, Masaru1,2(1Institute for Advanced Biosciences, Keio University, Kanagawa, Japan, 2Department of Environment and Information Studies, Keio University, Kanagawa, Japan, 3Department of Policy Management, Keio University, Kanagawa, Japan)

Theheartdevelopsandgainsnewfunctionswhilecontinuouslypump-ingblood,andheartabnormalitiesduringtheearlydevelopmentalstagesprogresstocongenitalheartmalformations;therefore,thede-velopmentalprogramoftheheart, includingtheexpressionofthegenesresponsible forvarious ionicchannels, is likelytobetightlyregulated.Here,we integrateddevelopmentalchanges in1) ionicsystemsand2)energymetabolismonthemathematicalmodels.1)Thequantitativechangesinindividualionicsystemswererepresentedasrelativecurrentdensities.Weswitchedtherelativecurrentdensitiesof9ioniccomponentsbetweenearlytolateembryonicstagesintheKyotomodel,andshowedthattheincreaseininwardrectifiercurrentbeforethedisappearanceoffunnycurrentwaspredictedtoresultinabnormallyhighintracellularCa2+concentrations.2)Thechangesinglycolyticenzymaticactivitiesandthoseinconcentrationsofglycogenandtotalcreatinewere implementedaccordinglytothemodeltorepresentspecificdevelopmentalstages.WethensimulatedeffectsofhypoxicconditiontodynamicchangesincontractileforceandATPconcentration.Asaresult,ourmodelshowedthatthefetalventricularcellsmaintainedATPforlongerperiodsoftimethantheadultven-tricularcells,whichisconsistentwiththereporteddynamicsunderhypoxiccondition.NoCOI.

3O7J-4A novel physiological role of muscle fascia as a noci-ceptive sensory tissueTaguchi, Toru(Dept. Neurosci. II, Res. Inst. Environ. Med., Nagoya Univ., Nagoya, Japan)

Thesecondskeleton,fascia,isasheet-orlayer-likestructurewhichfunctionallybindsandseparateswholebodypartsfromheadtotoe,andmakesitpossibleforanimalstoperformsmoothandcooperativemuscularwork.Thefasciaalsoprotects internalstructuresthat itcovers,andformspassagewaysforbloodvesselsandnerves.Exceptforitssupportiveandbiomechanicalproperties,however,physiologicalrolesoffasciahaveneverbeensubjectedtointensiveexplorationinthebiomedicalsciences,eventhoughithaslongbeenconsiderednotonlyanimportantsourceofnociceptionandpainbutalsoacriticaltargetforclinicaltreatmentofpatientswithmusculoskeletalpain.Hereweexaminedperipheralandspinalmechanismsoffascialnociceptioninrats,andfoundthat1)nociceptivenervefiberswithpeptidergicandnon-peptidergicaxonsandterminalsweredistributedinthefascia,2)peripheralafferents(Aδ-andC-fibers)respondingtonoxiousmechan-ical,chemical,andthermalstimuliexistedinthefascia,3)nociceptiveinformationfromthefasciawasprojectedmainlytolaminaeI-IIofthespinaldorsalhornwherethenociceptiveinputismainlyprocessed,and4)spinaldorsalhornneuronsreceivinginputfromthefasciaex-isted,andtheneuronsweredefinitelysensitizedinhyperalgesiccon-ditionof thetissue.Takentogether, theseresultsstrengthenthesuppositionthatmusclefasciaisnotjustasupportivetissuesurround-ingthemuscle,butisanociceptivesensorytissue/organ,andthatitshouldbeatargetoftreatmentinpatientswithmyofascialpainsuchasastiffneckandlowbackpain.COIproperlydeclared.

3O7J-5Hypothalamic neuropeptide Y-induced GABA inhibi-tion of the rostral medullary raphe to inhibit sympa-thetic outflow to brown adipose tissueNakamura, Yoshiko1; Nakamura, Kazuhiro1,2(1Career-Path Promotion Unit for Young Life Scientists, Kyoto University, Kyoto, Japan, 2PRESTO, JST)

InjectionofneuropeptideY(NPY)intotheparaventricularhypotha-lamicnucleus(PVH)orthelateralventriclelowersmetabolismaswellascausesstronghyperphagia.However,theneuralmechanismoftheseobesogeniceffectsofhypothalamicNPYisunknown.WehypothesizedthatNPYsignalingfromthehypothalamusmayinhibitsympatheticpremotorneuronsintherostralmedullaryraphe(rMR)thatcontrolthermogenesisinbrownadiposetissue(BAT).Inthisstudy,weexam-inedthishypothesisbyrecordingBATsympatheticnerveactivity,BATtemperatureandotherparametersinanesthetizedrats,whosetrunkwascoveredwithawaterjackettocontroltheskintemperature.Skincooling-evokedincreasesinBATsympatheticnerveactivityandBATtemperaturewereeliminatedbyinjectionofNPYintothelater-alventricleorPVH.NPYinjectionintothePVHalsoeliminatedBATthermogenesisevokedbyglutamatereceptorstimulationintherMRwithanNMDAnanoinjection.Incontrast,NPYinjectionintothePVHdidnotaffectBATthermogenesisevokedbyantagonizingGABAreceptorsintherMRwithabicucullineinjection.TheseresultssuggestthatNPYsignalingfromthePVHactivatesaGABAergic inputtosympatheticpremotorneuronsintherMRtoinhibitBATthemogen-esis,leadingtoreducedenergyexpenditure.NoCOI.


3O7J-6Preoperative branched-chain amino acids administra-tion improved prognoses after hepatectomy in the el-derly rats with diabetes mellitusWakana, Noriaki1; Todoroki, Kikue1; Homma, Kazuhiro1; Tanaka, Etsuro1; Fukuyama, Naoto2(1Department of Nutritional Science, Tokyo University of Agriculture, Tokyo, Japan, 2Department of Physiology, Tokai University School of Medicine, Kanagawa, Japan)

Tostudywhetherpreoperativebranched-chainaminoacids(BCAA)administrationimprovesprognosesafterhepatectomyintheelderlyratswithdiabetesmellitus(DM),wedidexperimentsintheelderlyratswithDMinwhichBCAAwasadministeredbeforehepatectomy.Weused22elderlymaleFischer344ratsinducedDMbystreptozo-tocin.Theratswererandomlydividedintotwogroups;11ratswithBCAAsolution(Bgroup)and11ratswithwater(Cgroup)for7daysbeforehepatectomy.Then,70%partialhepatectomywasperformedinallratsaccordingtothemethodofHigginsandAnderson.Survivalrate,rateofbodyweightreduction,bloodsampledata(bloodsugar,AST,ALT,endotoxin,TNF-αandIL-6)andbromodeoxiuridine(BrdU)labelingindexbetweentwogroupswereevaluatedonpostoperativeday2.SurvivalrateinBgroupwassignificantlygreaterthanthatinCgroup(100vs55%,respectively,p<0.05).Rateofbodyweightreduc-tioninBgroupwassignificantlysmallerthanthatinCgroup(8.1±2.4vs12.1±1.2%,respectively,p<0.05).Therewerenosignificantdiffer-encesinbloodsugar,AST,ALT,endotoxin,TNF-αandIL-6betweentwogroups.BrdUlabelingindexinBgroupwassignificantlyhigherthanthatinCgroup(14.5±1.7vs11.7±0.8%,restrictively,p<0.05).Thus,preoperativeBCAAadministrationimprovedprognosesafterhepa-tectomyintheelderlyratswithDM.NoCOI.

3O7J-7Severe hypoxia-induced ventilatory depression ana-lyzed by spectrography of EEG and respiratory outputFukushi, Isato1; Kuniya, Mayu2; Maeno, Yoshimi2; Muraoka, Yoshihiro2; Takeda, Kotaro3; Okada, Yasumasa3(1Planning Division, Dental Support Co.,Ltd., Chiba, Japan, 2School of Human Sciences, Waseda University, Tokorozawa, Japan, 3Clinical Research Center, National Hospital Organization Murayama Medical Center, Musashimurayama, Japan)

Althoughmildhypoxiastimulatesbreathing,severehypoxiadepress-esventilationandthisphenomenonmayunderliethepathophysiologyofrespiratoryarrestinpatientswithseverehypoxia.Wehypothesizedthatseverehypoxia-inducedventilatorydepressionisaccompaniedbyadecreaseofcentralcommandfromthehigherbraintothelowerbrainstem.WeanalyzedtheresponsesofhigherbrainactivityandrespiratoryoutputtohypoxiainunanesthetizedadultmicebyEEGandwholebodyplethysmography,respectively.Totestthehypoxicresponse,micebreathedroomair,thenhypoxicgas(mild12%orsevere6%O2inN2)forseveralminutes,andagainroomair.TheEEG,respi-ratoryflow,andinspiredO2concentrationweremeasuredat400Hzsampling.TheEEGandflowdataweredetrendedandbandpassfilteredwith0.1–100Hz,and0.1–40Hz(6thorderButterworth),respectively.DigitalspectrogramsweregeneratedbyafastFouriertransformfromthefilteredwaveforms.Mildhypoxiapersistentlyincreasedventilation.Severehypoxiatransientlyincreasedventilationfollowedbyventila-torydepression.Changesinthespectrumpatternofrespiratoryfre-quencywerefoundinhypoxia.SeverehypoxiadepressivelyinfluencedbothEEGandtherespiratoryoutputpattern.Wesuggestthatseverehypoxiaaffectsthehigherbrainfunctionanddepressesventilation.NoCOI.

3O7J-8Endogenous ATP attenuates hypoxia-induced exci-tation of the RVLM neurons via P2 purinergic recep-tors in the in situ arterially-perfused preparation of rats.Koganezawa, Tadachika; Hoki, Ayaka(Department of Physiology, Division of Biomedical Science, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan)

Ithasbeenknownthatneuronsintherostralventrolateralmedulla(RVLMneurons)generatetheactivityofthecardiovascularsympa-theticnerve(SNA).Recently,wehavereportedthattheRVLMneuronssenseandareexcitedbycentralhypoxia.However,itisstillunclearhowtheRVLMneuronssensehypoxia.Inthisstudy,weexaminedparticipationofendogenousATPviaP2purinergicreceptorsinthehypoxia-sensingmechanismoftheRVLMneuronsinthein situarte-riallyperfusedpreparationofrats.WesystemicallyappliedaP2puri-nergicreceptorantagonist,PPADS(100µM),andanalyzedtheeffectonresponsesofSNAtotheapplicationofNaCNintotheRVLM(5mM,30nl)andthearterialchemoreceptors(0.03%,0.1ml).Asaresult,administrationofPPADSdidnotchangethebasalSNA,butcomplete-lysuppressedtheexcitationofSNAwhichwascausedbyNaCN-in-ducedactivationofthearterialchemoreceptors(control:158.4±35.6%,n=5;PPADS:-3.1±35.1%,n=5;p=0.027).Moreover,administrationofPPADSsignificantlyenhancedtheexcitationofSNAwhichwascausedbyinjectionofNaCNintotheRVLM(control:121.8±26.2%,n=5;PPADS:161.7±37.3%,n=5;p=0.036).TheseresultsmayindicatethatactivationofP2purinergicreceptorsbyendogenousATPisre-latedwithhypoxia-sensingmechanismofthearterialchemoreceptors,andalsosuppressivelyregulatestheexcitationoftheRVLMneuronstohypoxia.NoCOI.

Award Presentations (Poster)


Award Presentations (Poster)Hiroshi and Aya Irisawa Memorial

Award for Excellent Paper on Research in Circulation in the Journal

of Physiological Sciences

SP-1A new calpain inhibitor protects left ventricular dys-function induced by mild ischemia-reperfusion in in situ rat hearts.Takeshita, Daisuke; Tanaka, M; Mitsuyama, S; Yoshikawa, Y; Zhang, GX; Obata, K; Ito, H; Taniguchi, S; Takaki, Miyako (Department of Physiology II, Nara Medical University School of Medicine)

Ourpreviousstudiesshowedthatanewsolublecalpain inhibitor,SNJ-1945(SNJ),attenuatedcardiacdysfunctionaftercardioplegiaar-rest-reperfusionby inhibitingtheproteolysisofα-fodrin in invitrostudy.Nevertheless,toexplorerealistictherapeuticapproachesforclinicaluse,theinvivostudydesignisindispensable.TheaimofthepresentinsitustudywastoinvestigatewhetherSNJattenuatedleftventricular(LV)dysfunction(stunning)afterspeciallydevelopedmildischemic-reperfusion(mI-R)ininsiurathearts.SNJ(60µmol/l,5mli.p.)wasinjected30minbeforegradualandpartialcoronaryocclusionatproximalleftanteriordescendingartery(mI).ToconfirmLVdys-function,weexaminedcurvilinearend-systolicpressure-volumerela-tionshipbyincreasingafterload60minafterreperfusion.InthemI-Rgroup,specificLVfunctionalindicesatmidrangeLVvolume(mLVV),end-systolicpressure(ESPmLVV),andend-systolicpressure-volumearea(PVAmLVV):atotalmechanicalenergyperbeat,linearlyrelatedtooxygenconsumption)significantlydecreased,butSNJreversedthesedecreasestotimecontrollevel.Furthermore,SNJpreventedtheα-fodrindegradationandattenuateddegradationofCa2+handlingproteins(LTCCandSERCA2a)aftermI-R.OurresultsindicatethattherecoveryfromLVdysfunctioninducedbymI-Rinjuryisassociat-edwithinhibitionoftheproteolysisofα-fodrinininsiturathearts.WeconcludethatSNJcouldbeapromisingtooltoprotecttheheartfromthestunning.NoCOI.

Award Presentations (Poster)Aya Irisawa Memorial Promotion Award for Excellence by Women

Physiologists

SP-2From Sodium-Calcium exchanger to Shakuyaku-Kan-zo-toKimura, Junko(Department of Pharmacology, Fukushima Medical University School of Medicine, Fukushima, Japan)

Poster Presentations


Poster PresentationsIonic Channel, Receptor (1)

1P-001Quercetin diminishes the cAMP-stimulated Cl- secre-tion by blocking Na+,K+ -ATPase in epithelial cellsSun, hongxin1; Niisato, Naomi1,3; Marunaka, Yoshinori1,2,3(1Kyoto Pref. Univ. of Med., Kyoto, Japan, 2Kyoto Pref. Univ. of Med., Kyoto, Japan, 3Heian Jogakuin Univ., Kyoto, Japan)

EpithelialCl-secretionplaysanimportantroleinproductionofdrivingforceforwatermovement.Theaimofthepresentstudywastoinves-tigatetheactionofquercetin,aflavonoid,onCl-secretioninepithelialA6cells.QuercetinstimulatedepithelialCl-secretionunderbasalconditions,butdiminisheditundercAMP-stimulatedconditionsviamodificationofactivityofNa+-K+-2Cl-cotransporter(NKCC)contrib-utingtoCl-uptakeacrossthebasolateralmembrane.However,wehavenoideaonmechanismsofquercetinaction.AstheNa+,K+-pumpisessentiallyrequiredforproductionoftheNa+gradientgeneratingNKCCactivity,weassessedeffectsofquercetinonthepumpbymea-suringtheouabain-sensitivecurrentinapicallynystatin-treatedcells.Quercetinreducedthepumpcurrentabout50%,butquercetinstim-ulatedNKCC.Basedontheseobservations,wespeculatethefollowingpoints.1)Underbasalconditions,thepumpactivitywouldbemuchlargerthantheNKCCactivity.Therefore,evenifquercetininhibitedthepump, theNa+gradientwouldbestillkept largeenoughforquercetintofullyshowitsstimulatoryactiononNKCC,resultinginanincreaseofCl--secretion.2)Underforskolin-stimulatedconditions,thepumpactivitywouldnotbemuchlargerthantheNKCCactivity.Therefore,ifquercetininhibitedthepump,theNa+gradientwouldnotbekeptlargeenoughformaintenanceoftheforskolin-stimulatedactivityofNKCC,resultinginadecreaseofCl--secretion.NoCOI.

1P-002Moderate binding affinity of the Na+/H+ exchanger NHE1 for calcineurin is critical for downstream NFAT signaling.Hisamitsu, Takashi; Wakabayashi, Shigeo(Dep. of Mol. Physiol., Natl. Cer. Cardiovas. Ctr, Osaka, Japan)

Calcineurin(CaN),aCa2+-dependentphosphatase,isakeymoleculetogovernpathologicalcardiachypertrophy.CaNdephosphorylatesadownstreamtranscriptionfactorNFAT,whichinturninduceshyper-trophicgeneexpression.However,themechanismofCaNregulationisunclear,becauseCaNisnotactivatedbyincreaseincytosolicCa2+concentrationcausedbytheexcitation-contractioncouplingincardio-myocytes.CaNhasbeenhypothesizedtobeefficientlyregulatedviatheinteractionwithplasmamembraneCa2+-handlingproteinsinthelocalvicinity.Recently,wehavefoundthatapH-regulatingtransport-erNHE1activatedtheCaN-NFATsignaling,leadingtocardiomyocytehypertrophyviadirectbindingofCaNtothe6-residuesmotif(PVIT-ID)inthecytosolicdomainofNHE1.WehypothesizedthatlocalpHincreaseproducedbyNHE1enhancedtheactivityofCaNboundtoNHE1viasensitizingCa2+.However,itisunknownhowCaNsignalistransmittedfromNHE1toNFAT.Inthisstudy,weperformedthedetailedmutagenesisstudyforCaNbindingsiteofNHE1.SubstitutionofthePVITIDsequencewitheitherhigh (PVIVIT)or lowaffinitysequence (PVIAVN)bothabolishedtheCaN-NFATsignaling.Ala-nine-scanningmutagenesisrevealedthattheoriginalNHE1sequencewasthebest forsignalamplification,suggestingthatthebalancedaffinitybetweenNHE1andCaNiscriticalfortheefficientsignaling.WeconsiderthatsuchmoderateinteractionisimportantforremovalofCaNfromNHE1andrebindingtodownstreamtargetNFATafterNHE1-dependentactivation.NoCOI.

1P-003Stabilizing effects of G protein on the active confor-mation of the adenosine receptor type 1a differ de-pending on the type of G proteinTateyama, Michihiro; Kubo, Yoshihiro(Div. Biophysics & Neurobiol., Dept. Mol. Physiol., NIPS, Okazaki, Aichi, Japan)

WerecentlyreportedthatthestabilizingeffectsofGproteinbindingonthestabilizationoftheactiveconformationofthereceptordifferdependingonthetypeofreceptors.Inthepresentstudy,weaimedatinvestigatingtheeffectsofdifferenttypesofGproteinontheactiveconformationoftheGi/o-coupledadenosinetype1areceptor(A1aR),byusingafluorescenceresonanceenergytransfer(FRET)technique.Forthispurpose,examinedGαsubunitsweretheGαi1,GαoandthechimericGαqi5whichbindstotheA1aRandstimulatesGqsignalingpathway.YFPfusedattheC-tailoftheA1aRshowedtheagonist-in-ducedincreasesinFRETfromCFPattachedtotheGβ1subunitinthepresenceoftheGαi1andGαqi5butnotoftheGαo,suggestingthattheYFP-fusedA1aRinteractswiththeGαi1andGαqi5butnotwiththeGαo.ToexaminetheeffectsofthedifferentGαsubunitsontheactiveconformationoftheA1aR,wemadefunctionally intactA1aRFRETconstructswhicharefusedwithYFPandCFPatthethirdintracellularloopandC-tailoftheA1aR,respectively.TheFRETcon-structsshowedslightFRETdecreasesupontheagonistapplication,whichweresignificantlyenhancedbytheGαi1andGαqi5butnotbytheGαosubunits.Inaddition,theenhancingeffectoftheGαqi5ontheFRETdecreasewassignificantlylargerthanthatoftheGαi1.TheseresultssuggestedthateffectsofGproteinbindingonthestabilizationoftheactiveconformationoftheA1aRdifferdependingonthetypeofGprotein.NoCOI.


1P-004Structural rearrangements of the linker beta strands in P2X2 are coupled to the pore in a voltage dependent manner Keceli Batu; Kubo, Yoshihiro(National Institute for Physiological Sciences, Okazaki, Japan)

P2X2isanextracellularATPgatedcationchannel,whichshowsvolt-agedependentgatingwithnocanonicalvoltagesensor.Threeinter-subunitATPbindingsitesarelinkedtoporeformingtransmembrane(TM)domainsbyβ-strands.Weanalyzedvoltagedependentstructur-alrearrangementsof the linkerstrandsusinganengineeredthiolmodifiablesite.(1)WeobservedthatadoublemutantsofD315C&I67C(atβ-14andβ-1respectively)showsincreaseincurrent3-6foldbyreducingagent(DTT).WashoutofDTTandapplicationofCd2+in-ducedcurrentdeclineduetothebondformationbetweenD315CandI67C.ThiseffectwasabsentinWTorineithersinglepointmutants.(2)Cd2+ inducedcurrentdeclinewasanalyzedatdepolarizedandhyperpolarizedpotentialswithdifferentpulseprotocolsinthepresenceandabsenceofATP.(3)CurrentdeclinebyCd2+couldnotbeobservedintheabsenceofATP,butonlyinpresenceofATP,suggestingstatedependentmodification.(4)InpresenceofATP,Cd2+modificationwasfasterinhyperpolarizedconditionsthaninthedepolarizedcondition,suggestingvoltagedependentstructuralrearrangementsofthebetastrandslinkingATPbindingsitetoTMdomains.(5)Finallyweper-formedthesimilaranalyseswithaporemutantT339Swhichmakesthechannelconstitutivelyactiveatallmembranepotentials.WithT339SCd2+modificationratesweresimilarindepolarizedandhyper-polarizedconditions.Overallresultssuggestthatstructuralrearrange-mentsofthe linkerdomainsofP2X2arecoupledtothepore inavoltagedependentmanner.NoCOI.

1P-005Endothelin-1 induces contraction through endothelin receptor A coupled with Gq/11 in bovine ciliary muscleMiyazu, Motoi; Ishii, Nobuhito; Takai, Akira(Dept. Physiol., Asahikawa Med. Univ., Asahikawa, Japan)

Purpose:Todeterminereceptortypeassociatedwiththecontractileeffectofendothelin-1(ET1)onbovineciliarymuscleandinvestigatethesignallingmechanisminvolved.Methods:Isometrictensionwasrecordedindissectedciliarymusclebundles,usingaU-gaugetransducer.Isolatedmyocyteswereusedforrecordingwholecellcurrents.ExistenceandlocalizationofendothelinreceptorsAandB(ETRAandETRB),M3-muscarinicreceptor(M3R)andGq/11αwereexaminedbyimmunofluorescencemicroscopy.Results:ET1 (1-100nM)producedaslowlydevelopingcontractileresponse(τ=58±15min;n=12)inadose-dependentmanner(K=6±1nMandh=1.1±0.1;d.f.=15).Likecontractionevokedbycarbachol(CCh;0.1–10µM),ET1-evokedcontractionwasdependentonextracel-lularCa2+.ItwascompletelyinhibitedbyYM-254890(0.5µM),aGq/11-in-hibitorandpartiallyalsobyY-27632(10µM),aRhokinaseinhibitor.UnlikeCCh-evokedcontraction,however,ET1-evokedcontractionhadnorapidphasiccomponentandwaswashedoutslowly.ETRAantag-onistsdose-dependently inhibitedtheET1-evokedwhereasETRBantagonistsshowednoeffect.Underwhole-cellvoltageclampET1weaklyactivatedtwotypesofCa2+permeablenon-selectivecationchannel(NSCC)whichareknowntoopenuponM3Rstimulation.Im-munofluorescencemicroscopyrevealedadenseco-localizationofETRAandM3Rinthecellmembrane.Conclusion:TheET1-evokedcontractionofbovinecilarymuscle ismediatedbyETRAlinkedwithaGq/11signallingmechanismthatcommunicatesinsomemannerwithNSCCsandRhokinasepathway.NoCOI.

1P-006Area-specific D1 dopamine receptor expression in adult mouse astrocytesNagatomo, Katsuhiro1; Suga, Sechiko2; Yamamoto, Yoshio3; Yamada Katsuya1(1Dept. Physiol., Hirosaki Univ. Grad. Sch. Med., Hirosaki, Japan, 2Hirosaki Univ. Health & Welfare, Hirosaki, Japan, 3Lab. Vet. Biochem. & Cell Biol., Dept. Vet. Sci., Fac. Agr., Iwate Univ., Morioka, Japan)

Dopaminergicneuronsinthemidbrainnucleussubstantianigraparscompacta (SNc)releasedopamine (DA) fromtheirdendrites,whichextenddeeplyintotheadjacentsubstantianigraparsreticulata(SNr),consistingmostlyofGABAergicneurons.Indeed,realtimeRT-PCRandimmunohistochemistryshoweddiffuseexpressionofD1dopaminereceptor(D1R)intheSNr.However,mostacutelydissociatedGAB-AergicSNrneuronsshowednoresponsetoDAappliedinourperfo-ratedpatchexperiments.Interestingly,examinationofD1R-YFPtrans-genicmicerevealedthatSNrglialcellsexpressstrongD1R(i.e.YFP)signalintheirprocess.Inthepresentstudy,wecomparedD1Rex-pressioninacutelydissociatedneuronsandastrocytesinSNr,striatum,andcerebralcortexbydouble immunostainings.VesicularGABAtransporter(VGAT)-venusmicewereusedaswell.Fromthesestudies,wesuggestarea-specificD1Rexpressioninastrocytes.NoCOI.

1P-007N-glycosylation modulates AMPA receptor channel propertiesKandel, Munal Babu1; Wakazono, Yoshihiko1; Midorikawa, Ryosuke1; Oka, Shogo2; Takamiya, Kogo1(1Department of Neuroscience, Integrative Physiology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan, 2Department of Biological Chemistry, Human Health Sciences, Graduate School of Medicine, Kyoto University, Kyoto, Japan)

TheintracellularmolecularmechanismsunderlyingtheregulationoftheAMPAreceptorhavebeendramaticallyelucidatedinthepastfewdecades.Incontrast,theregulationoftheextracellulardomainremainsunclear.Here,wefocusedonN-glycosylationoftheAMPAreceptorintheextracellulardomainandtriedtoclarifytheir functionsbycombiningmolecularbiologicalandelectrophysiologicaltechniques.First,weexaminedtheeffectsofPNGaseF,whichisaglycosidasethatcleavestheN-glycosylationsiteatanasparagineresidue,onAMPAcurrentsinprimaryhippocampalculturedneuronsandHEK293cellsexpressingGluA1usingawhole-cellpatch-clamptechnique.Thedi-gestionofN-glycosylationinducedthere-sensitizationofAMPAcur-rentsintheboth.SixputativeN-glycosylationsitesarelocatedintheextracellulardomainofGluA1.Next,weperformedmutationstudiesofeachN-glycosylationsiteofGluA1toidentifytheN-glycosylationsiteresponsibleforthere-sensitizationinducedbyPNGaseFtreatment.TheseresultsindicatedthatN401wasacriticalsitefortheexpressionofre-sensitization.Finally,weanalyzedthepossibilityofre-sensitizationafterPNGaseFtreatmentusingacutebrainslices.SingleelectricalstimulationofSchaffercollateraldidnotshowthere-sensitizationinhippocampalpyramidalneurons,however,pairedpulsestimulationgeneratedthere-sensitization.NoCOI.


1P-008Metabotropic glutamate receptor-dependent slow Ca2+ oscillations in striatal neurons and astrocytes.Tamura, Atsushi1; Yamada, Naohiro2; Yaguchi, Yuichi2; Osanai, Makoto1(1Tohoku Univ, Grad. Sch. Med, Sendai, Japan, 2Grad. Sch. Eng, Osaka Univ., Suita, Japan)

Thestriatumplaysan importantrole in linkingcorticalactivitytobasalgangliaoutputs.GroupImetabotropicglutamatereceptors(mGluRs)areexpressedabundantlyinthestriatalprojectionneuronsandmaybeatherapeutictargetforParkinson’sdisease.ThegroupImGluRsareknowntomodulatethe intracellularCa2+signaling.TocharacterizeCa2+signalinginstriatalcells,spontaneouscytoplasmicCa2+transientswereexaminedinacuteslicepreparationsfromtrans-genicmiceexpressinggreenfluorescentproteinintheastrocytes.Inboththeputative-neuronsandastrocytesofthestriatum,spontaneousslowandlong-lastingintracellularCa2+transients(referredtoasslowCa2+oscillations),whichlasteduptoapproximately200s,werefound.Neitherthe inhibitionofactionpotentialsnor ionotropicglutamatereceptorsblockedtheslowCa2+oscillation.Depletionoftheintracel-lularCa2+storeandtheapplicationofantagonistsagainst inositol1,4,5-trisphosphate(IP3)receptorsandmGluR5blockedtheslowCa2+oscillationinbothputative-neuronsandastrocytes.Thus,themGluR5-IP3signalcascadeistheprimarycontributortotheslowCa2+oscillationinbothputative-neuronsandastrocytes.TheslowCa2+oscillationfeaturesmulticellularsynchrony,andbothputative-neuronsandas-trocytes participate in the synchronous activity. Therefore, themGluR5-dependentslowCa2+oscillationmayinvolveintheneuron-gliainteractioninthestriatum.NoCOI.

1P-009Comprehensive behavioral test battery analyses of the gene targeted mice of Prrt3, an orphan metabo-tropic receptorYamamoto, Tomomi1; Hattori, Satoko2; Kiyonari, Hiroshi3; Nakao, Kazuki4; Miyakawa, Tsuyoshi2; Kubo, Yoshihiro1(1Div Biophys and Neurobiol, Natl Inst Physiol Sci, Okazaki, 2Div Systems Medical Science, Fujita Health Univ, Toyoake, 3Lab for Animal Resources and Genetic Engineering, RIKEN CDB, Kobe, 4Lab of Animal Resources, Faculty of Med, Univ of Tokyo, Tokyo)

Prrt3 isanorphanmetabotropicreceptorwitha longN-terminusextracellularregion,anditbelongstofamilyCwhichmGluandGAB-ABreceptorsalsobelongto.TheexpressionofPrrt3mRNAinthebrainsuchasinhippocampusisshownintheAllenBrainAtlas,butthere isno informationabout its functionso far. ToapproachthephysiologicalsignificanceofPrrt3,weraisedknockout (KO)mice,usinggenetargetedEScellsobtainedfromKOMPRepository.ThehomozygousKOmiceweresmallinthebodysizeandmostofthemdiedwithin1weekafterbirth,demonstratingtheimportanceofthefunctionofPrrt3.AsitwasnotpossibletoobtainsufficientnumberofhomozygousKOmice,wecarriedoutcomprehensivebehavioraltestbatteryanalysisusingheterozygousKOmice. BytheBarnesprobetest,weobservedasignificantdecrease intheretentionofspatialmemoryafter4weeksintervalfromthecompletionoftraining.Bythefearconditioningtest,wealsoobservedasignificantdecreaseintheretentionoffearmemoryafter4weeks.AsheterozygousKOmiceofmostofthemetabotropicreceptorsinthebraindonotshowclearbehavioralabnormalities,itisnoteworthythatheterozygousKOmiceofPrrt3geneshowedclearphenotype,demonstratingthecriticalroleofPrrt3inthebrainfunctionsuchasmemoryretention.NoCOI.

1P-010Expression patterns of an orphan metabotropic recep-tor Prrt3 in miceYamamoto, Izumi1; Konno, Kohtaro2; Yamamoto, Tomomi1; Watanabe, Masahiko2; Kubo, Yoshihiro1,3(1Div Biophys and Neurobiol, NIPS, Okazaki, Japan, 2Dept Anatomy, Hokkaido Univ Grad Schl Med, Sapporo, Japan, 3Physiol Sci SOKENDAI, Hayama, Japan)

Metabotropicreceptors,alsoknownasG-proteincoupledreceptors(GPCRs)areinvolvedinmajorphysiologicalprocesses,andtheyaretargetsformodernmedicine.However,manystillremainsasorphanreceptors,andthediscoveryoftheir ligandsortheirphysiologicalfunctionshavebeenawaited.Prolinerichtransmembraneprotein3(Prrt3)isanorphanmetabotropicreceptor,anditsphysiologicalfunc-tionsincludingitsexpressionpatternshavenotbeenfullyunderstood.Here,weexploredtheexpressionpatternsofPrrt3 inmiceusingbiochemicalandhistochemicalmethods.OurWesternblotanalysisexperimentsusingmembranefractionsofvariousorgansfromWTandhomozygousPrrt3KOmice indicatedPrrt3 isabundantlyex-pressed inthebrain. In immunohistochemicalanalysisusingfreshfrozensectionsofmousebrain,weobservedspecificexpressionofPrrt3inhippocampus,thalamusandsubstantianigraofWTmice.BycloseanalysisthePrrt3expressionwasobservedbutnotinthesoma.Prrt3expressionindifferentsubcellularfractionswasalsoanalyzedusingWesternblot.TheresultsshowedPrrt3expressionwasinthesamefractionsassynaptophysinandnotPSD95,suggestingPrrt3isnotexpressedatthepostsynapticdensities.Synaptophysinisknowntoplayaroleinvesicleexocytosis,andexpressedinpresynapticter-minals.Takentogether,wespeculatePrrt3isexpressedinneurites,andpossiblyplayrolesatthepresynapticterminalinthebrain.NoCOI.

1P-011Voltage Sensing Mechanism in the Voltage-Gated H+ ChannelFujiwara, Yuichiro; Okamura, Yasushi(Integrative Physiology, Grad. Sch. of Med., Osaka University)

Voltage-gatedchannelsareresponsibleforsensingmembranepoten-tialandgeneratingelectricalimpulsesinmanyorgans.Inthevoltagesensingprocess,positivelychargedresidues inthevoltagesensordomainareknowntosensemembranepotentialandmoveacrossthemembraneelectricfiled,generatingatransient‘gatingcurrent’aheadofanioniccurrent.However,thevoltage-gatedH+channel(Hv)doesnotshowthegatingcurrentalthoughitbehavesasavoltage-gatedchannel.MolecularstructureofHvisalsounique,whereithasnocanonicalporedomain;andthevoltagesensordomainbearsbothvoltagesensingandH+permeation.Hence,theyareconsideredtobeinextricablyassociatedwitheachother,butthedetailshaveyettobeelucidated.HerewereportthatthegatingcurrentaheadoftheH+currentwasobservedinamutantHvchannel,andanadditionalmu-tation eliminated only the component of the H+ current. Thecharge-voltage(Q-V)relationshipofthegatingcurrentwasshiftedbychangingthepHo/igradientintherecordingsolutions,andthecon-ductance-voltagerelationshipoftheH+currentwellfollowedtheQ-Vshift.WealsoobservedthattheQ-VrelationshipwasshiftedtowardthedepolarizationdirectionbyapplicationofextracellularZn2+,wherethekineticsoftheon-gatingcurrentandthefollowingH+currentactivationweredecelerated;butthedeactivationkineticsofthegatingwasnot.TheseresultssuggestthatthevoltagesensingandtheH+permeationcanbeconsideredseparatelyintheHvchannel,andex-tracellularZn2+,aphysiologicalblocker,competesthevoltageon-sens-ing.NoCOI.


1P-012The role of conserved aromatic residues at the S4 voltage-sensor helix in the voltage-gated H+ channelOkuda, Hiroko; Okamura, Yasushi; Fujiwara, Yuichiro(Integrative Physiology, Grad. Sch. of Med., Osaka University)

Thevoltage-gatedH+channel (Hv) isavoltage-sensor likeproteinconsistingoffourtransmembranesegments(S1-S4).Thefunctionalunitisuniquelyadimer,andwerecentlyproposedthatthetwovolt-age-sensorS4helicesformedaninteractioninterfaceinthedimericchannel.TheaminoacidsequenceofS4iswellconservedinHv,anduniquely,Tryptophan(Trp)atthemiddleofS4 is100%conservedamongspecies.Trphasabulkysidechainandsoshouldbelesstol-eratedinpositionsthatinvolvedinprotein–proteininteractions.TounderstandfunctionalsignificancesoftheseeminglyunfavorableTrp,andwesystematicallymademutantsandexaminedtheirelectrophys-iologicalproperties. ThemutationofTrpsignificantlyacceleratedkineticsofdeactivation.AnalyzingtheeffectofTrpintroducedintothebackgroundofthemutantwhichhasnoaromaticresidueinS4,wefoundthatseveralTrpintroducedintoaroundtheoriginalTrppositioneffectivelyrecoveredtheslowdeactivationkinetics.Further-more,thedecelerationeffectbytheTrpintroductionwasnotobservedinthemonomericchannelmutants.MutationcycleanalysisindicatesthatthetwoTrpintheS4areenergetically-coupledindeactivationbutlesscoupledinactivation.TheseresultssupporttheideasoftheS4-S4interfaceinthedimericchannelandtherotationalmovementofS4duringthegating,andproposethatsterichindrancebetweentheoppositeTrpprovidestheuniqueslowdeactivation.

1P-013Chimeric study between sea urchin and mouse ortho-logs of voltage-gated proton channel revealed the critical sites for the activation speedSakata, Souhei1; Miyawaki, Nana1; Kurokawa, Tatsuki1; Oezkucur, Nurdan2; Okamura, Yasushi1(1Lab. of Integrative Physiology, Graduate School of Medicine, Osaka University, Osaka, Japan, 2Tufts Center for Regenerative and Developmental Biology,Tufts University, Boston, USA)

VSOP/Hv1isavoltage-gatedprotonchannelthatcontainsavoltagesensordomainlackingaporedomain.HumanandmouseorthologsofVSOP/Hv1exhibitsloweractivationkineticsthanothervoltage-gatedionchannels.SlowgatingisfavorableforNADPHoxidaseactivitiesinimmunecells.However,molecularmechanismsofslowkineticsofVSOP/Hv1remaintobefullyelucidated.WehaverecentlyfoundthatactivationkineticsofseaurchinVSOP/Hv1(Sp-VSOP)wasmorethan20-foldfasterthanthatofmouseVSOP/Hv1(mVSOP)uponheterolo-gousexpressioninHEK293cells.TounderstandmolecularbasisforrapidgatingofSp-VSOP,wemadechimerasbetweenmVSOPandSp-VSOP.TheactivationkineticsofSp-VSOPwasdeceleratedwhenthe3thtransmembranesegment(S3)andNterminalcytoplasmicregionweresubstitutedbythecorrespondingregionsofmVSOP.MutationinS3inSp-VSOP(R141H/S142H/S142F)deceleratedactivationkinet-ics.TolocalizeacriticalregionwithintheN-terminusfortheactivationkinetics,putativehelicalstructureintheNterminalregionofSp-VSOPwassubstitutedbythatofmVSOP.ThischimericconstructexhibitedsloweractivationkineticsthanSp-VSOP(WT).TheseresultshighlightS3andthehelicalstructureofNterminalregionascriticaldetermi-nantsoftheactivationkineticsofmVSOP.Mechanisticinsightsintogatingmechanismswillalsobeprovided.NoCOI.

1P-014Membrane topology analysis of the voltage-gated pro-ton channel at resting stateKurokawa, Tatsuki1; Okamura, Yasushi1,2(Grad. Sch. of Med., Osaka Univ., 2Grad. Sch. of Frontier Biosci., Osaka Univ.)

Thevoltage-gatedprotonchannel(Hv1)isaproteinthatcontainsthevoltagesensordomainbutnotporedomain.Thevoltagesensordomainplaysbothroleinvotagesensingandprotonpermeation.Theputativefourthtransmembranesegment(S4)ofmouseHv1(mHv1)hasthreepositivelychargedresidues.S4issuggestedtoundergoamovementduringactivationofthechannels,causingconformationalchangepos-sibly leadingto formationofproton-selectiveconductionpathway.However,detailedmechanismsofprotonpermeationofHv1areun-known.Inthisstudy,wetookanapproachofbiochemicalcysteinescanningtodefineresiduesfacingtheaqueousenvironmentofmHv1.Accessibilitiesof twocystene-bindingmolecules,N-ethylmaleimide(NEM)and4-acetamido-4’-maleimidylstilbene-2,2’-disulfonicacid(AMS),wereexaminedoncysteineintroducedintoindividualsites.OnlythefirstarginineonS4(R1:R201)wasinaccessiblebyNEMandAMSinmHv1,suggestingthattheaccessibilityprofilerepresentstherestingstateofmHv1.WealsofoundthatD108,acriticalresidueforprotonselectivity,canbeaccessedbothbyNEMandAMS,supportingamodelthatD108facesrelativelylargevestibuletowhichbothanionsandcationsareaccessible.Inaddition,R201,D181,E115werebothinaccessiblebyNEMandAMS,raisingapossibilitythatR201formssaltbridgewithD181orE115formingabarriertogetherwiththehydrophobiccoreregioninrestingstate.ThesefindingswillprovideinsightsintostructuralbasisforprotonpermeationandgatingofHv1.NoCOI.

1P-015Acceleration of the activation of the voltage-gated proton channel by the unsaturated fatty acidKawanabe, Akira; Okamura, Yasushi(Graduate School of Medicine, Osaka University, Osaka)

Theunsaturatedfattyacidsareimportantcomponentofthebiologicalmembranesandtheprecursorsofmediatorsofinter-andintra-cellularsignaling.Itiswell-knownthattheunsaturatedfattyacids,includingarachidonicacid(AA,20carbonsand4doublebonds),activatedorinhibitedtheionpermeationsofvariousionchannels.Thevoltage-gat-edprotonchannel(VSOP/Hv1)cancontroltheprotonconductancebymembranevoltageandpH.IthelpstheproductionofreactiveoxygenspeciesbyNADPHoxidaseinimmunocytes.TheenhancementoftheproductionofreactiveoxygenspeciesbyAAhasbeenreportedtobeaccompaniedbythe increaseoftheprotoncurrents inneutrophils,macrophages,andeosinophils.However,thedetailedmolecularmech-anismsofactionshaveremainedelusive.HerewereporttheeffectsofAAonmouseVSOP/Hv1heterologouslyexpressedinHEK293Tcellsbyinside-outpatchclamptechnique.TheadditionofAAwithrapid-perfusionsystemimmediatelyincreasedthemagnitudeoftheprotoncurrentsthroughmVSOP/Hv1thatareevokedduringoneseconddepolarizingstepby20times.AfterwashoutofAA,thecur-rentsrapidlyreturnedtotheoriginalcurrentlevel.Theanalysiswithsixtyseconddepolarizingpulseshowedthattheactivationkineticswasmorethan15timesacceleratedbyAA.WealsoexaminedtheeffectsofotherfattyacidsandconstructsofmVSOP/Hv1.Basedontheseresults,wewilldiscussthemolecularmechanismsandthees-sentialsitesfortheactionsofunsaturatedfattyacidsonVSOP/Hv1.NoCOI.


1P-016Metabolic Phenotype in Voltage-Gated Proton Chan-nels Knockout MiceKawai, Takafumi1; Okochi, Yoshifumi1; Imura, Yoshio2; Furukawa, Yuko1; Miyawaki, Nana1; Sakimura, Kenji3; Koizum, Schuichi2; Okamura, Yasushi1(1Lab. of Integr. Physiol., Grad. Sch. of Med., Osaka Univ. Suita, Japan, 2Dept. Neuropharmacol, Univ Yamanashi, 3Dept. of Cellular Neurobiology, Brain Research Institute, Niigata Univ.)

Voltage-sensoronlyprotein,VSOP/Hv1,consistsofthevoltage-sensordomain lackingporedomain. It functionsasvoltage-gatedprotonchannel(Sasakietal,Science.2006)anditsexpressionisspecifictomicrogliainmicebrain.Inphagocytessuchasneutrophil,macrophageandmicroglia,VSOPhasacrucialroleintheproductionofreactiveoxygenspecies (ROS)bycompensatingforcharge imbalanceuponelectronextrusionbyNADPHoxidase.Inthepresentstudy,wefoundthatfemaleVSOPknockoutmiceshowmetabolicphenotypeattheageof6monthandover.Bodyweightgainandincreasedfoodintakewasobservedinfemaleknockoutmice,andtheincreaseinvisceralfatismoreprominentthanthatofsubcutaneousfatinVSOPknockoutmice.Wealsofoundthatleptinlevelandinsulinlevelinbloodserumareincreasedin12montholdVSOPknockoutmice.Recently,ithasbeenreportedthatneuroinflammationinhypothalamusiscloselyas-sociatedwithobesity,suggestingthatVSOPdeficiencyinmicemi-crogliafacilitatestheinitiationofinflammationinmicehypothalamus.OurpreliminaryresultssuggestthatprimaryculturedVSOPknockoutmicemicrogliaappearstoshowdecreasedphagocyticability,whichmaybeinvolvedinthefacilitationofinflammationinthebrain.Inthepresentstudy,themechanismunderlyingthemetabolicphenotypeinVSOPknockoutmiceisdiscussed.NoCOI.

1P-017Analysis of the role of voltage-gated proton channel Hv1/VSOP in neutrophil chemotaxisOkochi, Yoshifumi1; Okamura, Yasushi1,2(1Integrative Physiology, Graduate School of Medicine, Osaka University, Osaka, Japan, 2Graduate School of Frontier Biosciences, Osaka University, Japan)

Neutrophilchemotacticmovementisnecessaryforpathogenelimina-tionandinflammatoryresponsesandthemotilityishighlyregulated.ChemoattractantssuchasN-formylpeptidefMLF,leukotrieneB4andIL-8activateneutrophil,leadingtoROSgenerationbyNADPHoxidase.Voltage-gatedprotonchannelHv1/VSOPdiscoveredinourlaboratoryhelpsROSproductionthroughtheregulationofNADPHoxidaseac-tivity.MouseneutrophilslackingHv1/VSOPgeneexhibitlessproduc-tionofROS,lowerpHincytoplasm,moredepolarizedmembranepo-tentialandlesscalciumioninfluxthanwild-typeneutrophilswhentheoxidaseisactivated.WetestedhowchemotaxisbehaviorisaffectedinHv1/VSOPdeficientneutrophils.Usingtranswellchamber,bonemarrowderivedneutrophilswerestimulatedwithfMLFandcountedthenumberofcellsmigratingtofMLF-containingwell.WefoundanabnormalchemotacticresponsetofMLFinHv1/VSOPdeficientneu-trophils:Hv1/VSOPdeficientneutrophilsshowednormalresponseto10µMfMLF,buttheyrespondedmorestronglyto1µMfMLFthanwild-typeneutrophils.TheseresultsmaysuggestthatHv1/VSOPregulatesthesensitivityforchemoattractant.Wearetryingtoidenti-fywhichfactorcausesabnormalchemotacticresponse.NoCOI.

1P-018Decreases in availability of voltage-gated proton channels through endocytic internalization induced by increases of intracellular pH in osteoclasts and microgliaKuno, Miyuki; Li, Guangshuai; Hino, Yoshiko; Moriura, Yoshie; Kawawaki, Junko; Sakai, Hiromu(Department of Physiology, Osaka City University Graduate School of Medicine, Osaka, Japan)

Voltage-gatedprotonchannels(H+channels)arehighlyproton-selectiveandcontributetonaturalimmunity.ThethroughputofH+channelsisregulatedprimarilybytrans-membranevoltage-andpH-gradients.Inaddition,thenumbersofH+channelsmaychangeinresponsetovaryingcellularconditions.Inthisstudy,weinvestigatedtheroleoftheintracellularpH(pHi)inregulatingtheavailabilityofH+channelsintwomacrophage-lineagecells,osteoclastsandmicroglia.Inwhole-cellclamprecordings,thepHiwaselevatedafterexposuretoNH4Clandreturnedtothecontrollevelafterwashout.However,theH+channelconductancedidnotrecoverfullywhenthepHiincreasewasprolonged.ThedecreasesintheH+channelconductanceswereaccompaniedbyreductionsinthecellcapacitance.ExposuretoNH4Clincreasedtheuptakeoftheendocytosismarker,FM1-43,confirmingthatpHiincreas-esfacilitatedendocytosis.ThepHiincreasesinducedbyV-ATPasesandH+channels,endogenousH+-transferringmechanisms, inpartfacilitatedendocytosis.Similarresultswereobserved inosteoclastsandmicroglia,butnotinCOS7cellsexpressingamurineH+channelgene.TheseresultssuggestthatpHiincreasesdecreasetheavailabil-ityoftheplasmamembraneH+channelsthroughfacilitationofdy-namin-dependentendocytosis inosteoclastsandmicroglia,andthatsignificantnumbersofH+channelsarenotinuseatphysiologicalpHi.NoCOI.

1P-019Proton permeation through the polytheonamide B channel: Single channel current recordings and a pro-ton permeation modelMatsuki, Yuka1; Iwamoto, Masayuki2; Matsunaga, Shigeki3; Oiki, Shigetoshi2(1Departments of Anesthesiology and Reanimatology, Faculty of Medicine Sciences, The University of Fukui, Japan , 2Departments of Molecular Physiology and Biophysics, Faculty of Medicine Sciences, The University of Fukui, Japan, 3Laboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan)

A48-merpeptidefromamarinespongeTheonellaswinhoei,polythe-onamideB (pTB), ishighlycytotoxicagainsteukaryoticcells.TheaminoacidsequenceofpTBisunprecedented,havingalternativeD-andL-aminoacidresiduesthroughoutthe48-merpeptide.ThepTBchannelformsaβ6.3-helixsimilartothegramicidinAchannel,andallowspermeationofmonovalentcations. InthisstudyweexaminedtheprotonconductionofthepTBchannelbyuseoftheplanarlipidbilay-ertechnique.Single-channelI-VcurveinHClsolutionexhibitedweakinwardrectification.Thesingle-channelamplitudeat1MHClwas～100pAat+200mV,whichismorethan40timeshigherthanthatofCs+.Thishighconductancesuggeststhatprotonspermeateviawaterchaininthechannel (theGrotthussmechanism).At lowHClconcentration,theI-Vcurvewassub-linearathighpotentials.Also,theconcentration-dependencyofsingle-channelconductancethroughthepTBchannel(pH-logIcurve)showedtheslopeofone.Theseresultsindicatedthattheprotonpermeationisdiffusion-limited.TheprotonpermeationthroughthepTBchannelwasmodeledwiththedis-crete-stateMarkovmodel.NoCOI.


1P-020Optically detected structural change in the N-terminal region of the voltage-sensor domainTsutsui, Hidekazu; Jinno, Yuka; Tomita, Akiko; Okamura, Yasushi(Laboratory of Integrative Physiology, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan)

Thevoltage-sensordomain(VSD)isafunctionalmodulethatundergoesstructuraltransitionsinresponsetomembranepotentialchangesandregulatesitseffectors,therebyplayingacrucialroleinamplifyinganddecodingmembraneelectricalsignals.Ion-conductiveporeandphos-phoinositidephosphatasearethedownstreameffectorsofvoltage-gat-edchannelsandthevoltage-sensingphosphatase,respectively.Upontransition, it isknownthattheVSDgenerallyactsontheregionC-terminaltoS4.However,ithasnotbeenaddresseddirectlywheth-ertheVSDinducesanystructuralchangesalso intheN-terminalregionofS1.Here,wereporttheexistenceofsuchanN-terminaleffect.Weusedtwodistinctopticalreporters,onebasedontheForsterresonanceenergytransferbetweenapairoffluorescentproteinsandtheotherbasedonfluorophore-labeledHaloTag,andstudiedthebe-haviorofthesereportersplacedattheN-terminalendofthemono-mericVSDderivedfromvoltage-sensingphosphatase.WefoundbothofthesereporterstobeaffectedbytheVSDtransition,generatingvoltage-dependentfluorescencereadouts.Wealsoobservedthatwhilethevoltagedependenciesof theN-andC-terminaleffectsappeartightlycoupled,thelocalstructuralrearrangementsreflectthewayinwhichtheVSDwasloaded,demonstratingtheflexiblenatureoftheVSD.NoCOI.

1P-021Regulatory role of arachidonic acid on human cardiac Kv1.5 channelsBai, Jiayu; Ding, Wei-Guang; Xie, Yu; Matsuura, Hiroshi(Dept. Physiol. Shiga Univ. Med. Sci., Otsu, Japan)

Arachidonicacid(AA)isanimportantconstituentofmembranephos-pholipidsandcanbeliberatedbyactivationofcellularphospholipaseA2.AAmodulatesavarietyofcardiacionchannelsviadiversemech-anisms,includingbothdirecteffectsbyAAitselfandindirectactionsthroughAAmetabolites.HumancardiacKv1.5channel (hKv1.5) isfunctionallyexpressedinatrialmyocytesandhasanimportantroleintheregulationofatrialrepolarizationofactionpotential.ThepresentstudywasdesignedtoinvestigatetheeffectofAAonhKv1.5wildtypeandmutantchannelsexpressedinCHOcellsusingthewhole-cellpatch-clamptechnique.AAdirectly inhibitedhKv1.5currents inaconcentration-dependentmanner(IC506.4µM).Theblockingactionwasfoundtoprogresswithtimeduringdepolarizingvoltagestep,suggestingthatAAisanopenchannelblocker.MutationsR487VandH463C(intheouterporemouth)reducedAAaction.I502A,V512A(intheS6domain)alsodecreasedAAeffect.Interestingly,themutationsofT479AandT480A(located inthebaseofselectivityfilter)haveoppositeactiononAAinhibition;T479AenhancedtheAAaction.AAinducedasimilarinhibitionofhKv1.5inthepresenceof4-Aminopyr-idine,aKv1.5blocker.Moreover,atalkaline(pH8.0),AAactionwasmarkedlyreduced.However,acidicpH(pH6.4) failedtoaffectAAaction.Ourfindingssuggestthatmultipleaminoacids (R487,H463,T479,T480,I502,I512)haveimportantroleindeterminingthechannelsensitivitytoAAandthealkalinesituationmaymodifythebindingsitesofAAonKv1.5channels.NoCOI.

1P-022The interaction between S4-S5 linker and C-linker regulates the slow deactivation of hERG channelKume, Shin-ichiro1,2; Nakajo, Koichi1,2; Kubo, Yosihiro1,2(1Div Biophys & Neurobiol, NIPS, Okazaki, Japan, 2Physiol Sci. SOKENDAI, Hayama, Japan)

hERGchannel,amemberofthevoltage-gatedK+channelfamily,isthemainsubunitoftherapidlyactivatingdelayedrectifierK+current(IKr)inhumanheart,andiswellknownforitsveryslowdeactivation.Ithassixtransmembranesegmentscomprisingformerfoursegments(S1-S4)formingthevoltagesensordomainandlattertwosegments(S5-S6)formingthechannelporedomain,andthesetwodomainsarelinkedbyS4-S5linkerincytoplasmicside.Furthermore,ithaslargecytoplasmicregions, includingeagdomain intheN-terminusandC-linker/cyclicnucleotidebindinghomologydomain(CNBHD)intheC-terminus. Thesecytoplasmicregionsareknownto interact toregulateslowdeactivation. Inthisstudy,weperformedthecyste-ine-cysteinebridgeformationexperimentusingamembrane-permeableoxidizingreagentTbHO2undertwoelectrodevoltageclampinXen-opusoocytes.Weobtainedthatthedeactivationspeedofthemutantcarryingtwointroducedcysteinesatglutamate544 inS4-S5linkerandarginine681inC-linkerafteradditionofTbHO2wereslowerthanthatbeforeadditionofTbHO2.Further,afteradditionofTbHO2,theconductance-voltagerelationshipofthemutantshiftednegatively.Thismodification was significantly enhanced when cells were volt-age-clampedataholdingpotentialof+40mV.Takentogether,theresultssuggestedthatS4-S5linkerandC-linkerinteracttoregulatetheslowdeactivationofhERGchannel,andthisinteractionchangesdynamicallybetweenopenandclosestate.NoCOI.

1P-023Functional regulation of β2-adrenoceptors on IKs cur-rent of sinoatrial node cells in guinea pigDing, Wei-Guang1; Bai, Jiayu1; Xie, Yu1; Toyoda, Futoshi1; Bellier, Jean-Pierre2; Matsuura, Hiroshi1(1Dept. Physiol. Shiga Univ. Med. Sci., Otsu, Japan, 2Molecular Neuroscience Research Center, Shiga Univ. Med. Sci., Otsu, Shiga Japan)

Theβ1-andβ2-adrenoceptors(ARs)arehighlyexpressedinmamma-lianheart,withthepredominanceoftheβ1ARssubtype.Theβ1/β2ARratiovariesamongdifferentcardiacregions;itishighintheventricles,relativelylowerinatriaandsinoatrialnode(SA).Directstimulationofsinoatrialβ2ARinmanhasapositivechronotropiceffect.Thepresentstudywasdesignedtoexaminetheactionofβ2ARstimulationontheslowcomponentofdelayedrectifierK+current(IKs)inSAcells.Zin-terolinthepresenceofCGP20712A(β1ARantagonist)andisoprotere-nolinthepresenceofICI118551(β2ARantagonist)wereusedforac-tivationofβ2andβ1ARs,respectively.β2ARstimulationincreasedIKselicitedby2-sdepolarizingpulsesinSA,atrialcells,butfailedtopotentiateIKs inventricularcells. InSAcells, theactionofβ2ARstimulationwasconcentration-dependentandthemaximaleffectwassignificantly largethanthatofβ1ARstimulation.Similartoβ1ARstimulation,zinterolsignificantlyshiftedcurrentactivationtonegativepotentials.Zinterol-inducedenhancementofIKswasmarkedlyattenu-atedbyapplicationwithproteinkinaseA(PKA)inhibitor,H89butwasnotaffectedbypretreatmentwithpertussistoxin.Theseresultsdemon-stratethattheeffectofβ2ARonIKsactivationismediatedthroughaPTX-insensitiveGproteinandPKApathway.Thus,β2ARsubpopu-lationisarelativelyimportantmediatorforIKsregulationinresponsetoβ-agoniststimulation.NoCOI.


1P-024H2O2 regulates IKs currents of sinoatrial node cells in guinea-pig via both CaMKII activation and oxidation processXie, Yu; Ding, Wei-Guang; Bai, Jiayu; Matsuura, Hiroshi(Department of Physiol. Shiga University of Medical Science., Otsu, Japan)

AccumulatingevidencessuggestH2O2regulatesseveralcardiacionchannels,suchastheenhancementonL-typeCa2+currentorNa+current inrabbitventricularmyocytesandinhibitiononexpressedhERGcurrent.TheslowcomponentofthedelayedrectifierK+current(IKs)playsanimportantroleinrepolarizationprocessofcardiacactionpotential.Inthepresentstudy,weexaminedtheeffectsofH2O2onIKscurrent insinoatrialnode (SA)cells isolatedfromguinea-pigheart.After4~5minofH2O2perfusion,IKscurrentwasstartedarapidin-creaseby60.45%fromcontroltoapeaklevelafter10minexposure.H2O2-enhancedIKswassignificantlyreducedafterminimumtheintra-cellularCa2+concentration[Ca2+]iofpipettesolution(frompCa7topCa11),suggestingtheinvolvementofCa2+intheregulationofH2O2onIKscurrentactivation.Next,wetestedhypothesisthatH2O2-en-hancedIKsispartiallyinducedbytheactivityofCaMKIIsignalpath-way.KN-93,aninhibitorofCaMKII,significantlysuppressedH2O2-in-duced increase inIKs (decreasedto10.8±4%,n=6);AIP,anotherCaMKIIinhibitor,appliedfrompipettesolutionalsoreducedtheeffectofH2O2onIKs(decreasedto24.1±3%,n=5).Finally,weinvestigatedthepossiblemodulationofH2O2oxidationonIKschannels.H2O2-inducedpotentiationonIKswasfullyreversedbyper-treatmentwithreducingagentdithiothreitol(DTT).ThesefindingsdemonstratethatH2O2-in-ducedIKsincreaseismediatedthroughbothCaMKIIsignalpathwayandoxidationprocess.NoCOI.

1P-025Ca2+ sensor proteins confer PIP2 sensitivity on KCNQ1 channelsInanobe, Atsushi; Tsuzuki, Chizuru; Kurachi, Yoshihisa(Dept. of Phamacol., Osaka Univ., Grad. Sch. of Med., Suita, Japan)

KCNQ1isapore-formingsubunitofcardiacK+currentwhichslowlyactivatesandcontrolsrepolarizationofactionpotential.ThesubunitphysiologicallyassembleswithaCa2+sensorproteinCalmodulin(CaM)ata1:1stoichiometryinaCa2+-independentmanner.ThebindingofCaMis thoughttostabilizeacytoplasmicdomainofKCNQ1andconferCa2+sensitivity.AlthoughthereareanumberofCa2+sensors,thespecificityofbindingtoKCNQ1remainsunclear.ThebindingsitesaremappedinthevicinityofasitebindingtoPIP2,alipidessentialforKCNQ1activation.However,itisunclearhowCaMbindingasso-ciateswiththePIP2sensitivity.Toaddressthesequestions,wedevel-opedadetectionsystemfortheKCNQ1-CaMcomplex.WepreparedGFP-fusedKCNQ1C-terminusandexpressedwithCa2+sensorsinE. coli.Thecelllysatewassubjectedtofluorescence-detectiongelfiltra-tion.Usingthissystem,wetestedthespecificityforthebindingofKCNQ1with8differentCa2+sensors.Amongthem,Calml3,theclosestrelativeofCaM,wasanonlymoleculetorestoretheKCNQ1folding.NextweexaminedtheeffectsofCa2+sensorsontheKCNQ1-PIP2in-teraction.Voltage-sensitivephosphatasewasexpressedwithKCNQ1andeitherCaMorCalml3inXenopusoocytestocontrolPIP2-contentbymembranepotentials.InthepresenceofCalml3,thecurrent-voltagerelationshipwasshiftedtorightandtheplateauwashigherthanthatinthepresenceofCaM.ThissuggeststhatCalml3augmentstheaf-finityofKCNQ1toPIP2.Therefore,Ca2+sensorsappeartoconferthePIP2sensitivityonKCNQ1viathedirectinteraction.NoCOI.

1P-026A pair of phenylalanine residues on the S4 and S5 segments create a physical and energy barrier for the voltage sensor before opening in KCNQ1/KCNE1 channelNakajo, Koichi1,2; Kubo, Yoshihiro1,2(Div. Biophys and Neurobiol, NIPS, Okazaki, Japan, 2Physiol Sci. SOKENDAI, Hayama, Japan)

Inhumanheart,KCNQ1,avoltagegatedK+channel,formsacomplexwithanauxiliarysubunitKCNE1.AmainroleofKCNE1isslowingthegatingofKCNQ1channel,andweandothergroupspreviouslyfoundthatKCNE1slowsdownthemovementofthevoltagesensordomain(VSD).However,howKCNE1changestheVSDmovementremainslargelyunknown.Here,weidentifiedtwoKCNQ1phenylal-anineresidues,F232ontheS4segmentandF279ontheS5segment,arecriticalfortheVSDmodulationbyKCNE1.WemutatedF232orF279intovariousaminoacidresiduesandfoundthatδG0(Gibbsfreeenergyofchannelopeningat0mV)isdependentonthebulkinessofthesidechainofthemutatedresidue,implyingthatF232andF279createaphysicalandenergybarrier forthevoltagesensorbeforechannelopening.Toconfirmthat,wenextdirectlytrackedtheVSDmovementbyvoltageclampfluorometry.Inthewild-typeKCNQ1/KCNE1,thefluorescencechangecorrespondingtotheVSDmovementwellprecededthecurrentactivationasseen intheothervoltagegatedK+channels. Ontheotherhand, intheF232AandF279Amutants,theVSDmovementandthecurrentactivationwerealmostoverlapped,indicatingthatthechannelimmediatelyenterstheopenstateoncethevoltagesensorreachestheupstate. TheseresultssuggestthatF232andF279createaphysicalandenergybarrierforthevoltagesensortoovercomebeforethechannelopening,andthatthisisthemolecularmechanismfortheslowgatingintheKCNQ1/KCNE1channels.NoCOI.

1P-027Stoichiometry and biophysical properties of the Kv4-KChIP complex change depending on the expression level of KChIP.Kitazawa, Masahiro1,2; Nakajo, Koichi1,2; Kubo, Yoshihiro1,2(1Div Biophys and Neurobiol, Natl Inst Physiol Sci, Okazaki, Japan, 2Dept Physiol Sci, SOKENDAI, Hayama, Japan)

Kv4isamemberofvoltage-gatedK+channelfamily.Itisexpressedinneuronaldendritesandcardiacventricularmyocytes,regulatingtheirexcitability.K+channelinteractingprotein(KChIP)isanauxilia-rysubunitforKv4whichisknowntoincreasethecurrentamplitudes,deceleratetheinactivationandacceleratetherecoveryfrominactiva-tionofKv4.However,themechanismhowKChIPmodulatesKv4re-mainslargelyunknown.WeconsideredthepossibilitythatKv4chan-nelbehaviorisregulatedbythestoichiometryofKv4-KChIPcomplex.WefirstinvestigatedhowtheamountofexpressedKChIP4changesKv4.2currentpropertieswithtwo-electrodevoltageclampandob-servedthattherecoveryfrominactivationofKv4.2wasgraduallyacceleratedwiththeincreaseintheexpressedamountofKChIP4.WenextmadetandemrepeatconstructsinwhichthestoichiometryofKv4.2-KChIP4complexwasstrictlycontrolledandcomparedtheirionchannelproperties.4:4(Kv4.2:KChIP4)channelshowedfasterrecoveryfrominactivationthan4:2channel.WenextdirectlycountedthenumberofKChIP4subunits inasingleKv4.2-KChIP4complexbycountingbleachingstepsusingsingle-moleculefluorescenceimagingandobservedthatthestoichiometryofKv4.2-KChIP4complexchang-esdependingontheexpressedamountofKChIP4.OurresultssuggestthatthestoichiometryofKv4.2-KChIP4complexisvariableandtherecoveryfrominactivationofKv4.2 iscontrolledbythenumberofboundKChIP4.NoCOI.


1P-028Effect of membrane lipids on the inactivation gating of the KcsA potassium channelIwamoto, Masayuki; Oiki, Shigetoshi(Dept. Mol. Physiol. Biophys., Univ. Fukui Fac. Med. Sci., Fukui, Japan)

Membranelipidsactasacofactorforfunctionoftheionchannelandspecificlipidmoleculesareindispensableformaintainingchannelac-tivities.FortheKcsApotassiumchannel,thepresenceofnegativelychargedphospholipidssuchasphosphatidylglycerol(PG)inthemem-braneisprerequisiteforthechannelactivity.Previouslywedemon-stratedthatthePGmoleculeontheinner(cytoplasmic)leafletrenderedtheKcsAchannelhighlyactive.WefoundthattheM0-helixlyingontheinnermembranesurfacechangeditsconfigurationwithalipid-de-pendentmanner,andmodulatedtheactivationgating.InthisstudywefocusedontheeffectoflipidsontheinactivationgatingoftheKcsAchannel.Asisthecaseforthevoltage-gatedchannels,theKcsAchan-neluponcytoplasmicacidificationexhibitsslowinactivationafterac-tivation. It issuggestedthatthe inactivationoftheKcsAchanneloriginates fromclosureof theselectivityfilterthat locates intheouterhalfofthetransmembranedomain.WeanalyzedtheinactivationrateoftheKcsAchannelinthevaryinglipidcompositionofthemem-branebyuseofartificial lipidbilayer.TheKcsAchannelshowedrelativelyslowinactivationinthenegativelychargedlipidscomparedtothatinneutrallipids.ByintroducingmutationsintotheM0helix,we further investigatedthemolecularmechanismunderlyingthelipid-dependencyoftheinactivationgating.NoCOI.

1P-029Gating-coupled clustering-dispersion behavior of the KcsA potassium channel in membrane: Direct obser-vation by atomic force microscopySumino, Ayumi1,2; Yamamoto, Daisuke3; Iwamoto, masayuki2; Dewa, Takehisa4; Oiki, Shigetoshi2(1JST/PRESTO, Saitama, Japan, 2Department of Molecular Physiology and Biophysics, Faculty of Medical Sciences, University of Fukui, Fukui, Japan, 3Department of Applied Physics, Fukuoka University, 4Department of Frontier Materials, Graduate School of Engineering, Nagoya Institute of Technology, Nagoya, Japan)

TheKcsApotassiumchannelisapH-dependentchannel,andtheac-tivationgateopensatacidicpH.Usingatomicforcemicroscopy(AFM),wehavecapturedtheopen-gatestructureofmembrane-embeddedKcsAchannelsbyremovingthepotentiallysight-obstructingcytoplas-micdomain(CPD).Here,werevealedpH-dependentclustering-disper-sionbehaviorofKcsAchannelsonthemembrane,whichoccurredconcurrentlywiththegatingconformationalchange.AtneutralpH,theclosedchannelsformedself-assemblednanoclusters.AtacidicpH,theopen-gatedchannelsweredispersedassingly-isolatedchannels.Paircorrelationfunctionofthechannelpositiongavethenearest-neigh-bordistanceofapproximately14nm,indicatingnon-contactingdisper-sion.High-speedAFMrevealedthattheclustering-dispersiondynam-icswerereversibleandcompletedwithinseveralminutes.TheseresultssuggestthattheKcsAchannelundergoconformationalchang-eswithintheclusterbutdonotopenuntilthechannelsaredispersed.Theinterplaybetweenthegatingconformationalchangeofindividu-alchannelsandthecollectivebehavioroftheclustering-dispersiondynamicsprovides insight intounderstandingmembrane-mediatedprotein-proteininteractionsandfunctionalcooperativity.NoCOI.

1P-030Functional consequences of a gain-of-function muta-tion in the Kir2.1 inward rectifier K+ channel associat-ed with short QT syndromeIshihara, Keiko; Itoh, Masayuki; Igata, Sachiyo; Takano, Makoto(Dept. Physiol., Kurume Univ. Sch. Med., Kurume, Japan)

Thedurationofactionpotentialdeterminestherefractoryperiodofthecardiacmuscle.TheshorteningoftheactionpotentialdurationoccursasshortQTintervalinECG,andincreasesthevulnerabilitytoarrhythmia.ThisshortQTsyndrome(SQTS)canbecausedbygain-of-functionmutationsintheKCNJ2geneencodingtheKir2.1subunit,which,togetherwithKir2.2and/orKir2.3,constitutesthecardiacstronginwardrectifierK+channel(IK1).Inthisstudy,weexaminedthefunc-tionalconsequencesoftheM301KmutationrecentlyidentifiedinaSQTSpatientbyperformingconventionalwhole-cellpatch-clampex-periments.Aspreviouslyreported,expressionofKir2.1bearingtheM301Kmutation(Kir2.1(M301K))alonedidnotgiverisetoanymea-surablecurrents,buttheco-expressionofKir2.1(M301K)withwild-typeKir2.1showedK+currentswithaweakinwardrectification.Co-ex-pressionofKir2.1(M301K)withKir2.2orKir2.3gaveessentiallythesameresults,supportingthattheKir2.1/Kir2.1(M301K)heterozygoteexpressesIK1withlargeoutwardcurrentsacceleratingtherepolariza-tion.Interestingly,inKir2.1/Kir2.1(M301K)heteromericchannels,thesensitivitytotheexternalK+(characteristicofIK1)wasaltered,andthesensitivitiestotheexternalBa2+andCs+blockage(ahallmarkoftheinwardrectifiers)wereincreased.TheseresultsprovideevidencethatthemutationinthecytoplasmicporeregionalterstheexternalionsensitivitiesofKir2.1channel.NoCOI.

1P-031Identification of palmitoylation enzymes for the HCN2 channel.Itoh, Masayuki; Takano, Makoto(Division of Integrated Autonomic Function, Department of Physiology, Kurume University School of Medicine, Kurume, Japan)

Wehavepreviouslydemonstratedthatamonghyperpolarization-acti-vatedcyclicnucleotide-modulated(HCN)channelfamily,HCN1,HCN2andHCN4,butnotHCN3,arethetargetofS-palmitoylation:Forex-ample,palmiticacidwascovalentlyattachedtomultiplecysteineresidues(C63,C69,C82,C89,C104)locatedinthecytoplasmicN-termi-nusofHCN2.S-palmitoylationreportedlyregulatesthetraffickingandthefunctionofvariousmembraneproteins.AsforHCNchannels,aninhibitorofpalmitoylation,2-bromopalmitatereducedthecurrentamplitudeofHCN2expressedinXenopusoocytesapproximatelyto45%ofcontrollevel.S-palmitoylationisareversiblepost-translationallipidmodification;palmitoylationanddepalmitoylationarecatalyzedbyproteinacyltransferases(PATs)andpalmitoylproteinthioesteras-es (PPTs),respectively. Inthepresentstudy,weaimedto identifytypesof theseenzymesthatregulatethepalmitoylationofHCN2channel.Weoverexpressedeach24typesofknownPATswithHCN2andfoundthatZdhhc3,Zdhhc9,andZhhc17significantly increasedthepalmitoylationofHCN2proteins. Incontrast,overexpressionofknownPPTssuchasAPT1,APT2,LYPLAL1andPPT1,didnotreducethepalmitoylationofHCN2,suggestingthatunknowntypesofenzymesmaybeinvolved.AlthoughourresultsdemonstratedthatHCNchan-nelsarethetargetofdynamicpalmitoylation-cycle,furtherstudiesareindispensabletoaddresswhethertheseenzymesexertphysiologicalroles inthesignal transductionsystemwhichmayregulateHCNchannels.NoCOI.


1P-032TMEM16F forms a Ca2+-activated Cl- channelShimizu, Takahiro1; Iehara, Takahiro1; Fujii, Takuto1; Okada, Yasunobu2; Sakai, Hideki1(1Grad Sch. Med. Pharm. Sci., Univ. Toyama, Toyama, Japan, 2Nat. Inst. Physiol. Sci., Okazaki, Japan)

Transmembraneprotein16(TMEM16),whichpossesseseightputativetransmembranedomainswiththeintracellularNH2-andCOOH-termi-naltails,isthoughttocompriseaCl-channelfamily.AlthoughithasbeenreportedthatTMEM16Aand16BfunctionasCa2+-activatedCl-channels (CaCC), the functionalpropertiesofTMEM16Fhavegreatlybeencontroversial. Inthepresentstudy,we investigatedelectrophysiological and pharmacological properties of humanTMEM16Fexpressed inHEK293Tcells. Inwhole-cellpatch-clamprecordings,applicationofaCa2+ionophore,ionomycin,orriseintheintracellularfreeCa2+concentrationto100µMinducedtransientac-tivationofmembranecurrentswithstrongoutwardrectificationinTMEM16F-overexpressingcellsbutnot inmock-transfectedcells.OutwardrectificationofTMEM16FcurrentswasnotaffectedbyanincreaseintheintracellularCa2+level.ReplacingextracellularCl-withaspartate-positivelyshiftedthereversalpotentialofTMEM16Fcur-rents,whereasnosignificantchangeof thereversalpotentialwasobservedbyreplacementofextracellularNa+withNMDG+,showingthattheTMEM16FcurrentisCl--selective.Consistently,TMEM16FcurrentswereinhibitedbyCaCCblockers,niflumicacidandtannicacid.AnionselectivitysequenceoftheTMEM16FcurrentwasI->Br->Cl->F-.Inaddition,aCaMKIIinhibitor,KN-93,hadnoeffectonCa2+-inducedactivationofTMEM16Fchannels.OurresultsindicatethatTMEM16FformsaCa2+-activatedCl-channelthatisnotregulat-edbyCaMKII.NoCOI.

1P-033Properties of persisitent Na+ current in Kenyon cells isolated from the mushroom body of the insect brainYoshino, Masami(Department of Education, Tokyo Gakugei University, Tokyo, Japan)

Manytypesofexcitablecellspossessanon-inactivatingorslowlyin-activating,persistentsodiumcurrent(INaP).TheINaPhasbeenshowntobemediatedbytetrodotoxin(TTX)-sensitiveNa+channels.Theaimofthisstudywastoinvestigatethebasicpropertiesofvoltage-depen-dentNa+channelcurrents in intrinsicneuronscalledKenyoncellwithinthemushroombodyofinsectbrain.UsingtheperforatedpatchclamprecordingsfromthecricketKenyoncells,weshowedthepres-enceoftwodifferenttypesofNa+currents,fasttransientcurrent(INaF)andslowsustainedorpersistentcurrent(INaP).INaFfirstappearedat-40mVwhereasINaPappearedat-50mV.Bothcurrentsreachedmax-imumat -20mV.INaFcompletely inactivatedduringtheperiodofmembranedeporalizationof150mswhereasINaPpersistedforseveralseconds.INaFhalfinactivatedat-46mV,whereasINaPhalfinactivatedat-30mV.INaFwasblockedmorepotentlybyTTXthanINaP.Further-more,INaPwasblockedby50µMCd2+whereasINaFwasnot.Riluzole,whichhasbeenproposedarelativelyspecificpersistentNa+channelblockerinhibitedbothINaPandINaF.UnderthecurrentclampconditionwhereINaFwasinhibitedbylowconcentrationofTTXorriluzole,themembraneoscillationappearedneartherestingmembranepotential.ThismembraneoscillationwasdisappearedbyadditionofCd2+,or1µMTTXindicatingthatINaPplaysaroleinthesubthresholdmem-braneoscillationandcouldbe importanttomodulateKenyoncellexcitability.NoCOI.

1P-034Functional analysis of Ci-CatSper channel in heterol-ogous expression systemArima, Hiroki; Okamura, Yasushi; Tsutsui, Hidekazu(Graduate school of Medicine, Osaka University, Osaka, Japan)

CatSperchannel,acationchannelexpressedspecificallyintestis,isessentialformalefertilizationinmouse.CatSperchannelisformedbyfouralphasubunits,CatSper1,CatSper2,CatSper3andCatSper4,asaheterotetramer.Eachsubunithassixtransmembranesegments(S1–S6).S4containsseveralpositivelychargedaminoacids,andtheaminoacidsequenceoftheputativeporeformingregionofS5–S6issimilartothatofvoltage-dependentcalciumchannel,whichsuggeststhatCatSper isavoltage-dependentcalciumchannel.However,detailedcharacteristicsaboutCatSperarestillunclearbecause functionalanalysisusingheterologousexpressionsystemhasbeenunsuccessful.Inthisstudy,wereportthattheregiononlyconsistingofS1–S4fromCi-CatSper3,correspondingtothevoltage-sensordomainofothervoltage-gatedionchannels,showsionicconductanceathyperpolariza-tion.WeexpressedaversionofCi-CatSper3truncateddownstreamofS4 inXenopusoocyte,anddetectedactivationofsubstantial ioniccurrentsuponhyperpolarization.Wefoundthatthecurrentislikelycarriedbyvariouscationsincludingsodiumandcalcium.Furthermore,eliminationofthepositivechargesinS4resultedindisappearanceofthe ioniccurrent, indicatingthattheS1–S4region itselfhas ionicpermeability.This is, toourknowledge,thefirstreportofCatSpercurrentrecordingsinaheterologousexpressionsystem.NoCOI.

1P-035Electric axon guidance in constant electric field cul-tureYamashita, Masayuki(Dept. Physiol. 1, Nara Med. Univ., Kashihara, Japan)

Inadditiontowell-knownmechanismsofchemicalguidance,growingaxonsinthenervoussystemaredirectedbyanextracellularelectricfieldinaprocessknownasgalvanotropism.Thegalvanotropicbehav-iorofnervecellsin vitrowasfirstdemonstratedaslongagoas1920.However,itremainsunknownwhetherembryonicnervetissuesgen-erateasimilarelectricfieldinordertoguidegrowingaxons.Yamashi-tahasrevealedthatanextracellularvoltagegradientexists intheembryonicretinaandthatthisgradientguidestheaxonsofnewbornretinalganglioncellstowardstheirtargets(BBRC431:280-283,2013).Theextracellularpotential isgeneratedbyepithelialNa+channels(ENaC)inneuroepithelialcells.Thesefindingsindicateanimportantroleforgalvanotropismintheinitialorientationofaxonsthatextendoverlongdistances,andprovideinsightintothemechanismsunder-lyingtheproperextensionofdevelopingaxonsintheembryonicbrain.Inthepresentstudy,aconstantelectricfieldculturesystemwasde-velopedinordertoinvestigatethemolecularmechanismsthatunder-liethegalvanotropicbehaviorofgrowingaxonsofretinalganglioncells.Retinalstrips(1mminwidth)weremadefromembryonicday6chickretinas.TheywereembeddedinMatrigelTMandculturedfor24hoursunderaconstantelectricfield(0.1–2.0mV/mm),whichwasestablishedbyanegativefeedbackcircuitandbymonitoringavolt-agedifferencebetweentwopoints(15mmapart)intheculturemedi-um.Itwasfoundthatthedirectionofgrowingaxonswasreversedbyreversingthepolarityofelectricfields.NoCOI.


Poster PresentationsHeart, Circulation (1)

1P-036Aortic baroreceptors play a greater role in baroreflex regulation of heart rate than carotid sinus barorecep-tors in unanesthetized, decerebrate ratsIdesako, Mitsuhiro; Matsukawa, Kanji; Ishii, Kei; Endo, Kana; Liang, Nan(Department of Integrative Physiology, Graduate School of Biomedical and Health Sciences, Hiroshima University)

Apreviousstudyusingdecerebrate,arteriallyperfusedratreportedthataorticbaroreceptorspredominantlycontributetobaroreflexreg-ulationofheartrate(HR),whilebothaorticandcarotidsinusbarore-ceptorssimilarlycontributetobaroreflexregulationofsuperiormes-enteric sympathetic outflow. To reexamine this issue usingunanesthetized,decerebraterat,weusedsurgicaldenervationofbi-lateralaorticnerves(AnD)orcarotidsinusnerves(CSnD),toidentifytherelativecontributionofeachbaroafferentlimbtobaroreflexregu-lationofHRandrenalsympatheticnerveactivity(RSNA).Meanarte-rialpressure(MAP)wasincreasedbyphenylephrinetoelicitbaroreflexbradycardiaandsympathoinhibition.Baroreflexsensitivity(BRS)wasestimatedfromthemaximalslopeofthebaroreflexcurvebetweentheresponsesinMAPandHRorRSNA.AnDreducedBRSforHRto31±5%ofthecontrolinintactcondition,whileCSnDreduceditto63±8%.Therewasasignificantdifference(P<0.01)intheattenuatedBRSforHRbetweenAnDandCSnD.Incontrast,BRSforRSNAwasreducedto50±5%followingAnDandsimilarlyto58±6%followingCSnD.Theseresultsindicatethataorticbaroreceptorshaveagreaterinfluenceonthecardiomotorlimbofarterialbaroreflex,whilesignalsofallfourbaroafferentsequallycontributetothevasomotorlimbofarterialbaroreflex.NoCOI.

1P-037Involvement of orexin system in the sympathetic nerve regulationMurakami, Manabu1; Ohba, Takayoshi2; Niwa, Hidetoshi3; Kushikata, Testuya3; Ono, Kyouichi2; Watanabe, Hiroyuki4; Hirota, Kazuyoshi3(1Department of Pharmacology, Hirosaki University, Graduate School of Medicine, Hirosaki, Japan, 2Department of Cellphysiology, Akita University School of Medicine, Akita,Japan, 3Department of Anesthesiology, Hirosaki University, Graduate School of Medicine, Hirosaki, Japan, 4Department of Internal Medicine, Akita University School of Medicine, Akita, Japan)

Orexinisaneuropeptidesecretedfromhypothalamicneuronsthatisknowntobeactivatedduringmotivatedbehaviorsandactivewaking.Beat-to-beatfluctuationinheartrate,aphenomenontermedHRvari-ability,hasbeenwidelyusedtoevaluateautonomicactivity.Inthepresentstudy,weexaminedtheimportanceoforexinsystemintheregulationofsympatheticnervesystemwithorexin/ataxin-3trans-genicrat,whichhasmarginalnumberoforexinneuron.RT-PCRanalysisshowedexpressionoforexinandorexinreceptor(OX1R)inthesuperiorcervicalganglionganglion,whileexpressionofanotherreceptor(OX2R)waslow.Orexin/ataxin-3transgenicratshowedin-creasedexpressionoftworeceptors,whileexpressionoforexinwasundetectable,suggestingcompensatory increaseofbothreceptors.OrexinA(10nM)increasedintracellularcalciuminthesuperiorcer-vicalganglionicneuron,suggestinginvolvementoforexinsysteminthesympatheticnervecontrol.IntheECGrecording,orexin/ataxin-3transgenicratsshoweddecreasedresponsivenesstoβ-blocker.Fur-thermore,theyshoweddeterioratedR-Rintervalregulation,indicatinginvolvementoforexinsysteminthesympatheticnerveregulation.NoCOI.

1P-038The effect of orexin-A on diurnal variation in arterial pressure in rats fed high-fat dietYamaguchi, Aoi; Abe, Chikara; Kitagata, Yuta; Nagai, Yuko; Obata, Koji; Morita, Hironobu(Gifu University School of Medicine, Gifu, Japan)

Thediurnalvariationofarterialpressure(AP),lowerAPduringrest-ingperiodandhigherAPduringactiveperiod,isobservedinrats.Sinceorexin isaneurotransmittertoregulatewakefulness,orexinmightparticipateindiurnalvariationinAP.Thisvariationisdisap-pearedinratsfedhigh-fatdiets(HFD),thusorexincontroldisorderisconsidered.Accordingly,weexaminedcircadianrelationshipbetweenorexinandAPinHFDrats.Ratswerefedwithnormal-fatdiets(NFD)orHFDfor11weeksfrompostnatalday28.WemeasuredAPcontin-uously for24hs.AftermeasurementofAP,bothorexin-AmRNAexpressioninhypothalamusandorexin-Aincerebrospinalfluidat1:00,7:00,13:00,and19:00weremeasured.Baselineorexin-AinHFDratswashigherthanthatinNFDrats.Thediurnalvariationoforexin-AmRNA,higherinactiveperiodandlowerinrestingperiod,wasob-servedinNFDrats.ThisvariationwascompletelyabolishedinHFDrats.Acuteinjectionoforexin-Aintotherostralventrrolateralmedul-lainducedsympathoexcitation(172±34%)andpressorresponse(36.0±5.7mmHg).Theseresults indicatethathigherandunchangingorexin-AmightparticipateindisappearanceofdiurnalvariationinAPinHFDrats.NoCOI.


1P-039The rostral nucleus of the solitary tract neurons re-ceive convergent inputs from the aortic depressor and lingual-trigeminal nerves.Ishizuka, Ken’Ichi; Satoh, Yoshihide(The Department of Physiology, The Nippon Dental University School of Life Dentistry at Niigata, Niigata, Japan)

Weexaminedresponsesoftherostralnucleusofthesolitarytract(NTS)neuronstostimulationoftheaorticdepressornerve(ADN)andlingual-trigeminalnerve(LTN),andalsoinvestigatedwhethertheseinputsconvergeontorostralNTSneurons inurethane-chloraloseanesthetizedrats.TheADNstimulationsignificantlyalteredtheirfiringratesinone-thirdoftherostralNTSneurons,anddidnot intwo-thirdoftheneurons.TheLTNstimulationsignificantlyalteredtheirfiringratesinlessthanhalfoftherostralNTSneurons,anddidnot inmorethanhalfoftheneurons.MajorityoftherostralNTSneuronswerefoundtoexhibitapulse-relatedactivityintheECG/ABP-triggeredcorrelationhistograms.Moreoverconvergent inputsfromADNandLTNwerefoundinlessthan20%oftherostralNTSneurons.Theseresultssuggestthattheseneuronsintegratetheinputsfrombaroreceptorandlingual-somaticafferents.Theseneuronalac-tivitiesmaybeinvolvedinthereflexcardiovascularresponsesinpart.NoCOI.

1P-040Downregulation of prepro-orexin gene expression in the NTS of SHR may be prohypertensiveGouraud, Sabine S; Waki, Hidefumi; Takagishi, Miwa; Kohsaka, Akira; Maeda, Masanobu(Department of Physiology, Wakayama Medical University School of Medicine, Wa kayama, Japan)

Sincethenucleustractussolitarii(NTS)isapivotalregionforregu-latingtheset-pointofarterialpressure,weproposedaroleforitinthedevelopmentofneurogenichypertension.OurpreviousfindingssuggestthattheNTSofpre-hypertensiveandhypertensivesponta-neouslyhypertensiverats (SHRs)exhibitsabnormal inflammatoryconditioncomparedtothenormotensiveWKYrats(Gouraudetal.JHypertens.2011,Gouraudetal.AutonNeurosci.2011).Whetherthischronicconditionaffectstheneuronalactivityandplasticity intheNTSremainsunknown.TounveiltheneuronalfunctionsintheNTSofSHRs,weinvestigatedtheexpressionofneurotrophintranscriptsinbothyoungandadultSHRs.Real-timequantitativePCRrevealedthattheexpressionofHcrt(hypocretin,transcriptforprepro-orexinthatformsorexin-A/hypocretin-1andorexin-B/hypocretin-2)isalteredintheNTSofbothadultandyoungSHRs.HcrttranscriptwasalsofounddecreasedinthehypothalamusofSHRbutnotinthecortexnorthemedullaoblongata.Wehavealsofoundthatorexin-Apeptidemi-croinjectedintotheNTSofanesthetizedSHRssignificantlydecreasedarterialpressureandthedepressoreffectswerelargerinSHRsthanWKYrats,suggestingthatitsdown-regulationintheNTSmaycon-tributetohypertensionintheSHRs.TheseprofilesmaybeinvolvedintheimpairmentoftheneuronalcircuitryregulatingcardiovascularautonomicactivityduringthedevelopmentofSHR.SupportedbyKAKENHI(25870639).NoCOI.

1P-041Cardiac vagal control in a knock-in mouse of dilated cardiomyopathy with a troponin mutationZhan, Dong-Yun1; Du, Cheng-Kun1; Akiyama, Tsuyoshi1; Morimoto, Sachio2; Shimizu, Shuji1; Kawada, Toru1; Shirai, Mikiyasu1(1Natl. Cereb. Cardiovas. Ctr., Suita, Japan., 2Fac .Med. Sci., Kyushu Univ., Fukuoka, Japan.)

Aims:Toinvestigatethecardiacvagalcontrolfunctionsinaknock-inmousemodelofdilatedcardiomyopathy (DCM)withδK210whichdevelopscardiacenlargement,heart failure,and frequentsuddencardiacdeath likeDCMpatients.Methods:Microdialysis techniquewasappliedtotheleftventricularmyocardiumofanesthetizedmiceandmyocardialinterstitialacetylcholine(ACh)levelsweremeasuredbyHPLCasanindexofAChreleasefromcardiacvagalnerveendings.Theeffectsofelectricalstimulationofcervicalvagalnervesat5and10Hzorα2-adrenergicstimulationbymedetomidine(0.1mg/kg,i.v.)onmyocardialinterstitialAChlevelswereexaminedinwild-type(WT)miceandδK210DCMmice.Results:Electricalvagalnervestimulationdecreasedheartrate(HR)andincreasedmyocardialinterstitialAChlevelsbothinWTandDCMmiceandtheresponsesofHRandmyo-cardial interstitialAChlevelswerenotdifferentbetweenWTandDCMmice.Incontrast,administrationofmedetomidinedecreasedHRandincreasedmyocardialinterstitialAChlevelsbothinWTandDCMmice,buttheresponsesofHRandmyocardialinterstitialAChlevelsweresignificantlysuppressedinDCMmicecomparedtothoseinWTmice.Conclusions:Inaknock-inmouseofDCM,peripheralcardiacvagalcontrolfunctionsincludingAChreleasefromvagalnerveendingsandpostsynapticresponsewerepreserved,butcentralvagalactivationthroughα2-adrenergicreceptorwasimpaired.NoCOI.

1P-042Characteristics of neural regulated vasoconstriction on guinea pig hepatic veinTakano, Hiromichi; Hashitani, Hikaru(Nagoya city university, Nagoya, Japan)

Ithasbeenreportedthattheincreaseofresistanceinhepaticvesselsregulatesthebloodcirculationinthebodythroughthevenousreturn.Asthecellularmechanismsundersuchavasoconstrictionhavenotbeenwellunderstood,weexaminedtheneuronalcontrolsofcontrac-tionontheguinea-pighepaticveins.Thetransmuralnervestimulation(TNS)evokedcontractiononthevessels.Asthe frequencyof theelectricalstimulation increased (10–100Hz), theamplitudeandthedurationofthecontractionwereincreased.ThesecontractionswereabsentinpresenceofTetrodotoxin(3µM).Phentolamin(3µM)inhib-itedtheinitialphaseofthecontraction.Inaddition,propranorol(3µM)increasedtheremainingcontraction.Guanethidine(10µM)alsoinhib-itedtheinitialphaseofthecontraction.Incontrast,atropine(1µM)shortenedthedurationoftheTNS-evokedcontraction.Theremainingcontractionisabolishedinpresenceof10µMguanethidine.1–10µMphenylephrineevokedvasoconstriction inadoseresponsemanner.Isoproterenol(3µM)evokedtherelaxationofphenylephrine-contract-edvessels.Y-27632inhibitedboththeTNS-andphenylephrine-evokedcontraction.Theseresultssuggestthattheadrenergicandcholinergicnervesregulatethecontractiononthehepaticvein.NoCOI.


1P-043Central serotoninergic system may be involved in mechanisms underlying anti-hypertensive effect of ex-ercise therapyWaki, Hidefumi; Takagishi, Miwa; Gouraud, Sabine S; Kohsaka, Akira; Maeda, Masanobu(Department of Physiology, Wakayama Medical University School of Medicine, Wakayama, Japan)

Wediscussedthecentralmechanismofanti-hypertensiveeffectofexercisetherapywithafocusonthenucleustractussolitarii(NTS),whichplaysanimportantroleinregulatingcardiovascularhomeosta-sis.Weidentifiedthatgeneexpressionoftheserotonin1A(5-HT1A)receptor,butnotothersubtypes, intheNTSwassignificantlyde-creasedafterlong-termdailywheelrunninginhypertensiverats(SHR).WealsofoundthatserotoninmicroinjectedintotheNTSofanesthetizedSHRsignificantlydecreasedarterialpressure(AP).Moreover,toiden-tifycardiovascularcontrolbytheserotonergicsysteminconsciousanimals,wemicroinjectedaconjugateofananti-serotonintransporter(SERT)antibodyandsaporin (anti-SERT-SAP) intotheNTStokillNTS-projectingserotonergicneurons.WefoundthatAPmeasuredinfreelymovingratssignificantlyincreasedafteranti-SERT-SAPinjec-tionsintotheNTS,andconfirmedthatserotoneregicneuronsinthedorsalraphenucleiwerereducedbyanti-SERT-SAPinjections.Theseresultsstronglydemonstratethattheraphenuclei-NTSpathwayde-creasesthebasallevelofAP.5-HT2AreceptorsintheNTSarepre-sumablyinvolvedinthecardiovascularresponses(LaguzziR.CellMolNeurobiol2003).Since5-HT1Areceptorscounteract5-HT2Areceptorsaction,wenowhypothesizedthatdown-regulationof5-HT1AreceptorsintheNTSmaybeinvolvedinthecentralmechanismofanti-hyper-tensiveeffectofexercisetherapy.ThisstudywassupportedbytheJSPS(24500793).NoCOI.

1P-044Renal and lumbar sympathetic nerve activity in re-sponse to repeated obstructive apnea over 4 days in ratsMiki, Kenju1; Sukeguchi, Chie1; Yoshimoto, Misa2(1Department of Integrative Physiology, Nara Women's University, Nara, Japan., 2Department of Cardiac Physiology, National Cerebral and Cardiovascular Center Research Institute, Osaka, Japan)

Theaimofthepresentstudywastoexamineresponsesofrenal(RSNA)andlumbarsympatheticnerveactivity(LSNA)torepeatedobstructiveapneaover4days.WistarmaleratswerechronicallyinstrumentedformeasurementsofRSNA,LSNA,andoxygensaturationofthebraintissue,andatrachealballoonforinductionofapneawasimplanted.Atleast3daysafterthesurgery,theratsweresubjectedtoobstructiveapneabyinflatingthetrachealballoonfor40seconds.Thisprocesswasrepeated6times/hourfor5hours/dayovera4-dayperiod.TheonsetoftheobstructiveapneaincreasedRSNA,LSNA,andarterialpressure,whilethoserecoveredtothepre-obstructionlevelsafterthecessationofobstructiveapnea.Thebasal levelofRSNAincreasedgraduallyandsignificantlythroughoutthe4-dayexperimentalperiod.ThemagnitudeoftheLSNAincreasewaslessthanthatofRSNA.Thebasallevelsofarterialpressureandheartrateremainedwithinthenormalrangesthroughouttheexperimentalperiod.ThesedatasuggestthatrepeatedobstructiveapneaactivatesRSNAinacumulativemanner,whichmayinparttriggerthedevelopmentofhypertensionobservedinsleepapneasyndrome.NoCOI.

1P-045Responses of renal and lumbar sympathetic nerve ac-tivity to chronic intermittent hypoxia in conscious ratsYoshimoto, Misa1; Tsuchimochi, Hirotsugu1; Miki, Kenju2; Shirai, Mikiyasu1(1Department of Cardiac Physiology, National Cerebral and Cardiovascular Center, Osaka, Japan, 2Department of Integrative Physiology, Nara Women's University, Nara, Japan)

Theaimofthepresentstudywastoexamineresponsesofrenal(RSNA)andlumbarsympatheticnerveactivity(LSNA)tochronicintermittenthypoxiainconsciousrats.Wistarmaleratswerechronicallyinstru-mentedwithbipolarelectrodeformeasurementsofRSNAandLSNAandatelemeterformeasurementofarterialpressure.Atleastaweekafterthesurgery,ratswereexposedto20cyclesofnormoxiaandhypoxia(5%O2atnadir)perhour,8houradayfor2weeks.Theshamgroupweresubjecttoalternatingcyclesofairunderidenticalexper-imentalconditionsinparallel.Whenratswereexposedtothehypoxia,RSNA,LSNA,arterialincreasedwhileheatratetendedtodecrease.However,basal levelsofRSNA,LSNA,arterialpressure,andheartremainedunchangedthroughoutthe2-weeksexperimentalperiod.ItisthereforewefailedtoobserveanysignificantchangesinRSNAandLSNAinresponsetochronicintermittenthypoxiaoverthe2-weeksperiodinrats.NoCOI.

1P-046Frequency-dependent effects of heel raising maneu-ver on orthostatic cardiovascular responsesNiizeki, Kyuichi; Onodera, Miki; Saitoh, Tadashi(Department of Biosystems Engineering, Graduate School of Science and Engineering, Yamagata University, Yonezawa, Japan)

Weinvestigatedtheeffectsofmusclepumpfunctiononcardiovascularautonomicresponsesbyheelraisingmaneuver(HRM)withfourdif-ferentfrequenciesof6(HRM6),7.5(HRM7.5),10(HRM10),and15(HRM15)cycles/min.TheR-R interval (RRI)andbeat-to-beatbloodpressure(BP)wereacquiredinhealthysubjects(8malesand5females,aged25.0±9.6).FromthecontinuousBPmeasurement,strokevolume(SV)wascalculatedbyapulse-contourmethod.Cardiacoutput(CO)andtotalperipheralresistance(TPR)werederivedasSVtimesheartrateandmeanBPdividedbyCO,respectively.Usingspectralanalysis,RRIandsystolicbloodpressure(SBP)variabilitywereestimatedforthelow-(LF)andhigh-frequency(HF)bands.Comparedwiththecontrolcondition,HRMledtoincreaseinthemeanSVduringstanding.Short-eningofRRIduringquietstandingwassuppressedbyHRMexceptforHRM15.TheincreaseinCOanddecreaseinTPRwereobservedinHRM10andHRM15.HRM6inducedincreasesinLFpowersofRRIandSBP,whileincreaseinHFpowerofRRIwasobservedonlyinHRM10.Theseresultsindicatethatthereisfrequency-dependenteffectofHRMoncardiovascularautonomicresponsestouprightposture.Ourresultscontributetodeterminingadequatefrequenciesofmusclecontractionthatdisplaysfavorablehemodynamiceffectofmusclepumpfunctionduringorthostaticstress.NoCOI.


1P-047Changes in prefrontal oxygenation associated with the Stroop interference task in elderly subjectsEndo, Kana; Liang, Nan; Ishii, Kei; Idesako, Mitsuhiro; Matsukawa, Kanji(Department of Integrative Physiology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan)

Agingmayleadtocognitivedecline,whiletheunderlyingmechanismisnot fullyunderstood. Ithasbeensuggestedthattheprefrontalcortexexertsasignificant influenceonthecognitivefunction.Theage-relatedchangesintheprefrontalcortexactivitymayplayaroleingenerationofcognitivedeclineinelderlysubjects.Toaddressthisissue,theprefrontaloxygenationduringaconsecutivecognitivetask(Stroopcolorwordtest;SCWT,100trials/session)wasexaminedwithatwo-channelnear-infraredspectroscopy(NIRS)inyoung(23±1yrs)andoldersubjects(62±1yrs).SCWTrequiredthesubjectstoanswerthedisplayedcolorofanincongruentcolorwordasquicklyaspossible.Theconcentrationofoxygenated-hemoglobin(Oxy-Hb)increasedsig-nificantlyinthebilateralprefrontalareasduringSCWTinbothgroups,whiletheextentof incrementswas larger inyoungsubjects.TheOxy-Hbresponsewascomparablebetweentherightandlefthemi-sphereinyounggroup,whiletheOxy-Hbresponseinoldergroupwasmarkedlybluntedontheleftside.Althoughthenumberoferrorswascomparablebetweenthetwogroups,thedurationforSCWTbecamelongerinelderlypeople.Theseresultssuggestthatthedecreaseintheregionalbloodflowoftheprefrontalcortex,especiallyontheleftside,mayleadtoloweroutcomeofthecognitivetask.Toexaminetheresponsesamongvariouscorticalareas,astudyusingamultichannelNIRShasbeenconductedandtheresultswillbediscussed.NoCOI.

1P-048Effect of exhaustive exercise on the increase re-sponse to visual stimulation in posterior cerebral ar-tery blood flowYamaguchi, Yuji1; Ikemura, Tsukasa1; Miyaji, Akane1; Hayashi, Naoyuki2(1Graduate School of Human-Environment Studies, Kyushu University, Fukuoka, Japan, 2Graduate School of Decision Science and Technology, Tokyo Insitute of Technology, Tokyo, Japan)

Neurovascularcoupling(NVC)isassessedasanincreaseresponsetovisualstimulationinthebloodflowvelocityintheposteriorcerebralartery (PCAv) (Aaslid1987).To investigatewhetheranexpectedchangeinPCAvaccompaniedwithexhaustiveexercisemodifiestheresponseinPCAvtovisualstimulation,wemeasuredthePCAvbytranscranialDopplerultrasoundflowmetryduringrestandexercisein13healthymaleswhiletheyperformedaleg-cycleexerciseat75%ofmaximalheatrateuntilexhaustion.NVCwasestimatedastherelativechangeinPCAvfromthemeanvalueobtainedduring20sofeyeclosingtothepeakvalueobtainedduring40sofvisualstimulationinvolvinglookingatareversedcheckerboard.Theconductanceindex(CI)ofthePCAwascalculatedbydividingPCAvbythemeanarteri-alpressure(MAP).Atexhaustion,PCAvsignificantlydecreasedby-4±1%(mean±SE)withadecreaseinPaCO2by-3±1%.PCAvresponsetovisualstimulationsignificantlydecreasedfrom16±1to11±1%.Comparedtotherestingbaseline,exhaustiveexercisesignificantlydecreasedtheincreaseresponseinMAPtovisualstimulationfrom5±2to0±1mmHg.TheCIresponsewasnotsignificantlychanged.TheseresultssuggestthatexhaustiveexercisedecreasesthePCAv,andthemagnitudeof theresponse inPCAvtovisualstimulation.(SupportedbyKozukiFoundation)NoCOI.

1P-049Activation of NTS histamine H1 receptors modulates the gain of baroreflex control of heart rate in rats.Takagishi, Miwa1,2; Waki, Hidefumi1; Gouraud, Sabine S1; Kohsaka, Akira1; Maeda, Masanobu1(1Department of Physiology, Wakayama Medical University, Wakayama, Japan, 2Department of Therapeutic Health Promotion, Kansai University of Health Sciences, Osaka, Japan)

HA-immunoreactiveneuronsarefoundexclusivelyinthetuberomam-millarynucleusandknowntoprojecttomanyareasofthebrain,in-cludingthenucleustractussolitarii(NTS),playsanimportantroleinregulatingcardiovascularhomeostasis.WehavepreviouslyshownthatactivationofhistamineH1receptors intheNTSincreasedarterialpressure(AP)andheartrate(HR),andattenuatedthecardiacbarore-flexgain.Inthepresentstudy,weexaminedwhetherH1receptorsintheNTScanaffectthereflexgainindependentlyofincreasesinthebasalAPandHR.Moreover,effectsofendogenoushistamineonthereflexgainwereexamined inurethane-anaesthetizedWistarrats.After2-Pyridylethylamine(25nmol),ahistaminereceptorH1agonist,wasunilaterallymicroinjectedintotheNTS,thegainofthecardiacbaroreflexwassignificantlyattenuatedwithoutanychangesinthesetpointsofAPandHR.AfterbilateralmicroinjectionsofH1-receptor-spe-cificantagonist(cetirizinehydrochloride)intotheNTS(100pmol),thebasalAPandHR,andthebaroreflexgainwerenotchangedfromtheircontrolvalues.ThesefindingssuggestthatNTShistaminemaymod-ulatecardiovascularsystemviaactivationofH1receptorsinthearous-alstageasahighlevelofactivityofhistaminergicneuronsthoroughH1receptorsisseeninthearousalstates,andwefurtherconfirmedthatactivationofH1receptorsintheNTSmodulatescardiovascularfunctions.NoCOI.

1P-050Role of T-type Ca2+ channel expression in cardiomy-ocytes exposed to hypoxic condition in the heartMorishima, Masaki1; Osagawa, Satoshi1; Inagaki, Tadakatsu2; Tsuchimochi, Hirotsugu2; Shirai, Mikiyasu2; Ono, Katsushige1(1Department of Pathophysiology, Oita University School of Medicine, Oita, Japan, 2Department of Cardiac Physiology, National Cerebral and Cardiovascular Center Research Institute, Osaka, Japan)

T-typeCa2+channelcurrents(ICaT)areinvolvedinautomaticactivityincardiaccells.TheCaV3.1andCaV3.2isoformsareresponsibleforICaTinnormalatrialandnodalmyocytesbutnotinventricularmyocytes.Inpathologicalcondition,bothisoformsarefunctionallyexpressedinventricularcardiomyocytes.However,adirectrelationbetweenhy-poxicstressandT-typeCa2+channelregulation isnotknown.WeinvestigatedmolecularmechanismsfortranscriptionalregulationoftheT-typeCa2+channel(CaV3.1andCaV3.2)inadultandneonatalratcardiomyocytesexposedtohypoxia.MaleWistarrats(10wk)weresubjectedtonormoxia(20%O2)orhypoxia(8%O2)for3h.mRNAexpressionoftheT-typeCa2+channelandtranscriptionfactors(Sp1,Id2,Csx/Nkx2.5,NRSF)wereassessedbyreal-timePCR.Down-regu-lationofCaV3.1mRNAinhypoxiccardiomyocyteswasaccompaniedbyanincreaseintranscriptionalrepressorId2throughtheactivationofhypoxia-induciblefactor-1α(Hif-1α).Ontheotherhand,expressionofCaV3.2,NRSF,andCsx/Nkx2.5mRNAwasnotchanged.Hypoxicstimulation(1%O2,6–24h)decreasedthebeatrateofcardiomyocytesassociatedwithreductionoftheCaV3.1isoform.TheseresultssuggestanovelsignalpathwayforthepathologicalexpressionofCaV3.1 incardiomyocytesdependingonthetranscriptionfactorsHif-1αandId2.NoCOI.


1P-051Overexpression of SERCA or sarcolipin does not alter Ca2+ induced Ca2+ release and Ca2+ leak in mouse myocardiumMorimoto, Satoshi1; Hongo, Kenichi1; Kusakari, Yoichiro2; Komukai, Kimiaki1; Kawai, Makoto1; Yoshimura, Michihiro1; Kurihara, Satoshi2(1Dvision of Cardiology, Jikei Univ. Sch. Med., Tokyo, Japan, 2Department of Cell Physiology, Jikei Univ. Sch. Med., Tokyo, Japan)

Sarcoplasmicreticulum(SR)playsanimportantroleincardiacexci-tation-contractioncoupling.Recently,decreasedCa2+uptakeintoSRbySRCa2+-ATPase(SERCA)hasbeenreportedtodecreasecardiaccontractioninheartfailure.However,therelationshipbetweenCa2+uptakeandSRfunctions [maximalCa2+content,Ca2+ inducedCa2+release(CICR)andCa2+leak]hasnotbeenfullyinvestigated.Wein-vestigatedtherelationshipbetweenCa2+uptakeintoSRbySERCAandotherSRfunctionsusinggeneticallymanipulatedmicehearts.Weusedsaponin-treatedthintrabeculaeobtainedfrommiceheartswithoverexpressionofSERCA(SERCA-TG)orsarcolipin(SLN-TG).Ca2+contentwasmeasuredbyreleasingallCa2+fromSRbycaffeine(50mM)afterloadingCa2+intoSRinthepresenceofATP(4mM)andvariousconcentrationsofCa2+forvariousdurations.CICRandCa2+leakwereestimatedbymeasuringtheremainingCa2+ inSRaftertreatingtheCa2+-loadedSRwiththesolutionsofvariousCa2+concen-trations(relaxingsolutionatpCa20forCa2+leak).Ca2+uptakeratewasfaster inSERCA-TGandslower inSLN-TGthanthat ineachnon-transgenicmouse(NTG).MaximalCa2+content,Ca2+leakandCICRinSERCA-TG,SLN-TGandeachNTGwerealmost identical.Theresultssuggest themodulationofCa2+uptakeratedoesnotaltermaximalCa2+content,CICRandCa2+leakatsteadystate.NoCOI.

1P-052Role of mitochondria on stretch-induced increase in calcium spark rate.Kaihara, Keiko; Naruse, Keiji; Iribe, Gentaro(Cardiovascular Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan)

Wehavepreviouslyreportedthatmyocardialdiastolicstretchinduc-esanincreaseinCa2+sparkrate.Ithasbeenreportedthatdiastolicstretchrapidlyproducesreactiveoxygenspecies(ROS)viaNADPHoxidase (NOX),andoxidatesryanodinereceptorstogiverisetoanincreaseinCa2+sparkrate.Mitochondriaarewell-knownasanothersourceofROSviaelectrontransportchaininATPproductionprocess.Inthepresentstudy,weinvestigatedtheinvolvementofmitochondri-alROSproductioninstretch-inducedacuteincreaseinCa2+sparkrateinmouseventricularmyocytes.Isolatedmouseventricularmyocyteswereexposedto10%axialstretchusingcomputer-controlledpiezo-ma-nipulatedcarbonfibers,attachedtobothcellends.Diastolicsparkrate,ROSproductionandmitochondrialmembranepotentialwerestudiedusingFluo-4,DCFandTMRE-loadedcells,respectively.AxialstretchsignificantlyincreasedCa2+sparkrate(n=19)andROSproduction(n=16).Applying5µMmitochondrialmetabolicuncouplerFCCPinthepresenceof5µMoligomycin(topreventATPdepletion)bluntedthebothresponse.Stretchsignificantlyhyperpolarizedmitochondrialmem-branepotential.ThepresentresultssuggestthatmyocardialstretchincreasesnotonlyNOX2-derivedROSbutalsomitochondrialROS,andbothareinvolvedinstretch-inducedincreaseinCa2+sparkrate.Stretch-inducedmitochondrialhyperpolarizationsuggeststhatstretchenhanceselectrontransportsystem,leadstoincreaseinmitochondri-alROSproduction.NoCOI.

1P-053CaV1.3 is involved in Ist in mouse SAN cellsToyoda, Futoshi1; Mesirca, Pietro2,3,4; Dubel, Stefan2,3,4; Ding, Wei-Guang1; Striessnig, Joerg5; Mangoni, Matteo E2,3,4; Matsuura, Hiroshi1(1Dept. Physiol., Shiga Univ. Med. Sci., Otsu, Japan, 2CNRS, IGF, Dept Physiol, Montpellier, France, 3INSERM, Montpellier, France, 4Univ Montpellier 1 & 2, France, 5Dept Pharmacol Toxicol, Ctr Mol Biosci, Univ Innsbruck, Austria)

Thesustained inwardNa+current (Ist)hasbeenput forwardasapotentialinwardcurrentdrivingthepacemakerdepolarization.How-ever,unknownmolecularcorrelatesforIstlimitstheunderstandingofitssignificanceintheSANpacemakeractivity.PreviousstudieshaverevealedclosesimilaritybetweenIstandICa,Lintheirpharmacologicalproperties,suggestingthatthesedistinctcurrentsshareacommonmoleculardeterminant.Todirectlyaddressthishypothesis,thepres-entstudyemployedtwogeneticallymodifiedmousemodels.Patch-clamprecordingsofIstinmouseSANcellswerecharacterizedbyanactivationondepolarizationinthepacemakerpotentialrange, littleinactivationduringdepolarizingpulses,Na+-permeabilityandsensitiv-itytovariousclassesofICa,Lmodulators.Inknock-outmicewithdis-ruptionofCaV1.3Ca2+channelsubunit,ICa,Lwassignificantlyreducedanditsvoltagesensitivityforactivationwasshiftedtopositivedirec-tion,whichwasaccompaniedbycompletelossofIst.Ontheotherhand,inknock-inmicecarryingapointmutationthatrendersCaV1.2dimin-ishedaffinityforbindingofDHPs,aconsiderablefractionofICa,Lex-hibitedresistancetotheinhibitionbynifedipine,whereasIstretainedtheDHPsensitivitycomparabletothatinwild-type.ThesefindingsclearlyshowthatCaV1.3subunitisinvolvedinthegenerationofnotonlyICa,LbutalsoIst.NoCOI.

1P-054Quantifying contributions of individual ion transport mechanisms to electric activities by using cardiac myocyte models.Yamaoka, Hiroyo(Graduate School of Life Sciences, Ritsumeikan University, Shiga, Japan)

Thecomputermodelsofcellularfunctionisdevelopedbyintegratingempiricalexperimentalequations(ordinarydifferentialequation),whichdescribeactivitiesofconstitutivemolecules.Thecellmodelshouldbefirstlyvalidatedbyreproducingvariousexperimentalfindings.Then,itisessentialtoanalyzemathematicalbehaviorofthemodeltoclarifytheprincipleofcellularfunction,aswellasthestabilityofthesystem.Ourgroupdevelopedthemethod‘leadpotentialanalysis’todeterminequantitativecontributionofeachionchannelsandtransporterstotheelectricactivitiesofthecellmodel,suchasthepacemakerdepolariza-tioninthesino-atrialnode,theventricularrepolarizationandabnormalbehavior.Inthepresentstudy,wefurtherrevisedthemethodanddemonstrateresultsofapplyingthemethodtorepresentativeventric-ularcellmodels.NoCOI.


1P-055C-terminal helix D of KCNQ1 contributes to normal IKs channel functionKomatsu, Takuto1; Kinoshita, Koshi2; Kimoto, Katsuya1; Hata, Yukiko2; Aonuma, Kei1; Tsushima, Eikichi1; Nishide, Kohki1; Hisajima, Nozomi1; Takahashi, Hiroyuki1; Fukurotani, Kenkichi1; Nishida, Naoki2; Tabata, Toshihide1(1Lab. Neural Info. Tech., Grad. Sch. Scis. & Eng., Univ. Toyama, Toyama, Japan, 2Dept. Legal Med., Grad. Sch. Med. & Pharm. Scis., Univ. Toyama, Toyama, Japan)

MutationsofKCNQ1whichencodestheαsubunitoftheslowdelayedrectifierK+(IKs)channelmaycausecardiacrepolarizationabnormalityandseverearrhythmia.WefoundthatapatientwithamildQTinter-valprolongationcarriesKCNQ1withamutation(A590T)inthehelixD,aregionsuggestedtobeimportantforsubunitmultimerizationandchanneltrafficking.Themutationatthenextposition(G589D)hasbeenreportedtodisrupttheinteractionofKCNQ1withYotiao(AKAP9),whichisresponsibleforcAMP-dependentIKsmodulationnecessaryfornormalchannelgating.WeexaminedwhetherandhowtheA590TandG589DmutationsaffectthemolecularandelectrophysiologicalpropertiesofIKschannelinHEK-293Tcells.ImmunoblotanalysisshowedthatboththemutantKCNQ1couldmultimerizebythemselvesorwithwild-typesubunit.Immunoprecip-itationanalysisshowedthatboththemutantKCNQ1could formcomplexeswithYotiaoandKCNE1.Patch-clampanalysisshowedthatanintracellularlyappliedcAMPanalogaugmentedbothIKsmediatedbyA590Tmutantchannelandwild-typechanneltosimilarextents.TheseresultsindicatethatKCNQ1helixDplaysimportantrolesinthefunctionalexpressionofIKschannelvianovelmechanismsinde-pendentofinteractionwithYotiao.NoCOI.

1P-056Testosterone abbreviates QT intervals and up-regu-lates KvLQT1 channel in cardiomyocytes through ge-nomic pathway Masuda, Kimiko; Wang, Yan; Ma, Fang Fang; Morishima, Masaki; Takanari, Hiroki; Ono , Katsushige(1Department of Pathophysiology, Oita University School of Medicine, Oita, Japan)

Afteradolescence,menhaveshorterQTintervalscompareswiththeseinwomen.ItispostulatedthatQTintervalsaremodulatedbysexhormones.Althoughithasbeenrecognizedthattestosteroneactivatesdelayedrectifierpotassiumcurrentincardiomyocytesbynon-genomeactionthroughNOS, the long-termactionsof testosteroneon ionchannelsarenotknown.Then,weinvestigatetheroleoftestosteronefocusingon itselectrophysiological long-termaction inrats. Afteradministrationoftestosteronetocastratedmalerats,QTintervalsweregraduallyshortened,andreachedsignificantdifferenceaweeklater(-12±4%).ExpressionofKvLQT1channelwasincreasedby43%inmRNAlevel,andby10%inproteinlevel.However,expressionofERG,Kir2.1,Kv4.3,Kv4.2,MiRP1,minK,andKChIP2wasunchanged.Thesimilarup-regulationofKvLQT1channelbytestosteronewasalsoconfirmedbyfemalerats’cardiomyocytes.ThisresultssuggestthattestosteronemodifiesQTintervalsthroughagenomepathway,whichmayresponsibleforthedifferencebetweenmenandwomeninar-rhythmogenicsubstates.NoCOI.

1P-057Effects of ibudilast on the GIRK channel in isolated atrial myocyte and atrial fibrillation induced by vagal nerve stimulation in ratKimura, Shingo1; Harata, Misato1; Fujita, Reiko2; Hirose, Masamichi3; Kubokawa, Manabu1(1Dept. of Physiology, Iwate Medical University, Morioka, Japan, 2Dept. of Chemistry, Ctr. Lib. Arts & Sci., Iwate Medical University, Morioka, Japan, 3Dept. of Molecular & Cellular Pharmacology, Sch. of Pharmacy, Iwate Medical University, Morioka, Japan)

Ibudilastisknownasanon-selectivephosphodiesteraseinhibitor,andhasbeenusedfortreatmentofbronchialasthmaorcerebralcontrac-tion. Inan isolatedatrialmyocyteundervoltageclamp, ibudilastnon-competitivelyandreversiblyinhibitedacetylcholine(ACh)-inducedG-protein-activated inwardlyrectifyingK+ (GIRK)current.Further-more,ibudilastalsosuppressedGIRKchannelcurrentelicitedbyin-tracellularapplicationofGTPγS,anonhydrolyzableanalogueofGTP.AlthoughintracellularapplicationofibudilastdidnotinhibittheACh-in-ducedGIRKcurrent,extracellularapplicationmarkedlyinhibiteditinthesamecell.Wefurtherexaminedtheeffectsofibudilastonthesinusrateandatrialfibrillationinanesthetizedrats.Electricalstimu-lationofbilateralcervicalvagalnerves frequency-dependentlyde-creasedthesinusrate,andatrialfibrillationwas inducedbyburstpacingoftheleftatriumduringvagalstimulation.Injectionofintra-venousibudilastsignificantlyrecoveredthedecreasesinthesinusrateinresponsetovagalstimulation,andsuppressedtheonsetofatrialfibrillationexceptoneof8ratsduringvagalstimulation.TheseresultssuggestthattheibudilastiseffectiveforpreventionortreatmentofatrialfibrillationbyinhibitingthecurrentofGIRKchannels.NoCOI.

1P-058The Effect of Nicorandil on Na/Ca Exchange current in Cardiac MyocytesWatanabe, Yasuhide1; Wei, Jia-zhang2; Takeuchi, Kazuhiko2; Yamashita, Kanna1; Kita, Satomi3; Iwamoto, Takahiro3; Watanabe, Hiroshi2; Kimura, Junko4(1Department of Health Science, Hamamatsu University School of Medicine, Hamamatsu, Japan, 2Department of Clinical Pharmacology, Hamamatsu University School of Medicine, Hamamatsu, Japan, 3Department of Pharmacology, Fukuoka University School of Medicine, Fukuoka, Japan, 4Department of Pharmacology, Fukushima Medical University School of Medicine, Fukushima, Japan)

Itwaspreviouslyreportedthatnicorandil,aK(ATP)channelopenermayexertantiarrhythmicactionsbyabolishingtriggeredactivity(Lathropetal.,1990).Thetriggeredactivityinducedbydelayedaf-ter-depolarization (DADs) isoneof themechanismsofventriculararrhythmiasassociatedwithintracellularCa2+overload.InthisstudyweexaminedtheeffectofnicorandilonNa/Caexchangecurrent(INCX)inisolatedguineapigventricularmyocytesusingthewhole-cellpatchclamptechnique.NicorandilenhancedINCXinaconcentration-depen-dentmanner.TheEC50valuesofnicorandilwere15.0µMand8.7µMfortheoutwardandinwardcomponentsofINCX,respectively.8-Br-cG-MP,amembranepermeableanalogofcGMPalsoenhancedtheINCXinaconcentration-dependentmanner.ODQ,aguanylatecyclaseinhib-itor(10µM)almostcompletelyabolishedtheeffectsofbothnicorandiland8-Br-cGMPonINCX.DADsinducedbyelectricalstimulationwithouabaindisappearedinthepresenceof100µMnicorandil.Wecon-cludedthatnicorandilenhancesthefunctionofNa/Caexchanger(NCX)viaguanylatecyclase,andthismaypartiallycontributedtothecardi-oprotectionofnicorandilbyacceleratingCa2+exitviaNCX.NoCOI.


1P-059Induced automaticity in ventricular myocytes from transgenic mice overexpressing HCN2Oshita, Kensuke1; Itoh, Masayuki1; Yanagi (Ishihara), Keiko1; Kuwabara, Yoshihiro3; Kuwahara, Koichiro3; Ushijima, Kazuo2; Takano, Makoto1(1Dept. of Physiol., Kurume Univ. Sch.of Med. Kurume, Japan, 2Dept. of Anesth., Kurume Univ. Sch. of Med. Kurume, Japan, 3Dept. of Cardiovasc. Med., Grad. Sch. of Med. Kyoto Univ. Kyoto, Japan)

HCNchannelsareexpressedintheventricleoffetalhearts,andaresilencedduringdevelopment.Thesechannelsarere-expressedinthehypertrophiedheart,andhavebeensuggestedtounderliearrhythmo-genesis.Totestthishypothesis,wegeneratedandanalyzedtransgen-icmiceoverexpressingHCN2specificallyintheirhearts(HCN2-Tg).Actionpotentials(APs)andmembranecurrentswererecordedusingwhole-cellpatchclampmethod.InallofHCN2-Tgmyocytes,0.3µMisoproterenol (ISO)significantlydepolarizedtherestingmembranepotential(RMP).In77%ofHCN2-Tgmyocytes,ISOinducedsponta-neousactionpotentials(SAPs).IntherestofHCN2-Tgmyocytes,thelaterepolarizationphaseofevokedAPswassignificantlyslowerthaninWTmyocytes.Analysisofmembranecurrentsandtime-differentialofAPsrevealedthatthesedifferencesareattributabletoHCN2tailcurrent.WhentheRMPwashyperpolarized in3mMK+bathingsolution, theRMPinHCN2-Tgwasmoredepolarizedthan inWTmyocytes,butthisdifferencewasnotobservedinthepresenceoftheHCNblocker,ivabradine.Moreover,theRMPofHCN2-Tgwasunsta-bleunderhypokalemiccondition,andSAPswas inducedin57%ofHCN2-Tgmyocyte.ThesefindingssuggestoverexpressionofHCN2increasesthelikelihoodofarrhythmia,particularlyunderpathologicalconditionssuchasexcessiveβadrenergicstimulationorhypokalemia.NoCOI.

1P-060Self-beating atypically-shaped cardiomyocytes (ACMs), a novel subpopulation of heart cells, origi-nate from cardiac progenitor cells expressing prion protein (PrP) in adult mouse heartNishino, Yuka; Nozuchi, Nozomi; Omatsu-Kanbe, Mariko; Matsuura, Hiroshi(Department of Physiology, Shiga University of Medical Science, Otsu, Japan)

Atypically-shapedcardiomyocytes(ACMs)areanewsubpopulationofspontaneouslybeatingheartcellswithapeculiarmorphologyiden-tifiedwithinacultureofcardiacmyocyte-depletedfractioncellsob-tainedfromadultmousecardiacventricles.ACMsgrowinsizeandstartbeatingwithin~3daysculturewithoutappreciableproliferationorexpressionofstemcellmarkerproteins,butstayintheheartuntilelderlystages.Ithasbeenrecentlyreportedthatprionprotein(PrP)servesasasurfacemarkerforisolatingcardiomyogenicprogenitorsfrommurineembryonicstemcells.ThepresentstudyexaminedthelocalizationoforiginalACMsinmouseheartusingPrPasacellmark-er.ACMswerefoundtoexpressPrPmostlyintheplasmamembraneevenintheveryearlystageofculture.ACMscouldbeisolatedbyflowcytometry,thoughtheyieldwasverylow.Immunohistochemicalanalysesrevealedthatthecellsco-expressingPrPandcardiactroponinT(cTnT)existedintheinterstitialspacesamongventricularmyocytes.Thesecellswereobservedassolitaryorclusteredcells.TheresultssuggestthatthePrP(+)/cTnT(+)cellsidentifiedinthemousecardiacventriclesaretheoriginsofACMsresidentascardiacprogenitorcellsinthepostnatalmouseheart.NoCOI.

1P-061Identification of a novel type of possible cardiac pro-genitor cells co-expressing prion protein (PrP) and cardiac troponin T (cTnT) in adult human heartNozuchi, Nozomi; Nishino, Yuka; Omatsu-Kanbe, Mariko; Matsuura, Hiroshi(Department of Physiology, Shiga University of Medical Science, Otsu, Japan)

Thepresentstudyexaminedtoseekanoveltypeofcardiacprogeni-torcells inadulthumanheart.Wehaverecently identifiedanewpopulationofheartcellsinadultmousecardiacventriclesthatspon-taneouslydevelopintobeatingcardiomyocyteswithapeculiarmor-phology,definedasatypically-shapedcardiomyocytes(ACMs).ACMsdidnotappreciablyproliferateorexpressstemcellmarkerproteins,butsurvivethelong-termpost-nataldevelopmentofcardiacventricles.Prionprotein(PrP)hasbeenreportedtoserveasasurfacemarkerforisolatingcardiomyogenicprogenitorsfrommurineembryonicstemcells,andwehavefoundthatmouseACMsstronglyexpressPrPmostlyintheplasmamembraneevenintheveryearlystageofculture.ToinvestigatewhethersuchPrP-positiveACM-likecellsareresidentinhumanheart, immunohistochemicalanalyseswereperformed inhumancardiacventriculartissuesfixedwithin2~3hrafterdeathselectedfrom5patients(2male,3female;aged41–86yearsold)whodiedofcancerorrespiratoryfailure.Wefoundthatsmallcellsco-ex-pressingPrPandcardiactroponinT(cTnT)existedintheinterstitialspaceamongventricularmyocytesinallhumantissuestested.ThesecellsdidnotexpresshematopoieticstemcellmarkerCD45.TheresultssuggestthepossibilitythatthesePrP/cTnT-positivecellssurviveduringlifeascardiacprogenitorcellsinhumanheart.NoCOI.

1P-062Mathematical analysis of end systolic force length re-lation of a linear approximated cardiac cell contrac-tion modelNakai, Shota; Amano, Akira(Dept. of Life Sciences, Graduate School of Life Science, Ritsumeikan Univ, Shiga, Japan)

ToelucidatethebehaviorofEmax,which is theslopeof theendsystolicpressurevolumerelation(ESPVR),itisusefultousesimulationmodels.Thisstudyadoptsthecardiovascularhemodynamicsmodelconsistsofacardiaccontractionmodel,NegroniLascano1996(NL96)model,acirculationmodel,andaleftventriculargeometrymodeltorelatetheNL96modelwiththecirculationmodel.ThesimulationmodelgivesagoodreproductionofthehighlinearityofESPVR.ToinvestigatetheparametersthataffectEmax,alinearapproximatedNL96modelwasemployedforthemathematicalanalysis.Withthismodel,analyticallyderivedendsystolicforcelengthrelation(ESFLR)showedthattheslopeofESFLRisproportionaltotherateparameterofcross-bridgeattachmenttoathinfilamentsiteandthecoefficientofoverlapfunctionthatrepresentstheoverlapratioofactinandmy-osintothesarcomerelength.Furthermore,itwasrevealedthattheslopeisinverselyproportionaltotherateparameterofcross-bridgedetachmenttoathinfilamentsite.Theseresultswereverifiedbythenumericsimulationresultsoftheoriginalmodel.NoCOI.


1P-063The effect of blood contact surface area during car-diopulmonary bypass—Biological body evaluation in a rat model—Fujii, Yutaka1; Shirai, Mikiyasu2; Takewa, Yoshiaki1; Tatsumi, Eisuke1(1Dept. Artif Organs., Natl.Cereb. Cardiovas. Ctr, Osaka, Japan, 2Dept. Cardiac Physiol., Natl.Cereb. Cardiovas. Ctr, Osaka, Japan)

Extracorporeallifesupport,suchasthecardiopulmonarybypass(CPB),preservethepatient’slifebyprovidingadequateoxygensupplyandbloodflowtovitalorgans.However,previousstudieshavesuggestedthattheinteractionofbloodandlargeartificialsurfacecontributestoinflammatoryresponseduringCPB.Asaresultofseriesofchainre-actions, thenumerouspowerful inflammatorymediators, includinghormonesandautacoids,areformedandreleased.WehypothesizedthatsmallCPBcircuitwhichreducesprimingvolumeandbloodcon-tactsurfaceareaattenuatesthesystemicinflammatoryresponsewithareductionofinflammatorycytokinelevelsandorgantissuedamageduringCPB.Ratsweredivided intothehighprimingvolumeCPB(primingvolume:15ml,surfacearea:0.044m2)groupandthe lowprimingvolumeCPB(primingvolume:7ml,surfacearea:0.036m2)group.CPBpumpflowwasmaintainedat80ml/kg/min.Bloodsampleswerecollectedbefore,and60minand120minafterinitiationofCPB.Pro-inflammatorymarkerssuchas(TNF-α)andbiochemicalmarkers(LDH,ALT,AST)weresignificantlyelevated inthehighprimingvolumeCPBgroupcomparedwiththelowprimingvolumeCPBgroupat60min.At120min,however,noneofthemarkerswasstatisticallydifferentbetweenthe2groups.Thesedatasuggestedthatinaddtiontothebloodcontactsurfaceareafactor,theCPBexposuredurationisalsoan important factor forcausingthesystemic inflammatoryre-sponse.NoCOI.

1P-064Effects of capsaicin treatment on rat left ventricular mechanical work and energetics in comparison with hyperthermia Obata, Koji1; Abe, Chikara1; Morita, Hironobu1; Takaki, Miyako1,2(1Dept. of Physiology, Gifu University Graduate School of Medicine, Gifu, Japan, 2Dept. of Molecular Pathology, Nara Medical University School of Medicine, Kashihara, Japan)

Wepreviouslyreportedthattheeffectsofhyperthermia(42°C)onleftventricular(LV)mechanicalworkandenergeticsusingtheexcised,cross-circulatedratheartmodel.Wenowinvestigatedtheeffectsofcapsaicin(aTRPV1agonist)onLVmechanicalworkandenergetics.WeanalyzedtheLVend-systolicpressure-volumerelation(ESPVR)andthelinearrelationbetweenthemyocardialoxygenconsumptionperbeat(VO2)andsystolicpressure-volumearea(PVA;atotalme-chanicalenergyperbeat) in isovolumicallycontractingratheartsduring infusionofcapsaicin (1–40ng/ml)under300-bpmpacing.Incapsaicintreated-hearts,LVESPVRshifteddownwardfromthecon-trolESPVRlikeinhyperthermic-hearts.TheslopeofVO2-PVArelationwasnotsignificantlydifferent.However, theVO2 interceptthat iscomposedofeachVO2 fractionconsumedinexcitation-contraction(E-C)coupling(mainlyconsumedforcalciumhandling)andforbasalmetabolism,wasdecreasedincapsaicintreated-heartsdifferentlyfromthatinhyperthermic-hearts.Inhyperthermic-hearts,theVO2interceptwasnotchangedbecauseofdecreasedE-CcouplingVO2andincreasedbasalmetabolicVO2.WesternblottinganalysisshowedthattheproteinlevelsofSERCA2,phospholamban(PLB)andphosphorylatedPLBwerenotchanged.TheseresultsindicatedthatcapsaicindecreasedtheLVcontractilitylikehyperthermiaduetothedown-regulationofthecal-ciumhandlinginE-Ccoupling.NoCOI.

1P-065Optical mapping study of the condition for circus movement of the excitatory waves in the rat isolated atrium preparationSAKAI, Tetsuro(Department of Systems Physiology, Graduate School and Faculty of Medicine, University of the Ryukyus, Okinawa, Japan)

Usingtheopticalmappingmethods,wehavestudiedthespatiotem-poralpatternoftheelectricalactivitie inthe isolatedratauricularpreparation. Each preparation was stained with a fast merocy-anine-rhodaninevoltage-sensitivedye(NK2761).Usingamulti-element(16×16)photodiodearray,weassessedopticallythespreadofexcitationbytimingthefeetofopticalactionpotentials.Wetriedtoevoketheeventof thetachyarrythmia (tachycardia-likeexcitation)withthecircusmovementofexcitatorywaves(re-entry)byapplyingtetanusstimulationunderseveralconditions.Asreportedpreviously,eventsoftcachycardia-like-excitationareobservedinthepreparationwithananatomicalobstacle.IntheCa2+overloadedcondition,there-entryofexcitatorywaveswasalsoobserved.Inthisstudy,wehavenewlyfoundthetcachycardia-like-excitationwithre-entryoftheexcitatorywavesbyapplyingouabainorryanodine.Inbothconditions,theblockedareasthroughwhichexcitationcouldnotpropagateappearedtran-sientlyduringtheformationofthere-entrypath.Theblockedareawithoutanatomicalobstaclesalsoappearedinthecenterofthere-en-trypath(singulararea).Allthesechemicalproceduresseemtodisturbthe intracellularCa2+dynamics,supportingourhypothesisthatthetcachycardia-like-excitationwithanomalouspatternsoftheexcitationspreadincludingthecircusmovementoftheexcitatorywavesisin-ducedbytheinhomogenousincreaseofintracellularCa2+concentration.NoCOI.

1P-066High-speed live imaging of single sarcomeres in the mouse heart in vivo.Kobirumaki-Shimozawa, Fuyu1; Oyama, Kotaro2; Hirokawa, Erisa3; Shimozawa, Togo4; Terui, Takako5; Minamisawa, Susumu1; Ishiwata, Shin'ichi2; Fukuda, Norio1(1Department of Cell Physiology, The Jikei University School of Medicine, Tokyo, Japan, 2Department of Physics, Waseda University, Tokyo, Japan, 3The Jikei University School of Medicine, Tokyo, Japan, 4Department of Physics, Gakushuin University, Tokyo, 5Department of Cell Physiology Anesthesiology, The Jikei University School of Medicine, Tokyo, Japan)

Toexplorethemolecularmechanismsofcardiacmusclecontractionunderphysiologicconditions,wedirectlyimagedthemotionsofsinglesarcomeresinthebeatingheartin vivoathighspatialandtemporalresolution.ThesarcomericZ-lineswerelabeledwithα-actinin-AcGFPviainfectionofrecombinantadenoviruses,andsarcomerelengthsweremeasuredusingaspinningdiskconfocalmicroscopeat~100fps(res-olution inXYplane,~20nm).Wesuccessfully imagedsarcomericmotions,simultaneouslywithelectrocardiogramandleftventricularpressure.Ouranalysisrevealedthatthereexistsapositivecorrelationbetweensarcomere lengthand leftventricularpressure (i.e., theFrank-Starling lawoftheheart).Likewise,Ca2+waves/transientswereobservedincardiomyocytesoftheisolatedheart.Atthemeeting,wewilldiscusshowcardiacexcitation-contractioncouplingisorganizedin vivo.NoCOI.


1P-067Simultaneous observation of single sarcomere length and local calcium in rat neonatal cardiomyocytes via expression of cameleon-Nano in Z-discsTsukamoto, Seiichi1; Oyama, Kotaro2; Shintani, Seine2; Kobirumaki, Fuyu1; Ishiwata, Shinichi2,3; Fukuda1, Norio1(1Department of Cell Physiology, The Jikei University School of Medicine, Tokyo, Japan, 2Department of Physics, Faculty of Science and Engineering, Waseda University, Tokyo, Japan, 3Waseda Bioscience Research Institute in Singapore, Waseda University, Singapore, Singapore)

Incardiacmuscle,contractionisregulatedbymicromolarconcentra-tionsofCa2+releasedfromthesarcoplasmicreticulum(SR)(i.e.,theCa2+-inducedCa2+releasemechanism),resultinginthebindingofCa2+totroponinCandsubsequentformationofcross-bridges.Inordertoenhanceourunderstandingofcardiacexcitation-contractioncoupling,weinthepresentstudydevelopedanovelexperimentalsystemforsimultaneousmeasurementofintracellularCa2+andsinglesarcomerelengthviaexpressionofyellowcameleon-Nano(i.e.,FRET-basedultra-sensitiveCa2+indicator)fusedtotheC-terminusofα-actinininZ-discsinprimary-culturedratneonatalcardiomyocytes.Thefluorescenceemissionratio(i.e.,YFP/CFP)oftheexpressedfusionproteinvariedinresponsetoachangein[Ca2+]i.Underadual-viewmicroscopy,wemeasured localCa2+changesby imagingYFP/CFPandanalyzedsinglesarcomerelengthbydetectingthedistancebetweentheYFPfluorescenceprofiles.Accordingly,wefoundthatsarcomere lengthvariedinresponsetoachangeinYFP/CFPinvariousregionsofthemyocyte.Theseresultssuggestthatthepresentexperimentalsystemwithyellowcameleon-NanoisusefulforthesimultaneousimagingoflocalCa2+andsinglesarcomerelengthincardiomyocytes.NoCOI.

1P-068The developmental changes in the contractile pro-teins of rat embryonic hearts around the period of the appearance of the heartbeatKobayashi, Takeshi; Maeda, Sachiko; Ichise, Nobutoshi; Miyakawa, Tsuyoshi; Ogon, Izaya; Yamada, Yoichi; Tohse, Noritsugu(Department of Cellular Physiology and Signal Transduction, Sapporo Medical University School of Medicine, Sapporo, Japan)

Wistarratheartbeginstocontractatembryonicday9.99-10.13,beforetheappearanceof the linearhearttube,andthebeginningof thecalciumtransientprecedesthe initiationofcontraction. Inordertoinvestigatetherelationshipbetweenthecalciumtransientandcon-traction,wetriedtodetectthedevelopmentalchangeinthecontrac-tileproteinamountofindividualembryonicheartbyhighsensitivity3-stepWesternblottingmethod.Wealreadyreportedthatmyosinregulatorylightchainofembryonicheartincreasedaroundtheperiodoftheappearanceoftheheartbeat.Inthisstudy,weshowedthede-velopmentalincreaseintroponinIduringthisperiod.Thisresultin-dicatedthatthedevelopmentalincreaseintroponinIalsoinvolvesintheinitiationofcontraction.NoCOI.

1P-069Preliminary analysis of cell cycle regulation in cardio-myocytes.Hashimoto, Ken; Ujihara, Yoshihiro; Mohri, Satoshi(First Dept. of Physiology, Kawasaki Medical School, Japan)

Earlyinthedevelopment,embryoniccardiomyocytes(CMs)activelyproliferate.Butthisactivityislostshortlyafterbirthinmammals,themechanismsofwhichareunknown.Atbirth,CMsareexposedtodramaticenvironmentalchanges,including(1)theabruptelevationofoxygentensionbytheonsetofbreathing(PaO2:20mmHginfetusesto100mmHginadults),(2)thetemporarystarvationduetothelossofmaternalbloodsupply,and(3)drastichemodynamicchanges.HerewefocusedontheeffectofelevatedO2tensiononCMproliferation.FetalCMsatembryonic(E)day17wereisolatedfromC57BL/6mice,withkeepinglowO2conditions(3%)duringisolation.TheexposureoftheseCMstoatmosphericO2(20%),thatmimicsbirth,inhibitedcellprolif-erationwhichwasevaluatedbothbydirectcellcountandtheexpres-sionofcellcyclemarkerKi67.ThissuggeststhatincreasedO2exposuretoCMsatbirthisthefundamentalsignaltoinitiatethedownstreamgeneprogramsforcellcycleexit,andlowO2milieumaybeessentialforfetalCMstoactivelyproliferate.ToidentifythecriticalgenestomediatetheO2-dependentcellcycleexit,weperformedmicroarrayanalysisusingtwodifferentsetofsamples.ThefirstwastheCMsfromfetus(E16)vsneonate(postnatalday2–3).Thesecondwastheisolat-edfetalCMsculturedunderlow(3%)vsnormal(20%)O2conditions.Whiletheformercoversthewholeeffectofcomplicatedchangesatbirth,thelatterdissectstheeffectofO2changesalone.Wearenowinthesteptoselectthecriticalgenes,andvalidatethefunctionsofeachgeneinknock-downexperiments.NoCOI.

1P-070Submembranous di-phosphorylation of myosin light chain and actin fiber formation play a critical role in thrombin-induced endothelial barrier disruptionHirano, Katsuya; Hirano, Mayumi(Department of Molecular Cardiology, Graduate School of Medical Sciences, Kyushu University)

Background:Thephosphorylationofmyosin lightchain (MLC) isafundamentalmechanismforendothelialbarrierdisruption.MLCisphosphorylatedatS19andT18.Anydifferentialroleofmono-anddi-phosphorylationofMLC(MLC-PandMLC-PP)was investigated.Methods:Thetrans-endothelialelectricalresistance(TER)wasmoni-toredasanindicationofbarrierfunctioninporcineaorticendothelialcells.ThelevelsofMLC-PandMLC-PPwerequantifiedwithPhos-tagSDS-PAGE.MLCTheMLCmutantswereexpressedbyusingadeno-viralvector.Results:ThrombininducedadecreaseinTERwithanincreaseinMLC-Pfrom24%to35%andMLC-PPfrom2%to35%.MLC-P localized inperi-nuclearregion,whileMLC-PP localizedatsubmembranousregion,whereactinfiberformationwasalsoinduced.Rhokinaseinhibitors(H1152andY27632)inhibitedthrombin-induceddecreaseinTERandsubmembranousMLC-PPandactinfiberforma-tion.TheexpressionofexogenousMLCsubstituted85%ofendogenousMLC.InthecellsexpressingMLCwt,thrombininducedsubmembranousMLC-PPanddecreasedTER.TheexpressionofMLCT18A+S19Aabolishedthrombin-inducedMLCphosphorylationandactinfiberformation,andadecreaseinTER.MLCT18AorMLCS19AabolishedMLC-PP,buthadmodesteffectonTER.Conclusions:MLC-PPandactinfiberformationinthesubmembranousregionplayacriticalroleinthrombin-inducedbarrierdisruption.MLC-Pissufficientforbarrierdisruption.InhibitionofMLC-PPisrequiredforbarrierprotection.NoCOI.


1P-071Cardiovascular function of marine mussels monitored by MRISeo, Yoshiteru1; Seo, Eriko2; Imaizumi-Ohashi, Yoshie1; Ohishi, Kazue3; Maruyama, Tadashi3; Murakami, Masataka4(1Dept of Regul Physiol, Dokkyo Med Univ Schl of Med, Tochigi, Japan, 2Div of Marine Life Sci, AORI, Univ of Tokyo, Kashiwa, Japan, 3Inst of Biogeosci, JAMSTEC, Yokosuka, Japan, 4Natl Inst for Physiol Sci, Okazaki, Japan)

InadditiontoNO,H2SandCOarenominatedasEDRF.However,toxicityofH2SandCOmakedifficulttoanalysetheireffects.Creaturesinthedeepsea,suchasBathymodiolus japonicus,liveinahighH2Senvironment.Therefore,thesemusselsmustbeusefulfortestingeffectsofH2Soncardiovascularfunction.Mytilus galloprovincialswereusedforsetupMRIexperimentsforanalysingcardiovascularfunction.Theheartratecouldbemeasuredusingthemotionghostoftheanteriorarteryorthebranchialvessels(1.1Hz,23°C).Cardiaccyclewasimagedbyretrospectivelyself-gatedfastlowangleshotsequence.Theend-di-astolic,end-systolicandstrokevolumeswere50%,21%and29%oftheheartvolume,respectively.Flowofhaemolymph intheheartandvesselsweremeasuredbyphase-contrastMRI.Thehaemolymphre-turnstotheauricleviatheanteriorobliquevein,andpassedthroughtheauriculo-ventricularvalve.Thestreamsfromtherightand leftvalvesmaymergetogetherattheposteriorendoftheventricle,thentheyareskewedtowardstheanteriordirectionbytheinnercurvatureoftheventricle,andflowintheventralsideoftheventricletotheanterioraorta.Thesevorticesinthehaemolymphstreammayaccountforthehighejectionfraction(58%)oftheheart,evenwithasingleoutlet.Now,westepforwardtoanalysethecardiovascularfunctionofBathymodiolus japonicus.NoCOI.

1P-072How cardiac system evolved in multicellular organ-ismsShimizu, Hiroshi(Dept. of Developmental Genetics, National Institute of Genetics)

HowcardiacsystemevolvedinmulticellularorganismsHiroshiShimizuNationalInstituteofGeneticsCardiacsystemplaysaroleincirculationofbloodthroughvascularsystemandtransportofnutrientsandwastematerials.Nkx-2.5orthologuesareexpressedinthecardiacmesodermofanimalsthathaveheart.Interestingly,however,thisgeneisfoundalsoinprimitiveanimalsthathavenoheart.Thetissuethatexpress-esNkx-2.5orthologuesshowsvariouskindsofpumpingmovement.Innematodes,itisexpressedinthepharynx.Inhydra,amemberofclasshydrozoaofphylumcnidaria,itisexpressedinthepeduncleoftheanimal.Sincethepumpingplaysaroleincirculationofgastricfluidinhydra,wepreviouslyproposedthatthecardiacfunctionappearedeveninthemostprimitiveanimalphylum.However,aproblemremainedunsolved.SincehydrozoaisthelatestclassofCnidaria,itremainedunclearhowthesituationisinprimitiveclassAnthozoathatincludesseaanemoneandcorals.Tosolvethisproblem,weextendedourbe-havioralanalysistotwomembersofAnthozoa,firstsessileseaanem-one (Actiniaria)andsecondburrowingseaanemone (Nematostellavectensis).Wefoundthat inAnthozoathepumpingmovementofNkx-2.5orthologueexpressingtissueplaysaroleasthegeneratorofhydropressure.Wealsoobtainedinformationthatinspiderstheheartpumpingplaystworoles,firstastheorganforcirculatoryfunctionandsecondasthegeneratorofhydropressure.Fromthisinformation,weproposeascenariothatNkx-2.5relatedpumpingstartedasthepressuregeneratorandgraduallydevelopedcirculatoryfunctionwithevolution.NoCOI.

1P-073Age-related effects of dexmedetomidine, an alpha-2 agonist, on coronary vasoactivity and cardiac function in guinea-pig heartsHongo, Maiko1; Fujisawa, Susumu1; Adachi, Takeshi1; Shinbo, Tomonori1; Shibata, Shigehiro2; Ohba, Takayoshi1; Ono, Kyoichi1(Cell Physiology, Akita Univ. Akita, Japan, 2Department of Critical Care Medicine, Iwate Medical Univ. Iwate, Japan)

Dexmedetomidine isapotentandselectiveα2agonist. Ithasbeenreportedthat,althoughthesubstancedoesnotappeartohaveanydirecteffectsonthemyocardialcontractility,itsometimescausesadecreaseinheartrateandadose-dependentdecreaseinarterialbloodpressure.Wehypothesizedthateffectsofdexmedetomidinemaydifferdependingonpostnatalages.Heartsfromyoung(<3wk)andadult(>6wk)guinea-pigswereisolatedandmountedonaLangendorffap-paratus,andasaline-filledballoonwasinsertedintotheleftventricle.Coronaryperfusionpressure(CPP)andtheleftventricularpressure(LVP)werecontinuouslymonitoredandtheelectro-fieldstimulation(EFS)wasappliedtostimulatesympatheticnerveterminals.Dexme-detomidinealmostcompletelyinhibitedtheincreaseofLVPinducedbyEFSinbothyoungandadulthearts.Ontheotherhand,theeffectonthecoronaryarteryresistancetodexmedetomidinealteredduringpostnataldevelopment.DexmedetomidinedecreasedCPPatallcon-centrationsinyounghearts,whereasitincreasedCPPatconcentrations>10nMinadulthearts.TheincreaseinCPPinadultheartswasin-hibitedbyprazosin,anα1antagonist.Thepresentfindingssuggestthatα1adrenoceptorisinvolvedintheincreaseinCPPinadultheartsinadditiontoα2adrenoceptor.Aging-associatedalterationofαadre-noceptorsubtypesmightbe linkedtocardiodepressanteffectsofdexmedetomidine.NoCOI.

1P-074Lactate impaired excitation-contraction coupling in the atrial myocardiumShimura, Daisuke1; Kusakari, Yoichiro2; Goda, Nobuhito1; Minamisawa, Susumu2(1Department of Life Science and Medical Bioscience, Waseda University, Tokyo, Japan, 2Department of Cell Physiology, Jikei University School of Medicine, Tokyo, Japan)

‹Background›Theeffectof thechange inmetabolicstatusonexci-tation-contraction (EC)coupling intheatrialmyocardiumispoorlyunderstood.Althoughthemyocardiummostlyutilizesfattyacidasanenergysource,wehavereportedthatmetabolicsubstrate(eqlactate)canbeusedforenergyproductionandthatthemetabolomicprofileisdifferentbetweentheatriaandtheventricles.Therefore,weaimedto investigatetheeffectof lactateexposureonECcoupling intheatrialmyocardium.‹Methods›Wemicro-injectedaequorinintosuper-ficialcellsoftheleftatriumisolatedfrommice(C57/BL6,15~17weeksofage),andsimultaneouslymeasuredintracellularCa2+concentrationandtension(1Hzat36°C).Weaddedlactate(~10mM)intoHEPES-Ty-rodesolution(pHwasadjustedat7.4).‹ResultsandConclusion›Lactateataconcentrationof10mMsignificantlydecreasedpeaktension(61.7±6.0%,n=3.p<0.05)andpeakCa2+concentration(78.8±4.8%,n=3.p<0.05).Sincepreviousstudiesreportedthatlactatedecreasespeaktension,butnotpeakCa2+concentrationintheventricularmyocytes,ourresultssuggestedthattheatriumhasdifferentcharacteristicofECcouplingfromtheventriclesinresponsetoanincreaseinlactate,ofwhichconditionisoftenobservedinmyocardialischemia.Simulta-neousmeasurementoftensionandintracellularCa2+concentrationintheatrialmyocardiumcanbeusefulanalysisoftheatrialphysiologicalproperty.NoCOI.


1P-075DNA microarray profiling identified cardiac fibrosis-re-lated genes following pressure overloaded hypertro-phyKusakari, Yoichiro1; Uesugi, Ken1; Nakai, Gaku1; Shimura, Daisuke1; Urashima, Takashi2; Kurihara, Satoshi1; Minamisawa, Susumu1(1Department of Cell Physiology, The Jikei University School of Medicine, Tokyo, Japan, 2Department of Pediatrics, The Jikei University School of Medicine, Tokyo, Japan)

Itisimportanttoidentifyamolecularinduceroffibrosisasathera-peutictargetforheartfailure.Aratmodelofcardiachypertrophyandfibrosiswasgeneratedbypulmonaryarterybanding(PAB).Histolog-icalanalyseswithMassonTrichromestainonshortaxissectionofrightventricularpapillarymusclesidentifiedthattheycouldbeclear-lydividedintotheinterstitialfibrosisgroup(PABF+)andthenon-fi-brotic,buthypertrophicgroup(PABF-), incomparisonwiththesh-am-operatedcontrol (Sham).Tension inPABF+wassignificantlysmallerthanthatinShamandPABF-.WecomprehensivelyanalyzedthemRNAexpressionof29215knownratgenesintherightventriclebyusingGeneChipRatGene1.0STArray(Affymetrix)tocompareageneexpressionprofileamongSham,PABF-,andPABF+(n=3each).Wefoundthattheexpressionlevelsofsomegenesthathavenotbeenrecognizedasafibrosis-relatedmoleculeweresignificantlyhigherinPABF+thanthoseinShamandPABF-.Amongthem,weconfirmedthatfibroblastgrowthfactor23(FGF23)[Sham;1.0±0.4,PABF-;0.7±0.3,PABF+;8.5±2.0]andneuraladhesionmolecule1 (NCAM1) [Sham;1.0±0.1,PABF-;1.7±0.7,PABF+;18.5±2.9]inPABF+weresignificant-lyhigherthanthoseinShamandPABF-byRT-PCR(n=6each).ThesedatasuggestthatFGF23andNCAM1arecriticalmolecularinducersofcardiacfibrosisfollowingpressureoverloadedhypertrophy.NoCOI.

1P-076Heart rate response to electrical muscle stimulation in the ovariectomized ratsShimoju, Rie1,2; Sadakiyo, Kaori2; Maruyama, Hitoshi2; Kurosawa, Mieko1,3(1Center Med. Sci., Intl. Univ. Health & Welfare, Otawara, Japan, 2Dept. Physical Ther., Intl. Univ. Health & Welfare, Otawara, Japan, 3Dept. Pharm. Sci., Intl. Univ. Health & Welfare, Otawara, Japan)

Ourpreviousstudyhasshownthatthepressorresponsestoelectricalstimulationofthemuscleareenhancedinovariectomized(OVX)rats,andthatacuteadministrationof17beta-estradiolrelievestherespons-es.Thepresentstudyaimedtoinvestigatewhetherheartraterespons-estothesamemusclestimulationarealsoaugmentedintheOVXrats,andwhetheracuteadministrationof17beta-estradiolinfluencestheresponses.Experimentswereperformedinurethaneanesthetized,artificiallyventilatedrats.Heartratewasmeasuredwithapulseratetachometer,whichwastriggeredbysystolicbloodpressurewaves.Electricalmusclestimulationwasdeliveredtothehindlimbmusclefor30satafrequencyof80Hzwithanintensityof1.5mA.17beta-estra-diolwasadministeredintravenouslyandtheeffectwasexaminedfor135minaftertheonsetoftheadministration.ResponsesofheartratewereaugmentedintheOVXrats,comparedwiththesham-operatedrats.Furthermore,17beta-estradiolsignificantlyreducedtherespons-esofheartratebetween45–135minaftertheonsetofadministration.ThepresentresultsdemonstratethattheresponsesofheartratetoelectricalmusclestimulationwereexaggeratedintheOVXratsandtheresponsescanberelievedbytheacutetreatmentwith17beta-es-tradiol.NoCOI.

1P-077Evidence for cholinergic vasodilatation in skeletal muscle during exercise in humansMatsukawa, Kanji1; Ishii, Kei1; Liang, Nan1; Endo, Kana1; Idesako, Mitsuhiro1; Hamada, HIronobu2; Kataoka, Tsuyoshi3; Watanabe, Tae3(1Department of Integrative Physiology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Japan, 2Department of Physical Analysis and Therapeutic Sciences, Graduate School of Biomedical and Health Sciences, Hiroshima University, Japan, 3Department of Health Care for Adults, Graduate School of Biomedical and Health Sciences, Hiroshima University, Japan)

Totestthehypothesis thatcentralcommandevokessympatheticcholinergicvasodilatationinskeletalmuscleduringexercise,wehaveattempted1) toexaminewhetherbloodflows incontractingandnon-contractingarmmusclesincreaseattheearlyperiodofone-armedcranking,2)toexaminewhetherbloodflowsinthebilateralmusclesincreaseduringmentalimageryoftheone-armedcranking,and3)toidentifywhetheratropinebluntstheincreasedflowresponses.Tensubjectsperformedvoluntaryone-armedcranking(at20–35%ofmax-imalvoluntaryeffort)andmentalimageryoftheexercisefor1min.Therelativeconcentrationsofoxygenated-hemoglobin(Oxy-Hb) inbilateralbicepsbrachiimusclesweremeasuredasanindexofmuscletissuebloodflowwithnear-infraredspectroscopy.Bothone-armedcrankingwiththerightarmandmentalimageryoftheexercisepro-ducedsubstantial increases intheOxy-Hbofthebilateralmuscles,whichwerebluntedbyatropine(10µg/kgiv).Thepresentfindingssuggestthatcentralcommandevokescholinergicvasodilatationequal-lyinbothcontractingandnon-contractingarmmusclesduringexercise.NoCOI.

Poster PresentationsNeuron, Synapse (1)


1P-078Modeling of the neocortical layer 5 pyramidal cell to predict the mechanisms of the slow oscillationNarita, Shinya; Kojima, Haruki; Mushiake, Hajime; Homma, Noriyasu; Osanai, Makoto(Tohoku Univ. Grad. Sch. Med., Sendai, Japan)

Theslowoscillationsofelectroencephalogram(EEG)orthelocalfieldpotential(LFP)werefrequentlyobservedduringslow-wavesleep.Itisthoughtthatthesynchronousmembranepotentialfluctuationbe-tweenUP-andDOWN-stateinthepyramidalneuronsgeneratestheslowoscillationsofEEGorLFP.Recentstudiesshowedthatslow-wavesleepmightberelatedtomemoryconsolidation.Therefore,theattempttorevealthemechanismsoftheslowoscillationshouldhelptoelucidatethememoryformation.TherearehypothesesthatslowoscillationisshapedbyCa2+dependentK+channel,andthatrecurrentexcitationregulatedbyinhibitorynetworks.However,thedetailedmechanismsremainunclear.Itisdifficulttoclarifythemechanismsinvolvedintheslowoscillationonlybyelectrophysiologicalexperiments.Thus,weconductedthecomputersimulationstudytoproposethemechanismsoftheslowoscillation,usingNEURONsimulator.Forthisaim,wedevelopedthemodelcell.Thefollowing ionchannelmodelswerecreatedand incorporatedtothemodelcell;voltagedependentNa+channels,voltagedependentK+channels,voltageactivatedCa2+chan-nels,Ca2+dependentK+channels,HCNchannel,existinginthepyra-midalcellsatcorticallayer5.WealsocreatedandincorporatedthemechanismsrelatedtotheCa2+dynamics.Usingthismodelcell,wereproducedthecurrentsoftheionchannelsandthefiringpropertiesoftherealpyramidalcell.Wewilldiscussthemechanismsconcerningtheslowoscillationwiththismodelcell.NoCOI.

1P-079Relationship between membrane properties and func-tional differentiation of neurons in the area postrema. Funahashi, Makoto; Sugeta, Shingo; Harima, Miki(Department of Oral Physiology, Graduate School of Dental Medicine, Hokkaido University, Japan)

Theareapostremaisoneofthecircumventricularorgansthatlackablood-brainbarrier.Previouslywereportedtwomajorcell typesclassifiedonthebasisofpresenceorabsenceofdisplayingthehyper-polarization-activatedcationcurrent (Ih).Acorrespondenceofeachtypeofcellstothephysiologicalfunctions,suchastheregulationoffoodintakeandtriggeringofnauseaand/oremesis,stillhaveremainedtobeunexplained.Todeterminethefunctionsofeachtypeofcells,weperformedthestudyusingapatch-clampmethod inthebrainslicesandabehavioralanalysisofnausea.Theresponsestocholecys-tokinin(CCK-8)andamylin,whichiswellknowasasatietyhormone,wererecordedfromtheelectrophysiologically-identifiedeachtypeofneurons.Wealso investigatedtheeffectsofZD7288,aselectiveIhblocker,ontheacquisitionofconditionedtasteaversion(CTA),inordertoevaluatethechangesintheexcitabilityofcellsexpressingIhontheinductionofnauseaand/oremesis.AllcellsrespondedtoamylinandthemajorityofcellsrespondedtoCCK-8werefoundincellsnotex-pressingIh.TheacquisitionofCTAwassignificantlysuppressedbythe i.p. injectionofZD7288, indicatingthesuppressionofapomor-phine-inducednauseaand/oremesis.ThesefindingsindicatethatcellsnotexpressingIhplayarolefortheregulationoffoodintake,whilecellsexpressingIh forthe inductionofnauseaand/oremesis.Wesuggestthefunctionaldifferentiationofeachtypeofneurons.NoCOI.

1P-080Optogenetic activation of parabrachio-amygdaloid pathway in the nociceptive amygdalaSugimura, Yae Kaito; Takahashi, Yukari; Watabe, Ayako M; Kato, Fusao(Department of Neuroscience, Jikei University School of Medicine)

Alargemajorityofneuronsinthesuperficiallayerofthedorsalhornprojecttothelateralparabrachialnucleus(LPB)ratherthantothethalamus.TheLPBneuronsthenprojecttothecapsularpartofthecentralamygdala(CeC).Thisnon-thalamocorticalpathwayplayspiv-otalroleinthenociception-emotionlink.TheLPB-CeCtransmissionispotentiatedinvariouspainmodels,asevidencedbyelectricalstimu-lationoftheafferentfibersintheamygdalaslices.However,duetothetechnicallimitations,ithasbeenunclearwhetherEPSCsrecordedinslicesresultedfrommonosynapticdirectglutamatergicconnectionsarisingfromtheLPBandnotfromglutamatergicfibersofnon-LPBorigin.Toaddressthisissue,weexpressedchannelrhodopsin(ChR2)intheLPBneuronsandtriggeredglutamatereleasefromtheiraxonterminalsintheCeCincoronalamygdalaslicesfromadultrats.5-6weeksafterthetransfectionofvirusvectors forsynapsin-drivenChR2(H134R)-YFPexpression,blue lightpulses (1–5ms)robustlytriggeredEPSCsthatwereabolishedbyCNQXandTTX.ThisEPSCwasfollowedbylargeandlong-lastingIPSCs,whichwereabolishedbypicrotoxinorCNQX,suggestingafeedforwardregulationofCeCneuronexcitabilitybyLPB.Moreover,all typesofCeCneuronsasclassifiedbytheirfiringpatternsexhibitedmonosynapticexcitatoryresponsestolightstimulation.TheseresultssuggestthatnociceptiveinputsfromLPBaffecttheexcitabilityofthecentralamygdalanetworkbymodulatingalargepopulationofCeCneurons.NoCOI.

1P-081Modulation of fear memory by dietary polyunsaturated fatty acids via cannabinoid receptorsYamada, Daisuke1,3; Takeo, Jiro2; Wada, Keiji1,3; Sekiguchi, Masayuki1,3(1Dept Degenerat Neurol Dis, Natl Inst Neurosci, Natl Cent Neurol and Psychiat, 2Fund Health Res, Cntl Inst, Nippon Suisan, 3JST, CREST)

Thoughtheunderlyingmechanismremainsunknown,severalstudieshavesuggestedbenefitsofn-3long-chainpolyunsaturatedfattyacid(PUFA)forpatientswithanxietydisorders.Elevatedfearisthoughttocontribute to thepathogenesisofparticularanxietydisorders.However, therearenostudiesontheeffectsofdietaryPUFAonconditionedfearmemory inexperimentalanimals.Theaimofthisstudywastoevaluatewhetherthedietaryω3toω6PUFA(3/6)ratioinfluencesfearmemory.Forthispurpose,theeffectsofvariousdietary3/6ratiosonfearmemorywereexaminedinmiceusingcontextualfearconditioning,andtheeffectsofthesedietsoncentralsynaptictransmissionwereexaminedtoelucidatethemechanismofactionofPUFA.Wefoundthatfearmemorycorrelatednegativelywithdietaryandbrain3/6ratiosinmice.Thelowfearmemoryinmicefedahigh3/6ratiodietwasincreasedbythecannabinoidCB1receptorantag-onist,reachingalevelseeninmicefedalow3/6ratiodiet.CB1recep-tor-mediatedsynapticplasticitywasfacilitatedinpyramidalneuronsofthebasolateralnucleusoftheamygdala(BLA)inmicefedahigh3/6ratiodiet.Theagonist-sensitivityofCB1receptorwasenhancedintheBLAofmicefedahigh3/6ratiodiet,andsimilarenhancementwasinducedbypharmacologicalexpulsionofcholesterolfrommem-braneinmicefedalow3/6ratiodiet.Theseresultssuggestthattheratioofω3toω6PUFAisafactorregulatingfearmemoryviacan-nabinoidCB1receptors.NoCOI.


1P-082Contextual memory encoding but not retrieval gener-ates wide diversity of post-synaptic current in hippo-campal CA1 neurons.Sakimoto, Yuya; Mitsushima, Dai(Graduate School of Medicine, Yamaguchi University, Yamaguchi, Japan)

Thehippocampusplaysacentralrole incontextual learningandmemory.Sincethelearningstrengthensbothexcitatoryandinhibito-ryCA1synapses,eachCA1neuronshowshighdiversityofpost-syn-apticcurrents(Mitsushimaetal.,Nature Commun,inpress).Inthepresentstudy,toexaminewhetherencodingorretrievalofmemorystrengthenstheCA1synapses,weanalyzedminiatureexcitatoryandinhibitorypost-synapticcurrents(mEPSCandmIPSC)inIA-trainedratsbeforeorafterthememoryretentiontest.Asalearningmodel,weemployed inhibitoryavoidance (IA)task,andacutebrainsliceswerepreparedforpatchclampanalysis.Untrainedratsshowedrela-tivelysmallmEPSCandmIPSCamplitudeswith lowdiversityofpost-synapticcurrents.Conversely,bothIA-trainedratsbeforeandaftermemoryretentiontestshowedhighertheamplitudeswithwidediversity,suggestingthatmemoryencodingratherthanretrievalenhancestheamplitudestogeneratediversity.Moreover,bathtreat-mentofCNQX(anAMPAreceptorantagonist,10µM)consistentlyblockedthemEPSCresponses.Incontrast,bathtreatmentofbicucul-linemethiodide (aGABAAreceptorantagonist,10µM)consistentlyblockedthemIPSCresponses.TheseresultssuggestthatcontextualmemoryencodingratherthanretrievaldrivessynapticdeliveryofAMPAreceptorsandGABAAreceptorsinCA1neurons.Theelectro-physiologicaldiversityineachCA1neuronmightencodeexperiencedcontextinrathippocampus.NoCOI.

1P-083Activity-dependent repression of spontaneous inhibi-tory synaptic current in rat hippocampusTaketo, Megumi; Matsuda, Hiroko(Dept. Physiol. 1, Facult. Med., Univ. Kansai medical, Osaka, Japan)

GABAAreceptor-mediated inhibitorypostsynapticcurrents (IPSCs)modifyexcitatorysynaptictransmissionthroughmodulationofactiv-ityofprincipalneurons.Comparingwiththeplasticityofexcitatorysynapses,theinhibitorysynapticchangeshavenotbeenwellcharac-terized.AlterationinefficacyofGABAAergictransmissionafterpost-synapticdepolarizationhasbeenreportedbyseveral investigators,butbothfacilitationandinhibitionwereobserved,andmechanismsoftheplasticityhavenotbeenelucidated.Inthepresentexperiments,effectof therepetitivepostsynapticdepolarizationonspontaneousIPSCswasdeterminedinacuteslicesofrathippocampus.Wholecellpatch-clamprecordingwasperformedtorecordGABAAergicIPSCsfromneonatalCA3neurons.RepetitivedepolarizationofpostsynapticneuronsalonedidnotcausemarkedalterationofthespontaneousIPSCs.Effectofsimultaneousactivationofpresynapticandpostsyn-apticneuronsontheIPSCswasthendetermined.Activationofpre-synapticneurons inducedtransient facilitationof thesIPSCs,butsucceedingrepetitivepostsynapticdepolarizationtransientlyreducedthefrequencyofthesIPSCs.Thoughapplicationofametabotropicglutamatereceptoragonistcouldnotreplacethepresynapticactivation,theIPSCinhibitioncausedbythesequentialpre-andpost-synapticstimulationwassuppressedbyspecificantagonistsofthemetabotropicglutamatereceptors.Theseresultssuggestthatpostsynapticdepo-larizationandfacilitationofsynapticalreleaseofglutamatetransient-lyinhibitthesIPSCs.NoCOI.

1P-084Inhibitory effects of 5-HT at excitatory synapses in the dentate granule cells.Nozaki, Kanako; Kubo, Reika; Furukawa, Yasuo(Grad. Sch. Integrated Arts and Sci., Hiroshima Univ., Hiroshima, Japan)

Thedentategyrus isagatewayforneuronal transmission inthehippocampal formation.Granulecellsareprimaryneurons inthedentategyrus,andreceiveexcitatoryinputsmainlyfromtheentorhi-nalcortexviaperforant-path.Serotonergicfibersfromtheraphenucleidistributeintothehippocampus,andpreviousstudiesshowthat5-HTmodulatestheactivityofGABAergic interneurons inthedentategyrus,andthereby,affectstheexcitatorytransmissionbetweentheperforant-pathandgranulecells.Apossibledirectmodulationoftheexcitatorysynapses is,however,notwellexamined.Inthepresentstudy,weexaminedactionsof5-HTontheexcitatorysynapses ingranulecellsundertheconditioninwhichGABAergicinputswereblockedbypicrotoxin.5-HT(1µM)decreasedtheinputresistance(IR)ofgranulecellsaround50%andfastenedthedecayofEPSPevokedbystimulationoftheperforant-path.ComputersimulationshowedthatsuchreductionofIRdecreasesEPSPsaround30%atmost.We,how-ever,observedmorethan50%reductionofEPSPsinsomeprepara-tions.Moreover,synaptictransmissionfailurewasoftenincreasedby5-HT,suggestingthat5-HTreducesreleaseprobabilityofthesynaps-es.Alloftheseeffectswereblockedbya5-HT1Areceptorantagonist,WAY100635.Mossycellsinthehilusofhippocampusalsomakeexcit-atoryconnectionstothegranulecells.However,EPSPsevokedbyselectivestimulationofmossycellaxonswerenotdecreasedby5-HT.Theseresultssuggestthat5-HTinducespathway-specificmodulationofexcitatoryinputstothedentategranulecells.NoCOI.

1P-085Pairs of stimuli modulate synaptic plasticity in hippo-campal CA1 areaUeda, Rika; Nakashima, Toshihiro(Department of Applied Biology, Kyoto Institute of Technology, Kyoto, Japan)

Thehippocampusplaysakeyrole inmemory.HippocampalCA1neuronsreceiveinputsviaatrisynapticpathway.Entorhinalcortexsendsthe informationtothedentategyrus (DG)viathepeforantpathway(PP).TheDGtransmitstheinformationtoareaCA3viathemossyfibers.CA3subsequentlysendsprocessedinformationtostra-tumradiatum(SR)inareaCA1viaSchaffercollateral(SC)pathway.CA1alsohasdirectexcitatoryconnectionswithentorhinalcortexviathePP.Thesedirectinputssynapseondistalpyramidalneuronden-drites instratumlacunosummoleculare (SLM).ThefunctionofthedirectPPinputsisnotwellunderstood,althoughrecentresearchin-dicatesthatthese inputshave importanteffectsonCA1pyramidalcells.ThetrisynapticpathhasalongerdelaytimesothatinformationarisingfromentorhinalcortexarrivesatSLM10–20mspriortothearrivalofinformationatSRinCA1.Inthisstudy,theeffectsofinter-actionsbetweenPPandSCinputsonfieldEPSP(fEPSP)inCA1areainbrainslicepreparationofratareinvestigated.Werecordedpopu-lationspikeamplitude(PSA)inCA1pyramidalcellandslopeoffEPSPatSRinCA1.Theresultsindicatethatsynapticplasticityismodulat-edbydifferentpairingintervals.Inadditiontothis,thepropertyofsynapticplasticityisdifferentbetweenPSAandfEPSPwhenthesamestimuliwereapplied.NoCOI.


1P-086Developmental changes in localization of the presyn-aptic cell-adhesion molecule neurexin-1β during hip-pocampal inhibitory synapse formationHanafusa, Manami; Sadamoto, Hisayo; Kuriu, Toshihiko; Konishi, shiro(Dept. Neurophysiol., Kagawa Sch. Pharm. Sci., Tokushima Bunri Univ., Sanuki, Japan)

Synapseorganizingmolecules,neurexins(NXNs)andneuroligins,areimplicated insynapseformation,maturationandmaintenance.It isunclear,however,howinhibitorysynapsesareformedandmodifiedduringdevelopmentandsynapticplasticity.Thus,wefirstdevisedamolecularprobeforvisualizinginhibitorysynapticcontactsites.Forthispurpose,weconstructedacDNAforNXN-1βofwhichC-terminalwastaggedwithmCherry(NXN-1β-mCherry).Whentransfectedintohippocampalneuronsunderdissociatedculture,NXN-1β-mCherrywasspecificallyaccumulated inaxonvaricositiesandco-localizedwithendogenousNXN-1β.Therefore,NXN-1β-mCherrycouldbeselective-lytransportedtosynapticsitesandserveasareliableprobeforvisu-alizingtheinhibitorypresynapticsiteunderlivingconditions.Wethenexamineddevelopmentalchanges inNXN-1βlocalizationataxonterminals.TheuseofVGAT-Venusmiceallowedustoeasilyidentifyaxonterminalsofhippocampalinhibitoryinterneuronsunderculture.Atanearlyculturestage(10DIV),discernibleclustersofNXN-1β-mCherrywerenotpresentinvaricositiesofVenus-positiveinhibitoryaxons,whereasNXN-1β-mCherryclusterscouldbedetectedatlaterculturestages(17and24DIV).Thus,theobservationsshowthatNXN-1βbeginstoaccumulate inpresynapticvaricositiesafter inhibitorysynapseformation,andsuggestthatthispresynapticcell-adhesionmoleculeplaysarole inmaturationandmaintenanceof inhibitorysynapses.NoCOI.

1P-087Suppression of GABAergic depolarization of hippo-campal mossy fibers by bumetanideOoura, Shunsuke; Kamiya, Haruyuki(Department of Neurobiology, Graduate School of Medicine, Hokkaido University, Japan)

IntracellularchlorideconcentrationinneuronsisregulatedmainlybychlorideextrudingK-ClcotransporterKCC2andchlorideuptakingNa-K-ClcotransporterNKCC1.ActivationofpresynapticGABAAre-ceptorsbymuscimoldepolarizesmossyfibers,possiblyduetohighchlorideconcentrationwithintheterminalsandtheaxons.Hereweexaminedtheeffectofbumetanide,ablockerofNKCC1,onmusci-mol-inducedenhancementofexcitabilityofmossyfibers.HippocampalsliceswereobtainedfromC57BL6Jmiceof12-17daysold.Antidrom-icpopulationspikes(ASs)wereelicitedbystimulationatthestratumlucidumintheCA3region,andwererecordedfromthegranulecelllayerofthedentategyrus.ApplicationofGABAAreceptoragonistmuscimol(1µM)increasedtheamplitudeofASs(to123±6.7%ofcontrol,n=7),possiblyduetorecruitmentofsubthresholdfibersbydepolarizationofmossyfibers.Bumetanideat10µMreducedtheeffectofmusimolonASs(to106±2.7%ofcontrol,n=7;P<0.05).Wealsousedafocalperfusionsystemtothestimulationsitetolocalizedrugapplication.Again,1µMmuscimolincreasedASs(to129±6.6%ofcontrol,n=6)and10µMbumetanidesuppressedtheeffectofmusci-mol(to116±6.7%ofcontrol,n=6;P<0.05).Theseresultsshowedthatbumetanide-sensitive transporterNKCC1 isexpressedat themossyfiberterminalsandaxons,andiscontributed,atleastpartly,tohigherintracellularchlorideconcentrationsinthepresynapticcom-partments.NoCOI.

1P-088The effects of propofol on inhibitory postsynaptic cur-rents evoked with paired-pulse stimulation in CA1 py-ramidal cells and dentate gyrus granule cells of rat hippocampal slices Ishiguro, Masanori1; Kobayashi, Suguru2; Nagamine, Takashi1(1Systems Neuroscience, School of Medical, Sapporo Medical University, Sapporo, Japan, 2Tokushima Bunri University, Faculty of Pharmaceutical Sciences)

Wehavereportedthatpropofol,oneofthemostpopularintravenousanestheticagents,effectsinGABA-mediatedinhibitorysynaptictrans-missiondifferentlyinCA1pyramidalcells(CA1-PC)anddentategyrusgranulecells (DG-GC).Propofolhas increasedtheamplitudesandprolongedthetimeconstantofIPSConlyinCA1-PCbutnotDG-GC.Inthisstudy,weexaminedtheeffectsofpropofol,ontheinhibitorypostsynapticcurrents(IPSC)evokedwithpaired-pulsestimulationinCA1-PCandDG-GCinrathippocampalslices.ThemonosynapticIPSCwereevokedbyelectricalstimulationatthe400msecinterstimulusintervalevery20secofGABAergicinterneu-ronsandrecordedfromCA1-PCandDG-GCbywholecellpatch-clamptechnique.Theeffectsofspecificconcentrationsof10µMpropofolontheIPSCinCA1-PCandDG-GCwereexaminedat-100mVmembranepotentials.Werecorded50 individualtraces ineachcellandpairedpulseratio(PPR)ofindividualtraceswascalculatedbydividingtheamplitudeofthesecondresponse(A2)bythefirst(A1),themean(A2/A1).PropofolwasobservedtosignificantlyincreasethePPRinCA1-PCbutnot inDG-GC.ThedatawasalsoanalyzedwiththemeanA2/meanA1ineachcell.ThemeanA2/meanA1wasalsoincreasedinCA1-PCbutnotinDG-GC.ThedatasuggeststhatpropofolmaychangeGABA-mediated inhibitorysynaptictransmissionpresynaptically inonlyCA1-PC.NoCOI.

1P-089Decreased expression of syntaxin 1A after experi-mental febrile seizure in mice hippocampal CA1 areaKaneko, Kentaro; Kato, Eiko; Fukushima, Teruyuki; Maekawa, Masao; Hori, Yuuichi(Dokkyo Med Univ, Sch of Med, Dept of Physiol & Biol Inf, Japan)

Febrileseizures (FS)arethemostcommontypesofconvulsionsininfantsandyoungchildren.However,itisnotclearwhetherFSresultin long-termsequencessuchastemporal lobeepilepsy (TLE).ThehippocampusofTLEpatientsischaracterizedbymossyfibersprout-ing,whichisalsoinducedbytheexperimentalFS.TheexperimentalFSelicitedthatthepup(postnataldays14–15)exposedtoaregulatedheatedairstreamcomingfromahairdryerplaced50cmabovethem.Inaddition,mossyfibersprouting isthoughttoresult inneuronalhyperexcitability.ToelucidatepathologicalmechanismsformossyfibersproutinginducedbytheFS,weinvestigatedtheeffectsoftheFSontheexpressionlevelofsyntaxin1A(STX1A),whichisknowntobeinvolvedinneuriteelongationandsprouting.Onpostnataldays14-15,ICRmiceweresubjectedtotheFS.AftertheFS,weisolatedmRNAfromthehippocampalCA1areaandperformedreal-timeRT-PCRanalysis.TheexpressionlevelofSTX1Adecreased8h,24h,and3waftertheFS.ItissuggestedthattheFS-induceddecreaseinex-pressionlevelofSTX1AmighthaveacausalrelationtoanincreaseinmossyfibersproutingobservedinTLE.WearenowinvestigatingwhethertheexperimentalFSleadstoanenhancedmossyfibersprout-inginthehippocampusbymeansofTimmstainingmethods.NoCOI.


1P-090The critical time window for dopamine actions on the dendritic spines of nucleus accumbens.Yagishita, Sho; Hayashi, Akiko Takagi; Watanabe, Satoshi; Kasai, Haruo(Department of Structural Physiology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Japan)

Inbehavinganimals,effectivereinforcementoccursonlywhenrewardscomeafteraneventwithinafewsecond.Aphasicactivityofdopamineneuronsmayworkasapositivereinforcer,enablinganimalstolearntheeventfromitsconsequence.Ithas,however,notbeenclarifiedwhetherdopamineactionsonneuronalcircuitsareselectivetothecriticaltimewindowforthereinforcement.Inordertoexaminethetemporalcharacteristicsofdopamineactionsonneuronalcircuits,weinvestigatedtheexcitatorysynapsesondendriticspinesofmediumspinyneurons (MSNs) innucleusaccumbens,acenterofpositiveemotionreceivingdensedopaminergicinnervations.Weinducedspineenlargementbytwo-photonuncagingofcagedglutamatespairedwithactionpotentialsinD1MSNsofacuteslicefromyoungadultmice,incombinationwithoptogeneticstimulationofdopaminergicfibersfromventraltegmentalarea.Wefoundthatdopaminegreatlypotentiatedtheenlargementofspinesshortly(within1.5s)afterglutamateuncag-ing,whereasstimulationofdopaminefiberseitherbeforeor5secondsafterglutamateuncagingdidnot.OurresultsalsosuggestedthatthecriticaltimewindowfordopamineactionswasshapedbyCa2+-depen-dentactivationofadenylatecyclaseIandefficientdegradationofcAMPbyphosphodiesterase10AinthindendritesofMSNs.Thus,thespinestructuralplasticityofMSNscanberesponsibleforthecriticaltimewindowforrewardactionsofdopamine.NoCOI.

1P-091Spatiotemporal analysis of firing prolongation in the corticostriatal networkOhta, Hiroyuki1; Morimoto, Yuji2; Tamura, Risa1; Sato, Yoshiaki3; Nishida, Yasuhiro1(1Department of Physiology, National Defense Medical College, Saitama, Japan, 2Department of Integrative Physiology and Bio-Nano Medicine, National Defense Medical College, Saitama, Japan, 3Teikyo Heisei University, Tokyo, Japan)

Wefoundthatstriatalneuronshaveatime integrativeproperty,showingprolongedfiringsafterrepetitivecorticaloptogeneticstimu-lationanditsresidualeffectafterstimulationintermission.Inadditiontothetimepropertiesoftheprolongation,wemeasuredspatialprop-ertiesoftheprolongedfiringsinthecorticostriatalslicebyusinganewlydevelopedoptogeneticmapping lightingsystemwithDigitalMicromirrorDevice (DMD)whichcanaccuratelycontrolboththelightingpatternandtiming.WeemployedtheacutesliceoftheWistarThy-1.2promoterChannelRhodopsin-2VenusRat.Striatalneuronalfiringswererecordedbyatetrodewithphotostimulationofthecortexandthecorpuscallosum.Striatalneuronsthatrespondedtorepetitivephotostimulationswith5secondsintervalshowedresidualfiringsforafewsecondsaftertheendofthe1sec-longphotostimulation.After5timesrepetitivephotostimulations,responsestotheotherareaofphotostimulationwere increasedordecreased.That is,repetitivephotostimulationshadasignificanteffectonthesubjectneuron’s“receptivefield”—groupingsofcorticostriatal inputfibers.Foreachstriatalneuronthefieldeithershrankorexpanded.NoCOI.

1P-092What does chronic methamphetamine treatment alter the activity of the medium spiny neurons in the stria-tum? Inoue, Ritsuko; Miura, Masami(Neurophysiology, Tokyo Metropolitan Inst. of Gerontology, Tokyo, Japan)

Thestriatumisamajornucleusofthebasalganglia,anddividedintotwocompartments,thestriosomesandthematrix.Thesecompart-mentsaredistinguishablebytimeofneurogenesis,cytochemicalmark-ers,andinput-outputcircuits.Recentstudiessuggestthatimbalancesbetweenstriosomeandmatrix functionsmightcontributetobasalganglia-relateddisorders.Stereotypyisapatternofbehaviorabnor-malities,whichcanbeinducedbychronicmethamphetamine(METH)treatment.Inthisstudy,weexaminedwhethertheeffectsofchronicMETHtreatmentonthephysiologicalactivityofstriatalneuronsdifferbetweeneachofthestriatalcompartments.Tovisuallyidentifythestriosomes,weusedaTH-GFPtransgenicmousestrainexpressingGFPinacompartment-specificmanner.METH(5mg/kg)orsalinewas injected intraperitoneallytwicedaily for5consecutivedays.Gradually,enhancedstereotypedbehaviorswereinducedbytheMETHtreatment.Wemeasuredstereotypyscoresaftertheinjectioninthemorning.Afterwithdrawalof2–7days,eachmousereceivedachal-lengewithMETHorsaline,andthenwecomparedc-Fosexpressionbetweeninthestriatalcompartments.Wealsomadewhole-cellrecord-ingsfrommediumspinyneuronsinslicepreparationtakenfromthemicetreatedwithMETHorsaline.Miniatureexcitatoryandinhibito-rypostsynapticcurrentswerecomparedtoexploretheimbalancedactivityofthestriatalcompartments.Thesestudieswouldhelpexplaintherolesof thecooperation instriosome-andmatrix-basedbasalgangliacircuits.NoCOI.

1P-093Serotonin-induced inhibition of glutamatergic trans-mission onto rat basal forebrain cholinergic neuronsMomiyama, Toshihiko1; Nishijo, Takuma1,2(1Department of Pharmacology, Jikei University School of Medicine, 2Department of Pharmacology, Keio University School of Pharmacy)

Whole-cellpatch-clampstudywascarriedouttoelucidatemodulatoryrolesofserotoninintheexcitatorytransmissionontocholinergicneu-ronsinthebasalforebrain(BF)oftherat.BFcholinergicneuronswereidentifiedwithstainingbyCy3-192-IgG.EPSCswereevokedundertheconditionwhereGABAA-,glycine-andNMDAreceptormediatedcurrentcomponentswerepharmacologicallyblocked.Bathapplicationofserotoninat10or30µMinhibitedtheEPSCsby30–40%.(R)-(+)-8-OH-DPAT,a5-HT1Areceptoragonists(30µM),orCP93129,a5-HT1BreceptoragonistalsoshowedinhibitoryeffectsontheevokedEPSCstosimilarextentcomparedwiththeeffectofserotoninatthesameconcentration.Theseresultssuggestthatserotonininhibitsnon-NMDAglutamatergictransmissionontoBFcholinergicneuronsvia5HT1Aand5-HTIBreceptors.NoCOI.


1P-094A long term HFS on STN induced LTP of GABAergic IPSC onto SNr GABA neurons evoked by stimulation on the internal capsule.Miyazaki, Takefumi(Dept. of Physiol., Tokyo Med. Univ., Tokyo, Japan)

Ahighfrequencystimulationonthesubthalamicnucleus(STN-HFS:constantcurrentpulse,amplitude;50–500µA,frequency;125Hz,du-ration;100µsfor20min)wasappliedintheratbrainslicepreparations.DuringSTN-HFS,thesubstantianigraparsreticulata (SNr)GABAneuronwaskeptinthecurrentclampmode.STN-HFSinducedaLTPintheGABAergicIPSCwhichwasevokedbyanelectricalstimulationonaninternalcapsule,includingaputativedirectpathway,inahalfofneurontested(9outof17neurons).At120minutesafterSTN-HFS,thenormalizedamplitudeincreasedto1.492±0.185(mean±S.E.M.).Undervoltageclampmodeattheholdingpotentialof-70mVduringSTN-HFS,HFSdidnotinducetheLTPofIPSC(n=5).Inthesolutionwithdopaminereceptorantagonists(1µMSCH23390and1µMSulpir-ide),STN-HFSinducedLTPinallneurons(n=8;2.048±0.336).Inthesolutionwith50µMAP-5(NMDAreceptorantagonist),STN-HFSinducedLTPonlyoneneuronoutof6.With10mMBAPTAintheelectrodesolution,LTPwasinducedalsoonlyonecelloutof6. Apairedpulseratiosbeforeand120minafterSTN-HFSarenotsignifi-cantlydifferent(n=6,before:1.581±0.126,after:1.356±0.054,p=0.123)intheneuronsLTPinduced.Theseresultssuggestthatthepost-synapticmechanisminducestheIPSC-LTPbySTN-HFSinSNrGABAneurons.Ontheotherhand,theinwardsynapticcurrentde-creasedinitsamplitudeforlongtermlikeLTD.Thissynapticplas-ticitymightbeonereasonwhythedeepbrainstimulationhasthebeneficialeffectsontheneuropsychiatricdisorders.NoCOI.

1P-095Intracellular distribution of AMPA receptors internal-ized upon LTD in cerebellar Purkinje cellYamaguchi, Kazuhiko(Lab. for Genetic Behaviology, BSI, RIKEN)

Asformolecularmechanismoflong-termdepression(LTD)atparallelfiber -Purkinjecell (PC)synapse,PKC-mediateddestabilizationofGluA2andfollowingendocyticeliminationofAMPAreceptorsfromthesynapticmembranehasbeenproposedasmechanismofLTD.However,estimatedenhancementofdestabilizationwasnotsufficienttoexplainphysiologicalmagnitudeofLTD.Adata-drivenkineticmodelofAMPAreceptortraffickingpredicteddecreaseintotalrecy-clingpoolsizeofAMPAreceptorsatLTD.Actually,densityofAMPAreceptorsinthedendriticspineofculturedratPurkinjecellwasre-ducedatLTDinducedbychemicalstimulation (HighK+andGlu).However,whetherthisdecreaseinAMPAreceptor-densityinspinewascausedbyproteolysisortranslocationwasnotclear.Here,weexaminedtheeffectofepoxomicin,ablockerofproteasome,andfoundthatitdidnotaffectdecreaseinAMPAreceptordensityinspineatchemicalLTD.ToconfirmtranslocationofAMPAreceptors,surfaceexpressedGluA2wastaggedbymonoclonalantibody,then,LTD-in-ducingchemicalstimulationwasappliedandcellswerefixedandstained.SurfaceexpressedGluA2wasactuallytranslocatedindendrit-icshaft,and itsdensity inspinewasvery low.TheseresultsalsosupportdecreaseinAMPA-receptordensityinspinewascausedbytranslocationofthemfromthespinetotheshaft.NoCOI.

1P-096Novel form of depolarization-induced synaptic plas-ticity of GABAergic transmission in the Purkinje cellsSatoh, Hiromasa; Saitow, Fumihito; Suzuki, Hidenori(Department of Pharmacology, Nippon Medical School, Tokyo, Japan)

Itiswellknownthatseveralformsofdepolarization-inducedsynapticplasticityofGABAergictransmissionoccurinthecerebellarPurkinjecells(PCs)suchasreboundpotentiation,depolarization-inducedsup-pressionofinhibitionanddepolarization-inducedpotentiationofinhi-bition.Inthisstudy,wefoundanotherformofdepolarization-inducedplasticityofGABAergictransmissioninPCs.Weperformedthewholecellvoltage-clamprecordingwithlowCl-pipettesolutionandrecord-edelectricalstimulationevokedIPSC(eIPSC)fromyoungratcerebel-larslices.ThedirectionofeIPSCchangedfromoutwardto inwardimmediatelyafterPCdepolarization.Thereafter,theamplitudeofeIPSCwasdepressed(Depolarization-inducedDepressionofInhibition:DDI)formorethan20min.BiophysicalandpharmacologicalstudiesrevealedthatreversalpotentialofeIPSCwaspositivelyshiftedbyCaMKII-de-pendentactivationofbothcalcium-activatedchloridechannelsandcation-chloridecotransporters.ThesedataindicatedthatDDIisinducedbypostsynapticmechanism.Further,weexaminedthephysiologicalrolesofDDIatPCsynapsesinrelationtoinstructivestimulationofclimbingfiber.Approximately50%ofclimbingfiberstimulation-induceddepolarizationofPCdecreasedtheimpactofexogenousGABA-medi-atedinhibitionofspontaneousspikeactivity.TheseresultssuggestedthatDDIhasasynergiceffectofexcitatorytransmission.ThisnovelformofDDImayprovideanotherregulatorymechanismofPCsintheneuronalcomputationofthecerebellarcortex.NoCOI.

1P-097Roles of developmental changes of GABAergic syn-aptic transmission on rat deep cerebellar nuclei neu-ronsSaitow, Fumihito; Nagano, Masatoshi; Suzuki, Hidenori(Department of Pharmacology, Nippon Medical School)

Activityofthedeepcerebellarnuclei(DCN)takesanimportantroleinoutputtingprocessedinformationfromthecerebellum.Ourpreviousstudiesreportthat5-HTshowstheinhibitoryeffectonGABAergictransmission:presynapticallyinhibitingthereleaseofGABA.Ontheotherhand,5-HTactsasanexcitatorymodulator:postsynapticallyelicitingslowinwardcurrent.Inthisstudy,weexaminedadevelop-mentalchangesofboththesynapticpropertiesandmodulatoryeffectsof5-HTonGABAergicsynapsesusingratcerebellarslices.Atayoungerstage(~P12),IPSCshadslowerkineticsandhighersuscep-tibilitytothe5-HT-inducedinhibitoryactionthanthoseatanolderstage (~P21).Onthecontrary,thepostsynapticmodulatoryactionshowednodevelopmentalchanges.Moreover,wefoundthatthereleaseprobabilityofGABAwasdecreasedwithdevelopment(orage).How-ever,theextentof5-HT-inducedinhibitoryactionwasnotassociatedwiththereleaseprobability.As forthedevelopmentalchange inpostsynapticsynapticproperties,wefoundthatthekineticsofIPSCswas largelydeterminedbyα1subunitexpression inpostsynapticGABAAreceptors.Insummary,5-HTreleasedontoDCNmaypre-andpostsynapticallyplayregulatoryrolesinboththemembraneexcitabil-ityandthegaincontrolofinhibitorysynapsesduringdevelopment.Especially,attheyoungerstage,thesemodulatoryeffectsof5-HTwouldbeimportanttocontroltheexcitabilityandtoformthenormalcerebellarfunctionintheadult.NoCOI.


1P-098Effect of inhibitory input in corticospinal synapse plasticityOhno, Takae1; Isoo, Noriko1; Isowaki, Mutsumi1; Fukuda, Satoshi1; Mishina, Masayoshi2; Sakurai, Masaki1(1Dept Physiol, Teikyo Univ Sch Med, Tokyo, 2Dept Mol Neurobiol & Pharmacol, Grad Sch Med, Univ Tokyo)

Neuronalplasticityisgenerallyactiveonlyinyoungageduringashorttimewindowsocalledcriticalperiod(CP),afterwhichisclosed,lossofplasticity limitsmajorremodelingofneuronalcircuitsandwillneverbere-openedthroughoutlife.MoleculesthatmayaltersuchCNSplasticity,however,stillremainspoorlyunderstood.BecauseGluN2B(2B)showmuchlongertimecoursewithlargerCainfluxthanGluN2A(2A),developmentalshiftof2Bto2Aishypothesizedtobeessentialforregulatingthistimewindow.Inthisstudy,usinginvitromodelofcorticospinalprojectionsystem,wetriedtoidentifymechanismthatclosestheCP.Inordertoselectivelyactivatethecorticospinalaxons,weinfectedthecorticalsliceswithAAV-EYFP-taggedChannelrho-dopsin(ChR2)andstimulatedwithLEDlight(465nm).2Bthatisknowntoshiftto2AduringdevelopmentdeclinedmarkedlytowardtheendofCPinthespinalcord.In2Aknockoutmice,whichexpress2BinhighlevelevenaftertheendoftheCP,showedextensionoftheCP.Partialreductionof inhibitory inputbystrychnine (0.2µM),whichmarkedlyenhancedCainfluxthrough2Bchannels,alsoextendedtheCP.Ourfindingsindicatethatlossof2BsubunitplaysanessentialroleinclosingtheCPandthatinhibitoryinputhasanimportantmodula-toryeffect.Furthermore2Bmaybethemoleculethatreactivatesthesynapticplasticityeveninmorematuredstageinthissystem.NoCOI.

1P-099Closure of the critical period in developmental corti-cospinal plasticity is determined by the decline of synaptic expression levels of GluN2B-NMDA recep-tors: Biochemical and live imaging studyIsoo, Noriko1; Ohno, Takae1; Isowaki, Mutsumi1; Kameda, Hiroshi1; Murabe, Naoyuki1; Mishina, Masayoshi2; Sakurai, Masaki1(1Dept. of Physiol., Teikyo Univ., Sch. of Med., 2Dept. of Mol. Neurobiol. & Pharmacol., Tokyo Univ., Grad. Sch. of Med.)

It isknownthatNMDAreceptor(NMDAR)subunitcompositionisshiftedfromGluN2Bto2Aduringdevelopment.Invitromousemod-elofcorticospinal(CS)projectionwithfunctionalsynapses,CSaxonsinnervatedentirespinalcordatanearlydevelopmentalstageandthenregressedmostlyfromventralsideinaGluN2B-dependentmanner.There iscriticalperiod (CP,6–11DIV) forthisactivity-dependentsynapseeliminationwithaxonalregression.Inthisstudy,toexaminetheroleofsynapticexpressionlevelsofGluN2B(synapticGluN2B)inthisregression,weemployedliveimagingofdevelopmentaldynamicsofEYFPtaggedChannelrhodopsin (ChR) -labeledCSaxons.WhenaxonalregressionwasblockedbyAPVapplicationduringtheCP,andthereafterAPVwasremoved,theCSaxonsontheventralsidewerenolongerregressedinwildtypemice(WT),whilethosewereelimi-natedinGluN2Aknockoutmice(2AKO).AttheendoftheCP,synap-ticGluN2Bin2AKOspinalcordsremainedhigherlevelscomparabletothoseinWTduringtheCP.Furthermore,evenaftertheCPwasclosed,upregulatingsynapticGluN2BbyproBDNFapplicationinducedtheaxonalregressioninWT.Togetherwiththesefindings,theclosureoftheCPinthisdevelopmentalplasticityintheformofregressionofCSaxonalprojection isdeterminedbythedeclineof thesynapticGluN2B.NoCOI.

1P-100The critical period in corticospinal plasticity was re-opened by upregulation of GluN2B: Electrophysiolog-ical and optical recording studyIsowaki, Mutsumi1; Ohno, Takae1; Isoo, Noriko1; Maeda, Hitoshi1; Fukuda, Satoshi1; Mishina, Masayoshi2; Sakurai, Masaki1(1Dept Physiol, Teikyo Univ Sch Med, 2Dept Mol Neurobiol & Pharmacol, Grad Sch Med, Univ Tokyo)

Inin vitrosystemofcorticospinal(CS)projectionusingsliceco-culturesofmicecerebralcortexandspinalcord(SpC),theCSsynapsesareonceformedthroughoutSpC,andtheneliminatedfromventralsideduringearlydevelopment.Thiseliminationoccursonlyfrom6to11daysin vitro(DIV),whichisdependentonNMDAreceptor(R)anditssubunit,GluN2B(2B).Thistermwascalledcriticalperiod(CP)inthissystem.BecauseofdevelopmentalshiftofNMDARsubunitfrom2BtoGluN2A(2A),itisassumedthatdeclineof2BmaydeterminetheclosingoftheCP.WestudiedmechanismsthatclosetheCPbyelec-trophysiologicalandopticalrecordings.CSaxonswerestimulatedatthedeeplayerofcerebralcortex.SpatialdistributionofCSsynapseswasstudiedbyopticalrecordingsofCS-EPSPs(optEPSPs)withvolt-age-sensitivedye.Wholecellrecordingsweremadetomeasure2BcomponentofCS-EPSCs.ToseewhethertheCPisclosedornot,wefirstappliedAPVuntil11DIVtoblockthesynapseeliminationandthereafterAPVwasremoved.WefoundthatoptEPSPsdecreasedontheventralsidein2Aknockout(KO)miceaftertheendoftheCP.2Bcurrentsin2AKOat11DIVwereequivalentWTinearlydevelopment.ProBDNFwhichareknownto increase2B,producedreductionofoptEPSPsontheventralsideaftertheendoftheCP;i.e.,theCPwasre-opened.Thesesuggest2BdeclineiscruciallyimportantforclosingtheCPintheCSsynapseelimination.NoCOI.

1P-101Quantitative analysis of postnatal development of corticospinal axons projecting to the spinal gray mat-ter (C7) in the mouseMurabe, Naoyuki; Sakurai, Masaki(Teikyo university school of medicine, Tokyo, Japan)

Rodentcorticospinal(CS)axonspostnatallyshowdynamicchangesofaxonandsynapsedistributionsinthespinalcord.Thisstudyaddress-espostnataldevelopmentofCSaxonsinthespinalgraymatteratthecervicalenlargementsegmentC7,which is involved incontrolofforelimbmovement.TovisualizeeveryCSaxon,weusedtransgenicmice,referredtoasCST-YFPmice,inwhichtheCSaxonsexpressyellowfluorescentprotein(YFP).DistributionoftheCSaxonsinthegraymatteratC7anditschangeduringpostnataldevelopmentwereinvestigatedwithalaserscanningconfocalmicroscope.GFP-immuno-reactiveaxonsranthroughtheventralmostofthedorsalcolumnwheretheCStract is located inrodentsandenterthegraymatter. Atpostnatalday7(P7),fineCSaxonsextendedradiallyandreachedthemarginofthegraymatter.Smallfusiform/granularvaricositieswereevenlyspacedalongtheCSaxons.Asdevelopmentproceeds,distri-butionofCSaxonsbecomessegregatedto fourregions.ThegraymatterreceiveddenseprojectionoftheCSaxonsdorsomediallyandlaterally,andmoderateprojectionsventromedially,andlightprojectionsventrolaterally.QuantificationofCSaxonsrunningventrolaterallyshowedanincreaseinnumberfromP7toP14.DensitiesofCSaxonspeakedatP14,andthendeclinedthereafter.Theseresults,togetherwithpreviousresults,suggestthatapopulationoftheCSneuronseliminatesynapsesafterinnervatinginfantgraymatter.NoCOI.


1P-102Homeostatic maintenance of the large-scale depolar-ization wave in the developing central nervous systemMomose-Sato, Yoko1; Sato, Katsushige2(1Dept Hlth & Nutr, Coll Human Enviro Studies, Kanto-Gakuin Univ, Yokohama, Japan, 2Dept Hlth & Nutr Sci, Fac Human Hlth, Komazawa Women's Univ, Tokyo, Japan)

WidelycorrelatedspontaneousactivityinthedevelopingCNSistran-sientlyexpressedand isconsideredtoplaya fundamentalrole inneuralcircuitformation.Thedepolarizationwave,whichspreadsoveralongdistancealongtheneuraxis,isanexampleofthisactivity.Al-thoughthewave is typically initiated inthespinalcord in intactpreparations,spontaneousdischargeshavealsobeendetectedintheisolatedbrainstem.Althoughthissuggeststhatthebrainstemhastheabilitytogeneratespontaneousactivity,but ispacedbyacaudalrhythmgeneratorofhigherexcitability,anumberofquestionsremains.Doesbrainstemactivitysimplyappearasapassiveconsequence,ordoesanyactivechangeoccurinthebrainstemnetworktocompensateforthisactivity?Ifthelatteristhecase,doesthiscompensationoccurequallyatdifferentdevelopmentalstages?Whereisthenewrhythmgenerator?Toanswerthesequestions,weopticallyanalyzedspa-tio-temporalpatternsofactivitydetectedfromthebrainstembeforeandaftertransectionattheobex.Theresultsrevealedthatthewavewashomeostaticallymaintained,whichwascharacterizedbyanin-creaseinexcitabilityand/orthenumberofneuronsrecruitedtothewave.Thewavewasmoreeasilymaintainedinyoungerembryos.Furthermore,wedemonstratedthattheabilityofbrainstemneuronstoperformsuchanactivecompensationwasnotlostevenatthestagewhenthewavewasnolongerobservedintheintactbrainstem.NoCOI.

1P-103Are motoneurons “star-shaped”?Fukuda, Satoshi; Maeda, Hitoshi; Yoshioka, Noboru; Murabe, Naoyuki; Kameda, Hiroshi; Sakurai, Masaki(Department of Physiology, Teikyo University School of Medicine)

Itisgenerallybelievedthatdendriticmorphologyofspinalmotoneuron(MN)is“star-shaped”.However,theanalysisoftheirdendriticpatternatanearlystagehasnotbeendone.ThusweinvestigatedtheshapeofratcervicalMNsinanearlydevelopmentalage(P6–8).TheMNswereidentifiedbyretrogradelabelingwithcholeratoxinBsubunitconjugatedwithAlexaFluor488whichwasinjectedintotheforelimbortrunk(pectralorserratusanteriormuscles)musclegroups.Follow-inga48-hoursurvivalperiod,transverseslicesofthecervicalcordwereprepared.Wholecellrecordingsweremadefromretrogradely-la-beledfluorescence-positiveMNs.NeurobiotinwasinjectedfromtherecordingpipetteswhichwerevisualizedwithTexasRed-avidin.Double-labeledneuronswereimagedwithtwo-photonlaserscanningmicroscopeandtracedwithNeurolucidasystem.TheMNsreceivedmonosynapticresponsestothestimulationofcorticospinaltractwerelimitedtothoseinnervatingforelimbmuscles.ThedendriteintheseMNsshowedpreferredorientationfordorsomedialdirectiontowardthedorsalcolumnwheretheCSTislocatedinrodents.ThedendritesofMNsinnervatingpectoralandserratusanteriormusclesshowedbipolarorientationsalongboundarybetweenthegrayandwhitematter.TheseobservationsmeanthatthedendriticpatternofmostMNsisgreatlydeviatedfromatypical“star-shaped”patternatleastatanearlydevelopmentalstage.NoCOI.

1P-104Transection of the whisker sensory nerve reorganizes topographical wiring of afferent fibers in the whisker sensory thalamus of miceTakeuchi, Yuichi1; Katayama, Yoko1; Miyata, Mariko1,2(1Dept. of Physiol., Sch. of Medicine, Tokyo Women 's Med. Univ., Tokyo, Japan, 2PRESTO, Japan Science and Technology Agency, Saitama, Japan)

Amaturerelayneuroninthewhiskersensorythalamus(VPm)typi-callyreceivesonlyonesensoryafferentfiber,whichoriginatesfromthewhisker-representingbrainstemnucleus(PrV2).Recently,wehavedemonstratedthattransectionofthewhiskersensorynerverecruitsmultipleafferentfibersontoarelayneuron.However,originsofnew-lyrecruitedfibers(PrV2ornot)andpotentialcompetitionbetweennewlyrecruitedandpreexistingfibersremainunexplored.Therefore,weheregeneratedtheKrox20-Cre;Ai14transgenicmice inwhichPrV2-originfibersarespecificallylabeledwithtdTomato(tdT).Usingthemice,PrV2-originandnon-PrV2-originfiberterminalsintheVPmwereanalyzedastdT(+)andtdT(-)VGluT2-immunoreactivepuncta,respectively.Afterthetransection, tdT(+)punctasignificantlyde-creasedcomparedwiththatintheshamgroup(4.8±1.5vs.6.1±1.2,mean±s.d,×10-3/mm2),whereastdT(-)punctaincreased(0.3±0.2vs.0.1±0.1).Densitiesoftotalpunctadidnotchange.Inaddition,thesizeoftdT(+)punctasignificantlydecreasedafterthetransection(2.0±0.2vs.2.3±0.2µm2).TheseresultsindicateconsiderableretractionandweakeningofPrV2-origin(whisker-related)fibersandinvasionofnon-PrV2-originfibers inthewhiskersensorythalamusafterthetransection.NoCOI.

1P-105Cortical feedback activity regulates connection pat-tern of afferent lemniscal synapses in the somatosen-sory thalamus.Narushima, Madoka; Miyata, Mariko(Department of Physiology, School of Medicine, Tokyo Women's Medical University, Tokyo, Japan)

Plentyofstudiesrevealedsynaptic/molecularmechanismsforinitialformationandneuronalactivity-dependentrefinementofsynapticconnectionsinearlydevelopment.However,mechanismsforsubse-quentmaintenanceofonceestablishedcircuitsinmaturedbrainarenotfullyunderstood.Inthesensorythalamus(VPmanddLGN),thesensoryafferentsynapses(lemniscalandretinogeniculate(RG)synaps-es)ontoathalamicneuronaremaintainedinanexperience-dependentmannerafterdevelopmentalsynapseelimination.Athalamicneuronreceivestwokindsofexcitatoryinputsfromafferentsandmassivefeedbackcorticothalamic (CT) inputs.Thus, the functional linkagebetweenthetwo inputsmayunderliethemaintenanceofthalamicafferentsynapses.Recentlywefoundthattype1metabotropicgluta-matereceptor(mGluR1)playsacriticalroleintheexperience-depen-dentmaintenanceofRGsynapses(Narushimaetal.,inpreparation).BecausemGluR1denselyexpressedatpostsynapticsiteofthefeedbackCTsynapses,wehypothesizedthatcorticalactivityregulatesmainte-nanceofafferentsynapsesaftermaturation.Herewereport thatpharmacologicalmanipulationofcorticalactivitytriggeredremodelingoflemniscalsynapses.TheresultssupporttheideathatCTfeedbackinputsheterosynapticallyregulatesmaintenanceofmaturedmono-in-nervationofafferentsynapsesinthesensorythalamus.NoCOI.


1P-106A novel approach to evaluate the anesthetic depth with somatosensory evoked potential elicited by dou-ble stimulationFujihara, Hiroaki; Fujiki, Nobuhiro(Department of Ergonomics, Institute of Industrial Ecological Science, University of Occupational and Environmental Health, Kitakyushu, Japan)

Evokedpotentialshavebeenusedinmanystudiesasanindicatoroftheneuralactivitytothesensoryinput.Weobservedanovelphenom-enonthataratiooftwoevokedpotentialselicitedbydoublesensorystimulationdynamicallychangesinresponsetothedepthofanesthe-sia.SmallscrewelectrodessetupforevokedpotentialmeasurementinmaleSDratskullundersevofluraneanesthesia.Twoconsecutiveelectricalstimulationswereappliedtotheupperlimb,andanamplituderatioofthefirstresponse(R1)andsecondresponse(R2)werecalcu-lated(R2/R1).Whiletheinter-stimulusintervalsarelongenough,R2/R1wasstableregardlessoftheanestheticdepth.Ontheotherhand,whenthe inter-stimulus intervalsareshort (300–500ms),R2/R1dy-namicallydecreaseinresponsetothedepthofanesthesia.TheseresultssuggestthattheR2/R1maybeanovelindicatorforevaluatingtheanestheticdepth.NoCOI.

1P-107Mechanisms of synaptic transmission mediated TRPM1 channels between retinal rod bipolar and AII amacrine cellsTamalu, Fuminobu; Watanabe, Shu-Ichi(Department of Physiology, Faculty of Medicine, Saitama Medical University, Saitama, Japan)

Inthemammalianretina,synaptictransmissionfromrodstorodbi-polarcellsismediatedbythemetabotropicreceptormGluR6.Inthedark,rodsreleaseglutamatebywhichrodbipolarcellshyperpolarize;conversely, inresponseto light,reducedreleaseofglutamatefromrodsopensanon-selectivecationchannel,namelyaTRPM1channel,anddepolarizestherodbipolarcells,fromwhichglutamateisreleasedtopost-synapticAIIamacrinecells(AIIcells).Wefoundthatanin-ter-eventintervalandamplitudeofEPSCsobservedinAIIcellsdra-maticallyincreasedwiththetemperatureincreaseandthatthecur-rent-timeintegraloftheEPSCsat35°Cwasabout3-foldgreaterthanthatat20°C.ThetemperaturedependentEPSCsshouldreflectglu-tamateinputfromrodbipolarcellsbecausetheEPSCswereblockedbyCNQXorL-AP4(anagonistofmGluR6)regardlessofthetempera-ture,butnotstrychnineandbicuculline,andthereversalpotentialwasnear0mV.Membranepotentialofrodbipolarcellsdepolarizedat22mV/10 °Cusingwhole-cellpatch-clamprecordings.AlthoughthetemperaturedependentdepolarizationwasnotdecreasedbyL-AP4,itwasdepressedusingperforatedpatch-clamprecordings.Thetem-peraturedependentEPSCsinAIIcellswerenotobservedinTRPM1KOmice.Theseresultssuggestthatglutamatereleasefromrodbipo-larcellsviamGluR6-TRPM1cascadeistemperaturedependentandthatbothadequatetemperatureandintracellularligandmightneces-saryforregulationoftheopenprobabilityoftheTRPM1channel.NoCOI.

1P-108"Electrophysiological Effects of IL-1β, IL-6 and TNF-α on Thalamic Dorsal Lateral Geniculate Nucleus Relay Neurons"Samios, Vinicius Nikolaos; Inoue, Takafumi(Department of Life Science and Medical Bioscience, School of Advanced Science and Engineering, Waseda University)

MajorinflammatorycytokineslikeInterleukine1β,Interleukine6andTumorNecrosisFactorαplayimportantrolesinthedevelopmentofneuropsychiatricconditions.Wepresentherehowelectrophysiologicalpropertiesofthalamicrelayneuronsareaffectedbyhigh levelsofIL-1β,IL-6andTNF-α.Patch-clamprecordingsofthalamicrelayneu-ronsfrombrainslicesfreshlydissectedfrommicewereperformed.IL-1β(1.4nM)inducedahyperpolarizationofrestingpotential(-64.1±1.35(IL-1β)vs.-61.3±1.28(control)mV,p<0.05),adecreaseinnormalizedIhamplitude(0.74±0.05vs.0.87±0.04,p<0.05),adecreaseinnormalizedactionpotentialthreshold(1.08±0.03vs.0.96±0.03,p<0.01),adecreaseinnormalizedmembraneresistance(Rm,0.80±0.05vs.0.97±0.03,p<0.01),adecreaseinspikenumbersinbursts(5.0±0.57vs.7.2±0.49,p<0.01)andincreasednormalizedburstlatency(1.09±0.06vs.0.95±0.02,p<0.05).IL-6(1nM)inducedadecreaseinnormalizedIhamplitude(0.73±0.05vs.0.87±0.04,p<0.05),adecrease innormalizedRm(0.67±0.09vs.0.97±0.03,p<0.01)andanincreaseinnormalizedburstlatency(1.36±0.16vs.1.06±0.06,p<0.05).TNF-α(1.2nM)inducednochangeintheevalu-atedparameters.TheseresultsshowthatIL-1β,IL-6,butnotTNF-αmayplayimportantrolesinelectrophysiologicalmodulationontha-lamicrelayneurons.Alteredneuronalbehaviorpromotedbythesecytokinescouldbeapossiblemechanismbehindconditionswheresensoryprocessingandconsciousnessareaffected,suchasindelirium.NoCOI.

1P-109Cholinergic gain control in the primary visual cortex of awake ratsYoshioka, Daisuke1; Soma, Shogo2; Suematsu, Naofumi3; Shimegi, Satoshi2,3(1Sch. Sci., Osaka Univ.,, 2Grad. Sch. Med., Osaka Univ., 3Grad. Sch. Front. Biosci., Osaka Univ. Osaka, JAPAN)

WerecentlyfoundtwoimportantrolesofAcetylcholine(ACh)invi-sualfunctions.First,AChadministeredtoV1causedaresponsegaincontrolinanesthetizedrats.Theresponsestovariousstimuluscontrastwerefacilitatedorsuppressedinproportiontothemagnitudeofthecontrolresponses,bywhichtheshapesoforiginalcontrast-responsefunctionswereunchanged.Second,thesystemicadministrationofacholinesteraseinhibitor,donepezil(DP),improvedthecontrastdetect-ability inbehavingrats.However, thedetectability improvementcannotbesimplyduetotheresponsegaincontrolinV1because1)DPactsatotherareasthanV1,2)anestheticmayinfluenceACh’sactions.Toclarifythosepoints,weperformedextracellularrecordingsfromV1ofhead-fixedratsunderawakeandanestheticconditionsandcomparedtheACh’smodulatoryeffects.DPwastopicallyadministeredontoV1.DPpredominantlycausedresponsegaincontrol inbothphysiologicalconditionsbutitalsoevokeddecreaseofcontrastgaininawakerats,suggestingahighresponsivenesstolowcontraststim-ulus.Therefore,ACh-evokedtwotypesofgaincontrolwithinV1seemtobeoneofthereasonsforDP-induceddetectabilityimprovementofawakerats.AllexperimentalprotocolswereapprovedbytheResearchEthicsCommitteeofOsakaUniversity,andallprocedureswerecarriedoutincompliancewiththepoliciesandregulationsoftheguidelinesapprovedbytheAnimalCareCommitteeoftheOsakaUniversityMedicalSchool.NoCOI.


1P-110Impaired visual working memory in mice with reduced clusters of protocadherinsKamatani, Daiki1,3; Watanabe, Kenji1; Hishida, Ryuichi1; Yagi, Takeshi2; Shibuki, Katsuei1,3(1Dept Neurophysiol, Brain Res Inst, Niigata Univ, Niigata, Japan, 2KOKORO-Biology Group, Grad Sch of Frontier Biosci, Osaka Univ, 3CREST, JST)

Clusteredprotocadherins (cPcdhs)areneurospecificcelladhesionmoleculescharacterizedwithmultipleclusters.Ofthe12clustersincPcdhα,cPcdhα1,12micehaveonlycPcdhα1andcPcdhα12,sothatcPcdhαdiversityisreducedinthesemice.However,noapparentab-normalityhasbeenfoundinthesemice.Inthepresentstudy,wereportthatvisualworkingmemoryisimpairedincPcdhα1,12mice.First,wetestedworkingmemoryregardingspatiallocationsofvisualcuesusingaT-maze.AlthoughcPcdhα1,12miceshowedagoodperformanceinavisually-guidedT-mazetask,theperformanceinamemory-guidedT-mazetaskwassignificantlyworsethanthat inwild-typemice(P<0.01).WedevelopedanM-mazeequippedwithadisplayfortestingshape-recognitionworkingmemory.Inacontroltask,acueshapeAorBwaspresentedatcenterofthedisplay,andthechoiceshapesAandBwerepresentedateitherbranchoftheM-mazewithoverlappedtiming.Ifmiceselectedthebranchwiththedifferentchoiceshapepresentedasthecueshape,theyobtainedareward.Bothwild-typemiceandcPcdhα1,12miceshowedagoodperformancewithasuccessrate>85%.Inaworkingmemorytask,thedelayperiodbetweenthepresentationofthecueshapeandthechoiceshapeswassetat20s.Inthisworkingmemorytask, theperformance incPcdhα1,12wassignificantlyworsethanthatinwild-typemice(P<0.003).TheseresultsindicatethatcPcdhαdiversityplaysanessentialroleinvisualworkingmemory.NoCOI.

1P-111Frequency dependent integration of excitatory syn-apses in the dendrites of auditory coincidence detec-tor neurons of birds.Yamada, Rei1; Ohmori, Harunori2; Kuba, Hiroshi1,3(1Dept. Cell. Physiol., Nagoya Univ. Grad. Sch. Med., Nagoya, Japan, 2Dept. Physiol., Facult. Med., Kyoto Univ., Kyoto, Japan, 3JST PRESTO, Saitama, Japan)

Differenceofsoundarrival timebetweentwoears (interaural timedifference,ITD)isamajorcueforsoundsourcelocalization.Inbirds,neuronsinnucleuslaminaris(NL)arethecoincidencedetectorofbi-lateralsynapticinputsandinvolvedinprocessingofITDs.Important-ly,NLneuronsaretunedtoaspecificfrequencyofsound(character-istic frequency, CF) and have several functional specializationsdependingonCF.Inparticular,thelengthofdendritesvariesalongthefrequencyaxisandneurons in low-CFregion (low-CFneurons)havedendrites7–20timeslongerthanhigherCFneuronshave.How-ever,thefunctionalrolesofCFspecificvariationofdendriticlengthfortheITDprocessingstillremainelusive.Inthisstudy,weanalyzedthedistributionofglutamatereceptorsalongtheNLdendriteswiththefocaluncagingofMNI-glutamateinthechickenbrainslices.Wefoundthatthedensityofglutamatereceptorswasheterogeneousinthelow-CFneuronsandincreasedtowardthedistalendofdendrites.Ontheotherhand,thistendencywasnotobservedinhigh-CFneurons.Inthelow-CFneurons,thepropertiesoffocal-evokedmEPSCsshowednocleardifferencesbetweentheproximalanddistaldendrites,sug-gestingthatthetypeanddensityofreceptorsunderthesynapticreleasesiteissimilaralongthedendrites.WewillfurtherexaminetheintegrationprocessatthefocaldendritestoaddressthefunctionalrolesofdendritesintheNLneurons.NoCOI.

1P-112Tonotopic variation of inhibitory synaptic transmission in nucleus magnocellularis of chickOnogi, Chikao1; Yamada, Rei1; Kuba, Hiroshi1,2(1Dept. Cell. Physiol., Nagoya Univ. Grad. Sch. Med., Nagoya, Japan, 2JST PRESTO, Saitama, Japan)

Neuronsinaviannucleusmagnocellularis(NM)generateactionpoten-tialsinresponsetoexcitatorysynapticinputsfromtheauditorynerve,andareinvolvedinarelayofauditorytiminginformationtohigherauditorycenter.NMneuronsaretunedtoaspecificfrequencyofsoundandarrangedtonotopicallywithinthenucleus.Itisknownthattem-poralprecisionofauditorynerveactivitydiffersalongthetonotopicaxis,anditdecreasestowardlowertuningfrequency.Tocompensateforthisvariation,NMneuronsdifferinthenumberandsizeofexcit-atoryinputsalongthetonotopicaxis;high-frequencyneuronsreceiveasinglelargeinput,whilelow-frequencyneuronsconvergemultiplesmallinputstogenerateaspike.However,itremainsunknownwheth-erNMneuronsalsovaryintheirinhibitoryinputsinamannerdepen-dentontuningfrequency.Inthisstudy,westudiedcharacteristicsofminiature inhibitorypostsynapticcurrent (mIPSC) inNMneuronsalongthetonotopicaxis,withwhole-cellpatch-clamptechniques inbrainslicesofposthatchchickens.WhatwefoundwasthatmIPSCshowedhigherfrequencyinlow-frequencyneuronsthaninhigh-fre-quencyneurons,withoutanydifferencesintheamplitudeandtimecourse.Thismaysuggestthatlow-frequencyneuronsreceivealargernumberofinhibitoryprojectionscomparedwithhigh-frequencyneu-rons.WewillfurtherrecordunitaryIPSCtoconfirmthisidea,anddiscusspossiblerolesofthisabundanceofinhibitoryprojectionsinthetemporalcodingoflow-frequencyNMneurons.NoCOI.

1P-113Homeostatic regulation of potassium channels in avi-an auditory neuronsKuba, Hiroshi1,2; Yamada, Rei1; Ishiguro, Go1(1Department of Cell Physiology, Nagoya University Graduate School of Medicine, Nagoya, Japan, 2PRESTO, JST, Saitama, Japan)

Appropriateadjustmentofneuronalactivityiscrucialformaintenanceandperformanceofneuralcircuits.Axoninitialsegment(AIS)isac-cumulatedwithvoltage-gatedNa+(Nav)channelsandisthesiteofspikeinitiationinneurons.WepreviouslyshowedthatdeprivationofauditoryinputsincreasedtheexpressionofNavchannelsattheAISandaugmentedexcitabilityofneurons inaviancochlearnucleus.However,howauditorydeprivationaffectsotherionchannels,suchasvoltage-gatedK+(Kv)channels,remainsunknown.Inthisstudy,weaddressedthisissuewithimmunohistochemistryandelectrophysiolo-gy,andfoundthatauditorydeprivationchangestheexpressionsofKvchannelsattheAISinasubtype-specificmanner;Kv1.1decreased,whileKv7.2increased,showingacomplementarychangeintheirex-pressions.Interestingly,auditorydeprivationcausedanegativeshiftofspikethresholdwithoutchangesintherestingmembranepotential.Kv1.1haslowthresholdandrapidkineticsforactivationandstronglyinhibitsfiring,whileKv7.2hasslowkineticsandcontributestosettherestingpotential.TheseindicatethatthedecreaseofKv1.1enhancedexcitabilityofneuronsbutitwasbalancedwiththeincreaseofKv7.2attherest.Thus,Kv1.1andKv7.2workcooperativelyattheAIS,andmaintainexcitabilityandrestingpotentialofneuronsduringdepriva-tionofafferentinputsinthecochlearnucleus.NoCOI.


1P-114Detrended fluctuation analysis reveals temporal pat-terns of spontaneous oscillatory activity in the olfac-tory center of the land slugKomatsuzaki, Yoshimasa1; Eto, Tamon1; Haga, Shouhei1; Tanaka, Yuichi1; Saito, Minoru2(1Department of Physics, CST, Nihon University, Tokyo, Japan, 2CHS, Nihon University, Tokyo, Japan)

Spontaneousoscillationofactivity inolfactorysystemiscommonlyobservedinvariousspeciesandisthoughttobeimportantforodor-in-formationprocessing.TheterrestrialslugLimaxvalentianushasahighlydevelopedolfactorycenter, theprocerebrum(PC), inwhichexhibitsoscillationof localfieldpotential (LFP)at0.7Hz.Here,weinvestigatedthestatisticalpropertyofspontaneousoscillationinthePCoveranextendedtimescale.Toanalyzetheautocorrelationofthetimeseriesofinterspikeintervals,weperformedadetrendedfluctu-ationanalysis,whichisascalinganalysistechniqueusedtoprovideaquantitativeparameter(scalingexponent,α).ThefluctuationofspiketimingofthePCpossessedalong-termcorrelationattimescaleslarg-erthan102spikes (α=1.21).Theapplicationofcychloheximide,aninhibitorofproteinsynthesis,inducedadecreaseinthescalingexpo-nentαatthelargertimewindow.Thisresultindicatesthatthefluc-tuationsofspiketimingsarecharacterizedbyadynamicsofmRNAtranslationinthePC.Furthermore,toinvestigatethecontributionofinnervationstospontaneousoscillation,wemeasuredtheLFPfollow-ingthetentacleamputation.Inthescalingregionforsmalln(n<102spikes),thescalingexponentαdecreasedfrom0.73to0.58,indicatingthatthefluctuationsofspiketimingsareuncorrelatedunderthisex-perimentalcondition.TheseresultssuggestthattheinnervationstoPCmaintaintheintrinsicdynamics.NoCOI.

1P-115Cholinergic system regulates oscillatory activity of the olfactory center in the slugKobayashi, Suguru(Kagawa Schl. Pharmaceut. Sci., Tokushima Bunri Univ., Sanuki, Kagawa, Japan)

Laminarstructureofsynchronousoscillatoryactivityiscommonintheolfactorysystemofbothvertebratesand invertebrates. Intheterrestrialslugs,periodicoscillationisrecordedfromthesurfaceofthelaminarstructureofprocerebrum(PC)anditsfrequencychangesaresuggestedtoencodetheolfactoryinformationandmemory.Ace-tylcholine(ACh)isknowntoincreasethefrequencyofthelocalfieldpotential(LFP)oscillationinthePC,andisoneofthecandidatesoftheneurotransmittersthatareinvolvedinsuchhighercognitivefunctions.Inthepresentstudy,wethusexaminedwhatrolecholinergicsystemplays inthePCoscillatorynetwork.First, theacetylcholinesterase(AChE)inhibitorenhancedtheexcitatoryeffectofACh,andfurther-more,AChEaloneincreasedfrequencyoftheLFPoscillationinthePC.Second,themRNAsfornicotinicAChreceptorswereexpressedwithnarrowdistributionofthePC.Nicotineinducedtheinwardcur-rentsinsomePCneuronsanditsestimatedreversalpotentialwas-55mV.FromthecalculatedchlorideNernstpotentialof -56mV,thisnicotine-evokedinwardcurrentisprimarilycarriedbyCl-.Thesere-sultssuggestthatAChcanfunctionasanexcitatorytransmitterforPCneuronspresentthePCviamainlynicotinicAChreceptorsacti-vation.Furthermore, theolfactorytentacleablationenhancedthenicotine-induced increase inLFPfrequencyofthePC.Thisresultadditionallysuggeststhepresenceof feedforward inhibition inthecholinergicafferentsfromtheolfactorytentacletothePC.NoCOI.

1P-116Spontaneous excitatory transmission enhancement and outward current produced by carbacrol in rat spi-nal substantia gelatinosa neuronsFujita, Tsugumi; Luo, Qing-Tian; Jiang, Chang-Yu; Kang, Qin; Ohtsubo, Sena; Matsushita, Akitomo; Kumamoto, Eiichi(Dept. Physiol., Saga Med. Sch., Saga, Japan)

Althoughtheoraladministrationofanessentialoilcomponentcarvac-rolproducesantinociception,cellularmechanismsforthisactionhavenotyetbeenexamined. Weexaminedtheactionofcarvacrolonglutamatergicspontaneousexcitatorytransmissionbyapplyingthewhole-cellpatch-clamptechniquetosubstantiagelatinosa(SG)neuronsinadultratspinalcordslices.Carvacrolsuperfusedfor2minproducedeitherincreaseinthefrequencyofspontaneousEPSCwithaminimalincreaseintheamplitudeoroutwardcurrentat-70mV.Thefrequen-cyincreaseandoutwardcurrenthadtheEC50valuesof0.69mMand0.55mM,respectively.TheformeractionwasinhibitedbyaTRPA1antagonistHC-030031butnotaTRPV1antagonistcapsazepine,whilethelatteractionwasunaffectedbytheantagonists.Current-voltagerelationshipoftheoutwardcurrent indicatedan involvementofachangeinthemembranepermeabilityofK+.Theoutwardcurrentwasinhibitedin10mM-K+butnotlow-Cl-andK+-channelblockers(TEAandBa2+)-containingKrebssolution. Inconclusion,carvacrolincreasedthespontaneousreleaseofL-glutamateontoSGneuronsfromnerveterminalsbyactivatingTRPA1butnotTRPV1channels.Carvacrolalsoproducedmembranehyperpolarization,whichwasmediatedbyTEA-andBa2+-insensitiveK+channels, inSGneuronswithoutTRPV1andTRPA1activation.Itissuggestedthatthehy-perpolarizingeffectofcarvacrolcouldcontributetoitsantinociceptiveeffect.NoCOI.

1P-117Cellular mechanisms for inward currents produced by oxytocin in adult rat spinal substantia gelatinosa neu-ronsJiang, Chang-Yu; Fujita, Tsugumi; Ohtsubo, Sena; Matsushita, Akitomo; Xu, Zhi-Hao; Kumamoto, Eiichi(Dept. Physiol., Saga Med. Sch., Saga, Japan)

Wehavepreviouslyreportedthatoxytocinproducesaninwardcurrentat-70mVbyactivatingoxytocinreceptors,resultingintheenhance-mentofGABAergicandglycinergicspontaneousinhibitorytransmi-sisoninspinalsubstantiagelatinosa(SG)neurons,acellularmechanismforantinociceptionproducedbyintrathecally-administeredoxytocin.Thepresentstudyexaminedcellularmechanismsfortheoxytocin-in-ducedinwardcurrentbyapplyingthewhole-cellpatch-clamptechniquetotheSGneuronsofadultratspinalcordslices.TheoxytocincurrentwasinhibitedbyaphospholipaseCinhibitorU-73122,anIP3-inducedCa2+-release inhibitor2-aminoethoxydiphenylborate. Ontheotherhand,aCa2+-inducedCa2+-releaseinhibitordantrolene,aproteinkinaseCinhibitorchelerythrine,anddibutyrylcyclic-AMPdidnotaffecttheoxytocinactivity.In31%ofneuronsexhibitingoxytocinactivity,anetofoxytocincurrent,estimatedfromadifferencebetweencur-rent-voltagerelationshipsintheabsenceandpresenceofthispeptide,reversedataroundtheequilibriumpotentialforK+.Theotherneuronsdidnotshowsuchareversal,andthenetoxytocincurrentwasinwardatnegativepotentials.Theoxytocincurrentwasdepressedinpeakamplitudeinhigh-K+andlow-Na+solution.Itisconcludedthattheoxytocin-inducedinwardcurrentinSGneuronsisduetoachangeinmembranepermeabilitiestoK+and/orNa+,whichispossiblymediat-edbyphospholipaseCandIP3-inducedCa2+release.NoCOI.


1P-118Optical survey of initial expression of synaptic func-tion in the embryonic chick trigeminal sensory nucle-usSato, Katsushige1; Momose-Sato, Yoko2(1Dept Hlth & Nutr Sci, Fac Human Hlth, Komazawa Women's Univ, Tokyo, Japan, 2Dept Hlth & Nutr, Coll Human Enviro Studies, Kanto-Gakuin Univ, Yokohama, Japan)

Weexaminedinitialexpressionofsynapticfunctionintheembryonicchicktrigeminalnucleususingvoltage-sensitivedyerecording.Brain-stempreparationswiththreetrigeminalnerveafferents,theophthal-mic (V1),maxillary (V2)andmandibular (V3)nerves,weredissectedfrom5.5-to6.5-dayoldchickembryos.Inourpreviousstudy(Satoetal.,1999),wedetectedslowsignalscorrespondingtoglutamatagicEPSPsandidentifiedtheprincipalsensorynucleusofthetrigeminalnerve(Pr5),spinalsensorynucleusofthetrigeminalnerve(Sp5)andtrigeminalmotornucleus(Mo5).Inthisstudy,weexaminedeffectsofremovingMg2+fromphysiologicalsolution,whichenhancedNMDAreceptorfunctioninthesensorynuclei.In6.5-dayoldembryos,theslowsignalwasobservedinthePr5andSp5onlywhentheV1nervewasstimulated,whereasitappearedinMg2+-freesolutionwitheverynervestimulation.In6-dayoldembryos,theslowsignalwasobservedonlyintheSp5withtheV1nervestimulation,andtheappearanceofsynaptic function inMg2+-freesolutionvarieddependingonnervesandpreparations.In5.5-dayoldembryos,synapticfunctionwasnotdetectedevenwhenexternalMg2+wasremoved.Theseresultsindi-catethattheinitialexpressionofsynapticfunctioninthetrigeminalsystemisearlierthanpreviouslyconsidered,andthatthedevelopmen-talorganizationofsynaptic function isdifferentbetweenthethreetrigeminalnervesandbetweenthetwosensorynuclei.NoCOI.

1P-119Attempts to record voltage sensitive FRET signal and extracellular spike current from a neuron using a pho-tometric patch electrodeHirai, Yasuharu1,2; Nishino, Eri1; Ohmori, Harunori1,2(1Dept. of Physiology and Neurobiology, Kyoto Univ., Kyoto, Japan, 2LIMS, Kyoto Univ., Kyoto, Japan)

Howneuronsgeneratespikesasaconsequenceofsynapticinputsisan important issuetounderstandtheflowofneuronal information,particularlyinadeepbraintissuewheremostofneuralactivitiesareoutofthereachofmodernelectrophysiologicaloropticalrecordingtechniquessuchaswholecellpatchrecordingortwophotonmicros-copy.Wehaveestablishedanewphotometricpatchelectrode(PME)recordingmethodthatdeliversandcollectslightfromatargetneuronbyusingapatchelectrodeasalightguide,whichenabledustoobtainfluorescentandelectricalsignalsfromaneuron.ByusingthismethodinadeepbraintissueoflivingchickswehavesofarsucceededtorecordCa2+sensitiveOregonGreenBAPTA-1fluorescencesimulta-neouslywiththefieldcurrentinresponsetosoundstimulusfromin-feriorcolliculusandField-L(avianauditorycortex).WeareapplyingthePMEtomonitorvoltagesignalsfromachickauditorybrainstemneuroninslicesbyusingDiO-DPAFRETtechnologythatwasrecent-lyreportedasafastandsensitivemembranepotentialindicator.Weareinterestedinapplyingthistechniqueeventuallytoauditorynucleiinvivo.ThePMErecordingtechniqueincombinationwiththevoltagesensitiveFRETrecordingmaycontributetoinvestigatetheprocessofsynapticintegrationasFRETresponsesandtheneuronaloutputascellattachedextracellularfieldresponses,whichallowsustoun-derstandtheflowofneuronalinformationinasinglecelllevelindeepbraintissues.NoCOI.

1P-120Generation of OXTR-IRES-Cre knock-in miceHidema, Shizu; Hayashi, Ryotaro; Hiraoka, Yuichi; Asayama, Emi; Ootsuka, Ayano; Miyazaki, Shinji; Nishimori, Katsuhikio(Graduate School of Agricultural Science, University of Tohoku, Sendai, Japan)

Theoxytocinreceptor (OXTR)andligandoxytocin (OXT)regulatereproductivefunction(i.e.parturitionandmilkejection),socio-sexualbehaviorsandsoon.WereportedthatOXTR-deficientmiceexhibitedpervasivesocialdeficits (1)andsoon. Successively,wegeneratedOXTR-Venusknock-inmicetolocateandanalyzetheneuronsexpress-ingOXTRbyvisualizingneuronsexpressingOXTR(2).Withthem,wedetectedtheexpressionofOXTRonGABAergicneurons,seroto-nergicneuronsandsoon.Inthepresentstudy,wenewlygeneratedOXTRcDNA-HA-IRES-Creknock-inmice,toexpressbothOXTRandCrerecombinaseunderthecontrolofOXTRpromoter,andtorestrict-edlymodifytheneuronsexpressingOXTR.Asinthepreviousstudy,wefoundanimpairedphenotypeinapartoftheirsocialbehaviorswithheterogonousOXTRKOmice(haploinsufficiency)(3),toavoidsucheffectcausedbyinactivationofonealleleofOXTRgenewithinsertionoftheothergene,andfurthertofacilitateanalysisofsubcel-lularlocalizationofOXTR,HA-tag-modifiedmouseOXTRcDNA,andfollowingIRESelementwereplacedattheupstreamofCresequenceinaknock-invector.Thislineisalsousefulformonosynapticanalysisofneuralcircuit,optogeneticanalysisofneuronsexpressingOXTR,and so on, by combinationwith ournewAAVvector system.1.Takayanagi,Y.etal.,ProcNatlAcadSciUSA102:16096(2005)2.Yoshi-da,M.etal.,J.Neuroscience29:2259(2009)3.Sala,M.etal.,JNeuroendo-clinol(2012)NoCOI.

1P-121Development of human iPSCs-derived neuronsOhara, Yuki1; Yamazaki, Hiroyuki1; Ootsu, Mao1; Sato, Kaoru2; Sekino, Yuko2; Shirao, Tomoaki1(1department of neurobiology and behavior, Gunma University Graduate school of medicine, Maebashi, Japan, 2Division of Pharmacology, National Institute of Health Sciences, Tokyo, Japan)

Differentiatedneuronsinducedfromhumaninducedpluripotentstemcells(hiPSCs)areexpectedtobeatoolfordevelopinganewmethodoftreatment forvariousneurologicaldiseases.However, thedetaildevelopmentalpropertiesofhiPSCs-derivedneuronshavenotyetbeenknown.Inthisstudy,weanalyzedneuronaldevelopmentofhiPSC-de-rivedneurons(iCellneurons,CellularDynamicsInternational)focusingontheirearlydevelopmentalstagescomparedwithratculturedneu-rons. In2days invitro (DIV)culture,weobservedthreedifferentstagesofratneurons,whichwerestages1,2and3indevelopmentalclassificationproposedbyDotti. Incontrast,weobservedonlyhiP-SCs-derivedneuronsofstage1and2.Thissuggeststhatdifferenceofthematurationspeedbetweenhumanandratneuronshasalreadyappearedatstage3.Moreover,totestwhethertherearedifferencesineffectonpharmacologicalagentsforF-actinandtranscription,wetreatedhiPSCs-derivedneuronsandratneuronswithCytocharasinD(CytoD)andHDAC-inhibitorvalproicacid(VPA).TheCytoDtreatmentcausedtranslocationsofdrebrinandF-actinfromthetransitionalzonetothedistaledgeofgrowthconeinbothiCellandratneurons.TheVPApromotedneuriteoutgrowthiniCellneuronslongerthaninratneurons.ThesedatasuggestthatthedevelopmentofiCellneuronsshowsdifferencesatdevelopmentalspeedandreagentreactivity.NoCOI.


Poster PresentationsSensory Function (1)

1P-122Effects of anion permeability of GABAA and GABAC receptors on surround response polarity in bipolar cells of the mouse retinaYin, Chengzhu; kaneda, makoto(Department of Physiology, Nippon Medical School, Tokyo, Japan)

Inthemouseretina,surroundresponsesinON-andOFF-bipolarcellsareregulatedbyGABA-mediatedchloride(Cl-)currentindendrities.AnoppositepolarityofsurroundresponsesbetweenON-andOFF-bipolarcellsisexplainedbythehypothesisthatreversalpotentialofCl-(ECl)inON-bipolarcellsishigherthanthatofOFF-bipolarcells.Theimmunohistochemichalfindings,electrophysiologicaldataandimagingoftheintracellularCl-concentrationsupportthehypothesis.However,thereversalofresponsepolaritybetweenON-andOFF-bipolarcellsisnotcompletelyelucidatedbythesefindings.Inthepresentstudy,weexaminedwhetherthepresenceofbicarbonateion(HCO3

-)shiftsEClinGABAAandGABACreceptorsbymeansofpatchclamptech-niques,sincedistributionofGABAAandGABACreceptorscancon-tributetotheformationofsurroundresponsesiftheyhavedifferentECl.WefoundthatthereversalpotentialofGABAAandGABACre-ceptorswithoutextracellularHCO3

-were-5±3.5mV(n=6)and-3.8±4.8mV(n=5),respectively.ThereversalpotentialofGABAAandGABACreceptorswithextracellularHCO3

-(24mM)were-4.5±5.7mV(n=6)and-2.6±3.4mV(n=5),respectively.Theseresultsruleoutthepossi-bilitythatthedistributionofGABAreceptorssubtypeswithdifferentHCO3

-permeabilityinbipolarcellscontributestotheformationofanoppositepolarityofsurroundresponsesbetweenON-andOFF-bipolarcells.NoCOI.

1P-123Mechanism of receptive field generation in early visu-al pathway: simultaneous recording of retinal ganglion cells and lateral geniculate cellsMiyoshi, Tomomitsu1; Suematsu, Naofumi2,3; Naito, Tomoyuki2; Sawai, Hajime1; Sato, Hiromichi2,3(1Dept Integr Physiol, Grad Sch Med, Osaka Univ, Suita, Japan, 2Lab Cogni and Behav Neurosci, Grad Sch Med, Osaka Univ, Toyonaka, Japan, 3Grad Sch Front Biosci, Osaka Univ, Toyonaka, Japan)

Retino-geniculatetransmissionhasbeenthoughttoberelativelysim-plebecausebothretinalganglioncells(RGCs)andrelaycellsofthelateralgeniculatenucleus(LGN)havecircularreceptivefields(RFs).However,wehaverecentlyreportedthatRFstructureofcatLGNneuronsisratherellipticalthancircular,givingthemmoderateorien-tationsensitivity(Suematsuetal.,2012;Naitoetal.,2013).ToclarifytheconnectionruleforgeneratingLGNRFs,wesimultaneouslyre-cordedpairsofsingle-unitactivitiesofRGCsandLGNneurons,whichshowedfunctionalmonosynapticconnectionswithcross-correlationanalysis.Wefoundthat1)spatialRFstructureofbothRGCsandLGNneuronswerecomparativelyelliptical,2)spatialRFstructuresofthepairswithsame-response-signwereoftenoverlappedandsimilarly-oriented,3)mostofthepairswithopposite-response-signexhibitedRFstructureswhichwerespatiallydisplacedandindependently-oriented,and4)thetemporalRFstructuresoftheRGCsweretightlycorrelatedwiththoseoftheirtargetLGNneurons.Theseresults indicatethatRFstructureofLGNneuron ismainlyinheritedfromthatofaprimary-projectingRGC,andthatstimulusfeatureselectivityofLGNneuronsissharpenedbymoreconvergentinputsfrommultipleRGCsthanpreviouslyconceived.NoCOI.

1P-124Trigeminal interpolaris/ caudalis transition neurons mediate ocular blood flow responses evoked by bright light in the ratTshiro, Akimasa1; Kemuriyama, Takehito1; Tandai-Hiruma, Megumi1; Ohta, Hiroyuki1; Hagisawa, Kohske1; Nishida, Yasuhiro1; Bereiter, David2(1Dept. Physiol. Nat.Def.Med. Coll. Saitama . Japan, 2Dept. Diagnostic and Biological Science, Univ. of Minnesota School of Dentistry, Minneapolis, MN, USA)

Abnormalsensitivitytobrightlightcancausediscomfortorpainandevokedparasympatheticreflexessuchaspupillaryconstriction,lacri-mationandvsodilation inthechoroid.Themechanismsunderlyingabnormalsensitivitytolightremainelusive;however,ithaslongbeenproposedthattrigeminalsensorynervesplayasignificantrole.Tri-geminalsensorynervesthatsupplytheeyeprojecttotwospatiallydistinctregionsofthelowertrigeminalbrainstemnuclearcomplex,subnucleusinterpolaris/caudalis(Vi/Vc)transitionaswellasthecau-dalis/uppercervicaljunction(Vc/C1)regions.Recentlywereportedthatlight-evokedVi/VcandVc/C1neuralactivityinvolvedincreasedparasympatheticoutflowconsistentwithavascular-linkedmechanismwith intheeye. However,since little isknownaboutunderlyingneuralcircuitryforreflexchoroidalbloodflow(CBF)responsestobrightlightstimuli,CBFwasmeasuredinaseparategroupofmaleratsaftersynapticblockadeofVi/VcorVc/C1regions.Brightlight(20klux)-increasedCBFwasmarkedlyinhibited(>80%)by10minafterCoCl2injectionintotheVi/Vcregion,wheresblockadeattheVc/C1hadnoeffect(<10%).TheseresultssupportthehypothesisthatlightresponsiveneuronsattheVi/Vctransitionregionarecriticalforoc-ularreflexfunctionsuchaschangeofintraocularbloodflow.NoCOI.


1P-125Orexin-A inhibits ocular-responsive trigeminal subnu-cleus caudalis neurons in the ratKatagiri, Ayano1; Iwata, Koichi2(1Dept. Diagnostic and Biological Sciences, Univ. of Minnesota Sch. of Dentistry, MN, US, 2Dept. Physiology, Nihon University Sch. of Dentistry, Tokyo, JP)

Orexin-A(OxA)issynthesizedexclusivelyinthehypothalamusandisassociatedwithhomeostaticregulationandpainmodulation.Previ-ouslywereportedthatincreasedposteriorhypothalamusoutflowre-ducedlight-evokedtrigeminalsubnucleuscaudalis/uppercervical(Vc/C1)neuralactivityandreflexlacrimation.TodetermineifOxAcon-tributedtoposteriorhypothalamusmodulationofocularnociception,OxAwasappliedtothedorsalbrainstemsurfacewhilerecordingfromocular-responsiveneuronsinsuperficiallaminaeattheVc/C1region.Vc/C1ocularunitswereactivatedbyexposuretobrightlightoroc-ularsurfaceapplicationofhypertonicsaline.OxAmarkedlyreducedboth light-andNaCl-evokedneuralactivity. Co-applicationof theorexin-1receptorantagonist,SB334867,reversedOxA-mediatedinhi-bitionofocular-evokedactivity.OxAalsoreducedthehighthresholdconvergentcutaneousreceptivefieldareaofocularunits.OxAdidnotalterthespontaneousactivityofVc/C1neuronsorrestingmeanarterialpressure.LocalapplicationofSB334867alonehadnoeffect.Co-applicationofOxAandtheorexin-2receptorantagonistdidnotpreventOxA-mediatedinhibition.TheseresultssuggestedthatOxAactedthroughorexin-1receptorstomodulatesomatosensory inputfromtheocularsurfaceanddeeptissuesoftheeyetoneuronsinsu-perficiallaminaeofthemedullarydorsalhorn.Acknowledgements:Univ.ofMinnesota,Prof.D.A.Bereiter.ThisworkwassupportedbyaGrantfromNIH(EY021447).NoCOI.

1P-126The effect of Menthol, Capsaicin and AITC on the thermal response of corneal primary afferent neuronsKurose, Masayuki1; Hatta, Azusa1; Wiersma, Jelle2; Oda, Masataka3; Yamamura, Kensuke1; Meng, Ian D2(1Division of Oral Physiology, Niigata University, Niigata, Japan, 2Department of Physiology, College of Osteopathic Medicine, University of New England, Biddeford, ME, USA, 3Division of Microbiology and Infectious Diseases, Niigata University, Niigata, Japan)

Previousstudieshavefoundthatcoldcellsinnervatingthecorneaaresensitivetotheocularfluidstatusofthecornealsurfaceandmayberesponsiblefortheregulationofbasaltearproduction.Inthepresentstudy,weexaminedtheeffectoftheTRPM8agonistmenthol, theTRPV1agonistcapsaicin,andtheTRPA1agonistallylisothiocyanate(AITC)oncold-evokedresponses inthesecornealprimaryafferentneurons.Extracellular,single-unitrecordingswereperformedinure-thane-chloraloseanesthetizedrats.Electrodespositionedinthetrigem-inalganglionwereusedto isolateandcharacterizecold-sensitivecornealneurons.Atlowconcentrations,Mentholincreasedthespon-taneousandenhancedthecold-evokedresponse.Onthecontrary,ahighconcentrationofmentholsuppressedthecoldcellactivity.Cap-saicinandAITCincreasedthespontaneousactivityandsuppressedthecold-evokedresponse.TheseresultsindicatedthatmostofthecoldcellhasnotonlyTRPM8channelbutalsoTRPV1andTRPA1channel.Menthol,thoughactionsatTRPM8,sensitizesownTRPM8channels.Capsaicin, thoughactionsatTRPV1,andAITC,thoughactionsatTRPA1maysuppressTRPM8channeldirectlyorbyotherintracel-lularmechanisms.Understandingofthemechanismsofthesedesen-sitizationofTRPM8channelmightbeuseful forknowingDryeyesyndrome.NoCOI.

1P-127Tyrosine hydroxylase immunoreactive amacrine cells in the gerbil retinaImada, Hideki1; Sakai, Kazuyoshi2; Miyachi, Ei-ichi1(1Dept. Physiol. Sch. Med. Fujita Hlth. Univ., Toyoake, Aichi, Japan, 2Dept. Anat. Sch. Hlth. Sci. Fujita Hlth. Univ., Toyoake, Aichi, Japan)

WehavefoundtwokindsofTH-IRamacrinecells(typeAandtypeB).ThetwotypeswereclearlydifferentintheshapeofsomataandstratificationofTH-IRdendritesintheinnerplexiformlayer(IPL)atP7.Type-ATH-IRcellshadmonostratifieddendritesextendingintheoutermostlayeroftheIPL,whiletype-BcellshaddendritesextendinginthemiddleofIPL.AtP7,type-Asomatawereobservedintheinnerpartoftheinnernuclearlayer(INL),itsdendritesextendedintotheouterpartoftheIPL.Type-BsomatawerelocatedintheinnerpartofthenewlyformedINL.ItsdendritewasobservedtospreadinthemiddleoftheIPL.AtP28,type-Asomatabecamesphericalandsig-nificantlylarger,andmorethicklystained,locatedinthevicinityofoutermostlayeroftheIPL.ThedendritesoftypeBextendedinthemiddleoftheIPL,wherethepoint-likelayerwasobserved.InadulttypeA,denselystained,round-shapedandlargesomatawerelocatedintheinnermostpartoftheINL,andtheirdendriteswithvaricositiesextendedintotheoutermostpartoftheIPL.Adulttype-Bsomata,ontheotherhand,werestainedweakly,withtheirthindendritespene-tratingtheouterpartoftheIPL.Theseresultssuggestthattwokindsofdopaminergicamacrinecellshavedifferentdevelopmentalpropertiesinthedevelopinggerbilretina.EyeopeningisanimportantperiodforthematurationofdopaminergicamacrinecellsandforthematurationoftheIPL.NoCOI.

1P-128Whisker-guided visual map shifts and formation of oc-ular dominance column-like structures in miceYoshitake, Kohei1,3; Tsukano, Hiroaki1; Tohmi, Manavu1; Hishida, Ryuichi1; Yagi, Takeshi2,3; Shibuki, Katsuei1,3(1Dept Neurophysiol, Brain Res Inst, Niigata Univ, 2 KOKORO-Biology Group, Grad. Sch. of Frontier Biosci, Osaka Univ, 3JST, CREST)

Micenavigatenearbyspaceusingtheirvisionandwhiskers.Therefore,youngmicemustlearntointegratetheseheterogeneousinputs,al-thoughthemechanismsareunknown.Wehavepreviouslyreportedthatcorticaldepressionwasinducedbyspatialmisalignmentbetweenvisualandwhiskerinputsintheprimaryvisualcortex(V1)ofyoungmicethathadwornamonocularprismgoggle.CorticaldepressioninV1alonedonoteliminatethevisuotactilespatialmisalignment.How-ever,partialdepressionofthevisualresponseswithspatialeccentric-ityandtheresultingmapshiftsmayalleviatethespatialmisalignment.Totestthispossibility,weinvestigatedthelocationofvisualrespons-eselicitedbyLEDstimuliplacedbetween0°and100°at20°intervalsincontrolmiceandmicethathadworntheprismgoggle.WefoundthatuniformmedialshiftsofcorticalresponsesinV1ofmicethathadworntheprismgoggle.Sofar,oculardominancecolumnhasnotbeenfoundinV1ofmice.However,theuniformmedialshiftsofvisualre-sponsessuggestsapossibilitythatoculardominancecolumn-likestruc-turescouldbeformedinthebinocularregionofV1afterprismwear-ing.Asexpected,thevisualresponseselicitedbyLEDstimuliplacedat0°viaeacheyewereclearlyseparated.Theseresultsindicatethatvisuotactilemisalignmentbetweenwhiskerandvisual inputscouldinducevisualmapshiftsandformationofocular-dominance-likestruc-turesinmice.NoCOI.


1P-129Pupillary reaction and eye movement during looking Benham's illusion figures with rotation or movementYanagida, Yurie1; Kobayashi, Takakazu2(1Graduate school of Engineering, Shibaura Institute of Technology and Science, Tokyo, Japan, 2Department of Electronic Engineering, Shibaura Institute of Technology, Tokyo, Japan)

Recentyears,studyofillusionhasbeenmainlycarriedoutagaininthefieldofpsychology.Insomeofopticalillusion,thereisfigureiscalledBenham’sdisk. Inspiteofdrawnbyblackandwhite inthefigure,wefeelthecolorat3–6round/sofrotationspeed.However,there is littleresearchonpupilreactionandeyemovementaboutBenham’sdisk.Therefore,weexaminedaboutthesewhenwearelookingatrotatingfigure.InadditiontorotatingofBenham’sdisk,weusedarectangularfiguredrawnsameBenham’sdiskpatternbutmoveshorizontally.Theheadwasfixedbythefixationdeviceofforeheadandchin.EyewastakenwithaninfraredCCDcamera,theeyemovementofthecenterofgravityandpupilareaweremeasuredandanalyzed. Asaresult, theareaofthepupilchangedwhethercolorisvisibleornot.Pupilareawaslargerintherotationspeedofcolorscanbeseencomparedtofastrotationspeedat8round/s.TheseoccurredregardlessofthedirectionofrotationandmovementspeedinBenham’sfigures.Further,itwasfoundthatinthemovementofrectangularfigureat1round/stherepeatedsmoothpursuitmovementandsaccadewereobserved.Thesedecreasedinthemovementbegintoseethecolor.Itisconsideredthatthesearecausedbecausecolorfigureisrecognizedasastillimage.Therefore,itisconcludedthatcolorofBenham’sillusionfigureisrecognizedasastillimageandinthecaseofrotationandmovementspeedatnottoofast.NoCOI.

1P-130Cooperative coding of unstationary images by multi-ple subtypes of retinal ganglion cellsTachibana, Masao; Matsumoto, Akihiro(Department of Psychology, Graduate School of Humanities and Sociology, The University of Tokyo, Tokyo, Japan)

Therearemanysubtypesofganglioncellsintheretina,anditisas-sumedthateachsubtypemaydetectaspecificvisualfeatureoftheretinalimage.However,itisnotclearwhethereachsubtypeprocess-esvisual information independentlyorcooperatively.Applyingthemulti-electrodearraytotheisolatedgoldfishretina,werecordedspikedischargesfrommultipleganglioncells.Dynamiclightstimuliwereprojectedontotheretinatomimictheretinal imagemotion.Thestimuluswascomposedofabrightsquaretargetandasurroundinglargebackgroundwithorwithoutrandomdots.Thetargetandthebackgroundwasjitteredorrapidlymovedseparatelyortogether.Basedonthereceptivefieldprofileestimatedbythespike-triggeredaverage,weclassifiedganglioncells intosixsubtypes (Fast/Slow,transient/medium/sustained).WefoundthattheFast-transientcellsfiredsynchronouslyandanticipatorywhenthetargetrapidlymovedtogetherwiththebackgroundwithrandomdots.Underthisstimuluscondition,cross-correlationanalysisofspiketrainsrevealedthatfunc-tionalconnectivitywasestablishedamongtheFast-transientcellsandotherspecificsubtypes.Thefunctionalconnectivitywasnotfixedbutchangeddependingonthelightstimuluspattern.Theseresultssuggestthatmultiplesubtypesofganglioncellsmaysendvisualinformationcooperativelytothebrain,andthattheFast-transientcellsmayplayakeyroleamongthesubtypes.NoCOI.

Poster PresentationsBehavior Science, Biorhythm

1P-131Age-dependent relationships between heart rate vari-ability and body acceleration in free-moving humanTaniguchi, Kentaro1,2; Shimouchi, Akito2; Seki, Junji3; Mizukami, Tomoe1,2; Jinno, Naoya2; Shirai, Mikiyasu2; Seiyama, Akitoshi1(1Human Health Sciences, Graduate School of Medicine, Kyoto University, Kyoto City, Kyoto, Japan, 2Department of Cardiac Physiology, National Cerebral and Cardiovascular Research Center, Suita, Osaka, 3Organization for Research and Development of Innovative Science and Technology, Kansai University, Osaka)

Theaimofthisstudy istoclarifyeffectsofagingonrelationshipsbetweenheartratevariability (HRV)andbodyaccelerationofthefree-movinghuman.R-Rintervalsandbodyaccelerationwererecord-edeveryoneminutesfrom65adults, including18young(20to39years),26middle(40to59years),and21elderlysubjects(60yearsormore)for24hoursduringtheirfree-movingdays.ForHRVanalysis,high-frequency(HF;0.15–0.4Hz)andlow-frequency(LF;0.04–0.15Hz)componentsofHRVwerecalculatedbyusingMemCalc,atimeseriesanalysistechniquethatcombinesanon-linear leastsquaremethodwithmaximumentropymethod.Thelagbetweentime-seriesofHRVandphysicalaccelerationwasdeterminedbythecross-correlationanalysis.Forthreetofourhoursbeforenightsleepandafterwake-up,thepercentratiosofthesubjectswithzerolagbetweenphysicalac-celerationandHRV(bothLF/HFandHF/TF)weresignificantlyde-creasedinanage-dependentmanner.Afterwake-up,theratiosofthesubjectswithzerolagtendedtobeloweredinallgenerations.Theseresultssuggestedthatthecoordinationbetweenphysicalactivityandautonomicnervoussystemwasdiminishedwiththeaging.NoCOI.


1P-132Circadian desynchronization disrupts heart mitochon-drial metabolism in miceHashimoto, Izumi; Kohsaka, Akira; Das, Partha; Nakao, Tomomi; Gouraud, Sabine S; Waki, Hidefumi; Maeda, Masanobu(Dept Physiol, Wakayama Med Univ, Wakayama, Japan)

Continuousshiftingofthesleep-wakecycle,oftenobservedinshiftwork,isassociatedwithincreasedcardiovascularmorbidityandmor-tality.However,molecularmechanismsunderlyingthedetrimentaleffectsofshiftinginthedailyphysiologicalrhythmoncardiacfunctionremainunknown.Toelucidateamolecularlinkbetweenthecircadianclockandcardiac function,weusedmicewhichdevelopedcardiachypertrophybyphenylephrineinfusion,andmaintainedtheseanimalsona12-hphaseshiftinthelight-dark(LD)cyclefor18days.WefoundthatchronicexposuretotheLDshiftprotocoldecreasedcardiacfunctioninmice.Inaddition,thereducedcardiacfunctionledbyLDshiftingwasaccompaniedbyalterationincardiacmitochondrialme-tabolism.Diurnalrhythmsofexpressionofgenesencodingkeycom-ponentsofmitochondrialoxidativemetabolismwereabolishedinmicemaintainedontheLDshiftprotocol. More importantly,disruptingphysiologicalrhythmicityinducedbyLDshiftingresultedinasignif-icantdecrease inenzymaticactivityofmitochondrialcomplexI inheart.Together,ourresultssuggestthatchronicdesynchronizationofthedailyphysiologicalrhythmwithexternalLDcyclesmayhaveadverseeffectsonheartfunctionthroughalterationinmitochondrialenergymetabolism.NoCOI.

1P-133Changes in the state of wakefulness in the locus coe-ruleus noradrenergic neurons-ablated miceTakahashi, Kazumi1; Konno, Kohei2; Kawamura, Naoki2; Itoi, Keiichi3; Kobayashi, Kazuto4; Eifuku, Satoshi1; Koyama, Yoshimasa2(1Dept Systems Neurosci., Fukushima Med. Univ., Fukushima, Japan, 2Dept. Sci. Technol., Fukushima Univ., Fukushima, Japan, 3Grad. Schl. Inf. Sci., Tohoku Univ., Sendai, Japan, 4Inst. Biomedical Sci., Fukushima Med. Univ., Fukushima, Japan)

Thenoradrenergic(NA)neuronsinthelocuscoeruleus(LC)arecrucialcomponentsinthesleep-wakingmechanisms.Toexploretherolesoftheseneurons,wespecificallyablatedthemwithimmunotoxin(IT)-me-diatedcelltargetinginmice,andassessedsleep-wakingbehaviorsandarousalresponsesinducedbyauditorystimuli.Thecorticalelectroen-cephalogram(EEG)andtheneckelectromyogram(EMG)werecontin-uouslyrecordedfromoneweekbeforetotwoweeksaftertheITin-jectioninthefreely-movingcondition.Duringboththelightperiodsanddarkperiods,therewerenodifferencesinthetotalamountsofwakefulness(W),lightslow-wavesleep(SWS),deepSWSandparadox-icalsleepbetweentheNA-LCablated (Ab)miceandnormalmice.However,from5to8daysaftertheinjection,thedurationofeachboutofWdecreasedandthenumber(frequency)ofWincreasedintheAbmice.Inresponsetothesoundstimuli(45or55dB,200ms)appliedduringsleep,theoccurrenceprobabilityofEEGdesynchroni-zation,thesignofW,wasunchanged,whilethatoftransientEMGactivitywithshortlatencywasincreasedintheAbmice.TheseresultssuggestthattheNA-LCneuronshavearoleinmaintainingorstabi-lizingthestateofWandexertinhibitoryinfluencestotheauditoryreflexpathwayinthebrainstem.NoCOI.

1P-134Optical imaging of spatially isolated solitary neuron of suprachiasmatic nucleus using micloisland culture methodHirata, Yoshihiro1; Honma, Sato2; Honma, Ken-ichi2(1Photonic.Bioimag.Sect., Hokkaido Univ. Sch. Med., Sapporo, Japan, 2Dept. Chronomed., Hokkaido Univ. Grad. Sch. Med., Sapporo, Japan)

Amastercircadianclockinmammalsislocatedinthehypothalamicsuprachiasmaticnucleus (SCN)which iscomposedofmultiple,sin-gle-neuroncircadianoscillatorcells.Recentstudieshavesuggestedthattheseneuronsareheterogeneousinnotonlycytochemicalbutalsotheiroscillatoryproperties.Inthisstudy,weexaminedcircadianpropertiesofthesolitarySCNneurononsmallspotsusingdissociatedcultureofSCNneurons.Culturedishwerepreparedbyopeningaholeinthebottomofa35mmpetridishandattachingITO(IndiumTinOxide)coatedglassslide.AcollagenlayerwasformedontheITOsurface,andaboveit,agarosethinlayerwasmade.Collagenislandswereformedbyaninfraredlaserbeamwhichmeltedagarosethinlayer.Thismethodenablesustomakesmallspotsofanysizeatanydensity.SCNneuronswerederivedfromtransgenicmicecarryingabioluminescentreporterforPer1orPER2.BioluminescencefromeachsinglecellwasmeasuredbyanEMCCDcameraeveryhourforatleast5days.WesucceededtodemonstratethatspatiallydissociatedsolitarySCNneuronsexhibitcircadianrhythmsofPer1expressionorPER2.Wealsodetectedcircadianoscillationofclockgene/proteinofglialcellsonislandswith/withoutSCNneurons.TheseresultssuggestthateachofsingleSCNneuronsexhibitscell-autonomouscircadianoscillation.Further,therhythmicactivityofglialcellsmayaffectcir-cadianexpressionofclockgene/proteinineachSCNneurons,orviceversa.NoCOI.

1P-135High plating density is necessary for robust AVP re-leasing rhythm of SCN cells in cultureWatanabe, Kazuto(Department of Physiology, Dokkyo Medical University, Mibu, Japan)

Inmammals,circadianrhythmsaredrivenbyapacemakerlocatedinthesuprachiasmaticnucleus(SCN)ofthehypothalamus.RecordingsofneuronalfiringofdissociatedSCNcellssuggestthatsingleSCNcellsarecompetentcircadianoscillators. TheSCNcellsalsoshowclearcircadianrhythmofargininevasopressin(AVP)releaseincellculturewhentheyareplatedathighdensity.WeexaminedtheeffectofplatingdensityonAVPreleasingrhythminculture. Thesamenumbersofthecellswereplatedondifferentsizeofareainawellofcloningplates.Whencellswereplatedinlargearea,AVP-releasingrhythmwasattenuated.BoththeamountofAVPreleaseandampli-tudeoftherhythmdependedontheplatingdensity.Co-culturewithcortexcellscouldnotrestorethelossofrhythmicityinlow-densityculture.Theseresultssuggestthatcell-cellcontactofSCNcellsisnecessaryforrobustAVPreleasingrhythm.NoCOI.


1P-136Involvement of gap junction in kainic acid-induced neuronal oscillations in anterior cingulated cortexShinozaki, Rina1; Hashizume, Miki1; Mukai, Hideo2; Murakoshi, Takayuki1(1Department of Biochemistry, Faculty of Medicine, Saitama Medical University, Saitama, Japan, 2Department of Computer Science, School of Science and Technology, Meiji University)

Neuronaloscillationisaprominentformofrhythmicactivityoccurringinthebrain.Fastneuronaloscillations(30–100Hz)arefrequentlyob-servedinthethalamo-corticalstructureduringwakefulnessandat-tentivebehavior.Abnormalitiesintheseoscillationsintheanteriorcingulatedcortex(ACC),amedialpartoftheprefrontalcortex,mightunderlieneuropsychiatric illnessessuchasschizophrenia. Inthecurrentstudy,toinvestigatetheroleofgapjunctionsintheneuronaloscillationintheACC,wedevelopedanin vitromodelofneuronaloscillationinaslicepreparationincludingACCfrommice,andexam-inedifthegap-junctionalcommunicationisinvolvedinthegenerationmechanismsoftheoscillation. Weevokedoscillationbyperfusing50µMkainicacid(KA)for10seconds.Oscillationactivitywasevalu-atedbypower-spectraldensityanalysis. Gapjunctionantagonist(150µM18β-glycyrrhetinicacid,100µMcarbenoxolone,100µMoctanol)wasadministeredbyperfusionfor30minbeforeKAactivation.Thesegapjunctionantagonistsmarkedlydecreasedoscillationpowerevokedbykainicacid.TheseresultssuggestthatgapjunctionscontributestogenerationoftheneuronaloscillationintheACC.NoCOI.

1P-137Neuronal activity of the insular cortex modulates ap-proach behavior to food in miceKusumoto-Yoshida, Ikue1,2,3, Liu, Haixin3; Chen, T. Billy2; Fontanini, Alfredo3; Bonci, Antonello2(1Dep. Physiol., Grad. Sch. Med.Dent. Sci, Kagoshima University, Kagoshima, Japan, 2National Institute on Drug Abuse IRP. Baltimore, MD USA, 3Stony Brook University, Stony Brook, NY USA)

Theinsularcortexisknownassensorycortexthatintegratesmultiplemodalitiessuchasvisceral,gustatoryandolfactoryinputsandplaysanimportantroleforrepresentingbodilystate.Ithasbeenreportedthatneuronsintheinsularcortexrespondtoreward-predictivecues,howeveritremainsunknownhowtheanticipatoryactivityshapestheanimalsreward-motivatedbehaviors.Here,weinvestigatetheinvolve-mentof insula intherewardapproachbehavior inmice.UsingaclassicPavlovianparadigm,adultmiceweretrainedtoformanasso-ciationbetweenthesensorycuesandsubsequentdeliveryofahighlypalatablefoodpellet.Localpharmacologicalinactivationoftheinsulasignificantlyreducedreward-approachbehavior,suggestingamodu-latoryfunctionoftheinsula.Singleunitrecordingfrombehavingmiceshowedsignificantchangesoffiringactivityduringcue-periodinabout30%ofrecordedneuronsinsameareaofinsula.Todetermineifinsu-laractivityisspecificallyimportantduringthecue-presentationperiodpredictingthesubsequentrewarddelivery,weemployedoptogenetictechniques.Usinghalorhodopsin,weinhibitedinsularactivityselec-tivelyduringthecueperiod. Insula inhibitionsignificantlyreducedapproachbehaviorduringthecueperiod.Thesedatasuggestthatactivityoftheinsularcortexduringthereward-predictivecuecon-tributesandmodulatestheexpressionofreward-approachbehavior.NoCOI.

1P-138Conditional knockout of the orexin 2 receptor gene specifically in GABAergic neuron reduce wakefulness Soya, Shingo; Sakurai, Takeshi(Department of molecular neuroscience and integrative physiology, Kanazawa Univ. Kanazawa, Japan)

Orexinneuronsprojectalmostallbrainregionswithespeciallydenseprojectionsbeingseen inmonoaminergicandcholinergicnuclei in-volved intheregulationofsleep/wakefulness.Thereareorexin1(OX1R)and2receptor(OX2R)expressedintheseneuron,andalsoGABAergicneuronsarelikelytofunctionaslocalinhibitoryinterneu-ronsanddistributedinmonoaminergicneuronandexcitedbyOX1RandOX2R.HoweverpreciseroleoforexinreceptormediatedGAB-Aergicneuronactivityinsleep/wakeregulationisunclear.Toaddressthis,wegeneratedconditionalOX2Rknockoutmiceespecially inGABAergicneuron,andanalyzesleep/wakefulnessstatesof thesemicemonitoredbysimultaneousEEG/EMGrecordings.Wefoundthatthesemiceshowedsignificantdecreaseofwakefulnesstime,accom-paniedbyincreasedNREMsleep.Also,thesemiceexhibitedsignificantdecrease inepisodedurationofwakefulness indarkperiod.TheseresultssuggestthatGABAergicneuronmediatedbyOX2Rplayanimportantroleinmaintainingwakefulnessindarkperiod.NoCOI.

1P-139Comparison of firing properties of sleep-related neu-rons in the mesopontine tegmentum and amygdalaKoyama, Yoshimasa; Nishimura, Kunihiro; Haruyama, Naoto; Aoyagi, Toshifumi; Matsuda, Shohei; Ito, Akira(Department of Science and Technology, Fukushima University)

Themasopontinetegmentalareahasacrucialrole inregulationofREMsleep.Alargepopulationofneuronsinthisareaincludinglat-erodorsaltegmentalnucleus(LDT)dischargespecificallyduringREMsleep(PSactiveneurons)ordischargehighlybothduringREMsleepandwaking.Ontheotherhand,theamygdala,acenterofemotionduringwaking,alsocontainsconsiderablenumberofPSactiveneurons.Althoughthesetwoareascontainsimilartypeofneurons(PSactiveneurons),functionalsignificanceofthePSactiveneuronsinthesetwoareasinregulationofREMsleephasneverbeencompared.Inthepresentstudy,singleneuronalactivityundersleepwakingcycleswasrecordedfromthemesopontinetegmentalareaandtheamygdalainhead-restrained,non-anesthetizedratsandfiringpropertiesof theneuronsinthesetwoareaswerecompared.Inthemesopontineteg-mentalarea,someofthePSactiveneuronsstartedtoincreasefiringbeforetheonsetofREMsleepandcontinuedtofireintonicfashionduringREMsleep,whilethePSactiveneuronsintheamygdalashowedphasicdischarge intermittentlyduringREMsleep,and increase infiringstartedaftertheonsetofREMsleep.Someneuronsinthemes-opontinetegmentalareahaveacorrelationwitheyemovementduringREMsleep,whilenoneoftheamygdalaneuronshavesuchcorrelation.BothintheLDTandamygdalasomeneuronshaveaclosecorrelationwithbloodpressurefluctuationduringREMsleep.RolesofthesetwoareasinrelationofREMsleepwouldbediscussed.NoCOI.


1P-140Influences of stress on sleep profiles and blood pres-sure fluctuation during REM sleepHaruyama, Naoto; Oonami, Hiroki; Nishimura, Kunihiro; Koyama, Yoshimasa(Dept Sci Technol, Fukushima Univ, Fukushima, Japan)

Thestressduringwakinghassomeinfluencesonsleep.DuringREMsleep,fluctuationofautonomicnervoussystemincludingthatofbloodpressure,heartrateorrespirationoccursanditishighlyprobablethatsucheventshavesomerelationswiththestressduringwaking.Inthepresentexperiment,toelucidatetheinfluenceofstressonsleepprofilesweexaminedtheeffectofstressonsleep-wakecyclesandonbloodpressurefluctuationduringREMsleep.Maleratsinashockchamberweregiven,everyoneminutefrom16:00to17:30,10secondstoneof10KHz(15times),whichwasassociatedwithelectricshock(0.7mA,1sec),andof500Hz(75times)withoutshock.Aftertheshock,ratsweremovedtotherecordingchambertorecordsleep-wakeprofilesandbloodpressurefrom20:00to8:00(darkperiod),thenfrom8:00to16:00(lightperiod).Duringdarkperiod,electricshockinduced72%increaseoftheamountofREMsleep(p<0.01),20%increaseoflightslowwavesleep (p<0.05),and7%decreaseofwakefulness (p<0.01)comparingwithcontrolanimals,whichweregivennoshockintheshockchamber.Duringlightperiod,amountofREMsleepincreasedto17%(p<0.05).Aftertheelectricshock,phasicdecreaseofbloodpressureoccurredmorefrequentlycomparingwithcontrolanimals(p<0.01),whileincreaseofbloodpressurehadnodifferencecomparingwithcontrolanimals.TheseresultssuggestthattheelectricshockstressassociatedwithsoundhasafacilitatoryroleonREMsleepandhassomeinfluencesonbloodpressurefluctuationduringREMsleep.NoCOI.

1P-141Relationship between sleep quality and menstrual cy-cles in healthy womenFujita, Sayaka(The University of Shimane, Izumo Campus)

Thepresentstudyaimedtoclarifytherelationshipbetweenmenstru-alcycleandsleepqualityinhealthywomen.Sleepqualityduringthemenstrualcyclesofsixwomen(agerange,19–28years)wasmeasuredforaboutonemonth.Basalbodytemperaturewasmeasuredwhilestillinbedinthemorning,andsleepwasassessedusingamat-typedevice.Basalbodytemperaturesduringtheovarianfollicular,thecorpuslu-teum,andthemensesphasesofthemenstrualcyclewere36.44±0.15°C,36.73±0.25°C,and36.53±0.19°C,respectively(p=0.00).Thetotalamountofsleeppernightwas300.48±109.17,302.83±74.70,and276.23±77.023min,duringthethreephases,respectively,andtheamountoftimespentinREMsleepwas62.62±33.20,59.30±14.27,and43.63±20.76min,respectively(p=0.05).Theamountoftimespentbeingawakeand inboth lightanddeepsleepdidnotsignificantlydifferamongthethreephases.However,theamountoftimespentinREMsleepsignificantlydiffered(p=0.04),indicatingthatqualityofsleepisaffectedbythemenstrualcycleinhealthywomen.NoCOI.

1P-142The role of ghrelin in entrainment of circadian rhythms to scheduled daily feeding in CS miceAbe, Hiroshi1,2; Ebihara, Shizufumi3; Okamoto, Tadao4(1Division of Psychology, Department of Morphological and Physiological Sciences, Faculty of Medical Sciences, University of Fukui, 2United Graduate School of Child Development, Osaka, Kanazawa, Hamamatsu, Chiba University and University of Fukui, 3Division of Biomodeling, Graduate School of Bioagricultural Sciences, Nagoya University, 4UCB Japan)

ThecircadianclockinCSmiceentrainstorestrictedfeedingschedule(RF).Behavioralrhythmandclockgenerhythmsinthesuprachias-maticnucleus(SCN,acentraloscillatorinthecircadianclocksystem)ofCSmiceshowclearentrainmenttoRF(foodentrainment).Toin-vestigatethemechanismsof foodentrainment inCSmice,presentstudywasdesignedtoclarifytheroleofghrelin,whichisapeptidecloselyrelatedtofeedingbehavior,inthefoodentrainmentinCSmice.Plasmaghrelinconcentrationwasmeasuredat6timepointswhilebehavioralrhythmwasentrainedtoRF.PlasmaghrelinconcentrationlevelsunderRFinCSmiceweresignificantlyincreasedpriortofeed-ingtime,whiletherewerenorhythmicchangesinadlibfeedingmice.Thechanges in freerunningbehavioralrhythmundercontinuousdarkness(DD)weremeasuredwhenghrelinwasperiodicallyadmin-istered.Periodicghrelinadministrationinducednochangesinfreerun-ningbehavioralrhythms.TheseresultsindicatethatghrelinisnotamajorcueforfoodentrainmentofcircadianclockintheCSmice.Thepresentresultssuggestthatghrelinmighthavearoleinthefood-en-trainableoscillatorwhichregulatesfoodanticipatoryactivityunderRF.NoCOI.

1P-143Effect of feeding patern on sleep depthOtsuka, Airi1,2; Shiuchi, Tetsuya1,2; Oura, Kanna1,3; Shimizu, Noriyuki1; Chikahisa, Sachiko1; Sei, Hiroyoshi1(1Department of Integrative Physiology, Institute of Health Biosciences, The University of Tokushima Graduate School, 2Department of Food Science, Institute of Nutrition and Bioscience, The University of Tokushima Graduate School, 3Student Lab, Faculty of Medicine, The University of Tokushima)

Weinvestigatedwhetherdifferentfeedingrhythmaffectssleep/wakeregulation.ThreegroupsofC57BL/6Jmiceweregiven labchowsfreelyduringdarkperiod(ZT12-24,Controlgroup),firsthalfofdarkperiod(ZT12-18;Morninggroup),orlasthalfofdarkperiod(ZT18-24,Eveninggroup)for2weeksrespectively.Off-linesleepscoringwasdoneonthecomputerscreenbyvisualassessmentoftheelectroench-phalogram(EEG)andEMGactivities,therebydistinguishingphasesofwake,rapideyemovement(REM)andnon-REM(NREM)sleep.TheEEGdeltaandthetafrequencybandwassetat0.5–4.0Hzand4.0–7.8Hz,respectively.Inthisstudy,powerdensityoftheEEGdelta(ratioofdeltatotheta)duringNREMsleepwasusedasaparameterofsleeppressure(slow-waveactivity,SWA).MiceinEveninggroupshowedlowerSWAthanthatofother2groups.Ontheotherhand,anamountofNREM,REMandwakein3groupsdidnotchange.Weobservedhighermonoamineconcentrations,whichactivateswakesystem, incerebralcortex inEveninggroup.Wealso found increasedmRNAexpressionoforexininhypothalamusinEveninggroup.Theseresultsindicatethatfeedingonlyinthelasthalfofdarkperiodalterssleephomeostasis.Thiseffectmaybepartlyinvolvedinincreaseoforexinexpressioninhypothalamusandelevatedmonoamineconcentrationincerebralcortex.NoCOI.


1P-144The effect of feeding condition and cold ambient tem-perature to synchronization on the rhythms of body temperature and sleep-wake patternSato, Nobuo1,2; Matsuda, Mayumi2; Marui, Shuri2; Ozaki, Makoto1; Nagashima, Kei2,3,4(1Dept. Anesthesiol, Tokyo Women's Med Univ, Tokyo, Japan, 2Body Temp. Fluid Lab., Fac. Human Sci., Waseda Univ, Japan, 3IABS, Waseda Univ., Japan, 4GCOE ActiveLife, Waseda Univ., Japan)

AimHomeothermicanimalshavesynchronizedcircadianrhythmsofbodytemperatureandsleep-wakepattern.Fastingandextremeam-bientconditionmodulatebodytemperatureandsleep-wakepattern;however,wedonotknowhowtherelationshipofthetworhythmsisaffected insuchconditions.Therefore, thepresentstudyaimedtoclarifytherelationshipduringfastingandcoldinmice.MethodsMaleICRmice(age,2–4m)housedat27°Cwithalightingcycleof12:12h(lights-on,7am)wereused.Usingaradiotransmitterdevice,bodytemperature(Tb),EEG,EMGandspontaneousactivitywasobtained.Micewereexposedto20°Cor27°Cfor30-hwithorwithoutfooddeprivation.ResultsTbdecreasedatfastingat27°Cby1.0±0.2°Cand0.9±0.2°Cinthelightanddarkphasescomparedwithfeedingat27°C,respec-tively.At20°Cexposure,thereductionsweredisappeared(0.1±0.1°Cand0.1±0.1°C);howeveraugmentedatfastingcondition(1.5±0.2°Cand2.2±0.2°C).Fastingconditionat27°C, totalsleeptimedidnotchange;however,bothfeedingandfastingat20°Ccoldcondition,totalsleeptimedecreasedinbothphases.Inallconditions,ultradianrhythmsofthebodytemperatureinthelightphasewerewellcorrelatedtowake-sleeppattern.ConclusionColdandfastingmodulatesbothrhythmsofsleepandTb,butdidnotaffectsynchronizedultradianrhythmsofTbandwake-sleep.NoCOI.

1P-1455HT1A receptors in orexin neurons play an important role in regulation of sleep/wake behaviorSaito, Yuki1; Etori, Keishi1; Tsujino, Natsuko3; Abe, Manabu2; Sakimura, Kenji2; Sakurai, Takeshi1(1Dept. Molecular Neuroscience and Integrative Physiology, Univ. of Kanazawa, Ishikawa, Japan, 2Dept. Cellular Neurobiol, Brain Research Institute, Univ. of NIigata, NIigata, Japan, 3International Institute for Integrative Sleep Medicine,Univ. of Tsukuba, Ibaraki, Japan)

OrexinAandorexinBare lateralhypothalamicneuropeptides.Aseriesofstudieshavesuggestedthatorexin-deficiencycausesnarco-lepsyinhumansandothermammalianspecies,highlightingrolesofthishypothalamicneuropeptideintheregulationofsleepandwake-fulness.Orexinswereshowntohaveastrongexcitatoryinfluenceonserotonergicneuronsintheraphenucleithroughbothorexin1andorexin2receptors.Conversely,orexinneuronsreceiveabundantinputfromtheserotonergicneuronsintheraphenuclei.Wealsofoundse-rotoninpotentlyinhibitedorexinneuronsthrough5HT1Areceptors,implyingthenegativefeedbackregulation.Thislinkagemightplayanimportantroleintheregulationofsleep/wakefulness.Toevaluatethishypothesis,wegeneratedmiceinwhichorexinneuronsspecificallylackexpressionof5HT1AreceptorsutilizingCre-loxPmediateddele-tionof5HT1Agene.Histologicalstudiesshowedspecificdisruptionof5HT1Areceptorexpressioninorexinneurons.Weexaminedsleep/wakecharacteristicsofthesemice,andfoundthatthesemiceexhib-itedseveralabnormalityinsleep/wakearchitecture,includingseverefragmentationofsleepstates.Thisobservationsuggeststhatseroto-nergicinhibitoryregulationoforexinneuronsplayanimportantroleinnormalmaintenanceofsleep/wakebehavior.NoCOI.

1P-146Serotonin is possibly involved in the anorexigenic and anti-depressant effects of estrogen in ratsNishimura, Yuri; Mabuchi, Kaori; Morimoto, Keiko; Takamata, Akira(Department of Environmental Health, Nara Women's University, Nara, Japan)

Estrogenattenuatesfoodintakeandenhancesc-Fosexpressioninthesuprachiasmaticnucleus(SCN)specificallyduringthelightphaseinovariectomizedrats.Inadditiontotheanorexigeniceffect,estrogenreportedlyhasantidepressanteffects.Serotoninisoneoftheagentsregulatingcircadianrhythmrespondingtolightandcontrollingfoodintake,andserotonindeficiencyisapathogenesisofdepression.Totestthehypothesisthatserotoninmediatesanorexigenicandantide-pressanteffectsofestrogen,weexaminedtheeffectofselectivesero-toninreuptakeinhibitor,fluoxetine(FLX)administrationonfoodintake,depression-likebehaviorandc-FosexpressionintheSCN.Ratswereovariectomizedandimplantedeitherwithestradiol(E2)orcholesterol(Veh)containingsilicontubingandaftersurgicalrecovery,weread-ministratedwithFLXorsalineatthebeginningofthelightphaseandthedarkphasefor10days.FLXattenuatedfoodintakespecificallyduringthelightphaseinthebothgroups,andreduceddepression-likebehaviorsinforcedswimtestintheVeh-group(on7thdayofadmin-istrationofFLX),andincreasedc-FoslikeimmunoreactivecellsintheSCNspecificallyduringthelightphaseonlyintheVeh-group.Thesesuggestthatserotoninisacandidatemediatingtheanorexigenicandantidepressanteffectsofestrogeninaphotostimulationdependentmanner.NoCOI.

1P-147Prolonged Bioluminescence Monitoring in Mouse ex vivo Bone Culture Revealed Persistent Circadian Rhythms in Articular and Epiphyseal CartilagesMinami, Yoichi1,2; Okubo, Naoki1,2,3; Kubo, Toshikazu2,3; Yagita, Kazuhiro1,2(1Department of Physiology and Systems Bioscience, Kyoto Prefectural University of Medicine, Kyoto, Japan, 2Department of Musculoskeletal Chronobiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan, 3Department of Orthopaedics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan)

Theboneisametabolicallyactiveorganwhichundergoesrepeatedremodelingcyclesofboneresorptionandformation.Inthisstudy,werevealedarobustandextremelylong-lastingcircadianrhythminexvivoculturemaintainedforoverninemonthsfromthefemoralboneofaPERIOD2Luciferasemouse.Furthermore,wealso identifiedrobustcircadianclocks inflatbones.High-or low-magnificationreal-timebioluminescencemicroscopicimagingrevealedthattherobustcirca-dianrhythmsemanatedfromthearticularcartilageandtheepiphy-sealcartilagewithinthegrowthplateofjuvenileanimals.Stimulationbyforskolinordexamethasonetreatmentcausedtype0phaseresetting,indicatingcanonicalentrainingpropertiesoftheboneclock.Together,ourfindingsfromlong-termex vivoculturerevealedthat“tissue-au-tonomous”circadianrhythminthearticularcartilageandthegrowthplateoffemoralbonefunctionsforseveralmonthseveninanorganculturecondition,andprovidedausefulinvitroassaysysteminvesti-gatingtheroleofthebiologicalclockinboneformationordevelopment.NoCOI.


1P-148Stability of whole-body rhythmic movement is en-hanced by self-vocalizationMiyata, Kohei; Kudo, Kazutoshi(Department of Life Science, Graduate School of Arts and Sciences, University of Tokyo, Tokyo, Japan)

Ithasbeenreportedthathumanrhythmicmovementarestabilizedbybeingentrainedtorhythmicauditorycue.Inpreviousresearch,rhythmicauditorycueshaveoftenbeengeneratedbyexternaldevicesuchasametronome.Ontheotherhand,wecangeneraterhythmicauditorycuebyself-vocalization.Usingself-vocalization insteadofexternalauditorycue,inprinciple,avarietyofrhythmicmovementcanbestabilizedbybeingentrainedtoself-voice.Therefore,weinves-tigatedwhetherentrainmentoccursbetweenself-vocalizationandwhole-bodyrhythmicmovement,andthestabilityofwhole-bodymove-ment.Thevoiceraterangedfrom80to180beatsperminutes(bpm)instepsof50bpm,and inonetrialonevoiceratewasproduced.Whole-bodyrhythmicmovementsweretwokindsofkneebendingmovement:knee-flexion-on-the-voiceandknee-extension-on-the-voice.Athighervoicerate,entrainmentbetweenknee-flexionandvocaliza-tionwasobserved.Thevariabilityofkneemovementintervalunderthevocalizationcoordinationwaslessthanunderthenon-vocalization.Werevealedtheentrainmentbetweenvocalizationandwhole-bodymovement,andthestabilityofwhole-bodymovementcausedbyself-vocalization.Thesefindingsindicatethatauditory-motorentrain-mentoccursregardlessof thesourcesofauditory information (i.e.,externalorinternal),suggestingthattheneuralpathwayunderlyingtheentrainmentbetweenrhythmicmovementandexternalauditorycuecanalsobeactivatedbyself-vocalization.NoCOI.

1P-149Acetaldehyde induces thirst sensation in hangoverUjihara, Izumi1,2; Hitomi, Suzuro1; Ono, Kentaro1; Kakinoki, Yasuaki2; Inenaga, Kiyotoshi1(1Div. Physiol., Kyyushu Dental Univ., Kitakyushu, Fukuoka, Japan, 2Div. Physiol., Kyushu Dental Univ., Kitakyushu, Fukuoka, Japan)

Inhangover,peopleexperienceheavythirst.ThemetaboliteofEtOHacetaldehyde(ACD)hasneverbeenconsideredtobeathirstinducingfactorinhangover.RecentstudiesshowthatACDactivatesmastcellsandelicitsreninrelease.WehypothesizedthatACDisafactorinduc-ingthirstsensationinhangover.MaleWistarratswereusedinthepresentstudy.EtOHsignificantlyincreasedwaterintake.Coadminis-trationoftheACDdehydrogenase inhibitorcyanamdewithEtOHincreasedbothwaterandsaltintakefurtherandearlier.ACDwithcyanamidemorerapidlyelicitedwaterandsaltintake.UrinationwaslessfoundintheearlystageevenintheadministrationofACDandcyanamide.Whenallowedtodrinkwaterandsaltsolution,urinevolumewasincreasedonlyafterdrinking,suggestingthaturinationisnotamaintriggerforinitiationofdrinkingbehavior.Theelicitedwaterandsaltintakeweresuppressedbyintraperitonealandintracerebroven-tricularinjectionsofAT1antagonistcandesartan.Thedrinkingbe-haviorwasalsosuppressedbythemastcellmembrane inhibitorscromolynanddoxantrazole.ImmunohistochemicalstudyshowedthatEtOH(andACD) increasedthenumberofc-Fos immunopositiveneuronsinthebrainregionsofthirstcenter.Takentogether,thirstsensationinhangoverofalcoholmaybeinducedthroughreninsecre-tionfrommastcellsandAT1receptoractivationofneuronsinthethirstcenterofthebraininrats.NoCOI.

1P-150Orexin receptors are involved in establishing cued and contextual fear memory.Kojima, Naoki; Soya, Shingo; Sakurai, Takeshi(Dept. Molecular Neuroscience and Integrative Physiology, Univ. of Kanazawa, Ishikawa, Japan)

Orexinsareneuropeptideswhichplayimportantrolesinregulatingsleep/wakefulness,energyhomeostasis,feedingbehavior,andrewardsystem.Toknowwhetherorexinreceptor1(OX1R)and2(OX2R)areimplicatedinestablishingcuedandcontextualfearmemory,weex-aminedphenotypesoforexinreceptordoubleknockoutmice(OXRD-KO),andprepro-orexinknockoutmice(pOXKO)usingcuedandcon-textualfearconditioningtest.WefoundthatOXRDKOandpOXKOmiceshowedmuchlowerfreezingtimethanthatofOX1RandOX2Rsingleknockoutmiceinbothconditioningandtestperiodofcuedandcontextual fear.Thesedatasuggestthatcuedandcontextual fearmemoryrequireOX1RandOX2Rfunction.Tofurtherdecipherthemechanismsbywhichorexinreceptorscontributetothefearmemoryconsolidation,weexaminedfreezingbehaviorofC57BL6Jmiceincuedandcontextual fearconditioningwithpharmacologicalblockadeoforexinreceptors.Weinjectedsuvorexant1hourbeforethetestperiodofcuedandcontextual fearconditioning.AcuteblockadeoforexinreceptorsusingsuvorexantreducedfreezingbehaviorintestperiodofcuedandcontextualfearconditioningtolevelscomparabletothatofOXRDKOandpOXKO.Theseobservationssuggestthatorexinsignalingplayanimportantroleincuedandcontextualfearmemoryconsolidationafterthetrainingperiod.NoCOI.

1P-151Circadian rhythms and the effect of glucocorticoids on clock gene Period2 expression in mouse nasal mu-cosaHonma, Aya1,2; Nakamaru, Yuji1; Fukuda, Satoshi1; Honma, Ken-ichi2; Honma, Sato2(1Department of Otolaryngology-Head and Neck Surgery, Hokkaido University Graduate School of Medicine, Sapporo, Japan, 2Department of Chronomedicine, Hokkaido University Graduate School of Medicine, Sapporo, Japan)

Thesymptomsofallergicrhinitis(AR)showmarkedcircadianchang-es,becomingworseintheearlymorning.Yet,noinformationissofaravailableastotheclockmechanismsinthenasalmucosa.IntranasalapplicationofglucocorticoidsiswidelyusedastopicaltreatmentofARwithoutknowingtheeffectsonthenasalclock.Inthepresentexperiment,weinvestigatedtheclockfunctionsinthemousenasalmucosaandtheeffectsofDexamethasone(DEX).WeculturednasalmucosaofadultmalePER2::LUCknock-inmiceandmeasuredbioluminescence levelsmorethan2weeks. Inaddition,100nMDEXwasappliedfor2hoursonthe5thdayofcultureat4differentcircadianphases.MousenasalmucosaexhibitedarobustcircadianrhythminPER2levelswiththepeak intheearlysubjectivenight.DEXphase-de-pendentlyphaseshiftedcircadianrhythmsofnasalmucosa,withthemaximalphasedelayatsubjectiveduskandthemaximaladvancesatmiddleofsubjectivenight.Ourresultsdemonstratedtheautonomiccircadianrhythmsinthenasalmucosaforthefirsttime.Sincethephase-shiftsduesolelytoDEXwereobservedatthephasewhentheserumglucocorticoidslevelisrelativelyhigh,theresultssuggestthatendogenousglucocorticoidscontroltheperipheraloscillatorofmousenasalmucosa.NoCOI.


1P-152Effect of mental task on taste perception and prefer-ence, and its relationship with daily eating behavior in young females.Someya, Nami1; Kudo, Fumiyo1; Kashima, Hideaki2; Fukuba, Yoshiyuki2(1Chiba Prefectural University of Health Sciences, Chiba, Japan, 2Prefectural University of Hiroshima, Hiroshima, Japan)

Itwasreportedthatrestrainedeaterswhointentionallyrestrictfoodintaketomaintainorloseweightconsumedmoreenergyunderstress-fulconditions.Wetestedthehypothesisthattasteperceptionandpreferencewereaffectedbybothmentaltaskanddailyeatingbehav-ior.Twenty-sevenfemales(18–22yr,BMI20.3±0.3)participatedinthisstudy.IndividualeatingbehaviorwasassessedbytheJapaneseversionof theDutchEatingBehaviorQuestionnaire (DEBQ).ThesubjectsperformedStroopcolor-wordtest(CWT)asamentaltask.BeforeandaftertheCWT,sweettasterecognitionthresholdwasmeasuredusingdifferentconcentrationofsucrosesolutions(0.2–100mM),andperceivedintensityofsweettasteofandpreferencefor200mMsucrosesolution,perceivedstressandappetitewereevaluatedusingvisualanalogscale.Thesemeasurementswerealsoperformedbeforeandafterrestingascontroltrial.TheCWTincreasedperceivedstressandappetite.Sweettasterecognitionthresholdwasdecreasedandperceivedintensityofsweettastewasincreasedaftertheresting,butthesewerenotchangedintheCWTtrial.Sweettastepreferencedidnotchange inbothtrials. Individualchange inthesweettastepreferencefrombeforetoaftertheCWTwasnegativelycorrelatedwiththescoreoftherestrainteatingscaleoftheDEBQ.Theseresultssuggestedthatsweettasteperceptionandpreferencecouldbemod-ulatedbybothmentaltaskanddailyeatingbehavior.NoCOI.

1P-153Genetic differences in glutamate appetite in mice de-pend on vagus-mediated postingestive effects of glu-tamateKitamura, Akihiko1,2; Inoue, Masashi1,3, Theodore M Nelson1; Maria L Theodorides1; Alexander A Bachmanov1(1Monell Chemical Senses Center, Philadelphia, PA, USA, 2Institute for Innovation, Ajinomoto Co., Inc., Kawasaki, Japan, 3 Lab of Cellular Neurobiol, Sch of Life Sci, Tokyo Univ of Pharm and Life Sci, Hachioji, Japan)

L-glutamate(Glu)iswidelyusedasaflavorsupplement(asmonosodi-umGlu,MSG).Gluelicitsumamitastesensation,and ingestedGluevokesmultiplephysiologicalresponses.Therefore,ingestivebehaviortowardsGlucanbeinfluencedbybothitssensoryandpostingestiveeffects.ThepurposeofthisstudywastounderstandthemechanismofGluappetite.Inoursurveyof28inbredmousestrains,wefoundthatmicefromtheC57BL/6ByJand129P3/Jstrainshadlargediffer-encesinvoluntaryMSGconsumption.TodevelopabettermodelforgeneticandphysiologicalstudiesofGluappetite,wehadintercrossedthesestrainsandthenusedselectivebreedingtoproducemousestrainswithhighand lowMSGintake (MSG-HandMSG-L,respectively).After14generationsofselectivebreeding,MSG-Hmicedrink6timesmore300mMMSGthanMSG-Lmice.WeusedMSG-HandMSG-Lmicetoexamine1)neuraltasteresponsestoaminoacidsandothertastants,2)behavioralresponsestoMSGinbrief-accesstestsofnaïveandMSG-exposedmice,3)bloodglucoseafterMSGgavage,4)effectofvagotomyonMSGconsumptioninpreferencetests,and5)flavorpreferencesconditionedbyoralMSG.Wefoundthatstraindifferenc-esinMSGintakemostlikelydependonpostingestiveeffectsmediatedbythevagusnerve.ThesedatasupporttheroleofthevagusafferentnervepathwayinGluappetite.NoCOI.

1P-154Calming responses during maternal carrying in human infants and mouse pups: comparative and ontogenic analysesYoshida, Sachine1,2; Esposito, Gianluca2; Kuroda, Kumi O2(1Laboratory of Neuroendocrinology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan, 2Unit for Affiliative Social Behavior, RIKEN Brain Science Institute, Wako, Japan)

Mother-infantbondingistheearliestandmostcriticalsocialrelationshipofmammalianinfants.Topromotethisbond,infantshaveinnatebe-haviorstoseekmaternalproximity.However,thephysiologicalmech-anismsunderlyingtheseinfantbehaviorsremainlargelyundefined.Hereweshowanovelsetof infantcooperativeresponsesduringmaternalcarrying.Infantscarriedbyawalkingmotherimmediatelystoppedvoluntarymovementandcryingandexhibitedarapidheartratedecrease,comparedwiththoseheldbyasittingmother.Further-more,we identifiedsimilarresponses inmousepupsasdefinedbyimmobilityanddiminishedultrasonicvocalizationsandheartrate.Usingmousepups,wefoundtheupstreamanddownstreamneuralsystemsregulatingthecalmingresponse.Somatosensoryandproprio-ceptiveinputsignalingarerequiredforinduction,andparasympathet-icandcerebellarfunctionsmediatecardiacandmotoroutput,respec-tively.Ontogenicanalysesrevealedthatthecalmingresponseoccurredwithinaspecificpostnataltimewindow.Inaddition,pupsshowedanincreasedpaintoleranceduringthecalmingresponse.Thelossofthecalmingresponsehinderedmaternalrescueofpups,suggestingafunctionalsignificanceforthisresponse.Thesefindingscollectivelyindicatethattheinfantcalmingresponseisacoordinatedsetofcentral,motor,andcardiacregulationsandisaconservedcomponentofmam-malianmother-infantinteractions.NoCOI.

1P-155CIN85 regulates child-rearing behaviors through do-pamine-prolactin signaling in the brainShimokawa, Noriaki; Sairenji, Taku; Takanashi, Yurie; Masuda, Shinosuke; Koibuchi, Noriyuki(Department of Integrative Physiology, Gunma University Graduate School of Medicine, Gunma, Japan)

Cbl-interactingproteinof85kDa(CIN85)isascaffold/multi-adaptorproteinimplicatedintheregulationofreceptorendocytosisandthecellularcytoskeleton.Recently,wereportedthatmicedeficientofCIN85expressionshowhyperactivephenotypes.Asamolecularex-planationofthisphenotype,theabsenceofstriatalCIN85causesde-creaseddopaminereceptorendocytosisinstriatalneuronsinresponsetodopaminestimulation.WeshowhereanotherphenotypebesidesthehyperactivityofCIN85knockout(KO)micethatofmaternalneglecttohernewborns.EventhoughthereisnodifferenceinthenumberoflivebirthsfromCIN85KOhomozygote,heterozygoteandwild-typemothers,respectively,almostallpupsborntoCIN85KOhomozygotemothershavediedwithintwodaysofbirth.ThiscouldbeexplainedbythefactthatCIN85KOmothersshowedsignificantlydecreasedarched-backnursing,akindofmaternalbehavior,andnewbornpupsfromCIN85KOmotherswereoftenfoundscatteredwithinthebedding.Importantly,whenmeasuringtheplasmalevelsofprolactin(PRL),wedetectedsignificantlydecreasedPRLlevelsinCIN85KOmicecom-paredtoheterozygoteandwild-typemice.PRLinjectioninCIN85KOmicecouldhoweverpartiallyrescuethedefectinmaternalbehaviorofthenextgeneration.OurfindingsindicateanimportantroleofCIN85intheregulationofthedopamine-PRLsysteminthebrainandprovidenewinsightintoamolecularexplanationformaternalbehavior.NoCOI.


1P-156Pathophysiological basis of maternal neglect in Cac-na1a mutant ratsKawakami, Nozomi; Ohmori, Iori; Kobayashi, Kiyoka; Michiue, Hiroyuki; Nishiki, Teiichi; Matsui, Hideki(Department of Physiology, University of Okayama, Okayama, Japan)

Cacna1ageneencodestheP/Q-typevoltage-gatedcalciumchannelpore-formingα1subunit.Groggyrat(GRY/Idr)withM251KmissensemutationinCacna1agenehasabsenceepilepsyandcerebellarataxia.Wenoticedthatgroggymotherratsoftenfailedtoraiseuppups.However,problemsofmaternalbehaviorsinCacna1amutantratwerelargelyunknown.Weaimtoclarifythepathophysiologicalbasisofmaternalneglectandattachmentdisorderingroggyrats.Wecom-paredmaternalbehaviorsandtheattachmentbehaviorsofthepupswithwildtype,heterozygous,andhomozygousrats.Assessmentofthematernalbehaviorsincludesnestbuilding,retrievingthepupstothenestandtimeinlickingthepupsclean.Toevaluatetheattachmentbehaviorsofthepups,ultrasonicvocalizationsat50kHzininfantratsat9dayswasmeasuredbyUltraVox(Noldus).Ultrasonicvocalizationsarerecordedwhenaninfantratisisolatedfrommotherandthesecondisolation.FemalehomozygousGRY/Idrratsshowedfailureofthenestbuilding,decreasednumberofthesurvivalofpups,prolongedretriev-al latencyandshort lickingduration.InfantheterozygousGRY/Idrratsexhibitedsignificantlylowerfrequencyofthevocalizationinthefirstandthesecondisolationfrommotherascomparedtowildtype.OurresultssuggestedthatCacna1amutationmayrelatedtoinsufficientmaternalbehaviorsandattachmentdisorderinpups.NoCOI.

1P-157Neurodevelopmental disorder like behaviors in Cac-na1a mutant ratsKobayashi, Kiyoka; Kitagawa, Yuichiro; Ohmori, Iori; Michiue, Hiroyuki; Nishiki, Tei-ichi; Matsui, Hideki(Department of Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan)

Voltage-gatedcalciumchannel,CACNA1Amutationshavelinkedtoabsenceseizures,cerebellarataxia,learningdisability,andhemiplegicmigraineinhuman.RatswithM251KmissensemutationinCacna1a(GRY/Idr)alsohaveabsenceseizuresandcerebellarataxia. Inthisstudy,weaimtoelucidatethatwhetherGRY/Idrratshaveneurode-velopmentaldisorderlikebehaviorsandhowitspathologicalmecha-nism. Toassessneurodevelopmentaldisorder likebehaviors,weperformedopenfieldtest,elevatedplusmaze,Barnesmazetest,threechambertestandnovelobjecttest.HeterozygousratswithCacna1amutation(GRYw/+)andwildtyperats(WT)wereexaminedfrom5to9weeksofage.WeconfirmedtherewasnodifferenceoflocomotoractivitybetweenGRYw/+andWTinopenfieldtest.Histologicalas-sessmentwasundertakenat8weeksofage.Striatum,nucleusac-cumbens,midbrainandfrontalcortexregionsweredissectedandregionalconcentrationof5-HTweremeasuredbyhigh-performanceliquidchromatography.GRYw/+ratsshowedlessanxious-likebehav-ior,learningdisabilityandcognitiveinflexibilitywithstatisticalsignif-icance.Theseresultsagreedwithsymptominpartofneurodevelop-mentaldisorderswhichwasobserved inpatientswithCACNA1Amutations.Theconcentrationof5-HTofsomeregionsinhomozygousGRY+/+ratsweresignificantlylowerthanthoseofWT.Neurode-velopmentaldisorderlikebehaviorsinGRYratscanbecausedbylowlevelof5-HTinthebrain.NoCOI.

Poster PresentationsNeurochemistry

1P-158Upregulation of leukemia inhibitory factor (LIF) during zebrafish optic nerve regenerationOgai, Kazuhiro1,2; Sugitani, Kayo1; Koriyama, Yoshiki1; Kato, Satoru1(1Dept. Mol. Neurobiol. Faculty Med. Inst. Med. Pharm. Health Sci. Kanazawa Univ. Ishikawa, Japan, 2Wellness Promotion Science Center, Inst. Med. Pharm. Health Sci. Kanazawa Univ. Ishikawa, Japan)

Fishretinalganglioncells(RGCs)canregeneratetheiraxonsfollowingopticnerveinjury,whereasmammalianRGCsfailtosurvive.Leukemiainhibitoryfactor(LIF),whichhadbeenoriginallydiscoveredasadif-ferentiationfactorofmyeloidcells,hasbeennowrecognizedashavingversatile functionssuchasaneffectonaxonalregeneration.ThesefindingsencouragedustoinvestigatethefunctionofLIFonzebrafishopticnerveregeneration.LIFmRNAexpressionwasstudiedbyRT-PCRandin situhybridization,andrevealedtobeupregulatedin1-5dayspost-injury(dpi)intheRGCs.ImmunohistochemistryandwesternblottingagainstLIFalsorevealedtheincreaseofLIFproteinat3dpiintheRGCs.NextweinvestigatedtheactivationofSTAT3,thedown-streammoleculeofLIF.Asexpected,STAT3wasactivatedin3-5dpiintheRGCs.TodeterminewhethertheactivationofSTAT3wasdi-rectlyevokedbyLIF,weemployedaLIF-specificmorpholinotoknockdowntheexpressionofLIF intheRGCs.AsaresultofLIFknockdown,STAT3activationwascancelled,implyingthatLIFdoesactivateSTAT3pathwayafter injury. Inaddition,LIFknockdownimpairedneuritesproutingfromretinalexplantsin vitro,decreasedGAP-43expression,narrowedtheoptictractwidthandunderminedthevisualfunctionrecoveryafternerveinjuryin vivo.Alltold,LIFisanindispensablefactorforestablishmentofopticnerveregenerationinadultzebrafish.NoCOI.


1P-159The functional study of Neuroglobin under oxidative stress in mouse retina. Sugitani, Kayo1; Sera, Mayuko3; Ogai, Kazuhiro1,2; Koriyama, Yoshiki2; Wakasugi, Keisuke4; Kato, Satoru2(1Div. Health Sci., Grad. Sch. Med., Kanazawa Univ. Japan, 2Dept. Mol. Neurobiol., Grad. Sch. Med., Kanazawa Univ. Japan, 3 Dept Clin Drug Info, Faculty of Pharm, Inst Med Pharm Health Sci, Kanazawa Univ. Japan, 4Dept. Life Sci., Grad. Sch. Arts and Sci., Tokyo Univ., Japan)

Neuroglobin(Ngb)wasidentifiedasanewmemberofhemeproteinswhichisexpressedathighconcentrationsincentralnervoussystemneurons.RecentstudieshaveshownthatmammalianNgbisinvolvedinneuroprotectionundervariousoxidativestress,suchasischemiaandreperfusioninsults.InRGC-5cells,murineretinalprecursorcells,overexpressionofNgbenhancedcellviabilityinculturecomparedtomockcontrolunderhypoxicstressconditions.Additionof100–500µMcinnamicacid,apotentinducerofNgb,increasedcellviabilitymorethantwo-foldsunderhypoxicconditions.ThesedatasuggestthatNgbpromotesneuronalprotectionandcellsurvivalunderoxidativestress.Followingmouseopticnerveinjury,Ngbclearlydecreasedfromret-inalganglioncells(RGCs)withinseveraldaysandmostofRGCsun-derwentapoptosis. Incontrast tomouse,zebrafish (zf)RGCscansurviveafteropticnerve injuryandNgbsignificantly increased indamagedzfretina.ToinvestigatetheroleofNgb,wemadechimericZHHHNgbproteininwhichmoduleM1ofhumanNgbisreplacedbythatofzebrafish.TheadditionofZHHHNgbproteininducedneuriteoutgrowthinRGC-5cellcultures.ThesedatasuggestthatNgbpro-motescellsurvivalandneuriteoutgrowthin vitro.NoCOI.

1P-160Functional characterization of Ftsj1, a X-linked men-tal retardation-related geneNagayoshi, Yu; Wei, Fan-Yan; Kaitsuka, Taku; Tomizawa, Kazuhito(Kumamoto University, Faculty of Life Sciences, Department of Molecular Physiology)

GeneticmutationsinXchromosome-linkedgeneshavebeenassociat-edwithmentalretardation(XLMR).Recently,linkageanalysesper-formedinBelgian,ChineseandJapanesefamilieshaveidentifiedFTSJ1geneasanovelcandidategene.FTSJ1shareshomologywithabac-terial23SrRNAmethyltransferaseFtsj.However,themolecularfunc-tionofFTSJ1anditspathologicalrelevanceinmentalretardationhaveremainedunknown.TounderstandthemolecularmechanismofXLMR,wehavegeneratedFTSJ1knockout(FTSJ1KO)mice,andinvestigat-edtheneurophysiologicalimpacts.WhiletheFTSJ1KOmousedevel-opednormally,weobservedadecreasedproteinsynthesis level inMEFcellsderivedfromKOmice.Therewasamarkeddysregulationofsynapticproteinsincludingglutamatereceptorsandsignalingmol-ecules.Furthermore,therewasanincreaseofimmaturedentricspinesincortical,striatalandhippocampalneurons.Thereresultssuggestthat lossofFTSJ1mighthaveaprofound impactontheneuronalactivityduetodysregulationofproteinsynthesisinneurons.NoCOI.

1P-161Mechanical hyperalgesia induced by repeated cold stress in SHRSP5/Dmcr rats: PCR-based cDNA sub-traction analysis for peripheral expression changes in pain related genesKozaki, Yasuko1; Umetsu, Rena1; Mizukami, Yukako1; Yamamura, Aya1; Kitamori, Kazuya2(1College of Pharmacy, Kinjo Gakuin University, Nagoya, Japan, 2College of Human Life and Environment, Kinjo Gakuin University, Nagoya, Japan)

Repeatedcoldstress(RCS)isknowntotransientlyinduceautonomicimbalanceassociatedwithhypotensionandhyperalgesia.Inthisstudy,weinvestigatedtheeffectsofRCSonthethresholdsforcutaneousmechanicalpainresponsesandonperipheralexpressionof“painre-latedgenes” inSHRSP5/Dmcr (Arteriolipidosis-prone,AL)rats.ALrat isderivedfromstroke-pronespontaneouslyhypertensiverats,thereforeitisconsideredtobesympathicotonia.RCSwasgivenbychangingtheenvironmentaltemperaturefrom24to4°Cat30minintervalsduringthedayand4°Catnightfor2days,andthen24°Cduringthedayand4°Catnightfor2days.Atimmediatelypost-RCS,themechanical thresholdwassignificantlydecreased (p<0.001vscontrolbyStudent’st-test).Thishyperalgesiaisthoughttobecausedinpartbyhypofunctionofdescendingpain inhibitorysystems,weconfirmedincreasedexpressionofcorticotropin-releasinghormoneinthehypothalamus(p<0.001).Todeterminethegeneexpressionchang-esaccompaniedbyRCSindorsalrootganglioncells(DRGs),weper-formedPCR-basedcDNAsubtractionofmRNAsfromDRGs.Wede-tectedup-regulationofTac1 (substanceP)anddown-regulationofS100a10(annexin2light),CatB(cathepsinB),andFstl1(follistatin-like1).AtleastsomeofthesegenesinDRGmayplaykeyrolestomechan-ical hyperalgesia induced by RCS. [Research funds: KAKENHI23590724]NoCOI.

1P-162Effects of environmental enrichment on a sensory-mo-tor cortex in miceInoue, Mai1; Odagawa, Maya1; Honma, Chihiro1; Yamada, Kazuyuki1; Akagi, Takumi1; Suzuki, Takayuki1; Murayama, Masanori1,2(1BSI, RIKEN, Saitama, Japan, 2Tokyo Tech, Tokyo, Japan)

Environmentalenrichment(EE)isknowntocauseneuroplasticchang-esinlower-ordercortices(e.g.,theprimarysensoryarea,S1)ofmice.Miceraised inEEconditions (EE-mice)alsoshowincreasedsocialability,asobserved inasocial interactiontest.Becausesociality islargelybasedonhigherbrainfunctions,theincreasedsocialityofEE-micesuggeststhatEEinducesneuroplasticchangesnotonlyinlower-butalsoinhigher-ordercortices.AlthoughsomecellularmechanismshavebeenproposedforEE-inducedneuroplasticchangesinlower-or-derareas,themechanismsunderlyingEE-inducedchangesinhigh-er-orderareasareunknown.OuraimwastoexaminetheeffectsofEEonneuroplasticityinalower-andahigher-ordercortex.Here,wespecificallyinterestedinareciprocallyconnectedsensory-motorcircuit,S1andasecondarymotorarea,M2.TounderstandeffectsofEEoftheS1-M2circuit,weraised2-week-oldmiceineitherEEconditionornormalenvironment(NE)for3weeks,andobservedcorticalactivityevokedbyhindpawstimulationusingavoltage-sensitivedye.NeuronalactivityinM2wassignificantlyincreasedinEE-micethaninNE-mice,suggestingplasticityintheM2underEEconditions.Totestthishy-pothesis,wemeasuredthenumbersofneuronsandsynapsesinEE-andNE-miceusingimmunohistochemistryandtransmissionelectronmi-croscopy,respectively.Here,wereportthedifferences invariousphysiologicalandanatomicalparametersbetweenEE-andNE-mice.NoCOI.


1P-163The neuroprotective effects of a novel nucleoside an-alogue (2Cl-C.OXT-A) on intracerebral hemorrhageLu, Feng1; Okabe, Naohiko1; Himi, Naoyuki1; Nakamura-Maruyama, Emi1; Shiromoto, Takashi1; Narita, kazuhiko1; Tsukamoto, Ikuko2; Maruyama, Tokumi3; Sakakibara, Norikazu3; Miyamoto, Osamu1(1The Second Department of Physiology, Kawasaki Medical School, 2Department of Pharmaco-Bio-Informatics, Faculty of Medicine, Kagawa University, Kagawa, Japan, 3Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Kagawa, Japan)

2Cl-C.OXT-A(COA-Cl)isanovelsynthesizednucleosideanaloguewiththemolecularweightof284.Itissoluble,stableandeasytosynthesize.IthasbeenreportedthatCOA-Clenhancesangiogenesis inhumanumbilicalendothelialcells (HUVEC).Thepreviousstudy inour labshowedtheneuroprotectiveeffectsofCOA-Clonischemiastrokebyreducinginfarctvolumeandattenuatingthebehavioraldeficits.ThepurposeofthepresentstudyistoevaluatetheneuroprotectiveeffectofCOA-Clonintracerebralhemorrhage(ICH),anothercommontypeof stroke, and investigate the potential mechanism of action.Sprague-Dawley (SD)ratswereused forthisstudy,andtheICHmodelswereperformedbytheinjectionofautologousbloodintotherightbasalganglia.COA-Clwas injected intracerebroventricularly10minafterICHwithadosageat30ug/kg.Weexaminedbrainedema1dayafterICHbywatercontenttest,andevaluatedthebehavioraldeficitsusingforelimbplacingandcornerturntest.COA-Clsignifi-cantlyreducedthebrainedema1dayafterICHand improvedthebehavioraldeficitsintheacutephaseofICH.OurreultsindicatedthatCOA-ClmayhaveaneuroprotectiveeffectonICH,anditmayprovideanewtherapeuticapproachfortreatinghemorrhagicstroke.NoCOI.

1P-164Quantification of brain dopamine and amino acids with high resolution mass spectrometry using novel stable isotope labeled reagent.Narita, Kazumi(Department of Integrative Physiology, Faculty of Medicine, University of Fukui, Fukui, Japan)

Weinventedapyrylium-basedlabelingreagent(PyII)formassspec-trometry.Thisagentinteractswithprimaryamines,suchascatechol-amine,aminoacidand lysineresidue inpeptides.Evennumberofstableisotope13Cbetween0and12arereplacedinthePyII.ThustherearesevenkindsofPyIIreagentofdifferentmass,weareabletoanalyzesevensamplessimultaneously.Afterlabelingindividually,eachsampleispooledandassayedbynanoLCMSsystem.Thoughlabeledaminesderivedfromdifferentsampleshavedifferentmassby2.006dalton,theselabeledaminesharesamechromatographicprop-erties,allowingbothamineidentificationandquantificationtobede-rivedfromthesameMSspectrum.TheprincipaladvantageofPyIIoverother labelingagent formassspectrometricanalysis is thatsevensamplescanbeanalyzedsimultaneouslyandthatPyIIandla-beledaminearequitestableinwater,therebyallowingforbiologicalapplication.Inthisstudy,usingthecombinationPyIIwithnanoLCMSsystem,dopamineandGABAareanalyzedinnanolittervolumeofsectionfromratbrain.Afterfixingtheratbrainbymicrowave,30micrometer-thicksectionweremadebyfrozenmicrotome.Fivehun-dredmicrometersquareoftissues,i.e.7.5nl,fordopamineanalysisand100micrometersquare,i.e.0.3nl,forGABAanalysisweredismountedbylasermicrodissection.AfterlabeledwithPyIIreagent,dopamineandGABAinthetissuewerequantifiedandmapped inthebrainatlas.NoCOI.

1P-165Relevance of RISE (Repetitive LTP-Induced Synaptic Enhancement) to memory consolidation in vivo.Nagaoka, Shogo; Urakubo, Tomoyoshi; Tominaga-Yoshino, Keiko; Ogura, Akihiko(Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan)

Memoryisconsolidatedafteracquisition,bythestructuralchangesofneuronsincludingformationofnewsynapses.Inthestableslicecul-turesoftherodenthippocampus,wepreviouslyreportedthattherepeatedinductionofLTPledtoalong-lastingsynapticenhancementcoupledwithnewsynapseformation.WenamedthisphenomenonRISE.AssumingRISEasaninvitrorepresentationofmemorycon-solidationinthebraininvivo,weareanalyzingittorevealcellularmechanismsunderlyingtheconsolidation.Inthisstudywetriedtoobtainsupportforthisassumption.WefoundthattheCa2+-permeableAMPAreceptors(CP-AMPARs)aretransientlyexpressedduringRISEdevelopmentandtheblockadeofCP-AMPARabolishedRISEestab-lishment.Makinguseofthesefacts,weadministeredaCP-AMPARblocker(JSTX;Joro-SpiderToxin)tothemiceduringcontextualfearconditioning.IntracerebroventicularinjectionofJSTXbeforeorinthedayofconditioningshowednoeffectinthemice’sfreezingresponseasassayed1daftertheinjection.However,theinjection1daftercon-ditioningsignificantlyinterferedwiththefreezingresponseasassayed1dafterinjection.TheseresultssupporttheassumptionmentionedabovethatRISEisrelevanttothememoryconsolidationinvivo.NoCOI.

1P-166Differential role of kinase activity of Ca2+/calmod-ulin-dependent protein kinase IIα in hippocampus- and amygdala-dependent memoryYamagata, Yoko1,2; Yanagawa, Yuchio3; Imoto, Keiji1,2(1Natl Inst Physiol Sci, Okazaki, Japan, 2SOKENDAI, Okazaki, Japan, 3Gunma Univ Grad Sch Med, Maebashi, Japan)

Ca2+/calmodulin-dependentproteinkinaseIIα(CaMKIIα)isoneofthemostabundantproteinkinasesinthecentralnervoussystem,andisthoughttobeakeymediatorforhippocampalsynapticplasticity.ToexaminewhetherCaMKIIαissimilarlyinvolvedinamygdala-dependentmemory,justasinhippocampus-dependentmemory,wemadeuseofourkinase-deadknock-inmouseofCaMKIIα(CaMKIIα-KI) in fearconditioning,inwhichcontextualmemoryisbothhippocampus-andamygdala-dependent,whereascuedmemoryisamygdala-dependent,butnothippocampus-dependent.Afterreceivingonepairingoftoneandfootshock,wild-type(WT)miceshoweddistinctcontextual(con-text-dependent)andcued(tone-dependent)fearmemory.Ontheotherhand,homozygousCaMKIIα-KImiceshowednocontextualmemory,butcuedmemorywasformedtoacertainextent.Whenintensetrain-ingwasperformed,CaMKIIα-KImiceshowedincreasedfearnotonlyintheconditioningchamber,butalsoinacompletelydifferentchamber,indicatingthattheycouldnotdiscriminatecontextualdifference,whileWTmicecould.CuedmemorywasevidentinCaMKIIα-KImice,al-thoughtoalesserextentthaninWTmice.Combinedwithbiochem-icalanalyses,theseresultsindicatethatkinaseactivityofCaMKIIαisdifferentiallyinvolvedinhippocampus-andamygdala-dependentmem-ory.SupportedbyGrants-in-AidforScientificResearch‹KAKENHI›fromJapanSocietyforthePromotionofScience,JAPAN(#22500301).NoCOI.


Poster PresentationsAutonomic Nervous Systems (1)

1P-167Roles of GABAergic and glutamatergic neurons in the tachycardia caused by activating forebrain beta adre-noceptorsYamaguchi, Ken'ichi; Iwasawa, Takahiro; Umeda, Kazuyoshi(Department of Homeostatic Regulation and Development, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan)

Wepreviouslyreportedthatbetaadrenoceptor(B-ADR)activationindifferentbrainregionselevatesheartrate(HR),at least inpart,bypotentiatingsympatheticactivity.However,itisnotclearhowactiva-tionofB-ADRsmayexcitethecerebralmechanisminvolvedinstim-ulatingsympatheticneuronsinthespinalcord.ThisstudyaimedtoclarifyrolesofGABAergicandglutamatergic (Glu)neurons inthetachycardiaduetostimulationofB-ADRsintheanteroventralthirdventricularregion(AV3V),becauseearlierdatasuggestpivotalcon-tributionofthoseneuronsincontrollingautonomicfunction.Experi-mentswereperformedinconsciousrats,monitoringbothcardiovas-cularandplasmavariables.Thefollowingresultswereobtained:1)StimulationofGlureceptorsbyAV3Vapplicationsofagonistscausedthetachycardiacapableofbeinginhibitedbythoseofantagonists.2)AV3VapplicationsofaGABAagonist(Mus)didnotalterHR,where-asthoseofitsantagonistevokedtachycardiacapableofbeingblockedbypreapplicationsofMusorGluantagonists.3)AV3VinjectionsofaB-ADRagonistisoproterenolcausedtachycardiaandrisesinplasmanoradrenaline,withoutaffectingbloodpressure.ItstachycardicactionwasinhibitedbyAV3VinjectionsofMusoraGluantagonistorsys-temicinfusionswithhexamethonium.Theseresultssuggestthatacti-vationofAV3VB-ADRsmaycausetachycardiathroughinhibitionoflocalGABAergicneuronsandexcitationofGluneurons.NoCOI.

1P-168Study of the re-activated endogenous microglia for treatment of chronic spinal cord injury.Hamanoue, Makoto1,2; Ogata, Toru3; Nakajima, Kazuyuki4; Takamatsu, Ken1,2(1Department of Physiology, Toho University School of Medicine, Tokyo, Japan, 2Div.Chronic Infla. Dis, Adv. Med. Res. Center, Toho Univ. Sch. Med, 3Dept. Rehab. for Mov. Func., Res. Inst., Natl. Rehab. Cent., 4Dept.Bioinfo., Facul. Engin., Soka Univ.)

Afterspinalcordinjury(SCI),inflammatorycellsandmicrogliaplaymajorrolesonmaintaininglocalcircumstancesbysecretingseveralgrowthfactorsandbyremovingcelldebris.After1week,microglialactivation,however,wasreduced,andlosttheactivityforregeneration.Inthisstudy,we investigateweatherp38MAPkinase (p38)couldre-activaterestingmicrogliatoremodelspinalcordstructureafterchronicSCI.Stimulatedmicrogliaobtainedfromratperinatalspinalcordwerestimulatedbykinase-active(p38a)andkinase-dead(p38d)recombinantp38protein.After16h,Glialcell-linederivedneurotroph-icfactor(GDNF)andVascularendothelialgrowthfactor(VEGF)RNAexpressionwadelevatedbyadditionofp38a,butnotbyp38d.En-hancementofproteinexpressionofGDNFandVEGFandphagocyticactivityofmicrogliaarealsoenhancedbythetreatmentofp38a,butnotbyp38d.Furthermore,continuousp38injectionenhancesCD68andIba1expressionintheadultratspinalcordsliceculture.TheseresultssuggestthatrestingmicrogliaduringchronicSCIcanalsobere-activatedbyp38aproteinasanextracellularstimulator.NoCOI.

1P-169Erythropoietin released from astrocyte protects the oligodendrocyte precursor cell against hypoxic and reoxygenation injuryAoyama, Mineyoshi1; Kato, Shin1,2; Nagaya, Yoshiaki1,2; Tamura, Tetsuya1,3; Kakita, Hiroki1,2; Hida, Hideki4; Asai, Kiyofumi1(1Department of Molecular Neurobiology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan, 2Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan, 3Department of Anesthesiology and Medical Crisis Management, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan, 4Department of Neurophysiology and Brain Science, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan)

Thehypoxiaresponsivecytokineerythropoietin(EPO)providesneu-roprotectiveeffectsinthedamagedbrain.ThepurposeofthepresentstudyistoevaluatetheEPO/EPO-receptor(EPOR)systembetweenastrocyteandoligodendrocyteprecursorcell(OPC)underhypoxia.WereportelevatedEPOmRNAlevelsandproteinreleaseinastrocytesfollowinghypoxicstimulationbyRT-PCRandELISA.Furthermore,EPORexpressionsweredetectedinOPCsbyRT-PCRandcellstain-ing.ToevaluatetheprotectiveeffectofEPOfromastrocytetoOPC,EPO/EPORsignalingwasblockedbyEPOsiRNAorEPORsiRNAgenesilencing.ThesuppressionofEPOproductioninastrocytesbyEPOsiRNAdecreasedtheprotectiontoOPCagainsthypoxicstress.OPCwithEPORsiRNAhadlesscellsurvivalafterhypoxic/reoxygen-ationinjury.ItsuggestedthatEPO/EPORsignalingfromastrocytetoOPCcouldpreventOPCdamageunderhypoxic/reoxygenationcon-dition.OurpresentfindingoftheinteractionbetweenastrocytesandOPCsmayproposethenewtherapeuticapproachtoOPCsagainstcellularstressandinjury.NoCOI.


1P-170IGF-1 and phosphorylated S6 protein increase in up-per motor neurons in zebrafish brain after spinal cord injury.Hisano, Suguru1; Ogai, Kazuhiro2; Mawatari, Kazuhiro2; Koriyama, Yoshiki3; Sugitani, Kayo2; Kato, Satoru3(1School of Health Sciences, College of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Ishikawa, Japan, 2Division of Health Science, Graduate School of Medical Science, Kanazawa University, Ishikawa, Japan, 3Department of Molecular Neurobiology, Graduate School of Medical Science, Kanazawa University, Ishikawa, Japan)

Fishcanregenerateitsmotoraxonsandregainlocomotivefunctionafterspinalcordinjury(SCI).Motorneuronsofadultzebrafishcanbeclassifiedintotwo:(1)uppermotorneuronsinthebrainstemand(2)lowermotorneurons inthespinalcord.UppermotorneuronsareknowntosurviveviaAkt/Bcl-2pathwayafterSCI,butthemechanismofaxonalelongationisnotwellknown.Infishopticnerveregeneration,wehavepreviouslyreportedthatinsulin-likegrowthfactor-1(IGF-1)inducedneuriteoutgrowthandsurvivalthroughAkt/Bcl-2pathway.Therefore,weinvestigatedthetimecourseofexpressionofIGF-1intheuppermotorneuronsfollowingSCI.IGF-1increasedat1–3dayspost-injury(dpi).Tofindwhetherthemammaliantargetofrapamycin(mTOR)pathway,knownasadownstreammoleculeofIGF-1andakeytoaxonalelongation, isactivated inaxotomizeduppermotorneurons,weperformedanimmunohistochemistryofphospho-S6(p-S6)protein,aresultantmoleculeofmTORpathwayactivation.Thep-S6isupregulatedafterSCI.Furthermore,Temusirolimus,themTORinhib-itor,inhibitedtheaxonalelongationofuppermotorneuronsinculture.Thesedata indicatetheaxonalelongationofuppermotorneuronsinducedbyIGF-1andmTORpathway.NoCOI.

1P-171Western blot analysis of tau phosphorylation on close-ly-located phosphorylation sites.Hashiguchi, Mitsuko1; HASHIGUCHI, TOSHIO2(1Department of Physiology, School of Medicine, Tokyo Medical University, Tokyo, Japan, 2Kuretake College of Medical Arts and Sciences, Kuretake School of Integrative Medicine, Saitama, Japan)

TheimmunoreactivityofAT8hadbeenknowntoexpressinthebrainsoftheearlystageofAlzheimer’sdiseasepatients.AT8isusedtovi-sualizephosphorylatedtauproteinsintheneurofibrillarytangles.TheepitopesofAT8consistofmultiplephosphorylationofadjacentSer/Thrmotifs,Ser199,Ser202,andThr205.HoweverthemodulatorymechanismsofAT8epitopeshavenotbeenclarified.WeareshowingphosphorylationmechanismfortheAT8specificepitopes,especiallyinC-terminaldomainofSer/ThrsitesinvitrokinasereactionbythemultiplecombinationsofCDK5-35orCDK5-p25,or,GSK3beta,sequen-tialorsimultaneousapplications.PhosphorylationwasanalyzedbyWesternblot(AT8,anti-phosphoSer202tauantibody(pS202),pT205,pS199,andpS199/202)andtwodimensionalphosphopeptidemaps.EvenCDK5andGSK3betasharetheiractivityonphosphorylatablesites,phosphorylationofAT8epitopeswasregulatedbyCDK5-p25andtheparticularcombinationsofCDK5-p35andGSK3beta.Ontheotherhand,wefoundprotectiveregulationofAT8epitopes.WewillalsoshowthediscrepancyofWesternblotsignalbyantiphospho-tauantibody,whichrecognizesasinglyphosphorylatedsite.NoCOI.

1P-172HPC-1/STX1A and STX1B contribute to regulation of social behaviorFujiwara, Tomonori1; Kofuji, Takefumi2; Mishima, Tatsuya1; Akagawa, Kimio1(1Department of Cell Physiology, Kyorin University School of Medicine, Mitaka, Tokyo, Japan, 2Radio Isotope Laboratory, Kyorin University School of Medicine, Mitaka Tokyo, Japan)

Twotypesofsyntaxin1 isoforms,HPC-1/syntaxin1A (STX1A)andsyntaxin1B(STX1B),areexpressedinneuronsandregulatevesicleexocytosisast-SNAREs.Previously,wegeneratedSTX1Ageneknock-outmiceandSTX1Bgeneknockoutmice.Interestingly,STX1Anullmutantmice(KO)normallydeveloped,whereasSTX1BKOwerebornalivebutdeadwithin14daysafterbirth.Thestudiesforneuropsy-chologicalpropertiesrevealedthatSTX1AKOshowedautistic-likebehavioralabnormalities(Fujiwaraetal2010)andSTX1Bheterozygousmutantmice(HT)showedschizophrenia-likebehavioralabnormalities(Fujiwaraetal2012).Inthisstudy,wefocusedonthesocialbehaviorbothinSTX1AKOandSTX1BHT.WeobservedthatSTX1AKOexhibitedimpairmentofsocialdiscrimination(ISD),andSTX1BHTshowedsocialavoidancebehavior(SA)whichisnotobservedinWTmice.InSTX1AKO,ISDwasimprovedbyadministrationofoxytocinorDAtransporterinhib-itorbutnotbydesipramine.Incontrast,SAinSTX1BHTwasim-provedbydesipraminebutnotbyoxytocin.Theseobservationsindi-catethatSTX1AandSTX1Bdifferentlycontributetoregulationofsocialbehavior.Additionally,inWT,weobservedthatchronicsocialdefeatstressdecreasedbothofSTX1AandSTX1Bproteinexpressionandcausedabnormalsocialbehavior.Theseresultssuggestthatde-creaseofSTX1Aand/orSTX1BgeneexpressioninCNSmayinduceabnormalsocialbehavior.NoCOI.

1P-173GTP cyclohydrolase 1 and its regulatory protein (GFRP) in the rat brain stemBellier, Jean-Pierre1; Ding, Wei-Guang2; Sakaue, Yuko3; Yasuhara, Osamu4; Matsuura, Hiroshi2; Tooyama, Ikuo1(1Molecular Neuroscience Research Center, Shiga University of Medical Science, Otsu, Japan, 2Department of Physiology, Shiga University of Medical Science, Otsu, Japan, 3Department of Pediatric, Shiga University of Medical Science, Otsu, Japan, 4School of Human Nursing, The University of Shiga Prefecture, Otsu, Japan)

Tetrahydrobiopterin(BH4)isacofactorrequiredforthebiosynthesisofseveral importantneurotransmitterssuchasthecatecholamines,serotoninandnitricoxide.TheenzymeGTPcyclohydrolase1(GCH1)isthefirstandrate-limitingenzymeinthemetabolicpathwayofBH4.In vitro,theGCH1feedbackregulatoryprotein(GFRP)maymediatesthefeedbackinhibitionofGCH1activitybyBH4.Here,weinvestigat-edthephysiologicalfunctionofGFRPin vivointheratbrainstem.ThedistributionofGFRPandGCH1wereexaminedbyimmunohisto-chemistryusinghome-madeantiseraonparaformaldehyde-fixedsec-tions. InteractionbetweenGFRPandGCH1wasexaminedusing2D-SDS-PAGEandproximityligationassay,whileactivityoftheBH4metabolicpathwaywasmonitoredusingHLPC-ECD-UV.Ourprelim-inaryresultsindicatedthatin vivo,GFRPandGCH1arelocalizedinserotoninergicandcatecholaminergicneurons.Expressionlevelofthetwoproteins,however,seemstovarygreatlyinfunctionoftheneu-ronaltype.MoreoverinteractionsbetweenGFRPandGCH1aswellasthemodulationofBH4levelappeartobedifferentdependingontheneuronalstructurestudied.ThoseresultssuggestthattheregulationofBH4biosynthesisbyGFRPoccursdifferently in functionof thestructureandneuronaltype.NoCOI.


Poster PresentationsMuscle Physiology (1)

1P-174Roles of Epac in masseter muscle hypertrophy in-duced by chronic stimulation of β2-adrenoceptorOhnuki, Yoshiki; Saeki, Yasutake; Okumura, Satoshi(Department of Physiology, Tsurumi University School of Dental Medicine, Yokohama, Japan)

ToelucidatetherolesofEpac(exchangeproteindirectlyactivatedbycAMP) inthepathogenesisofmusclehypertrophyandslow-to-fastfiber-typetransition inducedbyclenbuterol (CB),aβ2-adrenoceptor(β2-AR)agonist,weexaminedtheeffectsofchronicCBtreatment(i.p.,2mg/kg/dayfor3weeks)onmusclemass,fiberdiameterandmyosinheavychain (MyHC)composition inmassetermuscle (theprincipaljawcloserinrodents)ofwild-typecontrols(WT)andEpac1knockoutmice(KO).Massetermassandmusclefiberdiameterweresignificant-lyincreasedbyCBtreatmentinWTwhilenotinKO.Ontheotherhand,CBtreatmentsignificantlyincreasedtheproportionofMyHCIIbattheexpenseofthatofMyHCIId/xinbothWTandKO,indi-catingthatdisruptionofEpac1didnotaffecttheMyHCtransitiontowards faster isoforms. Inaddition,weexaminedwhetherEpacmediatedCB-inducedhypertrophythroughAkt/mTORpathway,apossibledownstreampathway influencingmusclephenotype.Phos-phorylation levelsofAktand itsdownstreamtargets,P70S6Kand4EBP,wassignificantlyincreasedbyCBtreatmentinWT,buttheincreasewasabrogated inKO.Theseresultssuggestthatchronicstimulationofβ2-ARinducesmassetermusclehypertrophythroughEpac/Akt/mTORpathway.NoCOI.

1P-175Sarcomere length nanometry in cardiomyocytes ex-pressing α-actinin-AcGFP in Z-discsShintani, Seine A.1; Oyama, Kotaro1; Ohki, Takashi1; Ishiwata, Shin’ichi1,2; Fukuda, Norio3(1Department of Physics, Faculty of Science and Engineering, Waseda University, Tokyo, Japan,, 2Waseda Bioscience Research Institute in Singapore, Waseda University, Singapore, Singapore,, 3Department of Cell Physiology, The Jikei University School of Medicine, Tokyo, Japan.)

Incardiacmuscle,achange insarcomere length (SL)by~100nmcausesadramaticchangeincontractility(i.e.,theFrank-Starlingmech-anism),indicatingtheneedforthesimultaneousmeasurementofSLandintracellular[Ca2+]iincardiomyocytesathighspatialandtemporalresolution.Toaccuratelyanalyzethemotionsofindividualsarcomereswithnanometerprecisionduringexcitation-contractioncoupling,weinthepresentstudyappliednanometrytechniquestoprimary-culturedratneonatalcardiomyocytes.First,wedevelopedanexperimentalsystemforsimultaneousnano-scaleanalysisofsinglesarcomeredy-namicsand[Ca2+]ichangesviaexpressionofAcGFPinZ-discs.Wefoundthatfollowingtreatmentwithionomycin,myocytesexhibitedspontaneoussarcomericoscillations(Cell-SPOC)atpartialactivationwithblockageofsarcoplasmicreticulumfunctions,andthewaveformpropertieswereindistinguishablefromthoseobtainedunderelectricfieldstimulation.Finally,we interpretedthepresentexperimentalfindingsintheframeworkofourmathematicalmodelofspontaneoussarcomericoscillations.Thepresentexperimentalsystemhasabroadrangeofapplicationpossibilitiesforunveilingsinglesarcomeredynam-icsduringexcitation-contractioncoupling incardiomyocytesundervariousconditions.NoCOI.

1P-176Imaging of sarcomere dynamics in neonatal rat car-diomyocytes expressing F-actin-containing stress fi-ber-like structuresFujii, Teruyuki1; Shintani, Seine A2; Tsukamoto, Seiichi1; Ishiwata, Shin’ichi2,3; Fukuda, Norio1; Minamisawa, Susumu1(1Department of Cell Physiology, The Jikei University School of Medicine, Tokyo, Japan, 2Pure and Applied Physics, Faculty of Science and Engineering, Waseda University, Tokyo, Japan, 3Waseda Bioscience Research Institute in Singapore (WABIOS))

Inthepresentstudy,weinvestigatedwhethersarcomericdynamicsisinfluencedbythedevelopmentofF-actin-containingstressfiber-likestructuresinneonatalratcardiomyocytes.Ventricularmyocyteswereisolatedfrom1-day-oldWistarrats,andculturedoncollagen-coatedglassbottomdishes. Stressfiber-likestructuresdevelopedwhenmyocyteswereculturedforthreedaysinthepresenceofbasicfibro-blastgrowthfactor(FGF-2,1.4nM).Likewise,stressfiber-likestruc-turesdevelopedwhenmyocyteswereculturedinthepresenceoftheactomyosin inhibitorN-benzyl-p-toluenesulphonamide (BTS,20µM).ThemagnitudeofsarcomericcontractionsdidnotsignificantlychangeupontreatmentwithFGF-2orBTS.Weherebyconcludethatinneo-natalcardiomyocytes,1)intracellularF-actin-containingstressfiber-likestructuresdevelopwiththesuppressionofactomyosininteraction,and2)thestressfiber-likestructuresdonotsignificantlyaltersarcomeredynamics.NoCOI.


1P-177The time course of Fyn and ROK activation in the signal transduction of abnormal vascular smooth mus-cle contractionKishi, Hiroko; Zhang, Ying; Takagaki, Ryodai; Miyanari, Kenji; Kimura, Tomohiko; Kobayashi, Sei(Department of Molecular Physiology and Medical Bioregulation, Yamaguchi University Graduate School of Medicine, Ube, Japan)

Rho-kinase(ROK)-mediatedCa2+-sensitizationofvascularsmoothmus-cle(VSM)playsacriticalroleforabnormalVSMcontractionssuchasvasospasm.Previouslyweidentifiedsphingosylphosphorylcholine(SPC)asanovelmoleculeto inducetheROK-mediatedCa2+-sensitizationleadingtoabnormalVSMcontractions.Furthermore,wealsoidentifiedFyn,amemberofSrc familytyrosinekinase (Src-TKs),asanovelsignalingmolecule forabnormalVSMcontractionmeditatingtheSPC-inducedROKactivation.Inthepresentstudy,weanalyzedthetimecourseofFynandROKactivation inhumancoronaryarterysmoothmusclecells.TodistinguishtheactivationofFynfromthatofothermemberofSrc-TKs,Fynwasimmunoprecipitatedanditsacti-vationwasanalyzedbywesternblotanalysistodetectitsautophos-phorylationofY420.ROKactivationwasanalyzedbythephosphory-lationofitstargetmoleculemyosinphosphatasetargetingsubunit1.TheresultshowedthatFynwasactivatedafter5minofSPCstimu-lationwhereasnoremarkableactivationofc-Src,anothermemberofSrc-TKs,wasdetected.After30minofSPCstimulation,theFynac-tivitywasbackto itsbasal level. Incontrast,ROKactivationwasdetectedafter5minofSPCstimulationandsustainedevenafter30min.TheseresultsshowthattheactivationofFynwastemporalwhereasthatofROKwassustainedafterSPCstimulationinthesignaltransductionofabnormalVSMcontraction.NoCOI.

1P-178Stretch speed-dependent muscle damage, functional loss, and mechanical hyperalgesia after lengthening contraction in ratsMori, Tomohiro1; Hayashi, Koei2; Agata, Nobuhide1; Taguchi, Toru2; Kawakami, Keisuke1(1Phys. Occupat. Ther. Progr., Nagoya Univ. Grad. Sch. Med., Nagoya, Japan, 2Dept. Neurosci. II, Res. Inst. Environ. Med., Nagoya Univ., Nagoya, Japan)

Muscledamage,functionaldeficits,andpainareoftenelicitedbyex-ercise,especiallyafterlengtheningcontraction(LC).HereweexaminedwhetherstretchspeedofLCcouldbeadeterminantleadingtothesechanges.Underisofluraneanesthesia,LCwasrepeatedlyloadedtotheratankleextensormusclesatadifferentstretchspeed(i.e.angularvelocitiesof50,100,200,or400deg./s)overafixedstretchrangeofmotion (i.e.90deg.).Thenumberofmusclefibers labeledwithEv-ans-bluedye,amarkerofmuscledamageor increasedmembranepermeabilityofcells,increasedastheangularvelocityofLCwasin-creased.ThenumberofmusclefiberswiththelargecrosssectionalareawasselectivelyandsignificantlydecreasedthreedaysafterLC(200deg./s).IsometrictorqueofdorsiflexionmeasuredtwodaysafterLCdecreasedastheangularvelocityofLCwasincreased.MuscularmechanicalhyperalgesiaafterLCwasaugmentedinanangularve-locity-dependentmannerwhilerepetitivestretchofthemusclealonewithoutcontractionhadnoeffectonthenociceptivethreshold.TheseresultsindicatethatthefasterangularvelocityofmusclestretchduringLCwasacriticaldeterminantleadingtoincreasedmembraneperme-ability,decreasedfunction,andfacilitatedmechanicalhyperalgesiainthemuscle.Thesefindingscouldbeusefulwhenprescribingexerciseforathletes,elderlypeople,andpatients.NoCOI.

1P-179Influence of the Connection between Skeletal Mus-cles on Muscle Shortening VelocityIshii, Yoshiki1; Sasai, Nobuaki3; Yamamoto, Hiroyuki1; Yamanaka, Yuki1; Mizuno, Tomohito1; Tsuchiya, Teizo2(1Faculty of Health Care Sciences, Himeji Dokkyo University, Hyogo, Japan, 2Faculty of Science, Kobe University, Hyogo, Japan, 3Faculty of Health Science, Suzuka University of Medical science, Mie, Japan)

Thepresentstudywas investigatedtoknowthe influenceof theconnectionbetweenskeletalextensormusclesbyfasciaeinkneejointontheshorteningvelocity.Wemeasuredtheforce-velocityrelation-ship,in vivo,inwholemusclepreparationsinkneeextensor,tricepsfemorismuscle(TFM),ofthefrog,Rana catesbeiana.TFMconsistsofthreemuscles,rectus femoris (RFM),vastusmedialis (VMM)andvastuslateralis(VLM).Frogswereanesthetizedbyinjectingurethaneintraperitoneally.ThenallthebranchesofsciaticnerveexceptfortheoneinnervatingVMMandVLMwerecut.Theactiveisotonicveloc-ityofVMMandVLMwasmeasuredatvariousstepsofloadat20±0.5°C.Experimentswereperformedonthreedifferentkindsofprepa-rations;inonepreparation,surfaceofTFMwastotallycoveredwithfascia(NF),inthesecondone,hamstringswasremovedfromTFM,and inthethirdone,RFMwasremovedfromNF.ThemaximumshorteningvelocityinNFwasthefastestamongthreeconditions.AndtheoutputofpowerinNFwasalsothelargest.Thetheseresultsin-dicatethatconnectionbetweenTFMandhamstringsandthatbetweenRFMandtheother inTFMhaveremarkable influenceonmuscleshorteningvelocityandmusclepoweroutput,suggestingthatoneofthe functionalrolesofconnectionbetweenmuscles is toproducecharacteristiccontractilepropertiesofmuscles.NoCOI.

1P-180Attenuating effects of β2-agonist clenbuterol on cast-immobilization induced atrophy of skeletal mus-cle fibers in rats: histochemical analysesSuzuki, Hideki1; Tsujimoto, Hisaya2; Shirato, Ken3; Mitsuhashi, Ryosuke3; Tachiyashiki, Kaoru4; Imaizumi, Kazuhiko3(1Dept. Health & Phys. Edu., Aichi Univ. of Edu., Kariya, Japan, 2Ins. Health & Sports Sci., Kurume Univ, Japan, 3Lab. Physiol. Sci., Fac. Human Sci., Waseda Univ., Japan, 4Joetsu Univ. of Edu.)

Usinghistochemicalmethods,weinvestigatedwhetherdailyadminis-tration(dose=1mg/kgbodyweight/day)ofclenbuterol(CLE)prevent-edcast-immobilization(IMM)-inducedatrophyofskeletalmusclefibers.AdultmaleSprague-Dawleyratsweredividedintocontrol(CON),CLE,IMM,andIMM±CLEgroups,andmaintainedfor9days.Theextensordigitorumlongus(EDL)andsoleus(SOL)muscleswereisolatedandanalyzedhistochemicallyafterATPasestaining.EDLandSOLmuscleweightsintheIMMgroupwerelowerthanthoseintheCONgroup.Theanalysesofcross-sectionalarea inmusclesrevealedthatEDLmuscleatrophywaslimitedtotypeIIfibers;SOLmuscleatrophywasmediatedbytypeIandtypeIIfibers.IntheIMM+CLEgroup,IMM-in-ducedatrophywasattenuatedintheEDLbutnotintheSOLmuscle.TheattenuatingeffectofCLEwasobservedintypeIIfibers.TheseresultssuggestthatCLEattenuatesIMM-inducedatrophyoftypeIIfibersinthefast-twitchEDLbutnotintheslow-twitchSOLmuscle.NoCOI.


1P-181Evidence for a new type of rigor actin-myosin linkages in skinned skeletal muscle fibersKarina, Hajar1; Kobayashi, Takakazu1; Sugi, Haruo2(1Department of Electronic Engineering, Shibaura Institute of Technology, Tokyo, Japan, 2Department of Physiology, School of Medicine, Teikyo University, Tokyo, Japan)

Itisgenerallybelievedthatmyosinheads(M)bindwithactinfilaments(A)toexertpowerstroke,associatedwithreactionAM.ADP.PitoAM+ADP+Pi.Thisschemeindicatesthat,attheendofpowerstroke,myosinheadstaketheformofrigororrigor-likeactin-myosinlinkagesAM.X-raydiffractionstudiesoncontractingskeletalmuscle,however,cannotdetectsuchrigorlinkages.Tosettletheproblem,wefirstfullyactivatedsingleskinnedskeletalmusclefibersincon-tractingsolution(pCa,4),putthefibersintohigh-Ca(pCa,4)orlow-Ca(pCa,>9)rigorsolution,andthenthefibersweresubjectedtoaseriesofrelease-stretchcycles(amplitude,5%offiberslacklength)atvarioustimesaftertransferofthefibersintorigorsolution.Ratherunexpect-edly,thefibersexhibitedsmallbutdistincttensionrecoveryfollowingtheinitialtensiondropcoincidentwithappliedrelease.Theextentofthetensionrecovery increasedmarkedlyby loweringtemperaturefrom20to10°C,anddecreasedinthepresenceofEDTA(5–10mM).Thetensionrecoverygraduallydecreasedwithtimeinrigorsolution,butwasstillobservableforatleast90min.ADP(upto10mM)hadnoappreciableeffectonthetensionrecovery.Theseresultsindicatethepresenceoftwodifferentkindsofrigoractin-myosinlinkagesinrigorfibers;onewith,andtheotherwithout,activetensionresponse.Thissuggeststhatmyosinheadsincontractingmuscledonotpassthroughrigorconfiguration.NoCOI.

1P-182ATP release from the rat skeletal muscle differs among muscle contraction formsMurase, Shiori; Mizumura, Kazue(Department of Physical Therapy, College of Life and Health Sciences, Chubu University, Kasugai, Japan)

Delayedonsetmusclesoreness(DOMS)isinducedbymuscularlength-eningcontraction(LC),butbyneithermuscularshorteningcontraction(SC)norstretch.Adenosinetriphosphate(ATP)iswellknowntobereleasedfromexcisedmuscleandwouldserveasatriggerforreleas-ingbradykinin-likesubstancethatstimulatesupregulationofnervegrowthfactor,whichinducesmechanicalhyperalgesiainDOMS.WeinvestigatedwhetherthereisanydifferenceinATPreleasefromtheskeletalmuscleamongLC,SCandstretch.WemeasuredATPreleasefromtheextensordigitorumlongus(EDL)muscleinvitro.Themus-cle-peronealnervepreparationwasputinasmallchamberandsuper-fused.InLCandSCgroup,thenervewaselectricallystimulatedfor1stoinducemusclecontractionandfollowedby3srestperiod.Thispatternwasrepeatedforatotalof10min.Thecurrentwassetatthreetimesthetwitchthreshold.Thestimulusparametertoinducetetaniccontractionwasafrequencyof50Hzwithpulsedurationof1ms.InLCgroup,themusclewasstretchedby2mmfromnaturallengthduringthecontractionperiod.Instretchgroup,themusclewasstretchedwithoutcontraction.Thesuperfusatewassampledat-6,-3,0(startofelectricalstimulation),0.5,1,1.5,2,5and10min.TheATPconcentrationwasmeasuredusingtheluciferin-luciferasemethod.ThelargestamountofATPwasreleasedbyLC.Thepeakwasaround1minafterLCwasstartedandthengraduallydecreaseddespitetheexistenceofmusclecontraction.Meanwhile,ATPreleasewasnotin-creasedbystretch.NoCOI.

1P-183Deficiency of Heat Shock Transcription Factor 1 Up-Regulates Interleukins in Regenerating Skeletal Muscle in MiceGoto, Katsumasa1; Nishizawa, Sono2; Koya, Tomoyuki2; Nakai, Akira3; Sugiura, Takao4; Ohira, Yoshinobu5; Yoshioka, Toshitada6(1Department of Physiology, Graduate School of Health Sciences, Toyohashi SOZO University, 2Department of Orthopaedic Surgery, St. Marianna University School of Medicine, 3Department of Biochemistry and Molecular Biology, Graduate School of Medicine, Yamaguchi University, 4Department of Exercise and Sports Medicine, Faculty of Education, Yamaguchi University, 5Osaka University, 6Hirosaki Gakuin University)

Skeletalmusclehasagreaterregenerativepotential.Duringinflam-matoryresponse followingmuscletrauma, interleukin-6 (IL-6)andIL-1βareup-regulated.IL-6andIL-1βarepotentiallymitogenicformyoblasts,aswellasinhibitorsofmyogenicdifferentiation.Heatshocktranscriptionfactor1(HSF1)suppressesinflammatorygenes.Howev-er,theinteractionbetweenHSF1andinflammatorygenesinskeletalmusclecellsremainunclear.Thepurposeofthisstudywastoinves-tigateaphysiologicalroleofHSF1geneonskeletalmuscleregenera-tion.Necrosis-regenerationcyclewas initiatedbythe injectionofcardiotoxinintotheleftsoleusmuscleofmaleHSF1-nullandwild-typemice.SlowerregenerationwasobservedinHSF1-nullmice,comparedwithwild-typemice.Up-regulationsofIL-6andIL-1βmRNAswereenhancedinHSF1-nullmice.HSF1-deficiency-associatedpartialdepres-sionofskeletalmuscleregenerationmightbeattributedtoup-regula-tionofproinflammatorycytokines.Thisstudywassupported,inpart,byJSPSKAKENHIGrantsNumbers22240071,24650411,24650407andthePromotionandMutualAidCorporationforPrivateSchoolsofJapan.NoCOI.

1P-184Screening of SESTD1 C-terminus binding proteins in skeletal musclesHanashima, Akira1; Kimura, Sumiko2; Murayama, Takashi1(1Department of Pharmacology, School of Medicine, Juntendo University, Tokyo, Japan, 2Department of Biology, Graduated school of Science, Chiba University, Chiba, Japan)

SESTD1isanovel79kDaproteinthatconsistsofSEC14domain,threespectrinrepeatsanduniquesequences.SomeresearchsuggeststhatSESTD1isinvolvedintheplanarcellpolaritypathwayduringmam-malianembryonicdevelopmentandtheotherresearchsuggeststhatSESTD1regulatestransientreceptorpotentialchannels insmoothmuscle.SESTD1isalsofoundinanothertissuesbutisnotanalyzed.Inthisresearch,weinvestigatedlocalizationandbindingproteinsofSESTD1instriatedmuscles.Westernblotexperimentsusinganti-SES-TD1monoclonalantibodyelucidatedthatSESTD1 isnotcardiacmuscleproteinbutskeletalmuscleprotein.Immunofluorescencemi-croscopyusingthatmonoclonalantibodyrevealedthatSESDT1islocalizedaroundZ-lineofsarcomereinskeletalmuscles.TosearchthebindingproteinsofSESTD1inskeletalmuscle,wecarriedouttheyeasttwo-hybridscreeningusingSESTD1C-terminusasbaitandmusclecDNAlibrariesaspreys.Severalproteins,locatedinZ-linesofsarcomere,suchasN-RAPandDesminwereobtainedascandidatesforSESTD1C-terminusbindingproteins.TheseresultsindicatethatSESTD1isthenovelZ-lineproteinofthesarcomereinskeletalmuscle.WearecurrentlyinvestigatingtheroleofSESTD1inmyofibrillogen-esis.NoCOI.


1P-185Interaction between water and proteins in skeletal muscleNakahara, Naoya1; Kimura, Masako2; Takemori, Shigeru1(1Dept. Molecular Physiol., Jikei Univ. Sch. Med., 2Dept. Integr. physiol., Kagawa Nutri. Univ.)

Magneticresonance(MR)imagesreflectnotonlywatercontent,butalsowaterstatesintissue.Inskeletalmuscle,MRdistinguishesfivewaterstateswhoselocalizationhasbeenclarifiedtakingadvantageofwell-organizedcrystallinestructureofsarcomere.Detailednatureofeachwaterstateis,however,notclarifiedyet.Interactionbetweenwatermoleculesandmacromoleculessuchasproteins isconsideredtorestricttheirmutualmotional freedomtorendercharacteristicstatetotheclustersofwatermolecules.Fromthis, it followsthatan increase inthermalmolecularmotionwithtemperature,waterandmacromoleculeswouldstoreentropicfree-en-ergythatcanbepartlyliberatedintheprocessofmusclecontraction.Withthisviewinmind,weobservedheatcapacityofskeletalmuscle(preparedfromsartoriusmuscleofRana Catesbeiana)withgradualincreaseintemperatureusingdifferentialscanningcalorimetry(DSC).Thefrozenpreparationofskinnedmuscleshowedatleasttwomus-cle-specificendothermicchangesat-50°Cand-25°C.Furthermore,theintegratedheatcapacity,uptophysiological temperature inwhichmusclecontractfrom-80°C,ofthefreshspecimenwas10J(~0.2mmolATP-hydrolysis)largerper1gspecimenthanthatofdenaturedspec-imen.Withthisabundantenergy,roleofwaterstateasanenergyreservoirissuggested.NoCOI.

1P-186Effects of heat stress on mTOR signaling and HSP expression in rat skeletal muscleKakigi, Ryo1; Naito, Hisashi2; Yoshihara, Toshinori2; Okada, Takao1(1Dept. of Physiology, Juntendo Univ. Faculty of Medicine, Tokyo, Japan, 2Dept. of Exercise Physiology, Juntendo Univ. Grad. Sch. Health & Sports Science, Chiba, Japan)

Heatstressiswellknowntoincreasetheexpressionofheatshockprotein(HSP)inskeletalmuscle.HSPplaysanimportantroleinchap-eroningnascentpeptidesduringtranslation.Meanwhile,themamma-liantargetofrapamycin(mTOR)signalingpathwayhasakeyroleinstimulatingtranslation initiation.ThepurposeofthepresentstudywastoinvestigatetheeffectsofheatstressonmTORsignalingpath-wayinratskeletalmuscle.Wistarmalerats(14wk-old)wererandom-lyassigned intotwogroups:sedentarycontrol (SC;n=4)andheatstressedgroup(HS;n=24).Afterovernightfasting,alegofratinHSgroupwasimmersedinahotwater(43°C)for30minunderanesthesiaandthegastrocnemiusmusclesinbothlegswereremovedatimme-diately(0min,n=6),30min(n=6),60min(n=6)and24hours(24h,n=6)aftertheheatstress.InSCgroup,4E-BP1andS6K1phosphorylation,andHSP72expressioninwhiteportionofgastrocnemiusmuscleweresignificantlylowerthanthoseofredportion.HeatstresssignificantlyincreasedAkt,mTOR,4E-BP1,S6K1andeIF2αphosphorylationat0mininredportionofthemuscle,whereasthesephosphorylationsig-nificantlyincreasedat0-60mininwhiteportion.HSP72expressioninredportiondidnotchange,whereasheatstresssignificantlyincreasedHSP72expressionat24hinwhiteportion.Theseresultssuggestthatheatstress increasesmTORsignalingandHSPexpression inratskeletalmuscle,whichmaybeaffectedbymusclefibertype.NoCOI.

1P-187Localization of L-type calcium channels in skeletal muscle is regulated by binding of the C-terminus of CaV1.1 subunits with junctophilins.Nakada, Tsutomu1; Flucher, Bernhard E2; Kashihara, Toshihide1; Komatsu, Masatoshi1; Yamada, Mitsuhiko1(1Department of Molecular Pharmacology, Shinshu University School of Medicine, Nagano, Japan, 2Department of Physiology and Medical Physics, Innsbruck Medical University, Innsbruck, Austria)

Inskeletalmyocytes,CaV1.1L-typecalciumchannels(LTCC)formafunctionalcomplexwithryanodinereceptorsatjunctionalmembrane(JM)wherethesarcolemmaiscloselyapposedtosarcoplasmicreticu-lummembranes.Junctophilins (JP)areknowntostabilizetheJMcomplexbybridgingthesarcolemmaandsarcoplasmicreticulum.AlthoughwepreviouslyshowedthatknockdownofJPsinhibitedtheproperJM-targetingandfunctionofLTCCinskeletalmyocytes,themolecularmechanismsunderlyingthesephenomenaisunclear.Inthisstudy,weinvestigatedtheinteractionofLTCCandJPswithbiochem-ical techniques.Co-immunoprecipitationstudyshowedthatCaV1.1interactedwithJP1andJP2inmouseskeletalmuscle.PulldownassaywithGST-fusionproteinsbearingcytosolicregionsofCaV1.1indicatedthattheC-terminusofCaV1.1specificallyinteractedwithJP1andJP2.PulldownassaywithGST-fusionproteinsbearingseveralpartialfragmentsoftheC-terminusofCaV1.1revealedthataminoacidresiduesbetween1545and1606intheproximalC-terminusarenecessaryforthebindingofCaV1.1andJPs.ThisJPbindingregionofCaV1.1includestheaminoacidstretchcorrespondingtotheJM-targetingmotifofCaV1.2identifiedpreviously,suggestingthatinteractionofthispartofCaV1.1andJPsisimportantfortheproperlocalizationofLTCC.NoCOI.

1P-188Neuromuscular transmission in the guinea-pig pros-tateLam, Hoi-Shung Michelle; Hashitani, Hikaru(Department of Cell Physiology, Graduate School of Medical Sciences, Nagoya City University, Japan)

Sympatheticneurogeniccontractionscontributetoanincreaseddy-namicprostatesmoothmuscletoneoftenseen inbenignprostaticenlargement.Blockadeofelectricalfieldstimulationwithα1adreno-ceptorantagonistsisonlypartial.Asecondarycomponentisspeculat-edtobepurinergicinorigin;therefore,thisstudyexaminestheexcit-atory junction potentials (EJPs) elicited during electrical fieldstimulationtodeterminethecontributionofpurinergicsignallingto-wardsneurogeniccontractionsintheprostate.α1adrenoceptorinhib-itorphentolamine (1µM)reducedtheamplitudeofEJPsto61%ofcontrol(n=4,P<0.05)whilealsoreducingtheEmaxofFRCsto56.7%(n=5,P<0.01) indicatingpartof thesympatheticneuromusculartransmission ismediatedbyadrenergicnerves.EJPs (n=4)weresuppressedbyα-βmethyleneATP(10µM)suggestingP2XreceptorsalsocontributetotheEJPsandtheEmaxofFRCswerereducedby44.5%(n=5,P<0.05).AlthoughPPADS(5µM),aP2X1receptorsubtypeinhibitor,reducedtheamplitudeofEJPsby64.8%(n=4,P<0.01),acombinationwithphentolamine(1µM)wasrequiredtoabolishEJPs.Itisapparentalargecomponentoftheneurogenicresponseintheprostateismediatedbypurinergicsignalling,wherebynerve-mediat-edATPreleaselikelyactsinconjunctionwithα1adrenoceptors.Thismayprovideanovelbasisforimprovedtherapeuticaltreatmentofanenhancedprostatetone.NoCOI.


1P-190The STZ-induced diabetes influences protein expres-sion of the sarcoplasmic reticulum in the fast-twitch muscle fiberEshima, Hiroaki; Kano, Yutaka(Dept. of Engineering Science, The University of Electro-Communications.)

Indiabetesmellitus (DIA), forceproductionandfatigue-resistant inskeletalmuscleisimpaired.WedemonstratedthatdiabetesimpairsintracellularCa2+([Ca2+]i)homeostasisandsarcoplasmicreticulum(SR)functioninratskeletalmuscle(Eshimaetal.AJP,2013).However,itisunclearwhetherthesefunctionaldeclineismusclefibertypespe-cific.Wetestedthehypothesisthatattenuationofcalcium-reguratoryproteindependsonmusclefibertype(slow-twitchvs.fasttwitchfiber)inDIA.AdultmaleWistarratsweredividedrandomlyintodiabetic(DIA:Streptozotocin i.p.)andhealthy (CONT)groups.Fourweekslateranimalswereanesthetizedandextensordigitorumlongus(EDL;fast-twitch)andsoleus(SOL;slow-twitch)wereisolated.Weexaminedmusclefibertypecompositions(TypeI,IIa,IIbandIIx)andcross-sec-tionalareabyimmunohistochemicalstaining,andcalcium-regulatoryproteinlevels(Ca2+release;RyR,Ca2+uptake;SERCA1andSERCA2)bywesternblot.InEDL,musclefibertypeswitching(-42.2%,IIb),at-rophy(-13.7%)andup-regulationofSERCA1andSERCA2(+38.7%and+54.7%,respectively)wereobservedcomparedwithCONT(P<0.05).Incontrast,RyRproteinlevelinDIAwaslowercomparedwithCONT(P<0.05).TheseseriesofalterationwasnotobservedinSOL.ThepresentinvestigationdemonstratestheSTZ-induceddiabetescausesmetabolicandmorphologicchanges for fast-twitchfibers.AlsothereciprocaladaptationsoftheSRfunctionwasfoundsuchasimpairmentofCa2+releaseandincreasedSRCa2+transportactivity.NoCOI.

1P-191Manual therapy and heating stimulation modulate mi-tochondrial activity following a lengthening contrac-tion of rat's gastrocnemius muscleUrakawa, Susumu1,2; Takamoto, Kouich1,2; Sakai, Shigekazu1; Matsuda, Teru3; Taguchi, Toru4; Mizumura, Kazue3; Ono, Taketoshi1,2; Nishijo, Hisao2,5(1Dept Judo Neurophysiotherapy, Grad Sch Med Pharmaceu Sci, Univ Toyama, Japan, 2JSPS Asian Core Program, 3Dept Physical Therapy, College of Life and Health Sci, Chubu Univ, Japan, 4Dept Neurosci II, Res Inst Environmental Med, Nagoya Univ, Japan, 5Dept System Emotional Sci, Grad Sch Med Pharmaceu Sci, Univ Toyama, Japan)

Physicaltherapies(e.g.,manualtherapybycompressionofthemuscleandheatingstimulation)havebeenusedasaneffectiveinterventionformuscularpain.Althoughwereportedthatthese interventionsamelioratedmechanicalhyperalgesiainducedbythelengtheningcon-traction(LC)ofrat’sgastrocnemiusmuscle,themechanismsofanal-gesiceffectremainunclear.Theaimofthisstudywastoassessmito-chondrialactivityasakeycomponentofmusclemetabolism.Sixweek-oldmaleSDratsatthebeginningwereused.Followingadequatehandling,LCwasappliedtotheleftgastrocnemiusmuscle.JustafterLC,heatingstimulationwasappliedtoexercisedmusclefor20minbyheatingpack(42°C).OnedayafterLC,manualtherapywithintermit-tentcompressionwasappliedtotheleftgastrocnemiusmuscleoftheawakeratsfor10min.AWesternblotanalysisrevealedthatexpres-sionofcytochromecoxidasesubunitIV, indicatorofmitochondrialactivity,graduallyincreasedfollowingthelengtheningcontraction,butsignificantlysuppressedbyheatingormanualtherapyassamelevelasnon-LCcontrol.Theresults indicatethatphysicaltherapycouldmodulatemitochondrialactivityfollowingLC.NoCOI.

1P-192Force-inhibiting effect of phosphatase inhibitors on bovine ciliary muscleIshida, Minori; Takeya, Kosuke; Akao, Teppei; Miyazu, Motoi; Takai, Akira(Dept. of Physiol., Asahikawa Med. Univ., Asahikawa, Hokkaido, Japan)

Ciliarymuscleisasmoothmusclecharacterizedbyarapidresponsetomuscarinicreceptorstimulationandsustainedcontraction.Ingen-eral,regulationofsmoothmusclecontractioninvolvesproteinphos-phorylation,suchasmyosinandotherregulatoryproteins.However,little isknownabouttheroleofproteinphosphorylation inciliarymusclecontraction.Inourpreviousstudy,okadaicacid,apotentPP2Ainhibitor,attenuatedtaeniacecumcontraction,butnotciliarymusclecontraction.Toaddresstheroleofproteinphosphorylation intheciliarymusclecontraction,weexaminedtheeffectsofselectivePP2Ainhibitorsonbovineciliarymuscleandguineapigtaeniacecum.Methods:Musclestripswerecontractedwithionomycin,andisometrictensionwasrecorded.Variousconcentrationsofphosphataseinhibitorswereadministeredtocontractedorrelaxedmusclestrips.Results:LowconcentrationofokadaicacidandFostriecin impairedionomycin-inducedcontraction intaeniacecum,butnot inciliarymuscle.Rubratoxin impairedcontractionboth intaeniacecumandciliarymuscle.Conclusion:Inthisstudy,weshowedthat1)someproteinphosphataseinhibitorshaddifferenteffectsontaeniacecumandciliarymuscle,2)force-inhibitingeffectsweredifferentamongtheexaminedPP2Ain-hibitors.TheseresultsmaybeattributabletothedifferencesinthespecificityofPP2Ainhibitors. Ifso, inciliarymuscle,unlikeothersmoothmuscles,force-inhibitingeffectofokadaicacidmaybemaskedbyactivatingcontractile-responsethroughinhibitionofanotherphos-phatase.NoCOI.

1P-193Role of T-type Ca2+ channel in generating sponta-neous Ca2+ transients in pericytes of the guinea-pig stomachHashitani, Hikaru(Dept.Cell Physiol., Grad.Sch.Med.Sci., Nagoya City Univ., Nagoya, Japan)

Objective:SpontaneousCa2+transientsofinterstitialcellsinvarioussmoothmuscletissuesprimarilyrelyonperiodicCa2+releasefromsarco-endoplasmicreticulum.Agrowingbodyofevidencesindicatesthatvoltage-dependentCa2+channelsalsocontributetothegenerationand/orsynchronyofCa2+transients.Methods:Inthemyentericlayeroftheguinea-piggastricantrum,spontaneousCa2+transientsindif-ferentcellpopulationswerevisualizedusingfluo-8fluorescenceCa2+imaging.Results:AnextensivenetworkofinterstitialcellsofCajal(ICC)generatedsynchronousspontaneousCa2+transients. BesidesICCCa2+transients,pericytesalsoexhibitedspontaneousCa2+tran-sientsthatweredevelopedalmostsimultaneouslyalongthelengthofthemicrovasculature. ICCandpericytesCa2+transientswerenotaffectedbynidefipine(3µM).PericytesCa2+transientswereprevent-edbycyclopiazonicacid(10µM),caffeine(1mM),Ni2+(10µM),mibe-fradil(10µM)ornominallyCa2+freesolutionbutnottetracaine(100µM).BlockadeofT-typeCa2+channelswithNi2+ormibefradilabolishedthe initialcomponentof ICCCa2+transients,but failedtodisruptsynchrony,althoughasignificantdelay inthepropagationofCa2+transientswasevident. Conclusions:FunctionalcouplingbetweencytosolicCa2+oscillatorandT-typeCa2+channelsappearstoplayacriticalroleinthegenerationofpericytesCa2+transients,whiletheroleofT-typeCa2+channelsinmaintainingintercellularcouplinginICCnetworkcouldbereplacedbyotherionicconductance.NoCOI.


1P-194Epithelium of seminal vesicle modulates neural activ-ity dependent contraction and originates stretch-sen-sitive contractionTakeya, Mitsue1; Itoh, Masayuki1; Ishihara, Keiko1; Hayashi, Tokumasa2; Nakamura, Kei-ichiro3; Takano, Makoto1(1Dept. Physiol., Kurume Univ. Sch. Med., Kurume, Japan, 2Dept. Urol., Kurume Univ. Sch. Med., Kurume, Japan, 3Dept. Anat., Kurume Univ. Sch. Med., Kurume, Japan)

Seminalvesicle(SV),amaleaccessorysexgland,hasbeenknowntocontractandexpeltheirsecretionintotheejaculatoryductinresponsetothesympatheticexcitation.AsnovelcontractilemechanismsofSV,wehavepreviouslysuggestedthattheepitheliumofSVoriginatesastretch-sensitivecontraction(SSC)anddepressesnoradrenaline-inducedcontraction.Inthepresentstudy,weexaminedtheinfluenceofepi-theliumonthecontractioninducedbyneuralexcitationofSVwall.Areversedringpreparation(RRP;~4mmlong)wasdissectedfromSVofguineapigandstretchedbyelevationofa forcetransducertomeasureitsisometriccontraction.Themagnitudeofthecontractionevokedbyelectricalfieldstimuli(EFS-C)wasincreasedinafrequen-cy-dependentmanner (5~100Hz).Blockadeofneuralexcitationbytetrodotoxin(1µM)abolishedEFS-CbutfailedtoaffectSSC.EFS-Cwasduetoexcitationofadrenergicandcholinergicnerves,becauseitwasdepressedbyapplicationofphentolamine,anα-adrenoceptorblocker,andfurtherdiminishedbyanadditionalapplicationofatropine.RemovalofepitheliumfromRRP,whichabolishedSSC,significantlyenhancedthemaximumamplitudeofEFS-C(2.86±0.50g,n=6)com-paredwiththatofepithelium-intactRRP(1.47±0.20g,n=6).Wecon-siderthattheepitheliumofSVplaysanunexpectedregulatoryroleonSVcontraction,althoughthemechanismremainsunsolved.NoCOI.

1P-195Comparative study of hyperalgesia in hindlimb- or an-kle-immobilization rat modelNakanishi, Takako; Kijima, Takeshi; Kitada, Masaki; Serada, Noriyuki; Hisamitsu, Tadashi(Department of Physiology, School of Medicine, Showa University, Tokyo, Japan)

Itisimportanttoestablishthecharacterizationofpainmeasurementinanimalmodel forelucidationofhyperalgesiamechanismand itstreatments.Inthisstudy,wemeasuredthethermalandmechanicalstimulationsensitivitiesinhindlimb(H)-orankle(A)-immobilization(IM)ratmodelsandwerecompared.InH-IM,righthindlimbwasimmo-bilizedbycast,andinA-IM,onlytherightanklewasimmobilizedwithanexternalfixation.Inbothgroups,hyperalgesiaandbodyweightweremeasuredeveryonceaweekafterimmobilizationfor4weeks.Thethermalsensitivitywasmeasuredbyplantertestandtheme-chanicalsensitivitywasmeasuredbyvonFreytest.Theplantertestdatashowed%ofright/lefthindlimbescapetime(second).VonFreytestdatashowed%ofright(g)/left(g)hindlimb:painthreshold.TwoweeksafterIM,inplantertest,theescapetimedecreasedto69.9%inH-IMand83%inA-IM,respectively.InvonFreytest,thepainthresh-oldsignificantlydecreasedto58%inH-IMand21%inA-IMafter2weeksIM,respectively (H-IMvsA-IM,P<0.05).ThebodyweightdecreasedinH-IM(-7.5g),butA-IMincreasedassameasintactmod-el(88g)4weeksafterIM.TheseresultssuggestthatA-IMmakegooduseofhyperalgesiamodel,becauseA-IMhaslowpainthresholdandlessstress.NoCOI.

Poster PresentationsOral Physiology (1)

1P-196Interaction of sympathoadrenal system and cervical sympathetic nerves mediated by adrenergic and NPY receptors on the hemodynamics in the jaw muscleIshii, Hisayoshi; Sato, Toshiya(Div. Physiol., Dept. Oral Biol., Sch. Dent., Health Sci. Univ. Hokkaido, Hokkaido, Japan)

Sympathoexcitationisknowntocausechangesineitherincreaseordecrease inthemassetermusclebloodflow(MBF).Althoughthereasonforthedifferencesintheeffectsisunclear,theinteractionbe-tweenneuralandhumoralmechanismsofMBFregulationmaybeimportantforthedifferencebecausesympathoexcitationinducesac-tivationofbothsympathoadrenalsystemreleasingcirculatingadren-alineandcervicalsympatheticnerves includingnoradrenalineandNPY.However,itisunclearwhetherthereisaninteractionbetweenthesesystemsintheMBFregulation.Weexploredthisquestionbyinvestigatingtheeffectsofelectricalstimulationofsplanchnicnerve(SPLN)ontheMBFforeitherintactorsectioningofsuperiorcervicalsympathetictrunk(CST)andadrenergicandNPYreceptorantagonists(BIBP3226)ontheresponsesinanesthetizedrats.TheSPLNstimula-tioncausedasignificantMBFincreasethroughβ2-adrenoceptorsintheanimalswithintactCST.SectioningoftheCSTchangedtheMBFincreasetodecreasebySPLNstimulation.TheMBFincreaseevokedbySPLNstimulationwasalmostabolishedbyphentolamine.AlthoughBIBP3226alonehadnosignificanteffectontheMBFincrease,BIBP3226incombinationwithphentolaminecausedasignificantMBFin-creaseevokedbySPLNstimulation.These indicate thatcervicalsympatheticnervesareinvolvedintheMBFincreasemediatedbysympathoadrenalsystem,andsuggestthatα-adrenoceptorsandNPYreceptorscontributetoMBFmodulationduringsympathoexcitation.NoCOI.


1P-197Functional expression of glutamate receptors in mouse odontoblastsNishiyama, A1; Sato, M2; Tsumura, M2; Katakura, A1; Tazaki, M2; Shibukawa, Y2(1Dept of Oral Med, Oral and Maxillofacial surg, Tokyo Dental College, 2Dept of Physiol,Tokyo Dental Coleage)

Odontoblastsarecellsthatreceivevariousstimulationappliedtothedentinsurface.Assensoryreceptorcells,theydrivethesensory-trans-ductionsequenceandform“reactionarydentin”.Inadditiontovarioustypesofmembranereceptorsexpressedinodontoblasts,suchasbra-dykinin,ATP,andmuscarinic-cholinergicreceptors,theexpressionofglutamatereceptorshasrecentlybeenreportedtobelocalizedontheodontoblastsmembrane.However,theirfunctionalroleanddetailedpropertiesremaintobeclarified.Glutamatereceptorsaredividedintotwogroups; ionotropic glutamate receptors (iGluRs:NMDAandAMPA/KAreceptors)andmetabotropicglutamatereceptors(mGluRswhicharesubdividedintoGroupI,II,andIII).Inthepresentstudy,weinvestigatedthefunctionalexpressionoftheseglutamatereceptorsbymeasuring intracellularCa2+concentration ([Ca2+]i)using fura-2fluorescenceandmRNAexpressionusingrealtimeRT-PCR.PCRanalysisrevealedtheexpressionofallmGluRandNMDAreceptorsubtypemRNAs,withanabundantexpressionofgroupIIImGluR.Applicationofmonosodiumglutamate (10nM–100µM) inducedatransientanddose-dependentincreasein([Ca2+]i).InthepresenceandabsenceofextracellularCa2+,theselectiveagonistsofGroupI,II,andIIImGluRsincreased([Ca2+]i),whileasimilarincreasein([Ca2+]i)wasnotobservedafteractivationofNMDAreceptorsintheabsenceofextracellularCa2+.TheseresultssuggestedthatodontoblastsexpressGroupI,II,andIIImGluRsaswellasNMDAreceptors.NoCOI.

1P-198Activation of P2Y12-BK receptor coupling disarms the cAMP-mediated inhibitory effect on ryanodine recep-tor channel-induced Ca2+ release in trigeminal gangli-on neuronsKawaguchi, A; Sato, M; Taza, M; Ichinohe, T; Shibukawa, Y(Tokyo Dental College, Tokyo, Japan)

PurinergicP2Y12receptors(P2Y12)areclassifiedasGi/o-protein-coupledreceptors.TheneuropathologicalrolesofP2Y12intrigeminalganglion(TG)neuronsremaintobefullyelucidated.WeinvestigatedP2Y12byimmunofluorescenceanalysisandmeasurementof intracellularfreeCa2+concentration([Ca2+]i) inprimaryculturedratTGneurons.WeobservedintenseimmunoreactivityforP2Y12inTGsomata,dendrites,andaxons,showing,co-localizationwithpan-neuronalmarker,neuro-filament200,isolectinB4,andsubstanceP.InthepresenceorabsenceofextracellularCa2+([Ca2+]o),applicationofaP2Y1,12,13agonist(2-MeS-ADP)andbradykinin(BK)transientlyincreased[Ca2+]i.Intheabsenceof[Ca2+]o,the2-MeS-ADP-inducedincreasein[Ca2+]iwasreducedbyapplyinginhibitorsofthesarcoplasmicreticulumCa2+-ATPaseandtheryanodinereceptorchannel.Applicationofanadenylatecyclaseinhib-itortransientlyincreased[Ca2+]i.Increasesin[Ca2+]icausedby2-Mes-ADPwere inhibitedbyaphosphodiesterase inhibitor.Both2-MeS-ADP-inducedandBK-inducedincreases in [Ca2+]iweresignificantlyinhibitedbyselectiveP2Y12antagonistsinthepresenceof[Ca2+]o.TheseresultsshowthatP2Y12aredistributedA-,non-peptidergicC-andpeptidergicC-neurons.ActivationofP2Y12inducesthereleaseofCa2+fromCa2+stores, triggeredbyadecrease in internalcAMPlevels.P2Y12-BKreceptorcouplingplayanimportantroleindrivingnocicep-tivefunctionbydisarmingcAMP-inducedinhibitionofCa2+mobiliza-tion.NoCOI.

1P-199Luminal entry of fluorescent dye is driven by solvent drag due to paracellular fluid secretion in the subman-dibular gland.Murakami, Masataka1; Wei, Fei1; Narita, Takanori2; Matsuki-Fukushima, Miwako3; Hashimoto, Sadamitsu4; Shibukawa, Yoshiyuki4; Sato, Masaki4(1NatI Inst Physiol Scis, Okazaki, Japan, 2Bioresource Sci, Nihon Univ, Fujisawa, Japan, 3Nihon Univ Dent, Matsudo, Japan, 4Tokyo Dent Coll, Tokyo, Japan)

Primarysalivaisamixtureoffluidsecretedthroughparacellularrouteandtranscellularroute.Byluminaldye-dilutionintheisolatedacini,thetranscellularfluidsecretionwasestimatedas25µl/g-minandtheparacellularfluidsecretionas60µl/g-minduringsustainedstimulation(2002).Inthepresentstudy,usingtheperfusedsubmandibularglandweexaminedwhetherfluorescentdyeenterstheintercellularcana-liculibysolventdrag.Theglandwassurgically isolatedfromtheWistarratunderanesthesia,andperfusedarteriallyonthestageofconfocalmicroscope.(5Live,Zeiss).Whenthefluidsecretionwasin-ducedbyα1-adrenergicstimulationwithphenylephrine,thefluores-cenceofintracellularcanaliculiincreaseddose-dependently.Combinedstimulationwithphenylephrineandxamoterol(β1agonist)increasedthefluorescencemorethanonlyphenylephrine.Singlestimulationwithisoproterenol(1µM)decreasedthefluorescenceintensityinthecan-aliculi,suggestingloweringfluidsecretionthroughpressure-dependentparacellularroute.Thepresentfindingsleadtheconclusionthatwecouldobserveentryoffluorescentdyefromintercellularspacetolu-minalspacebypressure-dependentparacellularroutebysolvent-drag,andthatthefluidsecretionthroughpressure-independentrouteisstillopenforfurtherstudies.NoCOI.

1P-200Effect of Danshen on paracellular fluid secretion in the isolated rat submandibular glandWei, Fei1,2; Murakami, Masataka2(1SOKENDAI, Life Scis., Physiol., 2NatI Inst Physiol Scis, Okazaki, Japan)

Danshen (DS) inducesasalivaryfluidsecretionwithoutanyothersectretagoguesinisolatedandarteriallyperfusedratsubmandibulargland (SMG).Thesecretoryeffectwasnotblockedbyatropine,aninhibitorofmuscarinicreceptors,orbyphentolamine,aninhibitorofalpha-adrenergicreceptor.ThesalivaryfluidsecretionbyDSincludestanscellularfluidsecretionbecausetheblockersofNa/K-ATPase(ouabain)andNa/K/2Cl-cotransporter(bumetanide)decreasedDS-in-ducedfluidsecretion.Inthepresentstudy,weexaminedwhetherDSinfluencestheparacellularfluidsecretion,bymeasuringsecretionofLuciferyellow(LY),afluorescentdyewithca500mwintheperfusate.ThenthesecretedsalivawascollectedalongtimecourseduringDSstimulation.BecausethecollectedsalivaonlybyDSwastoosmalltobemeasured,LYsecretionbyDSpluscarbamylcholine (CCh)wasmeasured.ThetimecourseofLYsecretionwasdifferentfromonlybyCCh.Thetimecourseofsalivaryfluidsecretionwasinparallelwithsecretionoffluorescentdyes,andshowedaninitialslowincreasefol-lowedbyaslowdecrease.ThepresentobservationsuggestedthatDScouldcontroljunctionalfluidpassagewithlargemoleculethroughtheparacellularroute.NoCOI.


1P-201Sex hormone-regulated expression of microRNAs in mouse submandibular glands: a putative Bio-Marker of DHT-dependent diseasesKurihara, Kinji1; Nakanishi, Nibuo2; Tomomura, Akito2; Muramoto, Kazuyo1(1Div. of Physiol., Meikai Univ., Sch. of Dent., Saitama, Japan, 2Div. of Biochem., Meikai Univ., Sch. of Dent., Saitama, Japan)

Mousesubmandibularglands (SMGs)havesexualdimorphismviaandrogenreceptor.MicroRNAs(miRNAs)aresmallnon-codingRNAsthatplaykeyrolesintheregulationofgeneexpression.WeexaminedeffectsofsexhormonesontheexpressionpatternofmiRNAsinmouseSMGs.SMGsofICRmicewere investigatedformiRNAsand42miRNAswereidentified.ExpressionpatternsofthesemiRNAsinmicewithvarioushormonaltreatmentswereanalyzedbyquantitativereal-timePCR.Amongofthe42miRNAs,miR-21aandmiR-143weremuchabundantandmiR-141slightlyabundantinmalemouseSMGs.CastrationcausedremarkabledecreaseintheexpressionofthesethreemiRNAs.DHTadministrationtothecastratedanimalsgreatlyincreasedmiR-21aandmiR-141butslightlymiR-143,resultingthemiR-141expressionsur-passedthemiR-143expression.TheseeffectsofDHToncastratedmiceweresimilartothoseofDHTonfemalemice.Ontheotherhand,DHTadministrationtonormalmalesincreasedthemiR-21a,miR-141andmiR143expressionwithalmostthesameratio,suggestingthatmiR-21aandmiR-141weremainlyregulatedbyDHTwhilemiR-143expressionwasdependentontestosterone.AhormonalconditionofcastratedanimalsthatwereadministratedDHTseemedtobesimilartothatofpartialandrogendeficiencyinagingmales(PADAM).ThechangeinexpressionpatternsofmiR141andmiR143wasexpectedtoapplyfordiagnosisofbenignprostatichyperplasia(BPH)andandro-geneticalopecia(AGA).NoCOI.

1P-202Expression of nestin in injured salivary glandYokoyama, Megumi; Katsumata-Kato, Osamu; Matsuki-Fukushima, Miwako; Fujita-Yoshigaki, Junko(Department of Physiology, Nihon University School of Dentistry at Matsudo, Chiba, Japan)

Objective:Itisknownthatsalivaryglandhasregenerativeability.Buttheoriginofregeneratedsalivaryglandcellsisstillunclear.Ourgoalistoidentifytheprecursorcellofregeneratedacinarcells.Wehavepreviouslyreportedthattissue injuries inducede-differentiationofcells,whichexpressedaneuralstemcellmarker,nestin,invitro.Inthisstudy,weexaminedwhethernestinisexpressedinundifferenti-atedsalivaryglandinvivo.Methods:Weperformedunilateralparotidduct ligationofmouseandextractedparotidglandat4,7,10daysafterligation.Wecomparedexpressionofnestinwithcontrolbyim-munoblotanalysis,HEstainingand immunohistochemicalstaining.Next,weremovedtheclipat7daysafterligation,andextractedpa-rotidglandin14dayslater.Results:Byimmunoblotanalysis,expres-sionlevelofnestinwassignificantlyincreasedat4and7daysintheligationsidecomparedtothecontrol.Morphologically,theatrophyofacinarcellswasobservedintheligationside.Intheatrophiedparotidgland,nestin-positivecellsweredetected.Theparotidglandthathasbeenreleasedfromductligationshowedhistologicalsimilaritytothecontrol.Conclusion:Becausenestinwasexpressedinthesalivaryglandthatchangedtoanundifferentiatedconditionbytissueinjury,itwassuggestedthatnestincanbeusedasaprecursormarkerinsalivarygland.NoCOI.

1P-203Localization and function of V-ATPase in salivary glands of mouse—analysis of the a3 subunit knockout mouse—Horie, Sawa1,2; Goto, Naomi3; Nakanishi-Matsui, Mayumi3; Sahara, Yoshinori2(1Dpt. Tumor Biol., Inst. Biomed. Sci., Iwate Med. Univ., Iwate, Japan, 2Dpt. Physiol., Iwate Med. Univ. Schl. Dent., Iwate, Japan, 3Dpt. Biochem., Iwate Med. Univ., Iwate, Japan)

VacuolarH+-ATPase(V-ATPase)islocalizedinintracellularmembranesoforganellesuchasthevacuole,lysosome,Golgiapparatus,synapticvesicle,andisknowntoacidifyintracellularcompartmentsaswellaspumpH+acrosstheplasmamembrane.V-ATPaseiscomposedoftwolargedomains,acytosolic(V1)domain,consistofA-Hsubunitscomplex,andatransmembrane(Vo)domain,consistofa,c,c’,c”,anddsubunitscomplex.Wepreviouslyreportedthata2,a3,d1,B2,C1,E2subunitisoformsarecommonlyexpressedinthemajorsalivaryglands,andB2subunitisoformislocalizedintheductcellsofeachsalivaryglands.Inthisstudy,wehavefurtheranalizedsubcellularlocalizationoftheB2subunitisoformusingastructuredilluminationmicroscopy(Nikon).Immunofluorecencewasfoundinapicalcellmembraneandcytoplasmofstriatedparotidductalcells.Inductalcellsofthesublingualandsubmandibularglands,immunofluorecencewasfoundinthebasolat-eralregionofcytoplasm.Byanalyzingphenotypesoftheknockoutmouseofthea3subunitisoform,therewaslittlesalivaproductionandthesizeofsalivaryglandswassmallerthanthatinthewildtypemice.IntraoralsalivarypHstendtobeslightlyacidified.TheseresultssuggestthatV-ATPasesplayacriticalroleforsalivaryductalfunction,suchas iontransports involved inepithelialelectrolyteabsorption,anionsecretionandmodificationofpH.NoCOI.

1P-204The localization of parasympathetic vasodilator fibers related to blood flow dynamics in rat salivary gland.Sato, Toshiya; Ishii, Hisayoshi(Div. Physiol., Dept. Oral Biol., Sch. dent., Health Sci. Univ. Hokkaido, Hokkaido, Japan)

Objective:Theparasympatheticbloodflowincreasewasreportedinratsalivaryglands.However,thelocalizationofvasodilatorfibersinsalivaryglandsarenotclear.Thesublingualgland(SLG)havemainlymucousacini,whilesubmandibulargland (SMG)haveamixtureofserousandmucousacini.Thissuggeststhatthehemodynamicmech-anismofbothglandsmaybedifferent. Inthisstudy,weanalyzedparasympatheticbloodflowincreaseofratSLGandSMGby laserspeckleimaging(LSI)techniquetoexaminelocalizationofparasym-patheticvasodilatorfibers.Methods:Theurethaneanesthetizedratsparalysedbypancuroniumbromidewereartificiallyventilated.Thecervicalvagiandcervicalsympathetictrunkwerecutintheneck.ThehemodynamicsofSLGandSMGwereanalyzedbyLSIwhenthe lingualnerve (LN)waselectricallystimulated.Results&Discussion:ThebasalbloodflowwashigherintheSLGthanSMG.TheregionaldifferencesofbloodflowincreasewerefoundamongSLGandsomepartsofSMGduringLNstimulation.Theseincreaseswerecompletelyinhibitedbyhexamethonium(10mg/kgi.v.).Althoughtheseincreaseswereinhibitedbyatropine(0.1mg/kgi.v.),inhibitoryeffectwasstrongerinSMGthanSLG.Therewasnosignificantdiffer-enceinbloodflowincreaseofeachglandbyintravenousadministrationofacetylcholine(0.01~1mg/kgi.v.).Theseindicatethatparasympa-theticbloodflowincreaseofSMGismainlyevokedbycholinergicfibersandthatofSLGisevokedbybothcholinergicandnon-cholinergicfibers.NoCOI.


1P-205Salivary functions change in experimental periodonti-tis model ratsNakamura, Mariko1,2; Ono, Kentaro2; Hitomi, Suzuro2; Matsuo, Kou3; Nakashima, Keisuke1; Inenaga, Kiyotoshi2(1Div. Periodontol., Kyushu Dental Univ., Kitakyushu, Japan, 2Div. Physiol., Kyushu Dental Univ., Kitakyushu, Japan, 3Div. Oral Pathol., Kyushu Dental Univ., Kitakyushu, Japan)

Thisstudywasdesignedto investigatethemechanismofsalivarydysfunctioninanexperimentalperiodontitisratmodelandtoexaminethe improvements insalivarysecretion followingtreatmentof theexperimentalperiodontitis.Intheperiodontitismodel,whichincludedaunilateralligaturefor4weeksaroundtheseconduppermolar,sev-eralsalivary functionswere investigated.Changes inthesalivaryfunctionwereevaluated4weeksafterremovaloftheligatureinsomerats.Theperiodontitismodelshowedsignificantreductions intheweightofthebilateralmajorsalivaryglandsandpilocarpine-inducedsalivarysecretion.Themodelalsoshowedanincreaseinthenumberofapoptoticcellsinbilateralsalivaryglands.AccordingtoCa2+imag-ingandWesternblotting,therewerenodifferencesinthemuscar-ine-inducedintracellularCa2+mobilizationinacinarcellsorintheM3receptorandAQP5expressionlevelsinthesalivaryglandsbetweentheshamandtheperiodontitismodel.Followingremovaloftheligature,differencesintheweightsofsalivaryglandsandpilocarpine-inducedsalivarysecretionbetweentheshamandtheperiodontitismodelani-malswerenot found.Theseresultssuggestthatexperimentalperi-odontitisleadstohyposalivationandthatrelieffromitimprovessali-varyfunction.NoCOI.

1P-206Maintenance of secretory proteins during maturation of rat parotid secretory granulesKatsumata-Kato, Osamu; Yokoyama, Megumi; Matsuki-Fukushima, Miwako; Fujita-Yoshigaki, Junko(Department of Physiology and Research Institute of Oral Science, Nihon University School of Dentistry at Matsudo, Chiba, Japan)

Thediameterofsecretorygranules(SGs)inratparotidacinarcellsisabout1µm,andthereforetheycanbeclearlyobserved.Thisisanadvantage in investigationofgranulematuration. ThematurationprocessofSGscomprisesenlargement, increase indensity,vesicletransport,andremodelingofmembrane.However,themaintenanceofsecretoryproteinsinSGsduringgranulematurationispoorlyun-derstood.β-adrenergicagonistisoproterenolwasinjectedintoratstoinducetheexocytosisofSGsfromtheirparotidglands,whichwereremoved2hrsaftertheinjection.Theisolatedacini,whichhadnoSGs,werethenincubatedwithLuciferYellow(LY)dyeasatracerfor3hrs.ThelocalizationofLYintonewly-formedSGswasestimatedbythefollowingmethods:(1)theco-localizationwithamylasebycon-focallasermicroscopy,(2)thesecretionofLYuponstimulation,(3)thedetectionoffluorescencefrompurifiedSGs,and(4)theintracellularlocalizationbyelectronmicroscopy.After2dayscellculture,new-formedSGsbecamelarger.Inaddition,co-localizationoffluorescencewithamylasewasobserved.Althoughthefusionandbuddingofsmallvesicleswerecontributedtoprocessofgranulematuration,LYstayedinSGsevenafterthematuration.Theseresultssuggestthatthese-cretoryproteinswhichhavenotransportsignalsuchasLYareretainedintoSGsduringthegranulematurationinparotidacinarcells.NoCOI.

1P-207A new relation between visceral sensation and nizati-dine, a gastric-acid inhibitorUeda, Hirotaka1; Suga, Mayu2; Yagi, Takakazu1; Matsuo, Ryuji3; Miyawaki, Shouichi2(1Department of Orthodontics, Kagoshima University Medical and Dental Hospital, Kagoshima, Japan, 2Department of Orthodontics & Dentofacial Orthopedics, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan, 3Department of Oral Physiology, Okayama University Graduate School of Medicine and Dentistry and Pharmaceutical Sciences, Okayama, Japan)

Nizatidinecollaterally increasessalivarysecretion inpatientswithgastroesophagealrefluxdisease(GERD).However,itisnotclearwheth-ernizatidineaffectscentralregulationofsalivation. Inthepresentstudy,wehypothesizedthatintracerebroventricular(thefourthven-tricle)administrationofnizatidinewouldcollaterallyenhancesalivarysecretionrelatedtothevagusnerve,focusingonthecentraleffectsofnizatidine.Weaimedtoclarifyinductionofsalivationbycentralniza-tidineand/orafferentsofthevagusnerveinrats.MaleWistarratswereusedforexperiments.Weperformedreal-timedetectionofsali-varysecretionfromsubmandibulargland.Vagusnerve-inducedsali-varysecretionwasmeasuredfor3minfollowingintracerebroventric-ularadministrationofnizatidine. Intheresults,1) thevagusnervestimulationincreasedsalivarysecretion,2)nizatidineenhancedsalivarysecretioninducedbythevagusnervestimulation.Totalsalivaryse-cretionwassignificantlyincreasedbynizatidineinadose-dependentmanner.Ourfindingssuggestthatcentralnizatidineenhancessalivarysecretioninducedbythevagusnervestimulationinrats.NoCOI.

1P-208Cytotoxicity of low-dose povidone-iodine and its ef-fects on rat oral mucosaMiyake, Masao1; Sato, So1,2; Prasedya, Eka Sunarwidhi1; Omori, Koichi2; Hazama, Akihiro1(1Dept.Cellular and Integrative Physiology, Fukushima Med.Univ.Sch.Med., Fukushima, Japan, 2Dept.Otolaryngology, Fukushima Med.Univ.Sch.Med., Fukushima, Japan)

Povidone-iodine(PVP-I)isascomplexofpolyvinylpyrrolidoneandiodinedevelopedasanoralcavitygargle. Itworksasabroadspectrummicrobicideagainstbacteriaandvirusbasedonoxidizingeffect.PVP-Iexposurecausesnephropathyandiododermaaspreviouslyreported,however,thereisfewquantitativestudies.Theaimofthisstudyistoevaluatethedose-andtime-dependenceofPVP-ItoxicityagainstHeLacellsandratoralmucosa.Wefoundgrowthinhibitionat10-2µM,increasedapoptosisat100µM,blister-likecytoplasmat101µM,andincreasednecrosisat102µMafter2-dayexposurewithPVP-I.AsclinicallyusedconcentrationofPVP-Iis103–104µM,relatively lowconcentrationofPVP-Imaybeabletodamagecellswithlong-timeexposure.IoncompositionofextracellularsolutionhadrelationshipwithPVP-Ieffect.Wealsofoundthinnerepitheliuminratoralmucosaafter1-dayexpo-surewith10-1µMofPVP-I,andithadmanyapoptoticcells.OurstudysuggeststhatrisksandbenefitsofPVP-Iapplicationforsterilizationandanti-canceragentshouldbereconsidered.NoCOI.


1P-209The mechanism of oral ulcer-induced pain hypersen-sitivity in rats.Hitomi, Suzuro1; Ono, Kentaro1; Kuramitsu, Sachiko1; Yamaguchi, Kiichiro1,2; Harano, Nozomu2; Miyano, Kanako3; Uezono, Yasuhito3; Matoba, Motohiro4; Inenaga, Kiyotoshi1(1Div. Pyisiology, Kyushu Dental Univ. Kitakyushu, Japan, 2Div. Dental Anesthesiology, Kyushu Dental Univ. Kitakyushu, Japan, 3Div. Cancer Pathophysiol., National Cancer Center Research Institute, Tokyo, Japan, 4Div. Palliative Med. and Psycho-Oncol. Palliative Care Team, National Cancer Center, Tokyo, Japan)

Oralulcersinducepainduringfoodconsumptionandspeaking.How-ever,becausenoanimalassaymeasuresintraoralpain-inducedbehav-iors,painmechanisms inoralulcersarepoorlyunderstood. Inthisstudy,weinvestigatedpainsensitivitiestochemicalandmechanicalstimulationsinoralulcersusingournewlydevelopedbehavioralassaysinconsciousrats.Treatmentwithaceticacidinthelabialfornixregionoftheinferiorincisorsdevelopedobviousoralulcers,showingsevereinfiltrationofinflammatorycellsandepidermolysisinhistology.Groom-ing-likebehaviorsfollowingtheadministrationofcapsaicinandcitricacidsolutionsweresignificantlyenhancedbyoralulcers.Theheadwithdrawalthresholdtomechanicalstimulationtooralmucosawassignificantlydecreasedinoralulcers.Inimmunofluorescence,oralul-cersproducedahighinfiltrationofsubstancescomparedtothehealthyoralmucosa,withoutchangesintheexpressionlevelsofTRPV1andASIC3intrigeminalganglion.Themechanicalallodyniainducedbyoralulcerswasnotsignificantlyinhibitedbyindomethacininjection.Theseresultssuggestthatoralulcersinducepainhypersensitivitytochemicalandmechanicalstimulations,mostlikelyduetotissuedamage.NoCOI.

1P-210Difference in modulation of jaw-opening reflex be-tween working and balancing side during fictive mas-tication in rabbitsMatsunaga, Tomoko; Morita, Takumi; Hiraba, Katsunari(Department of Physiology, School of Dentistry, Aichi Gakuin University, Nagoya, Japan)

Theaimofpresentstudyistoinvestigatethemodulationofjaw-open-ingreflex (JOR)responsesevokedby innocuousstimulationduringchewing,andassesstheextentofsuppressionbetweenworkingandbalancingsides.EMGactivityofthedigastricmusclewassimultane-ouslyrecordedwithmovementsoftheincisorpointofthemandibleduringfictivemastication inducedbyelectricalstimulationtothecorticalmasticatoryareaofanesthetizedrabbits.Thejawmovementsignalwasusedtodeliverthestimulitotheinferioralveolarnerve(IAN)atgivenjawpositionsasfollows:1)halfwayofjawclosingphase,2)endoftheclosingphase(ECP),3)midpointoftheocclusalphase,4)themaximumjawopenedposition.TheJORamplitudeatECPwasmostsuppressed(17.9%ofthecontrol)comparedwiththeotherposi-tions,andthesignificantdifferenceexistsbetweentheworkingsideandbalancingsideonECP.BecausethemandibularpositionatSOPwasexactlyjustbeforecontactingoftheopposingmolars,itiscon-ceivablethatsuchJORsuppressiondependingonthemasticationsideatECPdoesnotresultfromafferentinformationfromtheIANinducedbyteethcontactthatisuniquetotheworkingsideduringocclusalphaseofmastication. ItmaybeconcludedthatthestrongerJORsuppressionontheworkingsideatECPcouldbeadvantageoustodevelopstrongerbitingforceontheworkingatwhichthelowthresh-oldmechanoreceptorintheperiodontalligamentisinevitablyactivat-edbyteethcontact.NoCOI.

1P-211Spatial profile of neural excitation in rat somatosenso-ry cortex evoked by electrical stimulation of tooth pulps: an optical imaging studyNakamura, Hiroko1; Koshikawa, Noriaki2; Kobayashi, Masayuki2(1Department of Pediatric Dentistry, Nihon University School of Dentistry,Tokyo,Japan, 2Department of Pharmacology, Nihon University School of Dentistry,Tokyo,Japan)

Nociceptionisprocessedintheseveralregionsinthecerebralcortexincludingthesomatosensorycortex(SS).Thetopographicalrepresen-tationintheSSiswellidentified,however,itisstillanopenissuehowthenociceptionofthemolartoothpulpsisprocessedintheSS.Thisissueiscriticaltounderstandthemechanismoftoothpain,becausepatientswithtoothpainoftenclaimthattheycannot identifythediseasedtooth.Toexaminethepreciseregionsrespondingtotheelectricalstimulationof themaxillaryandmandibularmolartoothpulps,weperformedtheoptical imagingtechniquewithavoltagesensitivedye,whichenablesustovisualizeneuralexcitability inamacroscopicmanner.ElectricalstimulationofthemolartoothpulpsevokedneuralexcitationinthetwopartsoftheventralpartoftheSS:(1)therostroventralpartoftheSSadjacenttothetongueregion,and(2)theventralpartoftheSSaroundthemiddlecerebralartery.TheformerSSregionrespondedtoboththemaxillaryandmandibularmolartoothpulpstimuli.Ontheotherhand,thelaterSSregioncouldbefurtherdividedintorostralandcaudalsubregions,whichrespond-edtothemaxillaryandmandibulartoothpulpstimulation,respective-ly.TheseresultssuggestthatapartoftheSSrespondstoboththemaxillaryandmandibulartoothpulps,andthismightbeareasonfordifficultyinidenfyingadiseasedtooth.NoCOI.

1P-212Involvement of the red nucleus in the suppression in-duced by stimulation of the raphe magnus nucleus on the jaw-opening reflex Satoh, Yoshihide; Ishizuka, Ken’Ichi; Iwasaki, Shin-ichi(Department of Physiology, Nippon Dental Univertisy School of Life Dentistry at Niigata, Niigata, Japan)

Wefoundthatstimulationoftherednucleus(RN)modulatedthelow-andhigh-thresholdafferent-evokedjaw-openingreflexes(JORs).IthasbeenreportedthattheRNreceivesprojectionfibersfromtheraphemagnusnucleus(RMg),andstimulationoftheRMginhibitedtheno-ciceptiveJOR.ThepresentstudyaimedtoinvestigatetheeffectsofRMgstimulationbetween low-andhigh-thresholdafferent-evokedJORs,andwhetherRMg-inducedmodulationoftheJORisaffectedbyelectriclesionoftheRN.Theexperimentswereperformedonanes-thetizedrats.TheteststimulationwasappliedtotheinferioralveolarnervetoevoketheJOR.Thestimulusintensitywaseither1.2(lowthreshold)or4.0(highthreshold)timesthethreshold.Theelectromyo-gramswererecordedfromthedigastricmuscles.TheconditioningstimulationwasappliedtotheRMg.ThecontrolJORresponseswererecordedaswellasthemodulationinducedbystimulationoftheRMg.TheRNlesionwasmadebythepassageofelectriccurrent.TheeffectoftheRMgstimulationontheJORwastestedattheterminationoflesion.Stimulationof theRMgmodulatedthe low-thresholdaffer-ent-evokedJOR.StimulationoftheRMgsignificantlysuppressedthehigh-thresholdafferent-evokedJOR.ElectricallyinducedlesionsoftheRNreducedtheRMg-inducedsuppressionoftheJORevokedbythehigh-thresholdafferents.TheseresultssuggestthatthesuppressiveeffectofRMgstimulationontheJORismediatedinpartbyarelayintheRN.NoCOI.


Poster PresentationsEndocrinology (1)

1P-213Involvement of a neurosteroid 7α-hydroxypregneno-lone in the expression of courtship behavior of the male newtToyoda, Fumiyo1; Hasunuma, Itaru2; Nakada, Tomoaki3; Haraguchi, Shogo4; Tsutsui, Kazuyoshi4; Kikuyama, Sakae4(1Physiol Dept-I, Nara Medi Univ, Nara, Japan, 2Dept Biol, Faculty of Sci, Toho Univ, Chiba, Japan, 3Dept of Comp Behav Med, Nippon Veterinary and Life Sci Univ Tokyo, Japan, 4Dept of Biol, Waseda Univ, and Center for Med Life Sci of Waseda Univ, Tokyo, Japan)

Aneurosteroid,7α-hydroxypregnenolone,hasrecentlybeenfoundtoenhance locomotoractivity inthemalenewt,Cynops pyrrhogaster.Here,weshowthatthisneurosteroidisalsoinvolvedinenhancingtheexpressionofcourtshipbehavior.Intracerebroventricular(ICV)injec-tionof7α-hydroxypregnenoloneenhancedcourtshipbehaviordose-de-pendentlyinthesexuallyundevelopedmalesthathadbeenpretreat-edwithprolactinandgonadotropin,whichisknowntobringthemalestoasexuallydevelopedstate.Inthemalesreceivingnopriorinjectionsofthesehormones,however,theneurosteroiddidnotelicitthebehav-ior.ICVadministrationofketoconazole,aninhibitorofthesteroidogen-icenzymecytochromeP450s,suppressedthespontaneouslyoccurringcourtshipbehavior insexuallyactivemales.Supplementationwith7α-hydroxypregnenolonereversedtheeffectofketoconazoleintheseanimals.ItwasalsodemonstratedthattheeffectoftheneurosteroidonthecourtshipbehaviorwasblockedbyadopamineD2-like,butnotbyaD1-like,receptorantagonist.Theseresultsindicatethatendoge-nous7α-hydroxypregnenoloneenhancestheexpressionofthemalecourtshipbehaviorthroughadopaminergicsystemmediatedbyaD2-likereceptorinthebrain.NoCOI.

Poster PresentationsKidney, Urination

1P-214H+ secretion across the distal tubule in perfused bull-frog kidneyShiraiwa, Yuka; Daikoku, Eriko; Saito, Masahisa; Yamaji, Junko; Kubota, Takahiro(Department of Physiology, Faculty of Medicine, Osaka Medical College, Takatsuki, Japan)

Inthisstudy,weexaminedtheH+transportinthedilutionsegment(distaltubule)bymeasuringtransepithelialpotential(TP)withpHinthetubularfluid(pHTF)inperfusedbullfrogkidneywithdouble-barreledion-selectivemicroelectrodes.Futhermore,thesameexperimentswereconductedintheproximaltubule.DecreasingpHintheperitubularperfusionsolutionfrom7.7to6.7bydecreasingHCO3

-concentrationproducedtheincreaseintheTPfrom~+10mVto~+15mVwithtriphasicchangeinpHTF(initialslightincrease,seconddecrease,fol-lowedbytheincreaseinintheinthedistaltubule).Ontheotherhand,decreasingpHintheperitubularperfusionsolutionproducedthein-creaseintheTPby2mVwithbiphasicchangeinpHTF (aninitialslight increasefollowedbythedecrease inpHTF). TheperitubularperfusionwiththeacidsolutionproducedthedecreaseincytosolicpHinproximaltubulecellwithdepolarizationofperitubularmembranepotential.10-6MbafilomycincausedtheinitialdecreasefollowedbytheincreaseinpHTFinbothdistalandproximaltubule.TheseresultsindicatethatH+secretionsystemprimarilydependsonthesamemechanisms,suchasNa+/H+exchangerorH+-ATPaseintheluminalmembraneandelectrogenicHCO3

- transporter intheperitubularmembrane,inboththeproximalandthedistaltubule,probablyexceptH+extrusionmechanismintheluminalmembraneinthedistaltubule.NoCOI.


1P-215Urinary Bladder Mucosal Responses to Chronic Isch-emiaSunagawa, Masataka1,2; Wolf-Johnston, Amanda2; Nomiya, Masanori3,4; Sawada, Norifumi4,5; Andersson, Karl-Erik4; Hisamitsu, Tadashi1; Lori Ann, Birder2(1Dept. Physiology, Showa University School of Medicine, Tokyo, Japan, 2Dept. Medicine, University of Pittsburgh School of Medicine, PA, USA, 3Dept. Urology, Fukushima Medical University School of Medicine, Fukushima, Japan, 4Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, NC, USA, 5Dept. Urology, Interdisciplinary Graduate School of Medicine, University of Yamanashi, Yamanashi, Japan)

Chronicbladder ischemia (CBI)hasbeenshownto leadtobladderdysfunctions.Weinvestigatedtheexpressionofvariouscellularpro-teinswithintheurothelium(UT)andlaminapropria(LP)followingCBIintheraturinarybladder.UrinarybladderswereremovedfromadultSDratsthatunderwentaballoonendothelialinjuryoftheiliacarteries.Weexamineddistri-butionsofLP-vimentin-immunoreactive(IR)cellsandUT-connexins(Cx26,Cx43);andadditionalproteinsinvolvedinUTbarrierandsen-soryfunctions.CBIwasassociatedwithsignificantincreasesinvimentin-IRcellsandincreasedexpressionofthegap-junctionproteinsCx26andCx43ascomparedtoshamcontrol.CBIalsoresultedinchangesinexpressionlevelsofjunctionalmarkers(ZO-1)andNGFaswellasnorepinephrine.OurfindingsrevealthatCBIaltersanumberofproteinswithintheurotheliumandunderlyinglaminapropria.Theseproteinsareinvolvedinremodeling,repairaswellasintercellularcommunication.Inaddition,theincreasedexpressionofvimentin-IRcellsalsosuggeststhatchang-esincell-cellinteractionscouldplayaroleinCBI-inducedchangesinbladderactivity.NoCOI.

1P-216New methods for in vivo study of rapid homeostatic response of the kidney to calcium reduction in the mouse.Murata, Miyahiko1(1Department of Medical Care and Welfare Engineering, School of Industrial and Welfare Engineering, Tokai University, Kumamoto, Japan, 2Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan)

Calcium(Ca)homeostasisofthebodyfluidiswellknowntobestrict-lyregulatedbytwohormonalsystems,i.e.,parathroidhormoneandvitaminDsystems.ThekidneyisalsoassociatedwiththeCahomeo-stasisbyactiveresorptionofCaatthedistalconvolutedtubule,how-ever, itsautonomouscontributiontotheCahomeostaticresponse,especiallytotheacuteCadecrease,isunclear.Toelucidatetheacute,dynamicregulationofCahomeostasisbythekidney,Idevelopedatechniquetocollectbloodsimultaneouslyfromboththerenalarteryandveinintheanesthetizedmouse.ThistechniqueenableddifferentialmeasurementofCaconcentrationoftheinput-outputofakidney,byusingi-Stat(freeCaconcentration)and/orDriChem(totalCaconcen-tration)systems.Inaddition,forrapidloweringofCaconcentration,Iusedanovel“dilutionmethod”.Inthismethod,theintravenousinjec-tionofcalcium-freeperfusatelowersCaconcentrationtotheaimedlevelandthedilutionratecanbeconfirmedbymeasuringhematocrit.Astheresult,CaconcentrationintheoutputveinkepthigherthanthatintheinputarteryofthekidneyatallperiodsafterloweringCa.ThissuggeststhekidnyautonomouslyelevatesCaconcentrationinresponsetorapiddrop.NoCOI.

1P-217Effect of a hydrogen-enriched solution on the albumin redox of hemodialysis patients.Terawaki, Hiroyuki1,3; Zhu, Wan-Jun1; Terada, Tomoyoshi2; Matsuyama, Yukie2; Takahashi, Yasuhito1; Sakurai, Kaoru1; Kabayama, Shigeru3; Miyazaki, Mariko3; Itami, Noritomo3; Nakazawa, Ryoichi3; Ito, Sadayoshi3; Nakayama, Masaaki1,3; Era, Seiichi2(1Dialysis Center, Fukushima Medical University, Fukushima, Japan, 2Department og Physiology and Biophysics, Gifu University School of Medicine, Gifu, Japan, 3Electrolyzed Water Hemodialysis Study Group, Osaka, Japan)

Elevatedoxidativestress(OS)isassociatedwithseverecardiovasculardiseaseandprematuredeathamongpatientstreatedwithhemodial-ysis (HD).OS isenhancedbycontactbetweenbloodanddialysismembranesduringHDsessions.Thisstudyaimedtoclarifywhetherhydrogen(H2),whichisaknownantioxidant,iscapableofsuppressingincreasedOSinducedduringHDsessions.EightpatientsonregularHDtreatmentwerestudied.TwoHDsessionswereperformedinacross-overdesigntrialusingstandard(S-HD)andhydrogen-enrichedsolutions (H2-HD).Bloodsampleswereobtainedfromthe inletandoutletof thedialyzerduringHDtodeterminechangesinplasmalevelsofglutathione,hydrogenperoxide,andalbu-minredoxstateasamarkerofOS.Comparisonof inletandoutletbloodrevealedsignificantdecreasesintotalglutathioneandreducedglutathione,aswellassignificantincreasesinhydrogenperoxideinbothHDtreatments.However, themeanproportionofreversiblyoxidizedalbumininoutletserumwassignificantlylowerthanininletserumfollowingtheH2-HDsession,whereasnosignificantchangeswerefoundinS-HD,suggestingthatintra-dialyzerOSisreducedbyH2-HD.Inconclusion,theapplicationofH2-enrichedsolutionscouldameliorateOSduringHD.NoCOI.



2P-001Identification of amino acid residues involved in the TRPA1 inhibition by utilizing species specific differ-enceGUPTA, RUPALI1,2; SAITO, SHIGERU; TOMINAGA, MAKOTO1,2(1Division of Cell Signaling, Okazaki Institute for Integrative Bioscience (National Institute for Physiological Sciences), National Institutes of Natural Sciences, Okazaki, Japan., 2Department of Physiological Sciences, The Graduate University for Advanced Studies, Okazaki, Japan)

TransientreceptorpotentialankyrinA1(TRPA1)receptorisamem-berofTRPsuperfamilyandanexcitatorynonselectivecationchannel.AnincreasingbodyofevidencesuggeststhatTRPA1actsasanoci-ceptivereceptor forvariouschemicalsandphysicalstimuli.ManyTRPA1antagonistshavebeendevelopedandsomeofthemfunctionasanalgesicagents.ApreviousstudyshowedthatAP18,amammali-anTRPA1antagonist,didnotinhibitheterologouslyexpressedwesternclawedfrogTRPA1(fTRPA1).Here,IshowthatfTRPA1isalsoin-sensitivetoA967079(A96),oneofthemostpotentmammalianTRPA1antagonistwhich isstructurallysimilartoAP18.InaheterologousexpressionsystemwithXenopusoocytes,A96failedtoinhibitfTRPA1activationelicitedbyTRPA1agonists.Mutantchannelanalysesre-vealedthatspecifictwoaminoacidresidueslocatedwithinthefifthtransmembranedomainwereinvolvedintheinhibitoryactionofA96.SameaminoacidswerepreviouslyreportedtobeinvolvedintheAP18inhibition.Thesefindingsarebasedonspeciesdifferencesinsensitiv-ityofTRPA1antagonistsprovidenovelinsightsforstructural-functionrelationshipofTRPA1andusefulinformationinthesearchfornewanalgesicmedicinestargetedforTRPA1.NoCOI.

2P-002Heat and AITC activate green anole TRPA1 in a membrane-delimited mannerKurganov, Erkin1,2; Zhou, Yiming1,3; Saito, Shigeru1,3; Tominaga, Makoto1,2,3(1Division of Cell Signaling, National Institute for Physiological Sciences, 2The Graduate University for Advanced Studies, SOKENDAI, 3Okazaki Institute for Integrative Bioscience)

Transientreceptorpotentialankyrin1(TRPA1)isaCa2+-permeablenon-selectivecationchannelthatisactivatedbyvariousnoxiousstim-uli.TRPA1wasinitiallyidentifiedasapotentialmediatorofnoxiouscoldstimuliinmammaliannociceptivesensoryneuronswhileTRPA1sfromnon-mammalianvertebrates (snakes,greenanole lizardsandfrogs)wererecentlyreportedtobeactivatedbyheat,butnotcoldstimulus.Inthisstudy,weexaminedpropertiesforTRPA1channelofgreenanole(gaTRPA1)relatingtothermalandchemicalstimulationinwhole-cellandsingle-channelrecordings.HeatandAITCactivategaTRPA1bothsolelyandsynergistically inwhole-cell level. IntheabsenceofextracellularCa2+verysmallcurrentswereobserveduponheatstimulation,whilecurrentsizesanddesensitizationweresimilarboth inthepresenceandabsenceofextracellularCa2+uponAITCstimulation.gaTRPA1channelswerealsoactivatedbyheatandAITCinexcisedmembranepatcheswithan inside-outconfiguration. Bycomparingthekineticsofheat-andAITC-evokedsingle-channelcur-rents,wedefinedsimilaritiesanddifferencesofgaTRPA1channelresponsestoheatandAITC.Weobservedsimilarcurrent-voltagerelationshipandunitaryamplitutdesforheat-andAITC-evokedcur-rentswhilewefoundthatheat-activatedcurrentsshowedshorterdurationsofbothopen-andclosed-times.OurresultssuggestthatthegaTRPA1channelisdirectlyactivatedbyheatandchemicalstimuli.NoCOI.

2P-003Evolution of vertebrate TRPA1 channel as O2 sensorYamaguchi, Kaori; Takahashi, Nobuaki 1; Mori, Yasuo(Dept Technol & Ecol, Kyoto Univ, Kyoto, Japan)

Thefluctuations inatmosphericoxygen (O2) levelhavedriventheevolutionoforganisms.Approximately2billionyearsago,whenO2wasgeologicallyaccumulatedaftertheevolutionofoxygenicphoto-synthesisincyanobacteria,mitochondrionwasingestedintheprogen-itorcellofEukaryota,enablingthereductionofO2toxicityaswellastheefficientproductionofATP.Approximately200millionyearsago,whenO2wasgeologicallyreducedpresumablybytheglobalvolcanicaction,ancestorofmammalianacquiredviviparity,enhancingO2sup-plytotheirfetus.Furthermore,higherorganismshaveacquiredthesensingsystemsoflow-O2(hypoxia)andhigh-O2(hyperoxia)tosecureenergyproductionwhilediminishingtheriskofO2toxicity.However,itremainsunclearwhenanimalsacquiredtheO2sensingsystemsintheprocessofevolution.OurgrouphaspreviouslyshownthatmouseandhumanTRPA1channeldetectsO2availabilityinvagus.Inthisstudy,weexploredtheO2sensitivityofTRPA1channelsindifferentspecies(amphibia,avian,andmarsupial).Geneticanalysisshowedthatxenopus,chicken,andwallabyTRPA1didnotobtaintheprolylresidueresponsibleforhypoxiasensinginhumanTRPA1,whereasviviparaobtainedtheprolylresidueonTRPA1.Electrophysiologicalmeasure-mentsrevealedthatchickenTRPA1failedtorespondtohypoxia.Thus,it ishighlypossiblethatTRPA1acquiredO2sensingmechanismsaroundthesametimeastheacquirementofviviparityfunction,sug-gestingthathypoxicenvironmentapproximately200millionyearsagohavedriventheacquirementofO2sensingmechanismsaswellasthatofviviparityfunction.NoCOI.

2P-004Model Simulation of PLC-operated TRPC Currents Regulating by PI(4,5)P2-DAG SignalingMori, Masayuki X1; Itsuki, Kyohei2,3; Imai, Yuko2,3; Okamura, Yasushi4; Inoue, Ryuji3; Hase , Hideharu1; mori, yasuo1(1Department of Synthetic Chemistry and Biological Chemistry, Kyoto University, Kyoto, Japan, 2Kyushu University, Faculty of Dental Sciences, Fukuoka, Japan, 3Fukuoka University, Department of Physiology, Fukuoka, Japan, 4Osaka University, Department of Integrative Physiology, Osaka, Japan)

Transientreceptorpotentialclassical/canonical(TRPC)3,C6andC7arenon-selectivecationchannels,playingkeyrolesincardiovascular,gastrointestinal,renalandnervoussystemphysiology.Wehavere-centlyreportedthatthedepletionofphosphatidylinositol4,5-bisphos-phate [PI(4,5)P2] inhibitsTRPCchannelactivity, irrespectivetothepresenceofthechannelsactivator‘diacylglycerol(DAG)’.However,thecomplexregulationbyPI(4,5)P2andDAGtothePLC-coupledreceptorTRPCcurrentsisdifficulttoaddresshowtherespectiveregulatorsdifferentiallycontributetothesechannels.Toaidanintuitiveunder-standingofthiscomplexregulation,weherebuiltaPLC-coupledTRPCchannelmodelconsistingofPI-turnovermetabolism,channelgating,andbehaviorofPI(4,5)P2sensorproteins(CFP/YFPfusedPH-domain).Respectivesectionsofthismodelweremadebasedontheenzymaticreaction,theligand-gatedchannels,andprotein-ligandisotherms,re-spectively.Byaccordanceofthesimultaneousmeasurementsoflipiddynamics/TRPCcurrentsandthemodelsimulation,ourresultsdemonstratedthatthestrengthofPLCactivityiscriticaltotheap-pearancesofTRPCcurrents,suchasthecurrentamplitudes,thetotalionicinfluxandthepeaktimethroughthevariouschangesofPI(4,5)P2andDAG.NoCOI.


2P-005Abnormal receptor-response and mechanosensitivity of mutant TRPC6 channels associated with familial focal segmental glomerulosclerosis (FSGS) - involve-ment of cytoskeleton and podocinIchikawa, Jun; Inoue, Ryuji(Dep.Physiol., Fukuoka Univ.Sch.Med., Fukuoka, Japan)

Severalmutationsof transientreceptorpotentialcationchannel6(TRPC6)inhumanpodocytesareknowntocausetheinherentformsofglomerosclerosis,FSGS.WeinvestigatedthefunctionalimpactsofmurineTRPC6FSGSmutationsnearitsN-terminalankyrinrepeats(G108S,P111Q,N124S,M131T,N142S,R174Q)inheterologousexpres-sionsystemwithCa2+imagingandpatchclamptechniques.The111Q,131Tand142Smutantsshowedenhancedreceptorresponsestocar-bachol. Incontrast,exceptforsomeof131Tmutantsbeinghighlymechanosensitive,allFSGSmutantsshoweddiminishedmechanicalresponsesto2,4,6-trinitrophenolwhichisnormalinwild-typeTRPC6.Theenhancedmechanicalresponseof131Tmutantaswellasnormalmechanosensitivityofwild-typewereabolishedbydisruptingactincytoskeletonwithcytochalasinD.Incoimmunoprecipitationexperi-ments,simultaneousapplicationofreceptorstimulationandmechani-calstressenhancedthephysical interactionofactinwithTRPC6protein,whichwasvariablyaffectedbyFSGSmutations.Co-expressionofTRPC6withpodocin,aslitdiaphragmprotein,suppressedbothreceptorandmechanicalresponses.TheseobservationsindicatethatFSGSmutationsnearN-terminalankyrinrepeatdomainsalterthedegreeofreceptoractivationofTRPC6,anditsmechanosensitivityandinteractionwithactin.SuchdifferencesmaypartlyexplaintheetiologyofFSGSinwhichdegenerationofpodocytesleadstodisruptionofrenalfiltrationbarrier.NoCOI.

2P-006Redox signal-mediated sensitization of Transient Re-ceptor Potential Melastatin 2 (TRPM2) to tempera-tures affects insulin secretion from the pancreatic β-cellsKashio, Makiko1; Mori, Yasuo2; Tominaga, Makoto1(1Division of Cell Signaling, OIIB, NIPS, Okazaki, Japan, 2Kyoto Univ., Kyoto, Japan)

Pancreaticβ-cellhasauniquefunctioncouplingglucosesensingandmetabolismtoinsulinsecretion,avitalprocessinenergyhomeostasis.Inthepancreaticβ-cell,reactiveoxygenspecies(ROS)includingH2O2aregeneratedconcomitantlywiththeglycolyticandrespiratoryme-tabolism.Interestingly,expressionlevelsofantioxidantenzymesareextremelylowinthepancreaticβ-cellscomparedwithothertissues.ThisraisesthepossibilitythatROSproducedincidentaltobloodglu-coseelevationcouldservesignalingfunctionsinglucose-inducedinsu-linsecretionfromthepancreaticβ-cells.TRPM2isaCa2+-permeablecationchannelandexpressedinvarioustissuesincludingbrain,spleen,kidneyandpancreaticβ-cellswhereTRPM2issurroundedbybodytemperatures.WehavepreviouslyfoundanactivationmechanismofTRPM2wherebyitsheat-evokedresponseisdynamicallyelevatedbyH2O2,termed“sensitization”.TheregulatorymechanismbyH2O2isthoughttoenableTRPM2toactivateatbodytemperaturebyloweringtemperaturethresholdfromsupra-physiologicaltophysiologicallevels.InWTpancreaticβ-cells,H2O2enhancednotonlyheat-evoked[Ca2+]i-in-creasebutalsoglucose-induced [Ca2+]i-oscillationat37°C.However,theseeffectsofH2O2werenotobservedinTRPM2KOcells.Becauseanoscillatory increase in [Ca2+]i isakeyfeature inglucose-inducedinsulinsecretionfromthepancreaticβ-cells,wewillshowthedifferenceininsulinsecretionbetweenWTandTRPM2KOpancreaticislets.NoCOI.

2P-007Involvement of TRPM6/TRPM7 heterotetramers in maternal-fetal calcium transportSuzuki, Yoshiro1,2; Watanabe, Masaki1; Saito, Claire1; Fukuta, Naomi1; Tominaga, Makoto1,2(1Division of Cell Signaling, Okazaki Instutute for Integrative Bioscience (NIPS), Okazaki, Japan, 2SOKENDAI, Okazaki, Japan)

SerumCa2+levelinfetusshouldbefinelymaintainedinordertosus-tainthedevelopmentandfunctionofthebrain,muscleandothertis-sues. Inparallel,duringthe lasttrimester, fetusdevelopsbonestosurviveafterbirth.However,themolecularcomponentsaswellasthemechanismofregulationofthematernal-fetalCa2+transporthavenotbeenfullyunderstood. Inthisstudy,we identifiedTRPM6,aCa2+permeablecationchannelasacandidateapicalCa2+entrypathwayformaternal-fetalCa2+transport.TRPM6mRNAwasdenselylocalizedintheintraplacentalyolksac,whichwasreportedtobeimportantformaternal-fetalCa2+transportinrodents,anditsmRNAlevelsignifi-cantlyincreasedduringthelast4daysofpregnancy,whichcoincidedwithfetalboneformationinmice.Moreover,inprimarymousetro-phoblasts,usingCa2+-imagingandpatch-clampmethodsweobservedan intracellularCa2+ increaseandcurrentwithcharecteristicsofTRPM6/TRPM7heterotetramer.ThesecurrentswerealsoobservedinhumanTRPM6c-transfectedHEK293cellswhichexpresshumanTRPM7endogenously.However,thesecurrentswerenotobservedinmock-ormouseTRPM6-transfectedHEK293cells.TheseresultssuggestthatTRPM6isinvolvedinthematernal-fetalCa2+transport,formingtetramerswithTRPM7inplacentaltrophoblasts.NoCOI.

2P-008TRPM7 is highly expressed in the odontoblastsKatagiri, Chiaki1; Tsumuraya, Tomoyuki1; Okamoto, Fujio2; Koji, Okabe2; Matsushita, Masayuki1(1Dep of Molecular and Cellular Physiology, Grad Sch of Medicine, University of the Ryukyus, Japan, 2Section of Cellular Physiology, Fukuoka Dental College, Japan)

Transientreceptorpotential(TRP)ionchannelfamilyiswellknowntoplayaroleinasensorsuchastemperature,osmoticpressure,andredoxstatus.AmongTRPchannelfamily,TRPM7hasauniquestruc-tureorganizationthatcontainsaTRPchanneldomainwith6trans-membranesegmentsfusedtoanatypicalserine-threoninekinasedo-mainatitsCterminus.GeneticdeletionofTRPM7inmodelsystemsdemonstratesthatthischanneliscriticalforcellulargrowthandem-bryonicdevelopment.Inthisstudy,wefoundthatTRPM7ishighlyexpressedinodontoblastsinthedentalpulpbyinsituhybridizationofmouseembryo.Quantitativereal-timePCRanalysisrevealedthatexpressionofTRPM7inthetoothwasremarkablyhigherthananyothertissueofadultmouse.WealsoconfirmedthatTRPM7proteinisexpressedinodontoblastsbyimmunohistochemistry.ToinvestigatethephysicalfunctionofTRPM7inodontoblasts,weexaminedTRPM7channelactivitiesusingratodontoblastcell line.Theseresultssug-gestedthathigherexpressionofTRPM7playasanimportantroleinodontoblasts.NoCOI.


2P-009TRPM7 kinase activity is not necessary for oxidative stress-induced current inhibition.Inoue, Hana1; Murayama, Takashi2; Konishi, Masato1(1Department of Physiology, Tokyo Medical University, Tokyo, Japan, 2Juntendo Univ. Sch. Med. Tokyo, Japan)

TRPM7isaCa2+/Mg2+permeablenon-selectivecationchannelwhichcontainsakinasedomainat itscarboxylterminal.WepreviouslydemonstratedthatTRPM7currentwasinhibitedbyoxidativestress.Inthepresentstudy,weexploredthemechanismsforredoxsensingbyintroducingmutationsinTRPM7.Tetracycline-inducibleHEKcelllines forstablyexpressingwildtype (WT),kinasedomaindeleted(deltaK),M1596A(MA),andK1645R(KR)murineTRPM7wereestab-lished.Whole-cellrecordingsinWTrevealedthatTRPM7currentwasinhibitedbyoxidativestressinducedbyhydrogenperoxide(H2O2,500 µM) in a [Mg2+]i dependentmanner (2.8±2.7%, 26.3±1.4% or82.0±5.7%inhibitionunder0,7.9or232µM[Mg2+]iconditions,respec-tively).H2O2inhibited24.3±4.2%ofdeltaKcurrentintheabsenceofintracellularMg2+.However,Mg2+dependent inhibitioncouldn’tbeexamined,sincedeltaKcurrentexhibitedrundownwithMg2+-contain-ingintracellularsolutions.M1596isacorrespondingresidueofM1755ofTRPM6whichhasbeenreportedtobeoxidizedbyH2O2andinvolvedinthecurrentinhibition.WhilemutationofM1755toalaninemadeTRPM6resistanttoH2O2,TRPM7MAmutantremainedsensitivetoH2O2.MoreoverkinaseinactiveKRmutantexhibitedthecurrentin-hibitionbyH2O2similartoWT(12.0±1.5%,50.5±10.2%or81.8±2.3%inhibitionunder0,7.9or232µM[Mg2+]iconditions,respectively).ItisconcludedthatM1596andK1645arenotindispensableforinhibitingTRPM7currentinresponsetooxidativestress.NoCOI.

2P-010Investigation the effect of palmitoylation on TRP channelsZhou, Yiming1; Oku, Shinichiro2; Fukata, Masaki2; Tominaga, Makoto1(1Division of Cell Signaling, Department of Cell Physiology, National Institute for Physiological Sciences, Okazaki, Japan, 2Division of Membrane Physiology, Department of Cell Physiology, National Institute for Physiological Sciences, Okazaki, Japan)

S-palmitoylationisacommonreversiblepost-translationalmodificationofmanyproteins.Ithasbeenshowntocontrolproteinsurfaceexpres-sion,spatialorganization,protein-proteininteractions,andfunctionalactivity.DysregulationofS-palmitoylationhasbeenreportedtobeassociatedwitha largenumberofdiseases,suchasschizophrenia,mentalretardationandcancer.Recently,someionchannels,suchasAMPA,NMDA-typeglutamatereceptors,potassiumandsodiumchan-nelshavebeenproventoberegulatedandinfluencedbypalmitoylation.TRPchannelsarenon-selectivecationchannels.Theyareexpressedinmosttissuesandcelltypes,areactivatedbyawiderangeofstim-uliandplayimportantroles.ManystudieshaverevealedthattheTRPchannelsareimportantforsensationssuchastaste,pain,andtempera-ture,bothintheperipheralandcentralnervoussystems.DefectsandabnormalitiesinTRPchannelsareassociatedwithmanydiseasessuchaspaindisorder,polycystickidneydiseaseandcancer.ThisstudyisdesignedtoclarifywhetherandhowTRPchannelsareregulatedbypalmitoylation.Inaddition,thisstudyalsoexamineswhethertheab-normalpalmitoylationstateofTRPchannelsareassociatedwithanydiseasesinourbody.NoCOI.

2P-011Analysis of the propofol-induced mammal TRP chan-nel activation.Nishimoto, Rei1,2; Kashio, Makiko2; Tominaga, Makoto1,2(1Physiological Sciences, Sokendai, Okazaki, Japan, 2Cell signaling, OIIB, NIPS, Okazaki, Japan)

Propofol (2,6-diisopropylphenol) isoneofthe intravenousanestheticdrugsandcommonlyusedinclinicalfieldforgeneralanesthesia.Ad-ditionally,itcausesanintensepainuponinjection.Tounderstandthemechanismofpropofol-inducedpain,wefocusedontransientreceptorpotential(TRP)receptorsvanilloid1(TRPV1)andankyrin1(TRPA1),whichareimportantionchannelsforpainsensation.Weperformedcalcium-imagingsandwhole-cellpatch-clamprecordingsbyusingmousedorsalrootganglion(DRG)cellspreparedfromwild-type(Wt)andknockout(KO)mice.Inthecalciumimagingstudy,therewasnodifferenceinpercentageofthepropofol-responsiveDRGcellsisolatedfromWt,TRPV1KOandTRPA1KOmice.Propofol-inducedcalciuminfluxwerestillobservedintheDRGcellsfromTRPV1/A1doubleKOmice.Picrotoxin,anantagonistofGABAAreceptors,completelyinhibitedthepropofol-inducedcalciuminfluxobservedintheDRGcellsfromTRPV1/A1doubleKOmice.Inthewhole-cellpatch-clampstudy,propofolcausedinwardcurrentsintheDRGcellsfromTRPV1/A1doubleKOmiceandtheyalsorespondedtotheapplicationofGABA.TheresultsindicatethatpropofolmightcausecalciuminfluxintoDRGcellspartiallythroughGABAAreceptors.NoCOI.

2P-012Inhibition of the compound action potentials of frog sciatic nerves by aroma-oil compounds having vari-ous effects including TRP activationOhtsubo, Sena; Matsushita, Akitomo; Fujita, Tsugumi; Jiang, Chang-Yu; Xu, Zhi-Hao; Kumamoto, Eiichi(Dept. Physiol., Saga Med. Sch., Saga, Japan)

Wehavepreviouslyreportedthatcapsaicin,mentholandallylisothio-cyanate,whichactivateTRPV1,TRPM8andTRPA1,respectively,inhibit fast-conductingcompoundactionpotentials (CAPs)recordedfromthefrogsciaticnervewithoutTRPactivation.Thepresentstudyexaminedhowaroma-oilcompoundshavingvariousactionsincludingTRPactivationaffectfrogsciaticnerveCAPsbyusingtheair-gapmethod.Citral,whichactivatedTRPV1,TRPM8,TRPA1andTRPV3,attenuatedCAPpeakamplitudeswiththehalf-maximal inhibitoryconcentration(IC50)valueof0.48mM;thisactionwasresistanttoanon-selectiveTRPantagonistrutheniumred.Camphor,aTRPV1andTRPV3agonist,alsoreducedCAPpeakamplitudes(by30%at5mM)inamannerinsensitivetorutheniumred.Lavender-oilcompounds,linalylacetateandlinalool,reducedCAPpeakamplitudeswiththeIC50valuesof0.49and1.65mM,respectively.Citronellal,L-bornylacetateandroseoxidereducedCAPpeakamplitudeswiththeIC50valuesof0.50,0.65and2.0mM,respectively,whilemyrceneatahighconcen-trationsuchas5mMhardlyreducedCAPpeakamplitudes.Aneffi-cacysequenceofthearoma-oilcompoundsfortheCAPinhibitionswasaldehydes(citral,citronellal)>esters(linarylacetate,L-bornylacetate)>alcohols(linalool)>oxides(roseoxide)>>hydrocarbons(myrcene).Itissuggestedthataroma-oilcompoundsinhibitnerveconductioninamannerspecifictotheirchemicalstructureswithoutTRPactivation.NoCOI.


2P-013Interaction among crude medicines contained in dai-kencyuto in frog sciatic nerve compound action po-tential inhibitionMatsushita, Akitomo; Ohtsubo, Sena; Fujita, Tsugumi; Jiang, Chang-Yu; Xu, Zhi-Hao; Kumamoto, Eiichi(Dept. Physiol., Saga Med. Sch., Saga, Japan)

WehavepreviouslyreportedthattraditionalJapanesemedicineshaveanabilitytoreducethepeakamplitudeoffrogfast-conductingcom-poundactionpotential(CAP)andthatdaikencyuto(DTK)isthemosteffectiveininhibitingCAPsamongvariouskindsofthemedicine.DKTiscomposedofthreekindsofcrudemedicine,ginseng,Japanesepep-perandprocessedginger.ThepresentstudywasundertakentoknowwhethertheircrudemedicinesinhibitCAPsandifsowhetherthereisaninteractionamongtheirinhibitoryactions.Theexperimentswereperformedbyapplyingtheair-gapmethodtothefrogsciaticnerve.Wheneachofthecrudemedicinesat2mg/mlwastested,JapanesepepperandprocessedgingerreducedCAPpeakamplitudeby70%and30%,respectively,whileginsengradixhardlyaffectedCAPs.TheinhibitoryactionofJapanesepepperhadahalf-maximal inhibitoryconcentration(IC50)valueof0.77mg/ml.Ginsengradix(0.6mg/ml),whichhadnoeffectonCAPs,unaffectedtheinhibitoryactiononCAPsofprocessedgingerinarangeof0.2to2mg/ml,buthadatendencytoenhancetheinhibitionofCAPsbylow(<0.5mg/ml)butnothigh(>0.5mg/ml)concentrationsofJapanesepepper.Processedginger(1mg/ml)alsohadatendencytoincreaseCAPinhibitionbyJapanesepepperatlowbutnothighconcentrations.ItissuggestedthatthereisaninteractioninnerveconductioninhibitionamongcrudemedicinescontainedinDKTinamannerdependentontheirconcentrations.NoCOI.

2P-014Molecular basis of voltage-dependent inactivation of TRPP3 channelsHiguchi, Taiga1; Shimizu, Takahiro1; Fujii, Takuto1; Nilius, Bernd2; Sakai, Hideki1(1Grad. Sch. Med. Pharm. Sci. Univ. Toyama, Toyama, Japan, 2KU Leuven, Leuven, Belgium)

Wepreviouslyfoundthattransientreceptorpotentialpolycystin3(TRPP3)-expressingHEK293Tcellsexhibitdistincttailcurrents in-ducedbyrepolarizationafterdepolarization,suggestingthatTRPP3channelsatanopenstatecantransittoaninactivatedstateinresponsetomembranedepolarization.Howeverithasnotbeenclarifiedmolec-ularbasisofthistransition.InsomeKvchannels,N-typeandC-typeinactivationmechanismshavebeenreported:theporeisoccludedbymovementofintracellularN-terminusintheN-typemechanism,andtheperipheralstructureofaselectivefilterisalteredintheC-typemechanism.To investigatethe inactivationmechanismofTRPP3channels,weconstructedseveralmutants:ΔN,Δ2-90aminoacidresi-dues;R534A;R534K;R534Q;4A,NANR(531-534)-AAAA;3A,NANR(531-534)-AAAR).IntheΔNmutant,thetailcurrentswerestillobservedbyrepolarizationafterdepolarization.Ontheotherhand,nosignificanttailcurrentswereobservedintheR534A,R534Qand4Amutants,whilethecurrentswereretainedintheR534Kand3Amutants.TheseresultssuggestthattheintracellularN-terminusisnotinvolvedintheTRPP3inactivation,andthatpositivechargeoftheouterporeresidueR534 isessential forthevoltage-dependent inactivationofTRPP3channels.NoCOI.

2P-015Molecular basis for the species difference of TRPV1 channel property related to temperature adaptation in Xenopus speciesSaito, Shigeru1,3; Ohkita, Masashi2; Saito, Claire T.1; Fukuta, Naomi1; Ohta, Toshio2; Tominaga, Makoto1,3(1Okazaki Institute for Integrative Bioscience (NIPS), Okazaki, Japan, 2Faculty of Agriculture, Tottori University, Tottori, Japan, 3 The Graduate University for Advanced studies, Okazaki, Japan.)

Ouraimistoclarifythemolecularmechanismoftemperatureadap-tationespecially focusingonevolutionarychangesof temperaturesensationamongdifferentspecies.Forthispurpose,wefocusedontwospeciesofclawedfrog (Xenopus tropicalisandXenopus laevis).TheoptimaltemperaturerangeofX. laevisis16–22°C,whilethatofX. tropicalis is24–28°C.WefirstcomparedthethermalbehavioralresponsesandfoundthatX. laevis ismuchmoresensitivetoheatstimulationthanX. tropicalis.Thus,weclonedTRPV1,whichservesasaheatreceptorinawidevarietyofvertebrates,frombothspeciesandcomparedthechannelproperties.TRPV1channelpropertydifferedbetweenthetwospeciesinthatX. laevisTRPV1exhibitedalmostfullactivity fromthefirstheatstimulation,whileX. tropicalisTRPV1exhibitedonlyapartialactivityinthefirstheatstimulationandtheactivitygraduallyincreasedwithrepeatedheatstimulation.Bychar-acterizingTRPV1chimerasandpointmutantchannels,wefoundthatthethreeaminoacidsubstitutionsinTRPV1betweenX. laevisandX. tropicalisareinvolvedinthespeciesdifferencesofchannelproper-ty.Inconclusion,weidentifiedthefunctionalchangeofatemperaturereceptorrelatedtotemperatureadaptationandalsoclarifiedthemo-lecularbasisforthedifferencesofthetemperaturereceptorfunction.NoCOI.

2P-016Alanin scan analysis in the pore region of TRPV1 channelMurata, Yoshimichi; Kazama, Itsuro; Maruyama, Yoshio(Department of Physiology, Tohoku University Graduate school of Medicine)

TRPchannelscansenseandareactivatedbydiverseenvironmentalstimulisuchaschangesintemperature,membranevoltage,mechani-calforceandinternalorexternalchemicalligands.HoweveritisnotknownthatwhyTRPchannelscouldbeactivatedbysuchvariousstimuli. Wewould liketorevealthemolecularbasis fortheTRPchannelgating.ArethereanycommonmechanismstoTRPchannelgating?TRPchannelsappeartohavemanybulkyaminoacidresiduesinporeregionscomparetoKvchannels.WestudiedtheroleofthebulkyresiduesinTRPV1channelfunction.WemadeseriesofalaninreplacementmutantsinS5andS6ofTRPV1.TRPV1andtheirmu-tantswereexpressed inHEK293cellsandpatchclamprecordingswereperformedtoevaluatethechangeofgatingproperties.Inaddition,weexaminedthesensitivityofmutantchannelstocapsaicin.NoCOI.


2P-017Activation of TRPV2 may inhibit the differentiation of mouse brown adipocytesSun, Wuping1,2; Uchida, Kunitoshi1,2; Takahashi, Nobuyuki3; Suzuki, Yoshiro1,2; Zhou, Yiming1; Kawada, Teruo3; Tominaga, Makoto1,2(1Division of Cell Signaling, Okazaki Institute for Integrative Bioscience (National Institute for Physiological Sciences), Okazaki, Japan, 2Department of Physiological Sciences, SOKENDAI, Okazaki, Japan)

Transientreceptorpotentialvanilloid2(TRPV2)isaCa2+-permeablenon-selectivecationchannel,whichhasbeenreportedtobesensitivetotemperature,mechanicalforceandsomechemicals.TRPV2playsvitalrolesintheregulationofvariouscellularfunctions.However,themolecularidentityandfunctionofTRPV2remainsunexploredinmousebrownadipocytes.ThisstudyaimedtoclarifytheexpressionofTRPV2anditsfunctioninbrownadipocytes.WefoundthatTRPV2mRNAandproteinareexpressedinbrownadipocytes.ElectrophysiologicalresultsdemonstratedthatfunctionalTRPV2ispredominantlyobservedinbrownadipocytes.Moreover,theexpressionofTRPV2wasdramat-icallyincreasedduringthedifferentiationofbrownadipocytes.Non-se-lectiveTRPV2agonists,2-Aminoethoxydiphenylborate (2APB)andlysophosphatidylcholine (LPC) inhibitedthedifferentiationofbrownadipocyteswithadose-dependentmannerandthe inhibitionwasrescuedbyaTRPV2selectiveantagonist,SKF96365. Inaddition,mechanicalstimulation,whichactivatesTRPV2,alsostrength-de-pendentlyinhibitedthedifferentiationofbrownadipocytesandtheeffectwasrecoveredbySKF96365.ThusweconcludethatTRPV2mightbeinvolvedintheregulationofbrownadipocytedifferentiation.NoCOI.

2P-018Transient receptor potential vanilloid 4 (TRPV4) as a mechanotransducer in the mouse esophagusBoudaka, Ammar1; Boudaka, Ammar1,2; Mihara, Hiroshi2,3; Sugiyama, Toshiro3; Tominaga, Makoto2(1Department of Physiology, College of Medicine and Health Sciences, Sultan Qaboos University, 2Division of Cell Signaling, National Institute for Physiological Sciences, Okazaki, Japan)

MechanicalsensationfromtheesophagusisconveyedtotheCNSviavagalafferentsthathavespecialterminalstructuresintheesophagealwallcalledintraganglioniclaminarendings(IGLEs)andexpresspuri-nergicreceptors.Theexactmechanismthatmediatesthismechano-transduction isstill largelyunknown.Transientreceptorpotentialchannelvanilloid4(TRPV4)candetectvariousstimulisuchaswarmtemperature,stretchandsomechemicals.Itsexpressionandfunctionintheesophaguswasnotstudied.Here,weshowstructuralandfunc-tionalTRPV4expressioninmouseesophagusanditsinvolvementinATPrelease.TRPV4mRNAandproteinweredetectedinesophagealkeratinocytes.SeveralTRPV4activators,includingstretchstimuli,haveincreasedcytosolicCa2+concentrationsinWT,butnotinTRPV4KOkeratino-cytes.GSK(TRPV4agonist)andheatstimulusevokedTRPV4-likecurrentresponsesinWT,butnotinTRPV4KOcells.ThemRNAofanewlyidentifiedATPtransporter,VNUTanditsproteinwerealsodetectedinWTkeratinocytes.TRPV4activators(GSKandheatstim-ulus)increasedATPreleasefromWT,butnotfromTRPV4KOcells.Conclusion:TRPV4mediatesesophagealmechanotransductionviaATPreleasethatpossiblyactivatespurinergicreceptorsonIGLEs.NoCOI.

2P-019Involvement of L-type Ca2+channels in postnatal neurogenesis of the rodent adult hippocampal derived stem cellsTeh, Daniel Boon Loong; Ishizuka, Toru; Yawo, Hiromu(Dept Dev Biol & Neurosci, Tohoku Univ Grad Sch of Life Sci, Sendai, Japan.)

Dentategyrusofhippocampusisaregionofwelldocumentatedneu-rogenesis.NeurogenesisispresumedtoberegulatedbyL-typeCa2+channels(LTCCs),viathemitogenicandsurvivalsignalingpathways.Here,wetestedifLTCCsareinvolvedinneurogenesisusingPZ5,aneuralstemcellline.WedemonstratedthattheproliferationrateofundifferentiatedPZ5wasaffectedneitherbythegeneralLTCCagonist,BayK8644,Cav1.2/1.3specificagonist,FPL64176,norbytheLTCCantagonist,nimodipine,ascomparedtothecontrol.OnlyCav1.2andCav1.3LTCCssubsetswerepresenceatundifferentiatedPZ5cells.Retinoicacidinduceddifferentiationwascarriedfor12-daysinrespec-tiveLTCCagonistsandantagonist.BayKandFPLsignificantlyin-creasedthefractionofneuronalpopulation (βIII-tubulin+/MAP2+),whilenimodipineretarded it.Wealsonotedan inversepatternofoligodendrocytesfractionascomparedtotheneuronalfraction.Theastrocytesfractionremainedthesameinallgroups.ThenumberofNeuN+/βIII-tubulin+,appearedsignificantlyhigherinFPLandBayKascomparedtonimodipine,suggestingthatearlymaturationinLTCCactivecells.Theseneuronswereabletoinvokeactionpotential,sug-gestingitsfunctionality.ItissuggestedthatCav1.2andCav1.3promotePZ5differentiationintoneuronalfraction,attheexpenseoftheoligo-dendrocyticpopulation.NoCOI.

2P-020The lobe-specific interaction of calmodulin with the cardiac CaV1.2 channelShao, Dong-Xue1,2; Xu, Jianjun2; Zhao, Meimi1; Sun, Yu1; Minobe, Etsuko2; He, Guilin1; Feng, Rui1; Hu, Huiyuan1; Sun, Xuefei1; Guo, Feng1; Kameyama, Masaki2; Hao, Liying1(1Department of Pharmaceutical Toxicology, School of Pharmaceutical Science, China Medical University, Shenyang, China, 2Department of Physiology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan)

Toassessthecontributionoflobesinthecalmodulin(CaM)interactionwithcardiacCav1.2channel,CaMwasseparatedinto2partsatthemiddleofthemolecule:N-lobeandC-lobe (N-CaMandC-CaM).WeexaminedthebindingofN-CaMandC-CaMwiththefragmentsoftheputativeCaMbindingdomains (N-terminusandCT1intheCav1.2channel).WefoundthatbothN-CaMandC-CaMboundtoNterminusandCT1inaCa2+-dependentmanner,whilemutationofCa2+bindingsitesineachlobeabolishedCa2+-dependenceofbinding.Interestingly,althoughNterminusjustbound1moleculeoflobe,1moleculeofCT1bound3moleculesofC-CaMand1moleculeofN-CaM,respectively.CombinedwithourpreviousstudieswhichshowthatCaMinteractswithCav1.2in2:1manner,ourpresentstudysuggeststhatthatonemoleculeofCaMmaybindwithboth lobesboundtoC-terminalofCaV1.2,theothermoleculeofCaMbindswithonlyasingleC-orN-lobeboundtoC-terminalofCaV1.2andanotherN-orC-lobeboundtoN-terminalofCaV1.2.NoCOI.


2P-021The role of nucleotides in regulation of cardiac Cav1.2 channelLiu, Shuyuan1,2; Xu, Jianjun1; Minobe, Etsuko1; Kameyama, Asako1; Hao, Liying2; Kameyama, Masaki1(Department of Physiology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima,Japan., 2Department of Pharmaceutical Toxicology, School of Pharmacological Science, China Medical University, Shenyang, China)

OurpreviousstudiessuggestthatMgATPcoordinateswithcalmodulin(CaM) inmaintenanceofcardiacCav1.2channelactivity inthe in-side-outpatchesviadirectinteractionwithchannel.Sincethesimilar-ityofstructuresamongnucleotides,wespeculatethatothernucleotidescanalsoinduceCav1.2channelactivityinthepresenceofCaM.Toexaminethishypothesis,werecordedthesingleCa2+channelcurrentinisolatedguineapigventricularmyocytesusingpatchclamptech-nique.Afterthepatchesexcisedfromcellmembrane,1µMCaMto-getherwithnucleotides(ATP,GTP,CTPandUTP)wereapplied,wefound,althoughCaMalonehadminimaleffectonCav1.2activity,itsignificantlyinducedchannelactivityinthepresenceofeitherATP,GTP,CTPorUTP.TheworkingefficiencyofnucleotidesisATP>GT-P>UTP=CTP.ThisresultsuggeststhatthedirectdynamicinteractionofnucleotidesandCaMarerequiredforbasicCav1.2activity.NoCOI.

2P-022RIM proteins suppress voltage dependent inactiva-tion (VDI) through C-terminus of Voltage-dependent calcium channel(VDCC)-α1A subunit.Hirano, Mitsuru; Takada, Yoshinori; Yamaguchi, Kazuma; Mori, Masayuki X; Mori, Yasuo(Graduate school of Engineering, Kyoto University, Kyoto, Japan)

Ca2+influxintothepresynapticactivezonesthroughtheVoltage-de-pendentcalciumchannels(VDCCs)isimportantforneurotransmitterrelease.OurgrouphasbeendemonstratedthatinteractionbetweenVDCC-βsubunitsandRIMproteins,presynapticactivezonescaffold-ingproteins,enhancestheneurotransmitterreleaseviasuppressingofvoltagedependent inactivation (VDI)ofVDCCs,andanchoringsynapticvesiclesinthevicinityofVDCCs(Kiyonakaetal.2007,Uriuetal.2010).Recently,Sudhof’sgroupreportedthatPDZdomainofRIMproteinsdirectlyinteractswiththeC-terminusofα1Aandα1B,whicharepore-formingVDCCsubunits, tocontributethe localizationofVDCCstopresynapse(Kaeseretal.2012).However,thiscontributionofthe interactionbetweentheC-terminusofα1Aorα1BandRIMproteinsonVDIofVDCCsiscontroversial.Here,inadditiontotheVDIsuppressionbyβ-subunits,weextendourfindingsbyobservationthatRIM2αsignificantlyenhancesthesuppressionoftheVDIinlongα1Asplicingisoform,whichpossessPDZbindingmotif,comparedtotheVDIinshortisoformofα1A,whichlacksPDZbindingmotif.ThisenhancementwasobservedonlyinRIM2αexpressingcellsbutnotinRIM3γexpressingcells,whichcontainsVDCC-βsubunitsinteractingregionbutnotPDZdomain.Thus, functionalcouplingamongRIMproteinandβ-subunitandC-terminusofα1Amayenhancesneurotrans-mitterreleaseviasustainingCa2+influxthroughthesuppressionofVDI.NoCOI.

2P-023Effects of amino-terminal disease-associated muta-tions on the RyR1 channelMurayama, Takashi1; Kurebayashi, Nagomi1; Yamazawa, Toshiko2; Oyamada, Hideto3; Suzuki, Junji4; Kanemaru, Kazunori4; Takemori, Shigeru2; Oguchi, Katsuji3; Iino, Masamitsu4; Sakurai, Takashi1(1Dept. Pharmacol., Juntendo Univ. Sch. Med., Tokyo, Japan, 2Dept Mol. Physiol., Jikei Univ. Sch. Med., Tokyo, Japan, 3Dept. Pharmacol., Sch. Med., Showa Univ., Tokyo, Japan, 4Dept. Pharmacol., Grad. Sch. Med., The Univ. Tokyo, Tokyo, Japan)

Type1ryanodinereceptor (RyR1) isaCa2+releasechannel inthesarcoplasmicreticulumandplaysapivotalroleinexcitation-contractioncouplinginskeletalmuscle.RyR1isthemajortargetformuscledis-eases,e.g.,malignanthyperthermia (MH)andcentralcoredisease(CCD).Todate,over200mutationshavebeenidentifiedintheRyR1geneofMHandCCDpatients.ItiswidelybelievedthatMHandCCDmutationscausehyperactivationoftheCa2+-inducedCa2+release(CICR),resultinginabnormalCa2+homeostasisinskeletalmuscle.CICRshowsbiphasicCa2+dependence,thustheactivitycanbedeterminedbythreeparameters:sensitivitytoactivatingCa2+,sensitivityto inactivatingCa2+,andthegain(i.e.,peakactivity).However,itremainsunclearhowthedisease-associatedmutationsaffectstheseparameters.Inthisstudy,weexpressedseveralRyR1channelscarryingdifferentMH/CCDmutationsatamino-terminalregioninHEKcellsandtestedtheirCICRbylive-cellCa2+imagingand[3H]ryanodinebinding.Ourresultssuggestthattheamino-terminaldisease-associatedmutationsdivergentlyaffectstheparametersofCICRdependingonthesites formutation.Theunderlyingmolecularmechanismwillbediscussed.NoCOI.

2P-024Ca2+ release properties of type 2 ryanodine receptor mutants associated with ventricular arrhythmia and sudden deathKurebayashi, Nagomi1; Murayama, Takashi1; Suzuki, Junji2; Kanemaru, Kazunori2; Iino, Masamitsu2; Sakurai, Takashi1(1Dept. Pharmacol., Juntendo Univ. Sch. Med., Tokyo, Japan, 2Dept Pharmacol. Grad. Sch. Med.,The Univ. Tokyo, Tokyo, Japan)

Type2ryanodinereceptor (RyR2) isaCa2+releasechannel inthesarcoplasmicreticulumandplaysapivotalroleinexcitation-contractioncouplinginheart.RyR2isthemajortargetforarrhythmogenicdis-eases, i.e.,catecholaminergicpolymorphicventriculartachycardia(CPVT)andarrhythmogenicrightventricularcardiomyopathy(ARVC).Todate,over150mutationshavebeenidentifiedintheRyR2geneofCPVTandARVCpatients.AlthoughitiswidelybelievedthatCPVTandARVCmutationscausehyperactivationoftheCa2+-inducedCa2+release(CICR),thereasonsforthedifferencebetweenthetwodiseas-esarenotwellknown.CICRshowsbiphasicCa2+dependenceagainstcytoplasmicCa2+,thustheactivitycanbedeterminedbythreeparam-eters:sensitivitytoactivatingCa2+,sensitivitytoinactivatingCa2+,andthegain(i.e.,peakactivity).Inaddition,CICRisalsoregulatedbylu-minalCa2+;highluminalCa2+activatesCICR.Itremainsunclearhowthedisease-associatedmutationsaffecttheseparameters.Inthisstudy,weexpressedRyR2channelscarryingseveralCPVTandARVCmutationsinHEKcellsandevaluatedtheirCa2+releasepropertiesbylive-cellCa2+imagingand[3H]ryanodinebinding.Ourresultssuggestthatthedisease-associatedmutationsdivergentlyaffectstheparame-tersofCICRdependingonthesitesformutation.Thedifferencebe-tweentheCPVTandtheARVCmutationswillbediscussed.NoCOI.


2P-025A ventricular cell model incorpolating new Ca2+-in-duced Ca2+-releaseMemida , Hiraku(Dept. of Life Sciences, Graduate School of Life Sciences, University of Ritsumeikan, Siga, Japan)

AventricularcellmodelincorpolatingnewCa2+-inducedCa2+-relea-seDept.ofLifeSciences,GraduateSchoolofLifeSciences,RitsumeikanUniv.HirakuMemida,AkinoriNoma.ThegradedCa2+releaseviaRyRattheterminalcisternaofsarcoplasmicreticulum(SR)isdependentontheextentofactivationoftheL-typeCa2+channelcurrent(ICaL).ThislocalcontrolofCa2+releaseisstillnotreproducedinmostofcardiaccellmodelsdevelopedonthedesk-topcomputerlevel.ThisisbecausetheCa2+-inducedCa2+-releaseoccursinanall-or-noneman-nerwhenasinglecommonpoolisassumedforbothICaLandRyRCa2+fluxes.Weaimedat improvingourventricularcellmodelbyadoptingthetightcoupledLCC-RyRmodel (CaRU)basedon localcontroltheory(Hinchetal.2004).TheoriginalmodelofHinch,howev-er,failedtoreconstructthespontaneousCa2+releasefromSR,whichhasbeenwellestablishedinexperimentalstudies.WehavemadeanattempttoreconcilethisdifficultybyassumingalocalCa2+accumu-lationneartheCa2+releasingsite.Theioniccurrentswerealsoim-provedaccordingtonewexperimentaldata.Wewilldiscussrelevanceofournewanimalmodel instudyingmechanismsofphysiologicalfunctionsoftheventricularmyocytes.NoCOI.

Poster PresentationsHeart, Circulation (2)

2P-026Subcellular Na Channel Remodeling in Ischemic Bor-der Zone as a Mechanism of Reentrant Ventricular Tachyarrhythmia: In Silico StudyTsumoto, Kunichika1; Ashihara, Takashi2; Haraguchi, Ryo3; Nakazawa, Kazuo3; Kurachi, Yoshihisa1(1Osaka University, Osaka, Japan, 2Shiga University of Medical Science, Otsu, Japan, 3Nat Cereb and Cardiovasc Center, Suita, Japan)

Previousexperimentalstudieshavereportedthat inthe ischemicborderzone(IBZ)oftheinfarctedventricularmyocardium,thesodium(Na)channelswereredistributedwithinamyocyte,andthatthesub-cellularNachannelremodelingcontributedtoconductionslowing.However,theimpactof intracellularNachannelremodelingonthearrhythmogenicityinischemicmyocardiumisunclear.WerecentlyreportedinsimulationsthatthesubcellularNachannelremodelingobservedinIBZenhancedthesensitivityoftheischemicmyocytestoNachannelblockersinphysiologicalrelevantmyofibermodelincor-poratingtheelectricfieldmechanism(takingintoaccounttheinter-cellularcleftpotentials).Asaresult,NachannelblockadetendedtocauseconductionblockintheIBZthaninthenon-ischemiczone(NZ).Here,weextendthesimulationsintotheinvestigationofeffectsoftheNachannelblockadeinaring-shapedmyocardialmodelconsistingofNZandIBZonprematureventricularcontractions.ThenwefoundthatunidirectionalconductionblockbyNachannelblockadewasmorelikelytooccurattheborderbetweenIBZandNZ.Furthermore,wefoundthatsuchunidirectionalconductionblockwasinvolvedintheinitiationofreentranttachyarrhythmia.Ischemia-inducedsubcellularNachannelremodelingmightbepartlyresponsibleforthearrhyth-mogenicmechanismofclass-Iantiarrhythmicdrugsinpatientswitholdmyocardialinfarction.NoCOI.

2P-027Inhibitory effects of verapamil on bidirectional ventric-ular tachycardia in anesthetized ratsNomura, Hiroko; Oikawa, Shota; Nagata, Rina; Nishio, Miki; Yamasaki, Masao; Hata, Tadayoshi(Graduate School of Health Sciences, Fujita Hearth Univ. Toyoake, Japan)

Purpose:ThepurposeofpresentstudyistoinvestigatetheeffectofcaffeineontheoccurrenceofBVTandinhibitoryeffectofCa2+channelblocker,verapamil.Methods :AdultfemaleWisterratswereanesthe-tizedwithhalothanegas(1.2%),andECGwasrecordedwithaBiopacsystemMP-150.Protocol1:Weinjectedcontinuouslycaffeine(0.5mg/kg/min)throughtherightfemoralvein,andthenaddedepinephrine(10µg/kg/min)at15minafterthestartofcaffeine.WeobservedevokedBVTandevaluatedtheeffectofdrugsusingECGmeasurementsofRR,PR,QTc,JTp/JTandTp-e/QT.Protocol2:Weinjectedverapam-il(0.25mg/kg)atfiveminutesbeforethecaffeineadministration,andobservedECGcontinuously.Results:In12animalsofprotocol1,BVTwasobservedintenanimals.BVTwasnotebokedinanyof8animalsofprotocol2.TheadministrationofcaffeineelicitedprolongationofQTc,reducedTp-e/QTandincreasedJTp/JT.However,thepretreat-mentofverapamilinhibitedtheprolongationofQTcandJTp.Discus-sion: IthasbeenrecognizedthatBVTiscausedbydelayedafterdepolarizationandintracellularabnormalCa2+handling.Inthepresentstudy,BVTwaselicitedbycaffeinewhichincreasesCa2+releasefromthesarcoplasmicreticulum,ontheotherhand,BVTwasinhibitedbythevoltage-dependentCa2+channelblocker.WeconcludethattheinfluxofCa2+isoneofpathogenicmechanismofBVT,andanabnor-malityofCa2+handlingisrepresentedintherepolarizationcharacter-istic(JTp).NoCOI.


2P-028Dynamical mechanisms of early afterdepolarizations in long QT syndromes: insights from bifurcation anal-ysis of mathematical models for human ventricular myocytes"Kurata, Yasutaka1; Hisatome, Ichiro2; Tanida, Mamoru1; Shibamoto, Toshishige1(1Department of Physiology, Kanazawa Medical University, Ishikawa, Japan, 2Div. Reg. Med. Therap., Tottori Univ. Grad. Sch. Med. Sci., Yonago, Japan)

Wetheoreticallyinvestigateddynamicalmechanismsofearlyafterde-polarizations(EADs) in longQTsyndrome(LQTS)bystabilityandbifurcationanalyses,aswellasnumericalsimulations,usingmathe-maticalmodelsofsinglehumanventricularmyocytes(Kurataetal,BiophysJ,2005).LQT1-3modelcellsweredevelopedbyreducingtheslow(IKs)orrapid(IKr)componentofthedelayed-rectifierKchannelcurrent(IK)(LQT1,2)orincorporatinganon-inactivatingcomponentoftheNachannelcurrent(INa)(LQT3).EADswereinducedbyen-hancementofICaLintheLQTSmodelcells.Theirstabilityandbifur-cationsduringincreasesoftheL-typeCachannelcurrent(ICaL)orIKreductionsweredeterminedbybifurcationanalysestoconstructbi-furcationdiagrams:equilibriumpoints(EP),limitcycles(LCs),andtheirstabilityweredeterminedas functionsofbifurcationparameters.DecreasingIK,increasingICaLorincreasingnon-inactivatingINaleadstoEADgeneration inthevicinityofHopfbifurcation (HB)points.FurtherinhibitionofIKyieldedstableEPatdepolarizedpotentialsandquiescence.ThethresholdofEADgenerationduringIKdecreas-esorICaLincreaseswaslowerintheLQTSmodelcellsthaninthenormalcell,whichwasduetotheshiftinHBpoints.EADscouldberegardedastransientLCsinducedviaHB(EPdestabilization)atde-polarizedpotentialsduringtheslowactivationofIKs.NoCOI.

2P-029Channel dysfunction in ryanodine receptor mutants evoking fatal arrhythmiaUehara, Akira1; Yasukochi, Midori1; Murayama, Takashi2; Kurebayashi, Nagomi2; Honda, Akira1; Shioya, Takao3(1Fukuoka University, School of medicine, Fukuoka, Japan, 2Department of Cellular and Molecular Pharmacology, Juntendo University, Tokyo, Japan, 3Department of Physiology, Faculty of Medicine, Saga University, Saga, Japan)

Mutationsofthehumancardiacryanodinereceptorgene (hRyR2)mappedto1q42-q43areassociatedwithcatecholaminergicpolymorphicventriculartachycardia(CPVT).CPVTisaseveregeneticarrhyth-mogenicdisordercharacterizedbyexercise-andstress-inducedven-triculartachycardiamanifestingassyncopeandsuddendeath.FulllengthofmouseRyR2cDNA(accession:NM-023868)clonedbyKure-bayasiandMurayama,whichisdifferentfromthatoftheChen’sgroup(accession:AF295105),wasinsertedintoatetracycline-induciblemam-malianexpressionvector.EachmutationwasintroducedusingtheQuickChangesite-directedmutagenesiskit. Thestable inducibleHEK293celllinesweregeneratedfortheWild-typeormutants.HereweexaminedhowCPVT-associatedRyR2mutantsaredysfuctioned.First,thesingle-channelcurrentsfrompurifiedRyR2proteinswereactivatedbyCPVTmutations.Second,3H-ryanodinebindingfromthemicrosomesofHEK293expressedwithRyR2showedthattheRyR2channelactivitywasincreasedbyCPVTmutations.Third,in-tracellularCa2+oscillationsinHEK293cellsmeasuredbysingle-cellCa2+imagingusingfura-2occurredmorefrequentlyinCPVTmutantsthaninwild-type.Thus,ourCPVTmutantsconsistentlyexhibitedagainoffunctionactivities,suggestingthatCa2+ leakviaRyR2fromcardiacsarcoplasmicreticulumisenhanced.NoCOI.

2P-030Safety Mechanism of hERG-Targeting Class III Anti-arrhythmic AgentsFurutani, Kazuharu; Tsumoto, Kunichika; Kurachi, Yoshihisa(Department of Pharmacology, Graduate School of Medicine, Osaka University, Osaka, Japan)

Potassiumchannelsencodedbyhumanether-a-go-go-related gene(hERG)underlie therapidlyactivatingcomponentof thedelayedrectifyingpotassiumcurrent(IKr)inheartandplayanimportantroleinterminatingthecardiacactionpotential (AP).Recently,wehaveexperimentallydemonstratedadualeffectofsomeanti-arrhythmicagents,suchasnifekalant,onhERGchannel.BesidesblockinghERGchannel,thesecompoundscanfacilitateitsactivation.ToassesstheclinicalrelevanceofhERGchannelmodulationsbycompounds,weconductedsimulationsofcardiacAPinaPriebe-BeuckelmannmodelwithhERGchannelblockandfacilitation,usingourexperimentaldataofnifekalant.WefoundthatthehERGchannelblockprolongedactionpotentialduration(APD)intheblockconditionsbothwithandwithoutfacilitation. Inaddition, therefractoryperiod inbothconditions in-creasedfromcontrolconditionsothattheectopiccellexcitation issuppressed.Asinoursimulation,wecouldobserveanearlyafterde-polarization(EAD)whenbothIKrandtheslowlyactivatingcomponentofthedelayedrectifyingpotassiumcurrent(IKs)wereblocked.Impor-tantly,facilitationmechanismpreventshazardousprolongationofAPDandtheinductionofEADbyacceleratingtherepolarizationrateviaanincreaseinIKrduringprolongedphase3.Therefore,anti-arrhythmicagentsthathasbothblockandfacilitationeffectsonhERGchannelmayhavealowerriskforinducingEADsandtriggeredactivityandthusbemoresuitable forthetreatmentofarrhythmiasthanpurehERGblocker.NoCOI.

2P-031Modeling fluid exchange at the capillary, tissue and lymphatic capillaryHimeno, Yukiko; Amano, Akira; Noma, Akinori(Department of Life Science, Ritsumeikan University, Kusatsu, Japan)

Fluidexchangebetweenbloodandinterstitialfluidatthecapillary,andflowatthelymphaticcapillaryisoneofthemostindispensableissuesforthehomeostasisoftheinternalmilieu.However,complexinterdependencyofthepressures involved indeterminationofthefluidexchangemakesitdifficulttotacklethisissuewithoutmathe-maticalapproach. Inthepresentstudy,wedevelopedacapillarymodel,whichallowedustoclarifymechanismsregulatingtheinter-stitialfluidvolume.Thecapillary isassumedtobeacylinder.Thetranscapillaryfluidfluxiscalculatedfromthefluidpressureandcolloidpressuredifferencebetweenplasmaandinterstitialfluid.Wellknown3safetyfactorsagainstedema,1.Lowtissuecomplianceinnegativepressurerange,2.Increasedlymphaticflowand3.Proteinwashoutbythelymph,areincorporatedintothemodel.Whentheinterstitialfluidpressurewaswithinaphysiologicalrangeinourmodel,anin-creaseinbloodpressureinducedaninterstitialfluidvolumeincrease,whichinturnreducednegativetissuepressuretopreventedema.Thelymphflowalleviatestheedemabybothcarryingfluidofffromthetissue,anddecreasingtheinterstitialfluidcolloidpressure.Fromtimecourseoftheeffects,itwasfoundthatthetissuecomplianceandthelymphflowactasalong-termandashort-termregulatoroftheinter-stitialfluidvolume,respectively.Mathematicalmodelingofthefluidexchangeatthecapillaryenabledustocalculatefluidconvectionquantitativelyandestimatefunctionalimpactsofthetissuecomplianceandlymphflow.NoCOI.


2P-032Effects of period of sinusoidal leg exercises on bra-chial and middle cerebral artery blood flowsFukuba, Yoshiyuki1; Miura, Kouhei1; Kondo, Ayaka1; Watanabe, Sachiko1; Endo, Masako Y1; Kashima, Hideaki1; Hayashi, Naoyuki2; Fukuoka, Yoshiyuki3; Koga, Shunsaku4(1Dept Exerc Sci Physiol, Pref Univ Hiroshima, 2Tokyo Inst Technol, 3Doshisha Univ, 4Kobe Design Univ)

Toexplorethecontrolofuppernon-exercising limbandcerebralcirculationduringlegexercise,wemeasuredthedynamicsofbrachi-alartery(BA)andmiddlecerebralartery(MCA)bloodflow(BF)inresponsetosinusoidalworkrate(WR)protocolsofvaryingperiods.SevenhealthyyoungmalesubjectsperformeduprightlegergometerexercisewithaconstantWRfor30minfollowedbythreedifferentperiodsofsinusoidalWRexercises(numberoftherepetitions);6-min(3),4-min(4),and2-min(7)whereWRfluctuatedintherangeof20WtopeakWR(60%ofpeakVO2).Duringtheprotocols,wemeasuredpulmonarygasexchange,heartrate(HR),meanarterialbloodpressureandstrokevolume,bloodvelocity(BV)andcrosssectionalareaofBA,BVofMCA,andforearmandforeheadskinBFandsweatingrate(SR).Thevariableswerefittedasy(t)=M+A*sin((2π/T)*t-θ),wheret:time,A/M:relativeamplitude,T:period,θ:phaseshift.Almostvariablestracedthesinewaveadequately.ThephaseshiftsandA/MofBVinMCA(phase(º):6-min,44+/-13,4-min,69+/-20,2-min,112+/-33;A/M(%):6-min,4.5+/-1.5;4-min,4.8+/-1.9;2-min,3.3+/-0.7,mean+/-SD)weresimilarwithvariablesregardingO2delivery(e.g.VO2,HR).Contrari-ly,theresponseofBFinBAdisplayedananti-phaseandaconstantA/M(approximately28%)regardlessoftheperiod.Non-activelimbcirculatorycontrolisdifferentialtothemaincoordinationofO2-deliv-erytoactivemuscle.NoCOI.

2P-033Spontaneous and nerve-evoked constrictions of sub-mucosal venules in rat stomachMitsui, Retsu; Hashitani, Hikaru(Departement of Cell Physiology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan)

Objective:Venulesofthebladderanddistalcolonexhibitrhythmicspontaneousconstrictionsthatarelikelytopreventthestagnationofbloodevenduringtheirdistension.Hereweexaminedthespontaneousandnerve-evokedactivityofratstomachmicrovasculature.Methods:Changesindiameterofmicrovasculatureweremonitoredbyvideoimagingsystem,whileintracellularCa2+invenularsmoothmusclecells(VSMCs)werevisualisedusingfluo-8fluorescenceCa2+imaginginthesubmucosalpreparationsofratstomach.Results:VSMCsshowedsynchronisedspontaneousCa2+ transientsandassociatedvenularconstrictions.L-typeCa2+channelblocker(1µMnifedipine)disruptedthesynchronyofCa2+transientsandabolishedspontaneousconstric-tions.Theinhibitorsofsarcoplasmicreticulum(SR)Ca2+-ATPase(10µMCPA),IP3receptors(100µM2-APB)andCa2+-activatedCl-channels(100µMniflumicacid)abolishedspontaneousconstrictions.Transmu-ralnervestimulationimmediatelytriggeredpurinergic/α-adrenergicarterioleconstrictionwhileevokinglong-lastingα-adrenergicconstric-tionofvenules(1µMphentolamine).Alltheseresponseswereblockedbysympatheticnervetransmitterdepletion(10µMguanethidine).Theactivationofprimaryafferentnerves(300nMcapsaicin)dilatedvenulesbyreleasingCGRP.Conclusion:Spontaneousconstrictionsinsubmu-cosalvenulesoftheratstomachappeartoprimarilydependonCa2+releasefromSR.SynchronyofCa2+transientsamongstVSMCsmayrequireelectricalcouplingthrough‘regenerative’L-typeCa2+channelactivation.NoCOI.

2P-034Characteristics of spontaneous transient hyperpolar-izations in vascular endothelial cellsYamamoto, Yoshimichi1,2; Takano, Hiromichi2(1Laboratory of Physiology, School of Nursing, Nagoya City University, Nagoya, Japan, 2Department of Cell Physiology, Graduate School of Medical Sciences, Nagoya City University, Nagoya, Japan)

Ithasbeenreportedthatthevascularendothelialcellsalwaysreleasesmallamountofnitricoxide(NO)whichisplayinganimportantroleinthebasicregulationofthetotalperipheralresistance.Asthecellu-larmechanismsundersuchaspontaneousreleaseofNOhavenotbeenstudied,weexaminedendothelialspontaneousactivitiesusingtheendotheliallayerpreparationsacutelydispersedfromtheguinea-pigmesentericarteries.IntheCa2+-imagingofthesepreparations,wehavefoundthatspontaneoustransientincreasesinthe[Ca2+]ioccurinindi-vidualcells.Wethentriedtoexaminewhethersuchanincreasein[Ca2+]i inducedanyelectricalevents inthemembrane.Soapatchelectrodewasappliedtoacellwithinthepreparationandthemem-branepotentialwasobservedunderthecurrentclampcondition.Themembranepotentialwasnotconstantbuttransienthyperpolarizationsoccurredspontaneously.Theseactivitieswereblockedbyeithercha-rybdotoxin(10-7M),apamin(10-7M)orremovalofexternalCa2+.Ca2+-ac-tivatedK+-channelssensitivetobothcharybdotoxinandapaminseemedtobeinvolvedinthetransienthyperpolarizations.Ca2+influxmightactivatethesechannelsdirectlyorbenecessarytorefilltheintracel-lularCa2+stores,thereleasedCa2+fromwhichmightactivatethesechannels.NoCOI.

2P-035Implications of adenosine and muscarinic acetylcho-line receptors on bradycardia during systemic episod-ic hypoxia in ratsTsuchimochi, Hirotsugu; Yoshimoto, Misa; Inagaki, Tadakatsu; Sonobe, Takashi; Shirai, Mikiyasu(Department of Cardiac Physiology, National Cerebral and Cardiovascular Center Research Institute, Osaka, Japan)

Pronouncedbradycardiaisfrequentlyobservedinpatientswithob-structivesleepapnea,andhasbeenrecapitulatedinrodentssubjectedtosystemicepisodichypoxia.Toelucidateitsmechanism,maleWistarratspreviouslyimplantedwithtelemetrydevicestomonitorarterialbloodpressure(TRM54P,TR-Millar)andheartratewereexposedtoacuteepisodichypoxia(90-sroomair/90-sN2;5%O2atnadir)inasealedchamber.Acuteepisodichypoxiaelicitedamarkedpressorresponse,followedbysignificantbradycardia,aspreviouslyreported.Thiswasnotmediatedsolelybyabaroreflex,asα-1adrenergicrecep-torblockadewithprazosin(2mg/kg,ip)suppressedonlypressorbutnotbradycardiacresponsesduringepisodichypoxia.Undertheinflu-enceofprazosin,boththenon-selectivemuscarinicacetylcholine(ACh)receptorantagonistatropine(1mg/kg,ip)andtheselectiveM2AChreceptorantagonistAF-DX116(0.2mg/kg,ip)completelyblockedthebradycardiacresponsetoepisodichypoxia.Furthermore,boththenon-selectiveadenosinereceptorantagonistcaffeine(50mg/kg,ip)andtheA1-selectiveantagonistDPCPX(2mg/kg,ip)significantlyattenu-atedbradycardia.Inconclusion,non-baroreflexmediatedbradycardiaobservedduringsystemicepisodichypoxiaismediatednotonlybyM2AChreceptorbutalsobyadenosineA1receptor,suggestingthattheyshareacommonsignalingmechanismtomodulateheartrateinresponsetosystemicepisodichypoxia.NoCOI.


2P-036Prostaglandin E2-EP4 signaling-induced fibulin-1 may play a role in intimal thickening in the ductus arterio-susKumagaya, Shun1,2; Yokoyama, Utako2; Minamisawa, Susumu3; Inoue, Takafumi1; Ishikawa, Yoshihiro2(1Dept. of Life Science and Medical Bioscience, Waseda Univ., Tokyo, Japan, 2Cardiovascular Research Inst., Yokohama City Univ., Kanagawa, Japan, 3Dept. of Cellular Physiology, the Jikei Univ. School of Medicine, Tokyo, Japan)

Intimalthickening(IT)isessentialforclosureoftheductusarteriosus(DA).However,molecularmechanismsofITintheDAarenotfullyunderstood.SincewehavepreviouslyreportedthatprostaglandinE2

(PGE2)-EP4signalingpromotedhyaluronan-mediatedITintheDA,wesoughttouncoveranovelEP4-mediatedmechanismthatinduceITintheDA.MicroarrayanalysisandquantitativeRT-PCR(qRT-PCR)usingsmoothmusclecellsofratDA(DASMCs)onday21ofgestation(e21;near-term)revealedthatEP4stimulationfor24h increasedfibulin-1 (273-fold,p<0.001,n=10).WesternblottingalsoshowedthatEP4stimulationfor72hincreasedsecretionoffibulin-1fromDASMCs(21-fold,p<0.001,n=4).ImmunohistochemistryofhumanDArevealedthatfibulin-1washighlyexpressedintheareaofIT.Furthermore,fibulin-1mRNAex-pressionwas1.6-foldhigherintheratDAthanintheaortaone21(p<0.005,n=3-5),andwasdevelopmentallyincreasedduringday19to21ofgestation (1.6-foldvs.e19,p<0.005,n=5). MessengerRNAofADAMTS-1whichregulatesproteolysisofglycoproteinbyinteractingwithfibulin-1wasabundantlyexpressedintheratDAcomparedwiththeaortaone21(4.7-fold,p<0.001,n=4-5).Inconclusion,PGE2-EP4signaling-inducedfibulin-1mayplayaroleinITformationintheDAbyenhancingtheactivityofADAMTS-1.NoCOI.

2P-037Mechanisms of vasoconstriction induced by acetyl-choline on guinea pig vena cavaSato, Rei; Takano, Hiromichi; Mitsui, Retsu; Hashitani, Hikaru(Department of Cell Physiology, Nagoya City University Medical School, Nagoya, Japan)

Objective:To investigatethemechanismsunderlyingacetylcholine(ACh)-inducedcontractionoftheguineapigvenacava.Method:Iso-metrictensionrecordingsweremadefromringpreparationsofguin-eapigvenacava.Insomeexperiments,theendotheliumwasmechan-icallyremoved.Results:ACh(1or10µM)causedcontractionsofthevenacava,whiletransmuralnervestimulationorphenylephrine(10µM)failedtocontractthevessel.Nifedipine(10µM)abolished1µMACh-inducedcontractionsandsuppressed10µMACh-inducedcon-tractions.InnominallyCa2+-freesolutions,1µMACh-inducedcontrac-tionswereunexpectedlyenhanced.10µMACh-inducedcontractionwasattenuated,albeititwaslargerthanthatinnifedipine.L-nitro-ar-ginine(10µM)augmentedACh(1or10µM)-inducedcontractions.Inendothelium-denudedpreparations,theremovalofextracellularCa2+ornifedipinediminishedACh(1or10µM)-inducedcontractions.How-ever,unlikeendothelium-intactpreparations,ACh-inducedcontractionsinnifedipineweresimilartothoseinCa2+-freesolutions.Conclusion:TheseresultssuggestedthatproportionalcontributionsofCa2+influxandCa2+releasefrominternalstorestoACh-inducedcontractionsmaybedifferentdependingontheconcentrationsofACh.Inaddition,theendotheliumappearstocounteractvascularcontractilitybyreleasingnitricoxideuponAChstimulation.NoCOI.

2P-038Secretome-Based Identification of PGE2-EP4-Induc-ible Factors in Human Abdominal Aortic AneurysmIshiwata, Ryo; Yokoyama, Utako; Ishikawa, Yoshihiro(Cardiovascular Research Institute, Yokohama City University, Yokohama, Japan)

[Backgrounds]Abdominalaorticaneurysm(AAA)isacommondiseaseintheelderly.AlthoughtheruptureofAAAisfatal,thereisnoeffec-tivepharmacologicaltherapyavailable.WehavepreviouslyreportedthatprostaglandinE2(PGE2),viaitsreceptorEP4,exacerbatesAAAbyincreasingsecretionofInterleukin-6andMatrixMetalloproteinase-2(MMP-2)both inmousemodelsand inhumanAAAtissues.Inthepresentstudy,byapplyingmassspectrometricanalysis,weidentifiedsecretedproteinsincreasedbyEP4stimulationinhumanAAAtissues,whicharepotentialexacerbatingfactorsofAAA.[Materials & Methods]HumanAAAtissueswereobtainedatabloodvesselprosthesis implantationwithwritteninformedconsents.Thetissueswereincubatedinserum-freeDMEMfollowedbystimulationwithEP4agonistorPGE2+EP4antagonistfor24h.TrypsinizedproteinswerelabeledwithiTRAQandwereanalyzedbyLC/MS/MS.IdentifiedproteinswereclassifiedbyGOtermsandsecretedproteinswereextracted.[Results]Atotalof694proteinswere identifiedfrom3specimensamongwhich41proteinswereincreased(>1.3-foldvscontrol)byEP4agonist.Theseincludedneutrophil-relatedprotein,extracellularma-trices,proteoglycans,MMP-2,andMMP-9.Amongthem,PGE2-inducedMMP-2secretionwasattenuatedbyEP4antagonist.[Conclusion]MassSpectrometricAnalysisidentifiedpotentialexacer-batingfactorsofAAA,risingnewinsightsintoAAAprogressionbyPGE2-EP4signaling.NoCOI.

2P-039Catecholamine-induced cardiac automaticity in rat pulmonary veinsOkamoto, Yosuke1; Ono, Kyoichi2(1Laboratory of Cardiovascular Science, National Institute on Aging, National Institute of Health, MD, USA, 2Department of Cell Physiology, Akita Graduate School of Medicine, Akita, Japan)

Atrialfibrillation(AF)isthemostcommonsustainedarrhythmiaanditscrucialoriginispulmonaryvein(PV).However,thearrhythmogen-icnatureofPVislessclear.Werevealedthatnorepinephrine(NE)evokedaspontaneousactivityinratPVcardiomyocytesandthere-sponsiblemoleculesforthearrhythmogenecity.WeperformedCa2+imaging,path-clampandimmunocytochemistrytocharacterizephys-iologicalpropertiesofenzymaticallyisolatedPVcardiomyocytes.WhenweappliedNEtothesecells,theyshowedrepetitiveintracellularCa2+increaseaccompaniedbydepolarization,andautomaticactionpoten-tials.Weobservedthatanoscillatorycurrentgeneratingtheautoma-ticitywasalwaysinwardirrespectiveofthemembranepotential,in-dicatingthecurrentwasderivedfromtheNa+-Ca2+exchanger(NCX).Theautomaticitywassuppressedbyblocking1,4,5-inositoltriphosphatereceptor(IP3R)andtheNCXandIP3Rwereco-localizedalongT-tu-bules.ThesefindingssuggestthatafunctionalcouplingbetweentheNCXandIP3Rcausesarrhythmicexcitability.Furthermore,wefoundauniquehyperpolarization-activatedCl-currentwhosebiophysicalpropertiesdifferedfromClC-2currentinthereactiontoeachintracel-lularCl-,extracellularacidificationandosmoticstress.Cl-channelblockersthatattenuatedthecurrentinhibitedtheNE-inducedauto-maticity.Thesefindingssuggestthatthespecialchannelmaybein-volvedintheautomaticity.NoCOI.


2P-040Venous identity lost in jugular segment of carotid-jug-ular (C-J) shunt but arterial identity strengthened in pulmonary artery in adult rats with C-J shuntLU, Ping; SHIMURA, Daisuke; JIAO, Qibin; UESUGI, Ken; KAJIMURA, Ichige; OMORI, Eriko; NAKAI, Gaku; ONDA, Akiko; SHIRAISHI, Ryosuke; KOBIRUMAKI, Fuyu; AKAIKE, Toru; KUSAKARI, Yoichiro(Dept of Cell Physiology, Jikei University School of Medicine, Tokyo)

ObjectivesWeinvestigatedwhethervesselidentityisremodeledinadultratswithC-Jshunt.MethodsandResultsThehemodynamicsofC-Jshuntwascharacterizedwiththefeaturesofbothcarotidarteryandjugularvein.EphB4transcriptsweresignificantlydown-regulatedin jugularsegmentofpatentC-Jshuntandbrachiocephalicvein,whereasEphrin-B2transcriptswerestronglyinducedinpulmonaryartery.AdecreasingtrendincollagenItranscriptswithaconcomitantincreasingtrendinelastintranscriptswasfoundinjugularsegmentofC-Jshunt.Elastinvs.collagenratiomightbemodulated.Geneex-pressionchangesevokedinthejugularsegmentsofC-Jshuntwerefurtherrevealedbymicroarrayanalysis.Genesinvolvedinvascularremodelingwerehighlyregulated.ThearterialmarkerEphrinB2,andothermarkersofarterialidentitywerenotinducedinjugularsegment.ConclusionsMarkersofvesselidentityarenotquiescentbutplastic,andwillberemodeledtoadapttohemodynamicchangesinadults.Vessel identityremodelingwaspreferentiallydeveloped inoriginallow-pressurebloodvesselswhenexposedtohigh-pressureoxygenat-edbloodflowfromC-Jshunt.Whethervessel identityremodelingwouldbeimplicatedinstructureandcompositionremodelinginadultsdeservesfurtherexploration.NoCOI.

2P-041Intimal Cushion and Elastic Fibers Degradation were shownFormation Were Less Prominent in the Chicken Ductus ArteriosusOmori, Eriko1; Akaike, Toru2; Kajimura, Ichige2; Goda, Nobuhito1; Minamisawa, Susumu2(Departure of Lifescience and Medical Bioscience, Waseda University,Tokyo, Japan, 2Department of Cell Physiology, the Jikei University of Medicine, Tokyo, Japan)

BackgroundTheductusarteriosus(DA)isafetalarterythatclosesafterbirth.OurpreviousstudieshavedemonstratedthatthePGE2-EP4signalpathwaypromotesneointimathickeningandfragmentationoftheelasticfibersintherodentDA.TheavianDAalsoclosesafterbirth,althoughthePGE2-EP4signalpathwaymaynotplayasignificantroleinvascularremodelingintheavianDA,becausethechickhasnoplacentathatisasourceofPGE2.Weinvestigatedwhethervascu-larremodelingwasobservedinthechickenDA.Experience&ResultsWeisolatedtheDAfromchickenatembryonicday16,19,hatchingandwithinadayafterbirth.TheserialparaffinsectionswerestainedwithElasticavan-Giesonstaining.Wefoundthesparseandfragment-edelasticfibersinthemediumfromembryoday16,andimpairmentbecamemoreapparentwithdevelopment.Intimalcushionformationwasnotclearlyobservedevenafterexternalhatching.Real-timePCRanalysisrevealedthattheexpressionlevelsofelastinandlysyloxidasemRNAsweresignificantlylowerintheDAthanintheaorta.Inaddi-tion,genesrelatedwiththePGE2-EP4signalpathwaywerenotpre-dominantlyexpressedinthechickenDA.ConclusionInternalcushionformationwaslessprominentinthechickenDAthaninthemamma-lianDA.Ontheotherhand,elasticfiberformationwasseverelyimpairedinthechickenDA,whichwasunlikelyduetothePGE2-EP4signalpathway.NoCOI.

2P-042S1P1 receptors are necessary, but not sufficient, for the angiogenic responses induced by a novel nucleic acid analogue COA-ClIgarashi, Junsuke1; Kubota, Yasuo2; Shoji, Kazuyo2; Sakakibara, Norikazu3; Maruyama, Tokumi3; Takata, Maki4; Kosaka, Hiroaki1; Takuwa, Yoh5; Hashimoto, Takeshi1; Yamashita, Tetsuo1; Konishi, Ryoji4; Tsukamoto, Ikuko4(1Department of Cardiovascular Physiology, Kagawa University Faculty of Medicine, Kagawa, Japan, 2Department of Dermatology, Kagawa University Faculty of Medicine, Kagawa, Japan, 3Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Kagawa, Japan, 4Department of Pharmaco-Bio-Informatics, Kagawa University Faculty of Medicine, Kagawa, Japan, 5Department of Physiology, Kanazawa University School of Medicine, Kanazawa, Japan)

COA-ClisarecentlydevelopednucleicacidanaloguethatpromotesangiogenesisviatheMAPkinasesERK1/2.Wenowshowthatan-tagonistsfortheS1P1receptors,W146andVPC23019,abolisheffectsofCOA-ClatthelevelsofERK1/2activationandtubeformationinculturedhumanumbilicalveinendothelialcells.GeneticknockdownofS1P1withsiRNAattenuatesCOA-Cl-elicitedERK1/2responses.Thus,theS1P1receptorsarenecessaryfortheangiogenicactionsofCOA-Cl.SignalingpropertiesofCOA-ClshowedsimilaritieswiththoseofS1P,anendogenousS1P1ligand,whichweresensitivetopertussistoxin(Gαi/oinhibitor),BAPTA-AM(calciumchelator),andPP2(Srcinhibitor).However,heterologousoverexpressionofS1P1inCHO-K1cellsdidnotpromoteCOA-Cl-evokedERK1/2responses,suggestingthatthereceptorspersearenotsufficientfortheCOA-Cl.Insum-mary,resultspointouttheS1P1receptorsasakeymoleculeatwhichthenovelnucleicacidanalogueCOA-Clinducesangiogenicresponsesinvascularendothelialcells.NoCOI.

2P-043Tonic Rho-kinase mediated vasoconstriction contrib-utes to coronary vasomotor tone in early diabetic ratShirai, Mikiyasu1; Pearson, James2; Tsuchimochi, Hirotsugu1; Yoshimoto, Misa1; Fujii, Yutaka1; Inagaki, Tadakatsu1; Sonobe, Takashi1(1Department of Cardiac Physiology, National Cerebral and Cardiovascular Center Research Institute, Osaka, Japan, 2Department of Physiology, Monash University, Melbourne, Australia)

ActivationofRhoA/Rho-kinase(ROCK)isincreasinglyimplicatedinacutevasospasmandchronicvasoconstrictioninmajororgansystems.WeaimedtoascertainwhetherincreasedROCKactivityplaysaroleinthecoronarydysfunctioninearlydiabetes.Synchrotronradiationmicroangiographywasusedtodetermineinvivocoronaryresponsesindiabetic (3weekspoststreptozotocin65mg/kg ip)andvehicletreatedmaleSDrats.Changesinvesselnumberandcalibreduringvasodilatorstimulationbeforeandafterblockadeofnitricoxidesyn-thaseandcyclooxygenasewerecomparedbetweenrats.Diabeticratsshowednosignificantchangesinfibroticscores,mediatolumenratio,capillarydensityorbaselinevisiblevesselnumberorcalibre.Respons-estoacetylcholineweresimilarbetweengroups,butthediabeticgroupshowedmoresegmentalconstrictionsduringblockade,whichwerenotcompletelyalleviatedbyacetylcholine,butwerealleviatedbyfa-sudil(10mg/kgiv).Further,secondordervesselbranchesindiabeticratsweresignificantlymoredilatedafterfasudilcomparedtocontrolrats.ROCK2expressionwasborderlinegreater indiabetichearts.Basedonnitrotyrosinestainingoxidativestresswasnotsignificantlyelevatedindiabeticrats.WeconcludethattonicROCKmediatedva-soconstrictioncontributestocoronaryvasomotortoneinearlydiabe-tes.NoCOI.


2P-044Involvement of PAF, but not histamine or serotonin, in increased pulmonary vascular resistance during ana-phylactic hypotension in anesthetized miceWang, Mofei; Shibamoto, Toshishige; Yamamoto, Yuki; Tanida, Mamoru; Sun, Lingling; Kurata, Yasutaka(Department of Physiology II, Kanazawa Medical University, Uchinada, Ishikawa, Japan)

Systemicanaphylaxisaccompaniespulmonaryvasoconstriction,whichmaycontributetoincreasedrightheartafterload,andfinallyanaphy-lactichypotension.Recentlywereportedthatthesystolicrightven-tricularpressure,measuredasan indicatorofpulmonaryarterialpressure(PAP),didnotincreaseduringanaphylaxisinanesthetizedmice (Shinomiyaetat.ExpLungRes.2013).However,pulmonaryvascularresistance(PVR)wasnotexactlymeasuredinthatpreviousstudy.HereitwasdeterminedbymeasuringdirectlyPAP,leftatrialpressureandaorticbloodflow(ABF)inopen-chestsensitizedBALB/cmice.Wealsoclarifiedtherolesofplatelet-activatingfactor (PAF),histamineandserotoninintheresponses.Anaphylaxiswasinducedbyanintravenousinjectionoftheovalbuminantigen.Alongwiththeaforementionedvariables,meanarterialpressure(MAP),centralvenouspressure,andairwaypressurewerecontinuouslymeasured.Insensi-tizedcontrolmice,MAPandABFsubstantiallydecreasedfollowingatransientincreaseaftertheantigeninjection,whilePAPincreasedat1–3minafterantigen.PVRincreasedtothepeakof2.8-foldat20minandreturnedtowardsthebaselinelevels.ThePAFantagonistatten-uatedPVRelevation,althougheitherantagonistofPAForhistamineattenuatedhypotension.Inconclusion,PAF,butnothistamineorse-rotonin,isinvolvedinanincreaseinPVRduringanaphylactichypo-tensioninanesthetizedmice.NoCOI.

2P-045Experimental study on usefulness of second deriva-tive of central pressure waves in normal and heritable hypercholesterolemic rabbitsKatsuda, Shin-ichiro1; Takazawa, kenji2; Horikoshi, Yuko1; Kusanagi, Masahiko3; Hazama, Akihiro1(1Department of Cellular and Integrative Physiology, Fukushima Medical University School of Mededicine, Fukushima, Japan, 2Tokyo Medical University Hachioji Medical Center, Hachioji, Japan, 3Japan Laboratory Animals, Inc., Tokyo, japan)

Weinvestigatedhowsecondderivativeofcentralpressurewaves(SDCPW)reflectedcardiovascularhemodynamicsinnormalandKuro-sawa andKusanagi-hypercholesterolemic (KHC) rabbits aged 12months.Pressureandflowwavesattheascendingaortaweresimul-taneouslyrecordedusingacatheter-tiptransducer(3Fr)andanultra-sonicflowprobeunderpentobarbitalanesthesia (30mg/kg). Initialpositive (a)andearlynegative (b)wavesofSDCPWweredistinct,whereasc,dandewaveswerenotclearinbothstrains.Anamplituderatioofbtoawaves(b/a)was-0.71±0.06(mean±SD)and-0.48±0.05inthenormalandKHCrabbitgroups,respectively,whichwassignificantbetweentworabbitstrains (p<0.001).Asurrogate indexofaorticcompliance(SV/PP)wassignificantlysmallerintheKHCrabbitgroup(0.043±0.008) than intheage-matchedcontrolgroup (0.062±0.009)(p<0.001),whereSVandPPwerestrokevolumeandpulsepressure,respectively.Thevalueofb/ashowedweak (r=0.571,p<0.05)andstrong(r=0.819,p<0.001)negativecorrelationwithSV/PPinthenormalandKHCrabbitgroups,respectively.Wecanconcludethatb/aofSDCWPwasusefulforestimatingaorticcompliancelikethatofsecondderivativeofphotoplethysmogramwaveform.NoCOI.

2P-046Significance of Hairy-related transcription factor (Hrt) in ischemia-induced angiogenesisHayashi, Hisaki1; Nakagawa, Osamu2(1Physiology, Aichi Medical University, Nagakute, Japan, 2Cardiovasc. Res. Sys., Nara Med. UniV., Kashihara, Japan)

Angiogenesisisaprocesstoformnewbloodvesselsfrompreexistingvesselsunderphysiologicalandpathologicalconditions.TheNotchsignalingpathwayplaysacriticalrole inembryonicangiogenesis.Hairy-relatedtranscriptionfactor1(Hrt1)andHrt2areenrichedinthecardiovascularsystem,andtheirexpressionisdirectlyregulatedbytheNotchsignalactivation.Hrt1/Hrt2compoundKOmicedie inuterowithseverevascularabnormalities, indicatingthatthesetwoHrtgeneshaveessentialroles inembryonicvasculardevelopment.Notchsignalingisalsoimplicatedinpostnatalangiogenesis,butthesignificanceofHrt1andHrt2,especiallythatinpathologicalconditions,remainedunclear.Inamousemodelofpathologicalangiogenesisbyhindlimb ischemia (HLI)surgerywithfemoralarteryocclusion, themRNAexpressionofHrt1,butnotHrt2,wasmarkedly induced inischemicmuscle.DominantinductionofHrt1mRNAexpressionwasalsoobservedbythetreatmentofculturedendothelialcellswithVEGF,anactivatorofNotchsignal,suggestingthatHrt1isaprincipalmedi-atorofNotchactivationinendothelialcellsofischemicmuscle.BloodflowrecoveryinHrt1KOischemichindlimbafterHLIwassignificant-lybluntedcomparedtoWT.Revascularizationofischemichindlimbwasalsoattenuated inHrt1KOmice.Accordingly,theseverityofischemicinjurysuchastissuenecrosisandlossismuchhigherinHrt1KOmicethanWT.TheseresultssuggestthatHrt1playsimportantrolesinregulatingischemia-inducedangiogenesis.NoCOI.

2P-047Identification and characterization of disease-specific activator of G-protein signaling (AGS) proteins: explo-ration of AGS protein in the polycystic kidney diseaseSuzuki, Hiroko1; Sakima, Miho1; Mamun, Abdullah Al1; Hayashi, Hisaki1; Iwase, Satoshi1; Inukai, Yoko1; Nishimura, Naoki1; Sato, Maki1; Shimizu, Yuuki1; Frank, Park2; Sato, Motohiko1(1Department of Physiology, Aichi Medical University, Nagakute, Aichi, Japan, 2University of Tennessee, Memphis, USA)

CellsignalmediatedbyheterotrimericG-proteinplaysimportantrolesinmaintainingthehomeostasisofcardiovascularsystemagainstphys-iologicalstimuli.InadditiontoG-proteincoupledreceptor(GPCR)atcell-surface,otherentitiescandirectlyregulatetheactivationstatusofG-proteins,andtheyareinvolvedinthedevelopmentofdiseases.Asapartofapproachtoidentifysuchproteins,wehavepreviouslyidentifiedactivatorofG-proteinsignaling8(AGS8)fromtheischemicmyocardiumandAGS11fromthehypertrophicheart.Thus,AGS8wasinvolvedinthehypoxia-mediatedapoptosisofcardiomyocytesviaregulationofGβγandchannelproteinconnexin43.AGS11,isolatedfromthehypertrophicheart,translocatedGα16intothenucleusandincreasedthetranscriptionoftheclaudin14.Weexpandedtheap-proachtothepolycystickidneydiseasetoidentifydisease-specificAGSproteins.CDNAlibrary,generatedfromtheratmodelwithprogressivecysticenlargementofthekidney,wassubjectedtoyeast-basedfunc-tionalscreenforreceptorindependentactivatorsofGαi3,GαsorGα16.InadditiontopreviouslyreportedAGSproteins,we identifiedtwocandidatesforAGSproteinexpressingpolycystickidneydisease.Therolesoftheseproteins inthepathophysiologyofpolycystickidneydiseasewillbediscussed.NoCOI.


2P-048Mechanisms of cardioprotective effects of high con-centration of magnesium on hypoxia/reoxygenation injury in chronic magnesium deficient rat heart are dif-fer from normal rat heartWatanabe, Makino; Kakigi, Ryo; Iesaki, Takafumi; Okada, Takao(Department of Physiology, Juntendo University Faculty of Medicine)

Magnesium(Mg)deficiencyhasbeenreportedtobeassociatedwiththedevelopmentofthecardiovasculardiseases,suddendeathandmetabolicsyndrome.Wereportedthat1)extracellularhighMgcon-centration(12mM)duringhypoxiaprotectedtheheartfromhypoxia/reoxygenationinjuryinnormalrat,anditsprotectiveeffectwasin-ducednotonlybysuppressionofenergyconsumption,butalsobyacceleratedopeningofmitochondrialKATPchannel,2)inchronicMg-de-ficientmicehearts, tolerancetohypoxia/reoxygenation injuryde-creased.Aimofthisstudywastodeterminewhetherhighconcentra-tionofMgduringhypoxiacouldprotectthehypoxia/reoxygenationinjuryinchronicMg-deficientratheartusingLangendorffperfusedratheartmodel.RatswerefedanMg-deficientdietfor8weeks.Theheartsunderwenthypoxiafor30min,followedbyreoxygenationfor30min.InMg-deficientgroup,therecoveryofcardiacfunctionfromhypoxia/reoxygenation injurywassignificantlyworsethanthatofcontrolgroup.WhenextracellularMgconcentrationincreasedfrom1.2to12mMduringhypoxia,inMg-deficientgroup,recoveryofPRPwassimilartothatofcontrolgroup.5-hydroxydecanoicacid(5-HD),amitochondrialKATPchannelspecificblocker,partiallyinhibitedtheprotectiveeffectofhighMg inMg-deficientgroup.TheseresultssuggestthatcardioprotectiveeffectsofhighMginMg-deficientratarenotonlyopeningthemitochondrialKATPchannelbutalsounknownothermechanism.NoCOI.

2P-049Application of 4-hydroxybenzoic acid as a trapping agent to monitor hydroxyl radical production during myocardial ischemia and reperfusionInagaki, Tadakatsu; Akiyama, Tsuyoshi; Zhan, Dong-Yun; Du, Cheng-Kun; Shirai, Mikiyasu(Dept. of Cardiac Physiol, National Cardiovascular Center Research Institute, Osaka, Japan.)

Background:Hydroxylradical (OH) is involved inthemyocardialischemia/reperfusion injury.Hydroxylationofaromaticcompoundshasbeenusedasan indirectmarkerof invivoproductionof .OH.Purpose:Todetecttheproductionof.OHduringmyocardialischemia/reperfusionbyapplying4-hydroxybenzoicacid(4-HBA)asatrappingagent.Methods:Usingmicrodialysis technique,dialysisprobewasimplantedintheleftventricularmyocardiumofanesthetizedratsandperfusedwithRingersolutioncontaining4-HBA(5×10-4M)at2µl/min.Dialysatesweresampledatbaseline,during30min-coronaryocclusionandafterreperfusion(180min).Asamplingperiodwas10min.Dialy-sateconcentrationofthe4-HBAhydroxylationproduct,3,4-dihydroxy-benzoicacid (3,4-DHBA)wasmeasuredusingHPLC-ECD.Results:Dialysate3,4-DHBAconcentrationwas1.5±0.3nMatbaseline.Aftercoronaryocclusion,dialysate3,4-DHBAconcentrationgradually in-creasedandreached3.0±0.4nMat20-30minaftercoronaryocclusion.Afterreperfusion,dialysate3,4-DHBAconcentrationfurtherincreasedto4.9±0.8nMat0-10minafterreperfusionandthenreachedthepeaklevel (6.0±0.7nM)at10–20minafterreperfusion.After20minofreperfusion,dialysate3,4-DHBAconcentrationgraduallydeclinedandalmostrecoveredbaselinelevelat120minafterreperfusion.Conclusion:Detectionof.OHwith4-HBAcouldbelesscomplicatedandmorere-liablethanthatwithotheraromaticcompounds.NoCOI.

2P-050Myocardial interstitial serotonin (5-HT) and 5-hydroxy-indole acetic acid (5-HIAA) levels during isch-emia-reperfusionDu, Cheng-Kun; Zhan, Dong-Yun; Akiyama, Tsuyosh; Inagaki, Tadakatsu; Shirai, Mikiyasu(Dept. of Cardiac Physiol., Natl. Cereb. Cardiovas. Ctr., Suita, Japan.)

Background:Ithasbeenreportedthat5-HTaccumulatesintheheartduring ischemia-reperfusionandcontributestothecardiomyocyteinjury.Littleinformationis,however,availableontheinvivo5-HTkineticsduringmyocardialischemia-reperfusion.Purpose:Toelucidatethe invivo5-HTkinetics includingaccumulationanddegradationduringmyocardialischemia-reperfusion.Methods:Microdialysistech-niquewasappliedtotheleftventricularmyocardiumofanesthetizedrats,anddialysate5-HTand itsmetabolitebymonoamineoxidase(MAO),5-HIAAconcentrationsweremonitoredasindicesofmyocar-dialinterstitial5-HTand5-HIAAlevelsduring30min-ischemiaand45min-reperfusion.Results:Invehicle,5-HTlevelscontinuedtoincreaseduring ischemiabut5-HIAAlevelsdecreasedandthenrecoveredduringischemia.Afterreperfusion5-HTlevelstransientlyincreasedandthendeclined.5-HIAAlevelsincreasedafterreperfusionandkepthighlevelby45minafterreperfusion.Localadministrationofpargyline(1mM),aMAOinhibitordidnotinfluencetheincreasein5-HTlevelsandthedecreasein5-HIAAlevelsduringischemia.Pargylineenhancedthe increase in5-HTlevels immediatelyafterreperfusionandsup-pressedthe increase in5-HIAAlevelsafterreperfusion.Conclusion:Simultaneousmonitoringof interstitial5-HTand5-HIAAlevels isuseful for understanding 5-HT kinetics during myocardial isch-emia-reperfusion.NoCOI.

Poster PresentationsNeuron, Synapse (2)


2P-051Dynamin isoforms decode action potential firing for synaptic vesicle recycling in sympathetic neuronsTanifuji, Shota; Mochida, Sumiko(Dept Physiol, Tokyo Med Univ, Tokyo, Japan)

Presynapticnerveterminalsmustmaintainstableneurotransmissiondespiteencounteringwidefluctuationsinthenumberandfrequencyof incomingactionpotentials (APs).Here,weexaminedtheroleofdynamininactivitysensinginthepresynapticsuperiorcervicalgan-glion(SCG)neurons.Dynamin1,2or3expressedinSCGneuronswasacutelyknockeddownbymicroinjectionofthesiRNA.3dayslater,eachdynaminexpressioninthesomawasapproximately60%andthesynaptictransmissionwithvariousAPfiringpatternwasimpaired.Paired-pulserecordingswithAPintervalof20and30msshowedthatknockdown(KD)ofdynamin2or3reducedtheamplitudeof thesecondexcitatorypostsynapticpotential (EPSP)morethancontrol.WithAPintervalof50–1000ms,eachdynaminKDsimilarlyreducedthesecondEPSPamplitude.EPSPrecordingduringandafterhighfrequencyAPfiringat5,10or20Hzshowedthatdynamin1,2KDinducedseveresynapticdepressionduringandafterAPfiring,where-asdynamin3KDinduceditduringAPfiring.Incontrast,withlowfrequencyAPfiringat0.05and0.2Hz,dynamin3KDreducedgrad-uallysynaptictransmission,whereasdynamin1or2KDreduceditat0.2Hz.Afterdepletionofsynapticvesiclesinthereadilyreleasablepool,dynamin1or2KDdelayedthefastrecovery,whiledynamin2or3KDdelayedtheslowrecovery.Theseresultssuggestthatthethreeisoformsofdynamin,anessentialendocyticprotein,workindi-viduallytomatchvesiclereusepathways,havingdistinctrateandtimeconstantwithphysiologicalAPfrequencies.NoCOI.

2P-052Temporal Ca2+ regulation of synaptic vesicle release efficacy following action potential firing at the release siteMori, Michinori; Tanifuji, Shota; Mochida, Sumiko(Dept Physiol, Tokyo Med. Univ., Tokyo, Japan)

Atthepresynapticactivezone,anactionpotential(AP)triggersCa2+influxandenteredCa2+causessynapticvesicles(SVs)exocytosis.Here,weshowtemporalCa2+regulationofreleasereadySVs(RRSVs)inthereadilyreleasablepool(RRP)followingAPfiringinpresynapticsym-patheticneurons incultureapplyingrapid-on-rateandslow-on-rateCa2+chelators.UnderreducedbasalreleaseprobabilitybymembranepermeableO,O’-1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’- tetraaceticacid,tetraacetoxymethylester(BAPTA)-AMorEthyleneglycol-bis(βam-inoethyl)-N,N,N’,N’-tetraacetoxymethylEster (EGTA)-AM,arapidsynapticdepressionwithin30msand30–120msaftergenerationofanAPwasreducedbyBAPTAandEGTA,respectively.Duringre-petitiveAPfiringwith50–200msinterval,EGTA,butnotBAPTA,delayedtheRRPreplenishment.AftertheRRPdepletionwithatrainofAPsfiring,BAPTAdelayedrapidandslowreplenishmentofSVsintotheRRP,whileEGTAdelayedtheslowRRPreplenishment.SlowCa2+regulationsupportedgenerationandmaintenanceapresynapticshort-termplasticity,augmentation.Unexpectedly,arapidCa2+regu-lationunderliesinductionofposttetanicpotentiation.Together,follow-ingAPfiring,thetemporalchangeinCa2+ levelatreleasesites, inadditiontospatialchangeinthepresynapticterminalandbeneaththeactivezoneaspreviousfindings,controlstheRRSVsreleaseefficacyforincomingnervesignals,andunderliessynapticplasticityresultedfromvariousfiringactivity.NoCOI.

2P-053Myosin IIB and VI regulates synaptic vesicle traffick-ing at presynaptic nerve terminals.Hayashida, Michikata; Tanifuji, Shota; Mochida, Sumiko(Dept physiol, Tokyo Med Univ, Tokyo, Japan)

Accumulatingevidencesuggeststhatmyosinregulatesvesicletrans-portinneurons.Wehaveshownthatamongmyosinisoformsprevi-ouslycharacterizedinneuronsmyosinIIBislocalizedtopresynapticnerveterminalofculturedsympatheticneuronsfromtheratsuperiorcervicalganglion.Recently,myosinVIisreportedtobeinvolvedinpostsynapticvesicleendocytosisinthebrain.HereweexaminethefunctionofmyosinIIBandVIfortraffickingofsynapticvesiclesinpresynapticnerveterminalsbymeasuringchangesinneurotransmit-terreleasebydisturbing functionofmyosinsbyspecificsiRNAs.SynaptictransmissiontriggeredbyactionpotentialswasinhibitedbymyosinIIBorVIsiRNA,butnotbyIIAorcontrolsiRNA.SynapticdepressionwithhighfrequencyAPtrainswasacceleratedbymyosinIIBorVIknockdown.KnockdownofmyosinIIBorVIknockdowndelayedrapidorslowrecoveryofsynapticvesiclesinthereadilyre-leasablepoolafterthedepletion,respectively.Thistraffickingstepishighlysensitivetoactivity-dependentcalciumtransient,consistentwithacalciumrequirementformyosinactivation.Theseresultssug-gestthatmyosinIIBandVIcontroladiscretecalcium-dependentsynapticvesicle traffickingtothereadilyreleasablepool throughdistinctrecyclingpathwaysinresponsetopresynapticfiringactivity.NoCOI.

2P-054Optical and electrophysiological measurements of synaptic exo-endocytosis at the rat calyx of Held syn-apseOkamoto, Yuji; Sakaba, Takeshi; Midorikawa, Mitsuharu(Graduate School of Brain Science, Doshisha University, Kyoto, Japan)

Atthenerveterminal,neurotransmitterisreleasedbythefusionofsynapticvesicleswithpresynapticplasmamembrane.Afterexocyto-sis,vesiclesareretrievedbyendocytosisandrecycledforreuse.Thecouplingofexo-andendocytosisofsynapticvesiclesisessentialforthemaintenanceofsynaptictransmission.Theexo-andendocytosishavebeenmeasuredbycapacitancemeasurement,orbyimaging(e.g.FMdyes,pHluorin).However,thenumberofstudiesthatapplybothtechniquesatthesametimeisverylimited,becauseofthetechnicaldifficultycausedbythesmallsizeofthenerveterminal(usuallyaround1µm).ThecalyxofHeldisalargesynapseintheauditorybrainstem.Becauseofitsterminalsize(~20µm),itallowsustoapplyelectrophysiologicalandopticalmethodsandanalyzepresynapticmechanismsreliably.Inthisstudy,wesimultaneouslymeasuredtheturnoveroffusionre-latedsynapticvesicleproteinsandplasmamembranebyapplyingimagingmethodandelectrophysiologicalrecordings,respectively.Welabeledsynaptotagmin-2(aCa2+censorforexocytosis)withanantibodycoupledtopH-sensitivefluorophorecypHer5Etomonitorthedynam-icsduringexo-andendocytosis.FluorescentchangesofcypHerweremeasuredsimultaneouslywithmembranecapacitancetocomparethecyclingofsynapticvesicleproteinsandthatofplasmamembrane.Byvaryingstimulusconditionsorbyapplyingpharmacologicalagentstomanipulatethetimecourseofendocytosis,wetestedhowthetimecoursesofbothmeasurementswerecorrelated(ornotcorrelated).NoCOI.


2P-055Synaptophysin-EGFP puncta slowly move retrograde-ly along axons in slice culture during the phase of en passant type synaptogenesisYoshioka, Noboru; Isoo, Noriko; Murabe, Naoyuki; Kameda, Hiroshi; Takahashi, Ichiro; Sakurai, Masaki(Department of physiology, Teikyo University School of Medicine)

Oneneuronmakesmultiplesynapticconnectionstopostsynapticcells,whichcanbeimplementedbyenpassanttypesynapses.Littleisknownabouttheprocessofenpassanttypesynaptogenesis.Oneofthemostimportantaspectsofsynaptogenesisistheassemblyofsynapticves-iclesonthepresynapticactivezone.Whatistheentirecourseofeventsforsynapticvesiclesuntilgettingtogetheratenpassantsynapses?Weattemptedtovisualizetheoverallprocessofsynapticvesicleas-semblyatenpassantsynapsesbywayofourstagetopCO2incubatorsystemequippedtoanuprightconfocalmicroscope.Weculturedcorticospinalslicecocultureinthisincubatorandchasedthefateofsynaptophysin-EGFP(Syp-EGFP)labeledvesiclesatvariousstagesforupto50hours.Duringtheearlyphaseof2–4daysinculture,EGFPpositivevesiclesappearedtoformclusterstoshowSyp-EGFPpuncta.Unexpectedly,thoseSyp-EGFPpunctashowedveryslow(lessthan100µm/hour),clearlyretrograde,andlong-distancemovement(morethan200µm).Themovementconsistedofconstant-velocitymovingphasesandpauses.Frequencyofmovementdeclinedtoward7DIV,whenactivecorticospinalsynapsesarethere.Atthisstage, largerdegreeofcolocalizationofactivezoneproteinBassoonwithSyp-EGFPpunctawasdetected. After formationofclusters, theclustersofvesiclesmaymoveslowlyandretrogradelyforlong-distancetofindthesiteofenpassantsynapses.NoCOI.

2P-056Effects of synaptotagmin binding on SNARE-mediat-ed fusion and SNARE complex formationKuroki, K; Masumoto, T; Ohmori, I; Michiue, H; Nishiki, T; Matsui, H(Dept of Physiol, Okayama Univ Grad School of Med, Dent & Pharm Sci, Okayama, Japan)

SNAREs(syntaxin,SNAP25,synaptobrevin)andsynaptotagminplayacentralroleduringCa2+-dependentneurotransmitterrelease.Recent-ly,wefoundthatsynaptotagmindissociatesfromsyntaxinbyCa2+.Here,we have studied the effects of synaptotagmin binding onSNARE-mediatedfusionand itscomplex formation.RecombinantSNAREswereexpressedinE.coli,purifiedandreconstitutedintoli-posomes.Inlipidmixingassay,incubationwiththecytosolicfragmentofsynaptotagmininhibitedSNARE-mediatedlipidmixingintheab-senceofCa2+.However,thisinhibitionwasreversedbyCa2+,indicatingthatCa2+releasedtheinhibitoryeffectofsynaptotagminfromSNAREs,whichtriggeredfusion.TheSNAREcomplexhasbeenproposedtoassemblelikeazipper,startingfromthemembrane-distalterminiandprogressingtowardthemembrane-proximalterminioftheSNAREmotifs.IfsynaptotagminsuppressesSNARE-mediatedfusionbypre-ventingSNAREcomplexesfromfullyzippering,SNAREsmayexistinahalfzipperedstatewhenboundtosynaptotagamin.TodeterminewhethersuchanintermediateSNAREcomplexexistsinthepresenceofsynaptotagmin,weemployedFRET.Thedataindicatethatbindingofsyntaxinandsynaptobrevinproceedstotheimmediatelyvicinityoftheirtransmembranedomainseveninthepresenceofsynaptotag-minwithoutCa2+.Theseresultssuggestthatsynaptotagminsuppress-esSNARE-mediatedmembranefusionthroughitssynatxinbindingbeforeCa2+triggerswithoutinhibitingtheinteractionbetweensyn-taxinandsynaptobrevinSNAREmotifs.NoCOI.

2P-057Domain-swapped oligomerization of SNAP25 for ul-trafast exocytosis at presynaptic terminalsSawada, Wakako; Takahashi, Noriko; Watanabe, Satoshi; Ohno, Mitsuyo; Kasai, Haruo(Structural Physiology, CDBIM, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan)

WeanalysedthemolecularstructureoftheSNAREcomplexusingintermolecularfluorescenceresonanceenergytransfer(FRET)with2-photon fluorescence lifetime imaging. We observed the do-main-swappedoligomerizationofSNAP25atthepresynapticterminalsofmousecorticalneurons.SNAP25isoneoftheSNAREproteinsatthetargetplasmamembraneanditcarriestwoα-helices,SN1andSN2.Wemeasuredthe intermolecularFRETratiobetweenTq-SNAP25andSN1-Venus-SN2andobservedahighbindingfraction(BF:26%)at the active zone. These data indicated the presence of a do-main-swappedmodel,whereSN1boundto theSN2of theotherSNAP25molecule,andformedanoligomer.Inaddition,weutilizedSNAP25knock-outmiceandarescuesystembyviraltransfectionsothatonlyfluorescent-labelledSNAP25 functioned.ThekineticsofCa2+-dependentexocytosiswasmeasuredusingIPSCtriggeredbyflashphotolysisofcagedCa2+compoundsorelectricalfieldstimulation.Transfectionbywild-typeFRETprobestriggeredIPSCwithatimeconstant(τ)of2ms.Furthermore,wedevelopedvariousfluorescentprobesoftheSNAP25mutantwithlongerlinkerbetweenthetwoα-helices (BF:12%,IPSCdelayed),ortheSNAP25mutantcarryingmutations(M71A,I192A),whichtriggerslowermembranefusion(BF:16%,noIPSC).C-terminaldeletionmutant(7or9AA)triggereddelayedIPSCandBFwasreduced.Therefore,thedomainswappingofSNAP25isnecessaryforrapidneurotransmitterrelease.NoCOI.

2P-058Unique Roles of Docking Proteins, SNAP-25 and Syn-taxin, in Short-Term Synaptic Plasticity Discovered by Botulinum Neurotoxins StudiesMaeno, Takashi1; Enomoto, Ko-ichi2(1Department of Physiology, Shimane Medical University, Izumo, Japan, 2Department of Physiology, Shimane University Faculty of Medicine, Izumo, Japan)

Tetanicenhancementandpost-tetanicpotentiationareconspicuousfeaturesofsynapsesinrespectoftheshort-termsynapticplasticity.Sincemembraneimpermeableproteinmodifyingreagentsalteredtheshort-termsynapticplasticity,itssystemwasthoughttobeeitherCa2+channelsordockingproteinslocatingintheactivezonesinsynapticterminalmembranepredominantly.Usingfrogneuromuscularprepa-rations,wemadeaCopernicanbreakthroughby investigatingtheshort-termsynapticplasticity fromdifferentangle: i.e. therolesofsyntaxinandsynaptosome-associatedproteinof25kDa (SNAP-25),plasmalemma-bounddockingproteins.Botulinumneurotoxinsarespecificenzymestoactonthedockingproteins.TruncationofSNAP-25byBotulinumneurotoxinserotypeA(BoNT-A)selectivelyabolishedtheslowpotentiation,whereascleavageofsyntaxinbyBotulinumneurotoxinserotypeC(BoNT-C)suppressedtheaugmentationstrong-ly.Further,doublepoisoningbyBoNT-AandBoNT-Celiminatedbothaugmentationandpotentiation.AlthoughsyntaxinandSNAP-25havebeenknowntocooperativelyactasadockingdevice,theresultsun-doubtedlyindicatethattheseexertuniquetemporallydifferentlateactionsinthemolecularprocessesleadingtotheshort-termsynapticplasticity.BoNTsstudieswereconductedin1997and1998attheDept.Physiol.,ShimaneMed.Univ.NoCOI.


2P-059Functional role of syntaxin 1B in the regulation of syn-aptic vesicle exocytosis and of the readily releasable pool at central synapsesMishima, Tatsuya1; Fujiwara, Tomonori1; Sanada, Masumi1; Kofuji, Takefumi2; Akagawa, Kimio1(1Department of Cell Physiology, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan, 2Division of Radioisotope Research, Kyorin University School of Medicine, Tokyo, Japan)

Twosyntaxin1(STX1)isoforms,HPC-1/STX1AandSTX1B,areco-expressedinneuronsandfunctionasneuronalt-SNAREs.However,littleisknownabouttheirfunctionaldifferencesinsynaptictransmis-sion.STX1Anullmutantmicedevelopnormallyanddonotshowabnormalitiesinfastsynaptictransmission,butmonoaminergictrans-missionsareimpaired.WepreviouslyreportedthatSTX1Bnullmutantmicediedwithin2weeksofbirth.Inthepresentstudy,inordertoexaminefunctionaldifferencesbetweenSTX1AandSTX1B,weana-lyzedthepresynapticpropertiesofglutamatergicandGABAergicsynapsesinSTX1BnullmutantandSTX1A/1Bdoublenullmutantmice.Wefoundthatthefrequencyofspontaneousquantalreleasewaslowerandthepaired-pulseratioofevokedpostsynapticcurrentswassignificantlygreater inglutamatergicandGABAergicsynapsesofSTX1Bnullneurons.DeletionofSTX1BalsoacceleratedsynapticvesicleturnoveringlutamatergicsynapsesanddecreasedthesizeofthereadilyreleasablepoolinglutamatergicandGABAergicsynapses.Moreover,STX1A/1Bdoublenullneuronsshowedreducedandasyn-chronousevokedsynapticvesiclerelease.OurresultssuggestthatalthoughSTX1AandSTX1Bshareabasic functionasneuronalt-SNAREs,STX1Bhasanessentialroleintheregulationofspontaneousandevokedsynapticvesicleexocytosisinfasttransmission.NoCOI.

2P-060Serotonergic fiber distribution and chronic pain-in-duced changes of the emotional behaviors in syntaxin 1A knockout miceMaekawa, Masao; Hori, Yuuichi(Dept of Physiol & Biol Inf, Dokkyo Med Univ Sch of Med, Mibu, Tochigi, Japan)

Serotonergicfibersaredistributedwidelythroughoutthebrainandareinvolvedinvarietyofbrainfunctions.Wehavereportedthatmicewithlargelyreducedserotonergicfibersdemonstrateimpairedper-formanceofwatermaze learningandreducedprepulse inhibition.Syntaxin1Aisinvolvedinthesynaptictransmissionanddysfunctionofthesyntraxin1Aresultsinabnormalbrainfunctioning.Recentworkindicatedthatserotonergicfunctionswereimpairedinthesyntaxin1Aknockoutmice.Tosupporttheideathatsyntaxin1Aaffectstheemotionalbehaviorsthrough impairingserotonergic functions,weinvestigatedtheserotonergicfiberdistributioninthebrainandtheemotionalbehaviorsofsyntaxin1Aknockoutmice.ThereductionoftheserotonergicfibersinthebrainofknockoutmicewasinvestigatedunderfluorescentmicroscopeusingtheBACtransgenicmouseex-pressinggreenfluorescentproteinunderthecontrolofpromoterofthetryptophanhydroxirase2.Weinvestigatedtheemotionalbehaviorsoftheknockoutmicewitharthritichindpaw.Inthearthriticmicegroup,CFA(0.05ml)wasinjectedintokneejointofthelefthindpawunderhalothaneanesthesia1-2daysbeforebehavioraltests.Ultrason-icvocalization (USV,50±4kHz)wasrecordedafternociceptivestimulationtothehindpaw.ArthriticknockoutmiceemittedUSVtoagreaterextentcomparedtoarthriticwildtypemice.Theresultssuggestthattheknockoutofsyntaxin1Aaffectchronicpaininducedemotionalbehaviorsthroughtheimpairmentoftheserotonergicsysteminthebrain.NoCOI.

2P-061Direct mechanical regulation of presynaptic functions by enlargement of dendritic spines in CA1 pyramidal neuronsWatanabe, Satoshi1,2; Takahashi, Noriko1; Noguchi, Jun1; Kasai, Haruo1,2(1Laboratory of Structural Physiology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan, 2CREST, JST, Kawaguchi, Japan)

Dendriticspinesofcorticalexcitatorysynapsesundergoextensiveenlargementduringlong-termpotentiation(LTP).Ithasbeen,howev-er,stillelusivewhetherLTPinvolvesalterationsofpresynapticfunc-tions.Forexample,retrogrademessengershavenotbeenidentified.Wethereforetestthepossibilitythattheenlargementofdendriticspinesmechanicallyfacilitatespresynapticfunctions.GlutamatereleasefrompresynapticterminalsonanidentifiedspinewasimagedusingGCaMP6sexpressedinCA1pyramidalneuronsinculturedhippocam-palslices.CalciumsignalsfromdendriticspinesshowedeithersuccessorfailureoftransmissioninresponsetoSchaffercollateralstimulation,allowingmeasurementoftransmitterreleaseprobability(Pr).WefoundthatPrwastightlycorrelatedwiththespinevolume intherest-ing-state,andmarkedlyenhancedduringartificialspineenlargementinducedbybluelightirradiationofphoto-activatableRac1(PA-Rac1).LocalapplicationofhypertonicsolutionalsoincreasedPr.Moreover,ourSNARE-basedFRETprobesdemonstratedimmediateincreasesinpresynapticSNAREcomplexbyspineenlargement.Theseresultssuggestthatspinestructuralplasticitycanregulatepresynapticfunc-tionsbydirectmechanicalinteractionsinrapidandsynapsespecificmanner.Thus,thespineenlargementmayrapidlypotentiatebothpreandpostsynapticfunctions,andeffectivelyfacilitatetheformationofcellassemblyincorticalnetworks.NoCOI.

2P-062Roles of Paranodal Potassium Channels in Cerebellar Purkinje Cell AxonsHirono, Moritoshi; Misonou, Hiroaki(Graduate School of Brain Science, Doshisha University, Kizugawa, Japan)

Voltage-gatedpotassiumchannelsareuniquelysituatedatandnearthenodeofRanvierandthoughttoplaycrucialrolesinregulatingactionpotentials(APs)intheaxon.Recentlyweobtainedresultsthatthecalcium-andvoltage-activatedSlo/BKpotassiumchannel isex-pressed intheparanodalregionofmyelinatedaxons incerebellarPurkinjecells(PCs).Therefore,weexaminedwhetherparanodalSlo/BKchannelsregulateAPsinPCaxons.Usingmousecerebellarslices,werecordedantidromicAPs,whichwereevokedbyelectricalstimu-lationofPCaxonsinthewhitematter,fromtheirsomabywhole-cellcurrent-clamprecordings.ShortsinglestimulationcausedasingleAPwithadelaydependingonthedistanceofthestimulationelectrodefromthesoma,suggestingthattheAPsweobservedareantidromicAPs.TotesttheroleofSlo/BKchannelsinAPpropagation,weex-aminedthefailurerateoftheantidromicAPsuponrepetitivestimu-lationat50-300Hz.Inthecontrol,failureoftheantidromicAPswasobservedwiththestimulationathighfrequenciesover100Hz.Puff-ap-plicationofaSlo/BKchannelblockernearthestimulationsiteintheaxonsignificantly increasedthefailurerate.Moreover, localaxonalapplicationsofnickelalso increasedthefailurerate.TheseresultssuggestthatparanodalSlo/BKchannelsimpactpropagationofanti-dromicAPs,particularlyathighfiringrates.Wewillalsodiscussax-onalcalciumtransients,whichactivateparanodalSlo/BKchannels,theionicmechanismofhowSlo/BKchannelsregulateAPs,andtheirpotentialimplicationsincerebellarPCs.NoCOI.


2P-063Estimation of the coupling distance between Ca2+ channels and exocytotic sensors using 3D diffusion modelImoto, Keiji1,2; Satake, Shin'Ichiro1,2(1Dept Information Physiol, Natl Inst Physiol Sci, Okazaki, Japan, 2Dept Physiol Sci, School Life Science, The Graduate Univ for Advanced Studies (SOKENDAI))

Neurotransmitterrelease isevokedbyCa2+enteringthroughCa2+channels.Ca2+spreadsbydiffusionandreachesexocytoticCa2+sensorswithin~100nmfromthechannel.Toestimatethecouplingdistanceandtheeffectofpaired-pulseactivationonthecouplingdistance,weconstructedasimplemodelofCa2+diffusionandreactionwithbuffers(fixed,mobile,andexogenous),andcombinedittotheCa2+-dependentstochasticreleasemodel.Simulationwasdonein2D-and3D-spacesusingthefinitedifferencemethodwiththe10nmgridsize.Whentherewasonechannel,aseparatemodelofconcentricmultilayeredshellswasalsoused.In2Dsimulation,thecouplingdistancewas~100nm.WhentwoCa2+channels(300nmapart)wereactivated,Pr(releaseprobability)atthemidpointwasenhancedbypairedpulseactivation.EnhancementwasclearlyreducedbyEGTA,withoutinfluencingPrnearthechannels.Theseresultswereconsistentwithourpreviousexperimentaldata,andsuggestthemicrodomainsignalinginpaired-pulsefacilitation.In3Dsimulation,diffusionbecamethepredominantdeterminant,andthecouplingdistancebecameshorter.TheEGTAeffectonPrbetweentheactivatedchannelswasnotobserved.Thissuggestsinvolvementofotherfactorstodeterminethecouplingdis-tance,suchasoccupationofyetanotherendogenousbuffers,hindranceofdiffusionbythe intracellularmatrix,sustainedCa2+bindingtoexocytoticsensors.Furtherscrutinyisrequiredforamorerealisticmodel.NoCOI.

2P-064Role of glutamate transporter in inhibitory synaptic transmissionIshibashi, Hitoshi1,2; Yamaguchi, Junya2; Nakahata, Yoshihisa2; Nabekura, Junichi2(1Dept. Physiol., Kitasato Univ. School of Allied Health Sci., Sagamihara, Japan, 2Dept. Develop. Physiol., Natl. Inst. Physiol. Sci., Okazaki, Japan)

Fast inhibitoryneurotransmission inthecentralnervoussystemismediatedbyGABAandglycine,whichaccumulateinthesamepre-synapticnerveterminalbyvesicularinhibitoryaminoacidtransport-er(VIAAT)andarethenco-released.However,themechanismsthatcontrolthepackagingofGABA+glycineintosynapticvesiclesarenotfullyunderstood.Inthisstudy,wedemonstratethedynamiccontroloftheGABA/glycineco-transmissionbytheneuronalglutamatetrans-porter,usingpairedwhole-cellpatchrecordingfrommonosynaptical-lycoupledculturedspinalcordneuronsderivedfromVIAAT-Venustransgenicrats.Shortstepdepolarizationofpresynapticneuronsevokedunitary(cell-to-cell)inhibitorypostsynapticcurrents(IPSCs).Undernormalconditions,thefractionalcontributionofpostsynapticGABAorglycinereceptorstotheunitaryIPSCdidnotchangeduringa1hourrecording.RaisedextracellularglutamatelevelsincreasedtheamplitudeofGABAergicIPSCsbyenhancingglutamateuptakeandreducedglycinerelease.Similareffectswereobservedwhenpresynapticneuronswere intracellularlyperfusedwithglutamate.Interestingly,high-frequencytrainsofstimulationdecreasedglyciner-gicIPSCsmorethanGABAergicIPSCs.ThepresentresultssuggestthattheenhancementofGABAreleasebyglutamateuptakemaybeadvantageousforrapidvesicularrefillingoftheinhibitorytransmitteratmixedGABA/glycinergicsynapsesandthusmayhelppreventhyperexcitability.NoCOI.

2P-065Simulation analysis of K+ buffering in astrocyteMurakami, Shingo; Kurachi, Yoshihisa(Dept. Pharmacol., Osaka Univ. Sch. Med.)

InresponsetoelevationofextracellularK+concentration([K+]out),as-trocytesclearexcessiveextracellularK+tomaintainproperenviron-mentforneuralactivity.TheK+clearancemechanisminastrocytestakestwoforms:K+uptakeandK+spatialbuffering.Thehigh[K+]outalsoinducesswellingofastrocyte,whichleadstoedemaandcelldeathinthebrain.HerewereportsimulationanalysisonthemechanismsoftheastrocyticK+clearanceandswelling.Astrocytemodelswereconstructedbyincorporatingintoacompartmentmodelvariousmech-anisms,suchasintra/extracellularionconcentrationsofNa+,K+andCl-,cellvolume,andmodelsofNa,K-ATPase,Na-K-Clcotransporter(NKCC),K-Clcotransporters, inwardlyrectifyingK+ (KIR)channel,passiveCl-current,andAquaporinchannels.Simulatedresponseofastrocytemodelstohigh[K+]outrevealedsignificantcontributionsofNKCCandNa,K-ATPasetotheincreasesoftheintracellularK+andCl-concentrations,andswelling.Moreover,weshowthattheKIRchannellocalizedatsynapticcleftabsorbstheexcessiveK+bydepo-larizingequivalentpotential forK+ (EK)abovemembranepotential,whiletheK+releasethroughKIRchannellocalizedatperivascularisenhancedbyhyperpolarizingEKanddepolarizingmembranepotential.FurtheranalysisofsimulateddrugeffectsshowsthatK+uptake,K+releaseandswellingcanbemodulateddifferentlybyblockingeachoftheionchannelsandtransporters.Thus,byidentifyingtheirdistinctroles,wehereshowthatastrocytescanbenewpotentialtargetindrugtherapyfordiseasesaccompanyinghigh[K+]out.NoCOI.

2P-066Manipulation of neuronal activities by non-invasive optogenetic approach to astrocytesYoshida, Keitaro; Xu, Ming; Tanaka, Kenji; Mimura, Masaru; Takata, Norio(Department of Neuropsychiatry, School of Medicine, Keio University)

Howandwheredofocalneuronalactivitiesspread?FunctionalMRIcanbeatooltomonitorspreadingmanners,however,therequirementsofbothtime-controlledactivationandreproducibleoutcomemaketheapplicationdifficult.Wethuschallengedtoestablishanexperimentalmodelthatsatisfiesabovepoints.Optogenticsisidealfortime-controlledmanipulation,butanopticfiberinsertiontobrainparenchymacausesaninjury,whichdisturbsareproducibleresult.Theusageoflight-sen-sitivechannelrhodopsinvariant,C128S,permittedsuccessfulphotoac-tivationofcorticalastrocytesbyanilluminationovertheskull,result-inginconsistentneuronalactivation.Wefurtherdemonstratedthataweakilluminationresultedinthefocalactivationbutastrongillumi-nationresultedinthepropagationofneuronalactivitythroughoutthehemisphere.Thesedata indicatedanestablishmentofnon-invasive,controllablemodelinneuronalactivity.NoCOI.


2P-067Developmental change in the nonuniform distribution of presynaptically silent synapses in single cultured hippocampal neuronKoyama, Miharu(The Fifth Department of Pharmacy, University of Fukuoka, Japan)

Aconceptofsilentsynapseiswidelyacceptedinthecentralnervoussystem.It iswellknownthatpostsynapticsilencingresults fromafailureofsynaptic transmissiondespitethatglutamate isreleasednormallyfrompresynapticterminals.Ontheotherhand,presynapti-callysilentsynapseisphysiologicallydeterminedasthefactthatthepresynapticterminalispresentbutisnotreleasingneurotransmitters.However,precisecharacteristicsofpresynapticallysilentsynapsesarenotfullyunderstood.Inthisstudy,presynapticallysilentsynapsewasidentifiedatexcitatoryglutamatergicsynapsesonsingleculturedhippocampalneuronat7–9,13–15and21–27daysinvitro(DIV)bydoublestainingwithastyryldye,FM1-43andanti-VGLUT1antibody.Synapticallocationwasdeterminedasafunctionofagivenareaofconcentriccirclesfromsoma.Wefoundthatthefractionofpresynap-ticallysilentsynapsessomehowincreaseddependingonthedistancefromsoma.Inadditiontothenonuniformdistribution,totalpercentageofpresynapticallysilentsynapseswithinasingleneuronwasdecreasedduringcultureperiods,indicatingthatpresynapticsilencingshiftstoactivestateinadevelopmentalstage-dependentmanner.Weconcludethatinformationprocessingwouldbefinelytunedbychangeinthefractionofpresynapticsilencingwithage.NoCOI.

2P-068Binomial distribution analysis of two components of short-term synaptic plasticity, facilitation and potenti-ation, at the frog neuromuscular junctionSuzuki, Naoya(Department of Physics, School of Science, Nagoya University, Nagoya, Japan)

Toinvestigatethemechanismoftetanicstimulationinducedenhance-mentoftransmitterrelease,weanalyzedtwocomponentsofshort-termsynapticplasticity,facilitationandpotentiation,usingbinomialdistri-butionwithtwoparameters,releaseprobability (p)andnumberofreleasablesynapticvesicles(n)havingthatreleaseprobability.Frogneuromuscularjunctionwasusedassynapsepreparation.Endplatepotentials (EPPs)andminiatureendplatepotentials (MEPPs)wereelectricallyrecordedwithan intracellularglassmicroelectrode inalowCa2+highMg2+Ringer’ssolution(0.50–0.75mMCa2+,5mMMg2+).Facilitationwasinducedby8stimuliwithintervalof25or30msec.Potentiationwasinducedby500stimuliat20Hzandsteadyenhancedstatewasmaintainedby350stimuliwithintervalof150–200msec.TheratioofvarianceincreasetomeanincreaseoffacilitatedEPPsdistributionwasalmost1.However,thatvalueofpotentiatedEPPsdistributionwassmallerthan1.Theseresultssuggeststhattheen-hancementoftransmitterreleaseduringfacilitationandpotentiationwascausedmainlybytheincreaseofnandp,respectively.Thedetailedbinominalanalysesofthechangeoftwoparameters,pandn,duringfacilitatedandpotentiatedEPPsdistributionsconfirmedthatincreaseofnandpcontributedlargelytotheenhancementoftransmitterre-leaseinfacilitationandpotentiation,respectively.NoCOI.

2P-069Effects of astaxanthin on neuronal functionsIsonaka, Risa; Katakura, Takashi; Kawakami, Tadashi(Department of Physiology, Kitasato University School of Medicine, Sagamihara, Japan)

Astaxanthin(AX)isacarotenoidpigment.Itiswidelydistributedinnature,which is included inmicroalgae,crustacean,andtheothermarineanimals.AXcouldeliminatereactiveoxygenspecies (ROS),therefore ithasattractedattentionasaprotectingagentagainstdamagecausedbyoxidativestress.SeveralexperimentalevidencesshowedthatAXprovidesantioxidanteffects,howeverthedirecteffectsofAXonneuronalcellhaverarelyreviewed.Inthisstudy,weinves-tigatedtheeffectsofAXonneuritegrowth inculturedratspinalneurons.Neurofilamentswere identifiedby immunocytochemistryusinganti-neurofilamentsantibody(SMI-31,SMI-32),andmeasuredneuritelength.Neuronspretreatedfor24hwithAXaloneandsubse-quentlytreatedfor72hwithAXandtheROSdonor.TreatmentwithAXalonewasnotsignificantlyeffectsonneuritegrowth,whereasitblockedagainstthegrowth inhibitoryeffectsof theROSdonor inneurons.Furthermore,weexaminedthedirecteffectsofAXonaxonaltransportincultureddorsalrootganglionneurons.NoCOI.



2P-070Cortico-cortical connections of the insular auditory field in miceWang, Chi; Takemoto, Makoto; Song, Wen-Jie(Dept of Sensory and Cognitive Physiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan)

Anauditoryfieldintheinsularcortexhasrecentlybeenidentified.Asthefirststeptounderstandthefunctionoftheinsularauditoryfiled(IAF),hereweexaminedtheconnectionofIAFwithotherareasofthecerebralcortexinmice.FirstwefocusedontheconnectionbetweenIAFandtheanteriorauditoryfield (AAF).Usingvoltage-sensitivedye-basedopticalimaging,weidentifiedIAFandAAFbytheirchar-acteristicfrequencygradient.Twofluorescenttracersindistinctcolorsweretheninjectedintoeachofthefieldsatfrequencymatchedsites.Afterasurvivalperiodofthreedays,retrogradelylabeledcellswerefoundinbothIAFandAAF,withintheinjectionsites,suggestingareciprocal,topographicconnectionbetweenthetwofields.Interesting-ly,wealsofoundretrogradelylabeledcellsinsensorimotorareasofthecortex.SuchconnectionsofIAFwithareasofdifferentmodalitiessuggestitsroleinlinkingsoundstoactions.NoCOI.

2P-071Quantitative frequency-position relationship in the pri-mary auditory cortex in guinea pigsSong, Wen-Jie1,2; Nishimura, Masataka1(1Dept of Sensory and Cognitive Physiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan, 2HIGO program)

Orderlyrepresentationofsoundfrequencyoverspaceisahallmarkoftheprimaryauditorycortex(A1).Aquantitativerelationshipbe-tweensoundfrequencyandcorticalpositionisyettobeestablished.HereweexaminedthisrelationshipinguineapigA1bypresentingstimulustonesinawidefrequencyrange,andrecordingtheevokedcorticalresponseswithahighspatialresolutionopticalimagingtech-nique.Wedeterminedthreebest-frequencypositionsasthecorticalpositionsinA1foreachtonefrequency:theonsetresponseposition,thepeakamplitudeposition,andthemaximumriseratepositionoftheresponseevokedbyatoneofthefrequency.Anonlinearlogfre-quency-positionrelationshipwasfoundforeachofthethreeindices,andthefrequency-positionrelationshipwasalwayswelldescribedbyaGreenwoodequation,withcorrelationcoefficientsgreaterthan0.99.Corticalmagnificationfactor,measuredinoctave/mm,wasfoundtobeafunctionoffrequency,notaconstant.Representationoffrequen-cy inA1wasmorespatiallyeventhan inthecochlea.OurresultsestablishaquantitativerelationshipforsoundfrequencyandcorticalpositioninguineapigA1,andshouldfindapplicationinanarrayofstudiesincludingmodelingoftheauditorycortex.NoCOI.

2P-072Spectral and temporal sensitivity of sustained and phasic neurons investigated for multiple sounds in the primary auditory cortex of awake catsChimoto, Sohei; Tazunoki, Shun; Sato, Yu(Department of Physiology, University of Yamanashi)

Inpreviousstudieswehaveshownthattheprimaryauditorycortex(A1)neuronsofawakeanimalsshoweddiversityof theresponsetime-coursesfromphasictosustainedpatternsduringpuretonestim-uli.FollowingstudiesalsoshowedthatA1neuronshavesuchresponsepatternsduringamplitudemodulation (AM), frequencymodulation(FM),andvowelsounds.Itisnotyetinvestigatedwhetherornotagivenphasic(sustained)neuronrespondstoanykindsofsoundstim-uliinthephasic(sustained)timecourse.Inthisstudyweinvestigatedresponsetimecoursestovariousartificialsounds (puretones, twotones,noises,clicktrains,AMsounds,andFMtones)andnaturalsounds(environmentalsounds,meow,vowels,andconsonants).Neuronswithphasicresponsestopuretoneshadwidefrequencyresponsefield(FRF)andshowedphasicresponsestoallkindsofsoundsinvestigated.Comparisonofpeakresponseamplitudesamongsoundsshowednosoundspecificity.NeuronswithsustainedresponsestopuretoneshadrelativelynarrowFRFandmanyneuronsshowedinhibitorysubfieldstotwotonesstimuli.Theyshowedsustainedresponsestoallsoundswhenthespectralfrequencyofthesoundwaslocatedatthecell’sFRFandnotlocatedattheinhibitorysubfields.Comparisonofmeandrivenrateamongsoundsshowednosoundspecificity.TheseresultssuggestthatasingleneuroninA1processessimilarinformationcontainedinanykindsofsoundsandeachneuronwithdifferenttime-coursescodesthedifferentacousticcharacteristicsofthosesounds.NoCOI.

2P-073Properties as associative memory circuits in the pri-mary auditory cortex of miceTsukano, Hiroaki; Hishida, Ryuichi; Shibuki, Katsuei(Dept of Neurophysiol, Brain Res Inst, Niigata Univ, Japan)

Inthepresentstudy,wefoundpropertiesofthemouseprimaryaudi-torycortex(AI)asassociativememorycircuits.Previously,wehavereportedthatresponsestofundamentalfrequency(f0)arerecordedinAIofmiceusingflavoproteinfluorescenceimaging,evenwhenspectralenergyatf0ismissing.Harmonicsoundsofsimultaneouslypresented20kHzand25kHz,whichproducedmissingf0perceptionat5kHz,activatedthe5kHzarea,whileinharmonicsoundsofsimultaneouslypresented19kHzand26kHzdidnot.Two-photonimagingconfirmedtheresultsobtainedbyusingflavoproteinfluorescenceimaging:singleneuronalactivitiesto20+25kHzsoundweresignificantlycorrelatedwiththoseto5kHz,buthadlittlecorrelationwiththoseto20kHzaloneor25kHzaloneinthe5kHzareaofAI.F0responseswerenotobservedinthemousestrainwhichexhibitedalmostnochirps,exceptinthecasethattheywererearedbynormalparentswithfrequentchirps.Inmicerearedinthepresenceof5+19+26kHz,the5kHzareainAIrespondedtoinharmonicsoundsof19+26kHz.Theseresultsindicateimportanceofexperienceforproducingf0responses.Further-more,anewhypothesisissuggestedthatrecallofmissingf0respons-esfromexperiencemaybeonlyapartofAIfunctions:AImayhavepropertiesasassociativememorycircuits. Inaccordancewiththispossibility,harmonicsoundsof4+8kHzactivatedthe20kHzarea,while inharmonicsoundsof5+7kHzdidnot.TheseresultsclearlyindicatethepropertiesofAIasassociativememorycircuits.NoCOI.


2P-074Responses of auditory fields sensitive to species-spe-cific pup calls in ratsKudoh, Masaharu; Nishida, Yoko; Ogawa, Go(Dept Physiol, Teikyo Univ Sch Med, Tokyo, Japan)

Rodentpupsemitlowerfrequencycallsotherthanisolationinducedultrasoniccalls.Wehavereportedthatlowfrequencypupcallsofratscanbedividedintothreegroups:harmonic-typecalls,noise-typecallsandpulsetrains.Ratpupsfrequentlyutterlowfrequencycallswhentheyarewiththemother,indicatingthatpupcallsareanimportantfactor inmaintainingaclosemother-puprelationship.Endogenousflavoproteinfluorescence imaging (excitation:450–490nm,emission:500–550nm)conductedinmotherratsafter3weeksofweaningperi-odandnulliparouscontrolratsshowsthatharmoniccallsevokeclearfluorescenceresponsesintheprimaryauditorycortex(AI).Incontrast,syntheticnoise-typecallsevokemarkedresponses intheposteriorpartofAIandposteriorauditoryfield(PAF).Inthepresentstudy,weinvestigatedplasticchanges inauditorycorticalresponses to lowfrequencypupcallsinmotherrats.FluorescenceresponsesofAItoharmoniccallsandthosetoartificialharmonicsounds(afundamentaland2ndand3rdovertones)werenotsignificantlydifferentinnullip-arousrats.However,fluorescenceresponsestoharmoniccallsweregreaterthanthosetoartificialharmonicsoundsinmotherrats,indi-catingAIofmotherratsrespondsselectivelytoharmonicpupcalls.SyntheticnoisecallsevokedstrongerfluorescenceresponsesinPAFofmotherratsthannulliparousrats.Thesefindingsindicatethatdif-ferentgroupsofauditoryfieldsaresensitivetodistincttypeofspe-cies-specificpupcallsinrats.NoCOI.

2P-075Distinctions in burst spiking between auditory cells in thalamic reticular nucleus projecting to first and high-er-order thalamic nucleiKimura, Akihisa; Imbe, Hiroki; Donishi, Tomohiro; Kaneoke, Yoshiki(Department of Physiology, Wakayama Medical University, Wakayama, Japan)

Thalamicreticularnucleus(TRN)playsapivotalroleingainand/orgatecontrolofsensory inputstransmitted fromthalamicnuclei tocorticalareas.TRNcontainstwotypesofauditorycellsprojectingtotheventral (MGV)anddorsaldivisions (MGD)ofmedialgeniculatenucleus,which,asfirst-andhigher-orderthalamicnuclei,areinvolvedinsensoryprocessingoflemniscalandnon-lemniscalsystems.Previ-ously,wereporteddistinctionsinauditoryresponsepropertiesbetweenthetwotypesofcells(TRN-MGVandTRN-MGDcells),whichweredetermined inanesthetizedrats,usingjuxta-cellularrecordingandlabelingtechniques.Inthepresentstudywefurtherexaminedspon-taneouscellactivityrecordedinastatewithasteadyambientnoiselevel(<45dB).TRNcellsexhibitedsinglespikesandbursts.BurstsofTRN-MGVcellsconsistedoflargernumbersofspikeswithshorterinter-spikeintervalsascomparedtothoseofTRN-MGDcellsinbothspontaneousactivityandauditoryresponseelicitedbynoiseburststimuli (intensity,70–80dB;duration,100ms).ThesedistinctionsinburstspikingwerecomparabletothoseobservedinthetwotypesofTRNvisualcellsprojectingtofirst-andhigher-orderthalamicnuclei(Neuroscience,2012).BurstspikingofTRNcellscouldhaveasignificantimpactonthalamiccellactivity.IntheloopconnectivitybetweenthecortexandthalamusTRNishighly likelytosubservedifferentialmodulationsofsensoryprocessing in lemniscalandnon-lemniscalsystems.NoCOI.

2P-076The effect of claudin-14 on maintenance in EP and endolymphatic ionic concentrationsDaikoku, Eriko1; Shiraiwa, Yuka1; Saito, Masahisa1; Yamaji, Junko1; Hasegawa, Keiko2; Haginomori, Shin-ichi2; Kawata, Ryo2; Kubota, Takahiro1(Dept. Physiol., Fac. Med., Osaka Med. Coll., Takatsuki, Japan, 2Dept. of Otolaryngol., Fac. Med., Osaka Med. Coll., Takatsuki, Japan)

WemeasuredK+,Na+,Ca2+,andH+concentrationsinendolymph([K]e,[Na]e, [Ca]e,pHe)withendocochlearpotential (EP) inbothwildtype(Cldn14+/+)miceandclaudin-14knockout(Cldn14-/-)miceat8weeksofage,usingdouble-barreledion-selectivemicroelectrodes.Wealsomea-suredtheauditorybrainresponse(ABR)inbothmice.Weobtainedthefollowingresults.1)pHe,[K]e,[Na]e,and[Ca]einCldn14+/+micewere7.98,155mM,~0.5mM,~100nM,respectivelyandthoseinCldn14-/-micewere7.85,154mM,1.2mM,~1µM,respectively. SignificantdifferenceinabovethosevaluesbetweenCldn14+/+andCldn14-/-micewereobservedinpHeand[Ca]e.EPwas+102mVinCldn14+/+and+90mVinCldn14-/-mice.Atmorethan14weeksofageinCldn14-/-mice,EPwasreducedto+30–50mV,pHewasdecreasedto7.51,and[Ca]ewas~0.1mM.SignificantcorrelationbetweenEPand[Ca]ewasobserved inbothCldn14+/+andCldn14-/-mice.ABRwasgraduallyreducedfrom4weeksto14weeksofageinCldn14-/-mice.ThesefindingssuggestthatCldn14+/+micehavetheEPmorethan100mV,highpHe,andlow[Ca]eandclaudin-14hassmall,butimportanteffectsonmaintenanceinEPandendolymphaticionicconcentrations.NoCOI.

2P-077Interaural canal affects the sensitivity of processing the interaural time difference detected in the nucleus laminaris in the chickOta, Naomi; Ohmori, Harunori(Department of Physiology, Graduate School of Medicine, Kyoto University, Kyoto, Japan)

Theinterauraltimedifference(ITD)isamajorcueforsoundlocaliza-tionandisfirstprocessedinthenucleuslaminarisinbirds.ITDisafunctionofaheadsizeofanimal,thusitisextremelysmallinsmall-head-edanimalssuchaschicks.IthasbeensuggestedthatITDisenhancedthroughtheacousticinterferenceofsoundbetweentwomiddleearcavitiesthroughtheinterauralcanal.However,theeffectoftheinter-auralcanalhasnotbeendirectlydemonstratedintheITDprocessinginanyauditorynuclei.Wethereforeconductedexperimentsthatoc-cludetheinterauralcanalwithanagarosegel,andexaminedtheeffectsofocclusionontheneuralactivityinthenucleuslaminaris.Aftertheocclusion,theITDtuningfunctionisshiftedalongtheITDaxis,thusthepropertyofITDprocessingwaschangedespeciallywithinthephysiologicalrangeofITD.Thesefindingsconfirmthatacousticcou-plingthroughtheinterauralcanalaffectsthesensitivityofITDpro-cessinginthenucleuslaminaris.NoCOI.


Poster PresentationsAutonomic Nervous System (1)

2P-078Regulation of energy metabolism and cardiovascular function by the hypothalamic AMP-activated protein kinaseTanida, Mamoru; Shibamoto, Toshishige(Department of Physiology II, Kanazawa Medical University)

Inmammals,AMP-activatedproteinkinase(AMPK)isextremelyes-sentialintheintracellularsignalingpathwayinvolvingactiveleptinreceptors.WeinvestigatedthepotentialroleofhypothalamicAMPKα2incardiovascularfunctionandenergymetabolismwithusinginvivosiRNAinjectiontoknockdownAMPKα2inrats.Inthepresentstudy,weproducereducedhypothalamicAMPKα2expressionby3daysinjectionofsiRNAintothethirdventricle.TheAMPKα2siRNA-treat-edratshad lowerpost-transfectionbodyweights,whereasbodyweightswereunchangedincontrolsiRNA-treatedrats.Inaddition,basallevelsofmeanarterialpressureofAMPKα2siRNA-treatedratsweresignificantlyhighercomparedwiththoseofcontrolsiRNA-treat-edrats.Basallevelsofbloodadrenaline,noradrenaline,glucoseandleptinweresimilarbetweenthetwogroups,butleptineffectsonbodyweight,foodintake,bloodpressure,heartrateandbloodFFAlevelswereeliminated inAMPKα2siRNA-treatedrats.Moreover, leptin-evokedenhancementsofthesympatheticnerveoutflowstothekidney,brownandwhiteadiposetissueswereattenuated inAMPKα2siR-NA-treatedrats.TheseresultssuggestthathypothalamicAMPKα2maybeinvolvednotonlyinappetiteandbodyweightregulationbutalsointheregulationofcirculationandenergymetabolism.Especially,AMPKmightfunctionasakeymoleculeinthecardiovascularandlipolyticeffectsofleptinthroughthesympatheticnervoussystem.NoCOI.

2P-079Effects of orexins on parasympathetic preganglionic neurons in the superior salivatory nucleus innervating the salivary glandsMitoh, Yoshihiro1; Sato, Tadasu2; Fujita, Masako1; Kobashi, Motoi1; Ichikawa, Hiroyuki2; Matsuo, Ryuji1(Department of Oral Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan, 2Division of Oral Craniofacial Anatomy, Tohoku University Graduate School of Dentistry)

Orexin(ORX)-Aand-Baresynthesizedbylateralhypothalamicneu-rons(LH)andareimplicatedintheregulationoffeedingandarousal.Meanwhile,neuroanatomicalstudiesrevealedthatthesuperiorsaliva-torynucleus(SSN)neuronsinnervatingthesubmandibularandsub-lingualsalivaryglands,whichistheprimaryparasympatheticcenter,receivesdirectprojectionsfromLHneurons.However,therearenoreportsthatneuronalactivityofSSNisinfluencedbyORXs.ThisworkaimedtoexaminingtheeffectsofORXsonneuronalactivityandex-pressionoforexinreceptor(ORXR)subtypes(ORXR1and2) inratSSNneurons.Whole-cellpatch-clamptechniqueswereappliedtoSSNneuronsretrogradelylabeledwithafluorescenttracerfromthechor-da-lingualnerve.At100nM,ORX-Ainduced inwardcurrentsandelicitedactionpotentials inmostofSSNneurons.AlthoughORX-Binducedinwardcurrentsin40%ofneurons,ithardlyelicitedactionpotentials. InthepresenceofanORXR1antagonist,SB-334867,thepeakamplitudeofinwardcurrentstoORX-Awerecompletelyinhib-itedinabout60%ofneurons.ImmunohistochemicalstudiesshowedthatORXR1and2expressionsinSSNneuronsare53%and40%,re-spectively.ThesedatasuggestthatSSNneuronsweremainlyexcitedbyORX-AviaORXR1.ORXsmaycontributetoabundantsalivarysecretionduringfeeding.NoCOI.

2P-080Immunohistochemical analysis of Nav1.9 sodium channel and transient receptor potential ankyrin 1 (TRPA1) in newborn rat large intestine enteric ner-vous systemSaiki, Chikako; Ide , Ryoji; Tamiya, Junko; Matsumoto, Shigeji(Department of Physiology, Nippon Dental University, School of Life Dentsitry at Tokyo, Tokyo, Japan)

Inthisstudy,weexaminedwhetherNav1.9sodiumchannelandtran-sientreceptorpotentialankyrin1(TRPA1)aredetectableimmunohis-tochemicallyinneonatalratentericnervoussystemoflargeintestine.Weusednewbornrat(aweekold).Aftereuthanasia,segmentsoftheratcolonwereremoved.Thetissueswerewashedanddehydratedingradedsucrosesolutions(10,20and30%)at4°C.Themucosawaseliminatedwithtweezersandtheremainingtissueswerefrozeninliquidnitrogen.Cryostatsectionswerecutat10µmthickness,andincubatedwithprimaryantibodiesdirectedagainstNav1.9sodiumchannel,TRPA1andPGP9.5.Fordoubleimmunostaining,incubationswiththethreecombinationsoftheantibodies (Nav1.9andTRPA1;Nav1.9andPGP9.5;TRPA1andPGP9.5)wereconductedforthreenightsat4°C.Thenthetissueswereincubatedwithsecondaryantibody(1hr,atroomtemperature)andwashedwithPBSbeforemountedonslides.WedetectedNav1.9orTRPA1expressioninthePGP9.5posi-tiveneurons,whicharedistributedinmusclelayers.Moreover,co-lo-calizationofNav1.9andTRPA1wasobservedamongthearea,whichiscorrespondingtothemyentericplexus.Inconclusion,ourresultssuggestthatat least innewbornratmyentericplexusofthe largeintestine functionaldependencemayexistbetweenNav1.9channelandTRPA1.NoCOI.


2P-081Characterization of ghrelin-sensitive neurons in the lumbosacral defecation center that are associated with facilitation of colorectal motility in rats.Naitou, Kiyotada; Sugita, Riko; Nakamori, Hiroyuki; Shiina, Takahiko; Shimizu, Yasutake(Veterinatry Science, Laboratory of Physiology, The United Graduate School of Veterinary Sciences, Gifu University, Gifu, Japan)

WehavepreviouslydemonstratedthatghrelinappliedtotheL6-S1spinalcord levelenhancescolorectalmotility. Insituhybridizationshowedthatneuronsinthespinalcordexpressghrelinreceptor.Inthisstudy,wecharacterizedtheghrelin-sensitiveneuronsinthelum-bosacraldefecationcenter,withparticular focusontheirsimilaritywiththoseinthehypothalamus.Ratswereanesthetizedwithalpha-chlo-ralose,andthedistalcolonandanuswerecannulatedtomeasuretheintracolorectalpressureandpropelledintraluminalliquidvolume.Theintrathecalinjectionoftetrodotoxinabolishedtheprokineticeffectofghrelin.NPY,whichisamajorneuropeptidereleasedfromghrelin-sn-sitiveneuroninthehypothalamus,didnotenhancecolorectalmotility.Consistently,NPY-Y1receptorantagonistfailedtoinhibittheactionofghrelin.Althoughleptinexertsoppositeeffectstoghrelinontheactivityofhypothalamicneurons,leptinhadnoeffect.Wethenexam-inedeffectsofAMP-activatedproteinkinase(AMPK)activationinthelumbosacraldefecationcenter,sinceAMPKisknowntoberelatedtoeffectofghrelinonfoodintake.Intrathecaladministrationofanacti-vatorofAMPK,AICAR,failedtoenhancethecolorectalmotility.Theseresultssuggestthatcharacteristicsoftheghrelin-sensitiveneuronsinthelumbosacraldefecationcenteraredifferentinthoseofthehypo-thalamus.NoCOI.

2P-082Serotonin enhances colorectum motility through an activation of lumbosacral defecation center in rats.Nakamori, Hiroyuki1; Naitou, Kiyotada2; Shiina, Takahiko1,2; Shimizu, Yasutake1,2(1Laboratory of Veterinary Physiology, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan, 2Department of Basic Veterinary Science, Laboratory of Physiology, The United Graduate School of Veterinary Sciences, Gifu University, Gifu, Japan)

Serotonin(5-HT)isoneofthetransmittersregulatinggutmotilityintheentericnervesystems.Inthepresentstudy,weexaminedeffectsofstimulationof5-HTreceptorsinthelumbosacraldefecationcenteroncolorectalmotilityinrats.Ratswereanesthetizedwithα-chloraloseandketamine,andcolorectalintraluminalpressureandpropelledin-traluminalliquidvolumewererecordedinvivo.Anintrathecaladmin-istrationofserotonintotheL6-S1regionofthespinalcordelicitedperiodicincreasesincolorectalintraluminalpressurethatwereasso-ciatedwithincreasesinfluidoutputthroughtheanalcannula.Similarenhancementofcolorectalmotilitywasinducedbyintrathecaladmin-istration of 5-HT2agonist,α-methylserotonin and5-HT3agonist,1-(3-chlorophenyl)biguanide.Incontrast,5-HT1or5-HT4agonisthadnoeffects.Applicationofeither5-HT2or5-HT3antagonistpartiallyblockedtheprokineticeffectofserotonin,whereastheserotonineffectwastotallyabolishedwhentheseantagonistswereappliedsimultane-ously.Itisconcludedthatserotoninpromotespropulsivecontractionsofthecolorectumthroughanactivationof5-HT2and5-HT3receptorinthelumbosacraldefecationcenter.NoCOI.

2P-083Firing properties of medullary expiratory neurons during defecationSasaki, Sei-Ichi1; Niwa, Masatoshi2(Ctr. Med. Sci., Ibaraki Pref. Univ. of Hlth. Sci., Ami-machi, Ibaraki, Japan, 2Dept. of Occup. Therp., Kyorin Univ., Hachioji, Japan)

Defecationisnormallyassistedbycontractionofdiaphragmandab-dominalmuscles.Experimentalevidencesuggeststhatexpiratory(E)neuronsofcaudalNRAclosely interrelatedwiththeactivitiesofneuronscontrollingrespirationandautomomicfunctions,becauseEneuronsextendtheiraxonsinthelumbarandsacralspinalcord.Thepurposeofthisstudyistoexaminethepossiblemechanismsinteract-ingwiththerespiratoryneuronsandthedefecation.Theexperimentswereperformedonadultcatsanesthetizedwithurethane-alphachlo-ralose.Thephrenicnerve,L1andL2abdominalnerveswerepreparedforrecording.Theanimalswereparalyzedandkeptonartificialven-tilation.Glassmicropipetteswereusedforextracellularrecordingsofrespiratoryneurons.Spinalprojectionoftherespiratoryneuronwastestedbymonopolarstimulationofthelumbarandsacralspinalcord.DefecationwasinducedbyinjectionofwarmRingersolutionintothesoftrubberballoonwhichwas inserted intherectum.Eneuronsshowedtherespiratoryrhythmintheexpiratoryphasesduringdef-ecation.ButfiringfrequenciesofEneuronshavingtheirdescendingspinalaxonstothe lowerthoracicand lumbarsegments increasedduringdefecation,butthoseofEneuronstothesacralsegmentsde-creased.TheseresultssuggestthatEneuronstothelowerthoracicand lumbarsegmentsmayexertexcitatorysynaptic inputstotheabdominalmotoneuronsandEneuronstothesacralsegmentssynap-ticinputstothepudendalmotoneuronsviasacrallocalcircuitsduringdefecation.NoCOI.

2P-084A method for recording of afferent and efferent mesen-teric sympathetic nerve activity in ratsKemuriyama, Takehito; Ohta, Hiroyuki; Tandai-Hiruma, Megumi; Tashiro, Akimasa; Hagisawa, Kohsuke; Nishida, Yasuhiro(Dept. Physiol., Nat. Def. Med. Coll., Tokorozawa, Japan)

Wepreviouslydemonstratedthatanexperimentalmodelforsimulta-neousmeasurementsofmesentericsympatheticnerveactivity(SNA),arteriolardiameter,andbloodflowinsmall intestinalarteriolesofSprague-Dawleyrats,toevaluateaquantitativerelationshipbetweenSNAandarteriolarvasomotionforbloodpressurecontrol.Theaimofthisstudywastodevelopamethodforrecordingofafferentandef-ferentmesentericSNA.Usingintraperitonealurethane(1.2g/kg),thesmallintestinewasexteriorizedthroughamidlineabdominalincisionandplacedonadish.Themesentericnervewasexposedbetweenthemesentericarteryandveinunderadissectingmicroscope.Bipolarsilverelectrodeswereputunderthenervetorecord.MesentericSNAwasrecordedwithapowerlabsystem.MesentericSNAwasrecord-edbeforeandaftercuttingtheproximalsideofthemesentericnerveforafferentrecordingsorcuttingthedistalsideofthenerveforeffer-entrecordings.MesentericSNAdecreasedaftercuttingtheproximalordistalsideofthenerve.EfferentmesentericSNAaftercuttingthedistalsidefluctuatedthanafferentmesentericSNAaftercuttingtheproximalside.Theseresultssuggestthatthemesentericnerveincludesbothafferentandefferentfibers,indicatingthatthismethodmaybeusefulforrecordingofafferentandefferentSNA.NoCOI.


2P-085Endogenously and exogenously newborn enteric neu-rons in the deep tissue of mouse small intestine un-derwent transection and anastomosisGoto, Kei1; Kato, Go2; Kawahara, Isao1; Kuniyasu, Hiroki1; Nabekura, Junichi2; Takaki, Miyako1(1Department of Molecular Pathology, Nara Medical University, Kashihara, Japan, 2Department of Developmental Physiology, National Institute for Physiological Sciences, Okazaki, Japan)

Nonlinearopticalmicroscopy,inparticulartwophoton-excitedfluores-cencemicroscopy(2PM),canprovidedeeperopticalpenetration(sev-eralhundredµm)inexvivoandinvivopreparations.Wehaveusedthisapproachandobtainedclearthree-dimensionalimagingofnewbornentericneuronsthatwereendogenouslygeneratedafterguttransec-tionandanastomosisinThy1-promoterYFPmouse.Neurogenesishasbeenpromotedbyoralapplicationofthe5-HT4-receptoragonist,mo-sapridecitrate(MOS).Mostneuronswerelocatedwithin100µmofthesurface.Toclarifythecellsourceofneurogenesis,mesenchymalstemcells (MSC) frombonemarrowsandneuralstemcells (NSC)derivedfromthehippocampusandsubventricularzonewerechal-lengedascandidates.MSCandNSCwereculturedwithMOSfor4–11days.OutgrowthofprotrusionwasfacilitatedinneurospheresformedbyNSCexvivo.TheNSCtransplantation fromthetailveinwasperformedaftertreatmentwithPKH26.One-twoweekslaterduringwhenMOSwasapplied,underastereomicroscopynervecell-likeredfluorescencewasvisible,butuncertain.2PMimagingmadeitpossibletoobserveexogenouslynewbornentericneuronsderivedfromtrans-plantedNSCshowingredfluorescenceamongendogenouslynewbornentericneurons (showinggreenfluorescence) inthedeeptissueofmousesmallintestineunderwenttransectionandanastomosis.NoCOI.

2P-086Habituation of sudomotor and vasoconstrictor compo-nents in bursts of skin sympathetic nerve activityTsukahara, Reiko1; Kuwahara, Yuko2; Iwase, Satoshi2; Shimizu, Yuuki2; Nishimura, Naoki2(1Institute for Developmental Reserach, Aichi Human Service Center, Kasugai, Aichi, Japan, 2Department of Physiology, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan)

Aburstofskinsympatheticnerveactivity(SSNA)evokedbyavarietyofarousalstimuliusuallycontainsbothsudomotorandvasoconstrictorspikes.Thepurposeofthisstudywastoexploredifferencesinhabit-uationprocessbetweensudomotorandvasoconstrictorcomponentsofSSNAburstsduringrepeatedarousalstimulation.SSNAsignalswererecordedbymicroneurographyfromthetibialnerveineighthealthysubjects.Simultaneously,skinsympatheticresponse(SSR)andskinbloodflowresponse (SKBR)weremonitoredonan innervatedareaoffootsole.Superficialelectricalstimulationofthemediannervewasusedtoinducearousalresponse.ByreferringtooccurrenceofSSRandSBFR,eachSSNAburstwasdividedintotwosegments:asudomotorsegmentandavasoconstrictorsegment.TimeaverageofSSNAwithineachsegmentwascalculatedassudomotorSSNAam-plitudeandvasoconstrictorSSNAamplitude.Onaverage,thesudo-motorSSNAamplitudeshowedsignificantreductionthroughout30stimuli.ThevasoconstrictorSSMAamplitudesignificantlydecreasedduringthefirst10stimuli,butdidnotchangeduringthesubsequent20stimuli.Sympatheticsudomotorandvasoconstrictordrivemaybedifferentlysubjectedtoneuralhabituationprocess.NoCOI.

2P-087Analysis for pathologic condition of facial hemihyper-hidrosisInukai, Yoko; Iwase, Satoshi; Kuwahara, Yuko; Shimizu, Yuuki; Nishimura, Naoki; Sato, Maki; Sugenoya, Junichi; Sato, Motohiko (Department of Physiology, Aichi Medical University School of Medicine, Aichi, Japan)

Facialhemihyperhidrosismaybeoftencompensatoryduetothecontralateralanhidrosis,butlittleisknownaboutthepathophysiolog-icalmechanism.Weexplainedindetailof11of14patientswiththissymptomaboutI:Unilateralhyperhidrosis:Cervicaldiskherniation,II:Segmentalhemihyperhidrosis:II-a:Harlequinsyndrome,andII-b:Probablecauseofcervicaldiskherniationinthis90thmeeting.Weexpressremaining3casesofsymptomaticsegmentalhemihyperhidro-sisthistime.WeanalyzedthesepatientsbydefiningthetotalbodysweatdistributionpatternsusingMinor’sstarch-iodinetest,totalskintemperaturedistributionusinginfraredthermography,andlocalskinbloodflowusinglaserDopplerflowmeterduringheating,andimagingtoconfirmthelesions.Results:Case1:Theexcessiveextensionoftheleftneck:A63-year-oldwomanwithhyperhidrosisintherightposte-riorregionofneck,andthe leftaxillasince40yearsoldand leftHornersyndrome.Case2:Lungcancerinrightupperlobeinvadingthedorsalrib:A44-year-oldmanwithhemihyperhidrosisoftherighthemitrunkandtherightbackpain.Case3:Rosssyndrome:A38-year-oldwomanwithanhidrosisattherighthemifaceandthelefthemitrunk,leftAdie’sTonicPupil,andareflexia,detectedregionalcardiacsym-patheticdenervationbyMIBGimaging.Forelucidationofthepatho-logicconditionforintroductionsofprecisetreatment,theexaminationofsweatingdistributionforlocalizeddiagnosesisessentialforfacialhemihyperhidrosis.NoCOI.

2P-088Renal afferent fibers regulate body fluid balance through controlling arginine vasopressin release.Kitagata, Yuta1; Nagai, Yuko1; Abe, Chikara2; Morita, Hironobu2 (1Department of Physiology, Gifu University Graduate School of Medicine, Gifu, Japan, 2Department of Physiology, Gifu University Graduate School of Medicine, Gifu, Japan)

Althoughitiswellknownthattherenalnerveincludesautonomicandsensoryfibers,propertyandfunctionsofrenalsensoryfibersarestillunclear.Wehypothesizedthattherenalafferentnervesmaycontrib-utebodyfluidhomeostasisthroughcontrollingrenalexcretoryfunc-tions.Toexaminethis,renalafferentnerveactivity(RANA),plasmaargininevasopressin (AVP)concentration,andurinevolumeweremeasured,whileintrarenalreceptorswereselectivelystimulatedusingdouble infusion (DI) technique.That is,hyperosmoticNaClsolutionwasinfusedintotherenalartery,whilehypoosmoticsolutionwasin-fusedintotheinferiorvenacava,thustotalloadedsolutionwasisoos-moticbuttherightkidneywasstimulatedbyhyperosmoticsolution.DIinducedincreasesinRANAandplasmaAVPconcentration,andadecreasedurinevolume.Theseresponseswerecompletelyabolishedbyrenaldenervation.Thesedataindicatethattherenalafferentfibersmightsensechangesinionicconcentrationorosmolarity,andalterrenalexcretoryfunctions,probablythroughcontrollingAVPrelease.NoCOI.


2P-089Renal afferent nerve activity and blood pressure re-sponse to intrarenal arterial infusion of ionic solutions in conscious rats.Nagai, Yuko1; Kitagata, Yuta1; Abe, Chikara2; Morita, Hironobu2 (1Department of Medicine, Gifu University, 501-1194, Gifu, Japan, 2Department of Physiology, Gifu University Graduate School of Medicine, Gifu, Japan)

Wehypothesizedthatthekidneysensesionicconcentrationinplasmaandsendthismessagetothecentralnervoussystemviatherenalafferentnerves.Toexaminethis,renalafferentnerveactivity(RANA)wasmeasuredinconsciousrats,whileasmallintrarenalarterialinfu-sionofhyperosmoticor isosmotic ionicssolutions.Toexcludethesympatheticnervecomponentfromtherenalnerverecord,hexame-thoniumwasadministeredinadvanceofRANArecording.Intrarenalarterialinfusionwasperformedviathechronicallyimplantedcatheterintotherightsuprarenalartery,whichallowedustomakeaselectivestimulationtotherightkidneywithoutcausingobstructionofrenalbloodflow.Therecordingelectrodeswerechronicallyimplantedontherightrenalnerve.InfusionofhighconcentrationsolutionofNaClorKCl(100µL/minfor1min)evokedaninstanttransientincreaseinRANAandariseinarterialpressure,while0.9%NaClor20%glucosesolutiondidnot inducetheseresponses.Administrationof losartanattenuatedthehyperosmoticNaClinfusion-inducedpressorresponsebutdidnotaffectRANA.OurresultssuggestthatkidneysensestheelevationofplasmaNa+,Cl-,orK+concentrationstoactivatetheaffer-entnerve,buttheconcomitantlyoccurredpressorresponsearemain-lymediatedviarenin-angiotensin-aldosteronesystem.NoCOI.

2P-090Arterial blood pressure response to the metabotropic excitatory amino acid receptor agonist L-AP4 injected into the caudal ventrolateral medulla of the ratTakemoto, Yumi; Tsuruda, Keiko(Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan)

Neuronsinthecaudalventrolateralmedulla (CVLM)playapivotalroleinthecentralcontrolofthecardiovascularsystemandaresensi-tivetothesulfur-containingaminoacidL-cysteinetoproduceade-pressorresponsethatisabolishedbybothantagonistsforionotropicexcitatoryaminoacids (iEAA)NMDAandnon-NMDAreceptors(Takemoto2013).Receptorbindingassaysusingsynapticmembrane(Pullanetal.1987)suggeststrongaffinityofL-cysteineforL-2-ami-no-4-phosphonobutanoate(L-AP4)sensitivereceptors.TheresponsetoL-cysteinecouldbeviaL-AP4relatedmetabotropicEAAgroupIIIreceptors. However, there isno informationaboutarterialbloodpressure(ABP)responsetoL-AP4intheCVLM.ThepresentstudythereforeexaminedABPresponsestomicroinjectionofL-AP4intheCVLMofurethane-anesthetizedrats,identifiedwithadepressorre-sponsetoL-glutamate(34nl,10mM).MicroinjectionsofL-AP4intheCVLMdose-dependentlydecreasedABP.AsinglepriorinjectionofanantagonistMK801(68nl,20mM)aloneforNMDAreceptors,oranotherantagonistCNQX(68nl,2mM)aloneforthenon-NMDAre-ceptorsabolishedthedepressorresponsetoL-AP4(34nl,5mM).TheresultsindicatethatL-AP4producedthedepressorresponseviabothNMDAandnon-NMDAreceptorspossiblyinaserialfashionintheCVLMoftherat,differentfromaparallelmodeofbothreceptorsinthedepressorresponsetoL-cysteine.NoCOI.

2P-091Effect of postural changes and vestibular lesions on responses of arterial blood pressure to neck flection in rabbitsMatsuo, Satoshi1; Nakamura, Yosuke2; Hosogai, Masae1 (1Department of Adaptation Physiology, Faculty of Medicine, Tottori University, 2Department of Otolaryngology, Head and Neck Surgery, Faculty of Medicine, Tottori University)

Ithasbeenshowninourpreviousstudythat45-degreehead-downposturalrotation (HDR) inducesatransientdropofarterialbloodpressure(ABP)inurethane-anesthetizedrabbits.WehypothesizethatvestibularorgansplayaroleinthedropofABPthroughthesuppres-sionofsympatheticnerveoutflowsbecausepeakactivationofaorticdepressornerve,anafferentforbaroreceptorreflex,occurredlaterthanthepeaksuppressionofsympatheticnerve.Totestthishypoth-esis,weexaminedresponsesofABPandsympatheticnerveactivitytoneckflection(NF)intheproneandlateralpositioninanesthetizedrabbitswithbilateralvagotomy.NFinthepronepositioninducedatransientdecreaseofABPwhichwasgreaterthanthatinthelateralposition.Thedecreasewasassociatedwithsuppressionofrenalsym-patheticnerve.NFdidnotaffectactivityofaorticdepressornerve.Investibular-lesioned (VL)animals,NFinducedasmalldropofABP,whichwassmallerthanthat inthecontrolrabbits.TheseresultssuggestthatnotonlyvestibularorgansbutalsoneckafferentscouldinducethetransientdropofABPthroughthesuppressionofsympa-theticnerve.NoCOI.

2P-092Reduction of plasma estrogen level affects circadian rhythms of heart rates and cardiac sympathetic nerve in female ratsMarui, Shuri1; Matsuda, Mayumi1; Sato, Nobuo1; Nagashima, Kei1,2,3(1Body Temperature and Fluid Lab., Fac. Human Sciences, Waseda Univ., Tokorozawa, Japan, 2IABS, Waseda Univ., 3GCOE Active Life, Waseda Univ.)

PurposeWeaimedtoclarifytheeffectofestradiol(E2)onheartrates(HR)andarterialpressure (AP),andmechanisms.MethodsFemaleWistarrats(n=9,ageof7–9wk)werebilaterallyovariectomized,andplacedaradiotransmitterforHRandAPmeasurementsintheab-dominalcavity.TwosilicontubescontainingE2weres.c.placedinonegroup(n=4,E2(+)),andemptytubesfortheother(n=5,E2(-)).Thetubeswereremoved10daysafterthesurgery(Day0).OnDay7or21,ratswerekilled.Theleftventricleofheartwastaken,andproteincontentsofβ1andβ2-adrenoreceptors(AR)wasdeterminedbyWesternblotting.ResultsOnDay0,HRwasgreater(P<0.05)intheE2(-)thanthatintheE2(+)group(388±15and337±13beats/min(bpm)inthelightphase;and450±12and390±12bpminthedarkphase,respectively).OnDay14,HRintheE2(+)groupincreased(377±15bpminthelightphase;and431±14bpminthedarkphase).OnDay21,HRbecamelower(P<0.05)intheE2(-)group(330±17bpminthelightphase;and388±20bpminthedarkphase).MeanAPwasnotdifferentbetweenthetwogroupsoneachday.Bothβ1-ARandplasmanoradrenalineweregreater(P<0.05)intheE2(-)thanE2(+)group,onDay0.OnDay7,bothβ1-ARandplasmanoradrenalinedecreased,howeveronDay21,plasmanoradrenalinebecamehigheragain.ConclusionAreductionofplasmaestrogenlevelmayaffectHR,whichisresultedfromgreat-ersympatheticactivityandβ1-ARexpressionintheheart.NoCOI.


2P-093Aging and plasma total homo-cysteine enhance the response of sympathetic nerve function to cold load-ingNakajo, Keisuke1; Matsumoto, Naoki1; Minamisawa, Susumu2; Toshima, Hiroko2(1Medical school student, Jikei University School of Medicine, Tokyo, Japan, 2Cell Physiology, Jikei University School of Medicine, Tokyo, Japan)

Purpose:Coldloadingisariskfactorofcardio-cerebrovasculardiseas-es(CVD),althoughitdoesnotinfluencehealthypeople.Westudiedthemodificationfactorofacoldpressortest.Method:Weevaluatedthesympathetic-nervefunctioninresponsetocoldloadingorcontroltestingbykeepingaplasticcupfilledwithicecoldwater(surfacetemperature;5.5°C)oracupwithcorrugatedpapersleeve(15°C),respectively.Wemeasuredheartratevariability,plasmacatecholamineconcentration,andtotalhomo-cysteine (t-HC):awell-knownriskfactorofCVD.Result:(1)Theeldergroup(n=10,49±5years)showedhigherLF/HFratiothantheyoungergroup(n=11,22±2years)incontrol(5.9±2.5vs.3.2±2.5,p=0.02)andcoldloadingstudy,(8.4±3.4vs.3.2±2.5,p=0.04).(2)When thesubjectsweredivided into thehigh t-HCgroup (n=3,21.2±11.5nmol/mL) (normalrange<13.5nmol/mL)andnormalt-HCgroup(n=7,10.0±2.1nmol/mL),LF/HFratioandplasmaepinephrineconcentrationinresponsetocoldloadingwerehigherinthehight-HCgroupthan inthenormalt-HCgroup (LF/HF:10.1±4.8vs.7.9±3.1,p=0.41;plasmaepinephrine:863±449vs.436±143pg/m,p=0.042,re-spectively).Conclusion:Theresponseofthesympatheticnervefunctiontocoldloadingwasmoresensitiveintheelderandhight-HCgroups,sug-gestingthattheirendothelialfunctionisimpairedandraisesariskofCVD.NoCOI.

2P-094GLP-1 inhibits reflex swallowing via the dorsal medul-la in the rat.Mizutani, Satoshi; Kobashi, Motoi; Fujita, Masako; Mitoh, Yoshihiro; Matsuo, Ryuji(Dept. Oral Physiol. Grad. Sch. Med. Dent. Pharma. Okayama Univ. Okayama, Japan)

Ourpreviousstudyrevealedthatappetite-enhancingpeptideorexin-Ainhibitedreflexswallowingviaorexin-1receptorssituated inthedorsalvagalcomplex(DVC).Glucagon-likepeptide-1(GLP-1),whichisanincretinhormonesecretedfromtheintestine,suppressesfoodintake.It isnotmuchclarifiedhowappetite-suppressingpeptidesactsonreflexswallowing.Inthepresentstudy,weexaminedtheeffectofGLP-1onreflexswallowingusinganaesthetizedratstoclarifyhowappetite-suppressingpeptidesactsonreflexswallowing.Swallowingwasinducedbytheelectricalstimulation(20Hz,20sec)ofthecentralcutendofthesuperior laryngealnerveandwas identifiedbytheelectromyogramleadpenetratedthemylohyoidemusclethroughbi-polarelectrodes.GLP-1wasinjectedintooneofthemedialDVCorthelateralDVC.ThemicroinjectionofGLP-1butnotvehicleintothemedialDVCsignificantlydecreasedswallowingfrequency.Themi-croinjectionofGLP-1intothelateralDVCdidnotchangeswallowingfrequency.PreinjectionofGLP-1receptorantagonistintothemedialDVCdisruptedtheinhibitoryresponseinducedbythemicroinjectionofGLP-1.TheseresultssuggestthatGLP-1inhibitsreflexswallowingviaGLP-1receptorsituatedinthemedialDVC.NoCOI.

2P-095The effect of playing a stringed-instrument ensemble on autonomic nerve functionIchihara, Kosuke1; Matsumoto, Naoki1; Tomita, Itsuka1; Minamisawa, Susumu2; Toshima, Hiroko2(1Medical school student, Jikei University School of Medicine, Tokyo, Japan, 2Cell Physiology, Jikei University School of Medicine, Tokyo, Japan)

Purpose:Itiswellknownthatmusichasgoodinfluenceonhealth.However,themechanismofhowmusicaffectsphysiologicalfunctionisnotunderstoodclearly.Therefore,westudiedtheeffectofplayingstringed-instrumentensembleonautonomicnervefunction.Method:Subjectsarenineundergraduate (UG) (20.8±1.0years)andsevengraduatestudents(GS)(25.5±1.9years)atamusiccollege.Theirspecialityisstringed-instrument:violins(n=4),violas(4),cellos(3),andcontrabass(1).WhiletheyplayedthestringquartetofMozart(EinekleineNachtmusikandDivertimento),theirECGandspirogramwererecordedbyusingLS-300(Fukuda,Japan).Wecalculatedheartrateviability(HFandLF/HF)byfrequencyanalysisofR-RintervalofECG.Result:Theheartrate(HR)andrespiratoryrate(RR)atrestwerenotdifferentbetweenUGandGS(HR:75±10vs.74±4/min)and(RR:17±2vs.15±4/min).TheGS’sHR,RR,andHFshowedtheperiodicityfluc-tuationatthetimeofplay(averageHR;86±6/min,RR;26±3/min,HF;149±154mS2)andrest(73±4,14±2,683±392).TheperiodicityfluctuationofHRandRRwasnotobservedintheUS.Conclusion:Thepresentstudydemonstratedthatrespiratoryrhythmunderplayingstringed-instrumentssynchronizedHRintheadvancedplayers,butnotinundergraduatestudents.Sincerespirationcanbeadjustedinvoluntary,itwassuggestedthatcirculationrhythmmightbeabletoadjustinsemi-voluntarybyRR.NoCOI.

2P-096Relationship between RR interval variability with gal-vanic vestibular stimulation and changes in arterial pressure upon head-up tiltTanaka, Kunihiko; Itoh, Yamato; Ikeda, Mayumi; Katafuchi, Tetsuro (Department of Radiological Technology, Gifu University of Medical Science, Gifu, Japan)

R-Rintervalvariability(RRIV)duringsupinepositionwithoutandwithgalvanicvestibularstimulation(GVS),changesinmeanarterialpressure(MAP)attheonsetof60°head-uptilt(HUT)withoutGVS,andtheirrelationshipwereanalyzed in25healthyyoungsubjects.MAPin-creasedordecreasedlessthan5mmHguponHUTin12subjects(UP),butMAPdecreasedmorethan5mmHgin13subjects(DOWN).Ap-plyingsinusoidalGVSof2mAattherandomfrequencybetween0.2and10HzdidnotchangemeanR-RintervalsandMAP.However,highfrequencycomponent(HF)ofRRIVincreasedinbothUPandDOWNgroups.TheincreaseinDOWNgroupwaslargerthanthatinUPgroup.RatiooflowfrequencycomponenttoHF(L/H)increasedinUPgroupwithGVS,butdidnotreachsignificantlevelinDOWNgroup.ThechangesinHFwithGVSweresignificantlycorrelatedwithchangesinMAPattheonsetofHUT,i.e.,thesubjectswhohadlargerincreaseinHFwithGVSshowedlargerdecreaseinMAP.Thus,GVSor input forthevestibularsystemduringHUTpossiblyactivatesvagalnerves,andthedominanceofexcitationinsympatheticorvagalduringvestibularstimulationisimportantforcontrollingMAPattheonsetofHUT.NoCOI.


2P-097Monitoring autonomic nerve activity using time-fre-quency analysis: A study of time shift widthTsutsui, Hiroshi; Ebina, Koudai; Niizeki, Kyuichi; Saitoh, Tadashi (Department of Bio-Systems Engineering, Yamagata University, Yonezawa, Japan)

Previousstudiessuggestthatautonomicnerveactivitycanbeevalu-atedbypowerspectrumanalysisofheartratevariability.Inthesestudies, theanalysiswasperformedforasetofdataonlyduringcertaindefinedconditions.Therefore,thisstudyaimedtoinvestigatewhetherpowerspectrumanalysisfordatasetswithashorttimeshiftwidthcanbeusedtoevaluateautonomicnerveactivityindexes,underanygivencondition.Aftera2-minrestingperiod,10healthymalevolunteersperformedamentalarithmetictask (MAT;additionormultiplicationin64squares),followedbyanother2-minrestingperiod.Electrocardiograph(ECG)monitoring,at1kHz,wasperformedthrough-outtheexperiment;R-Rinterval(RRI)datawerecalculatedfromtheECGdata.Thereafter,60-sRRIdatasetswereextractedfromthestarttotheendoftheexperiment.Thestartingpointofthedatasetswassetat10-sintervals,regardlessoftheparticipants’status.Alternative-ly, thestartingpointwassetat60-s intervalsduringbothrestingperiodsandduringtheMAT.Time-frequencyanalyseswereperformedfortheRRIdatasets,fromwhichthepowersofthelowfrequency(LF,0.04–0.15Hz)andhighfrequency(HF,0.15–0.4Hz)componentswerecalculated.MoredetailedchangesinpowersoftheLF/HFandHFcomponentscouldbeobservedindatasetswithashortertimeshiftwidth.Therefore,itispossiblethatpowerspectrumanalysiscanallowforreal-timemonitoringofthedynamicsofautonomicnerveactivityregardlessoftheoperatingconditions.NoCOI.

2P-098Blocking effect of peripheral adrenoceptors on the de-fensive cardiovascular reaction in the frogOhashi, Hiroki; Horiuchi, Takatoshi; Nagaoka, Yuya; ohmura, Ayano; Horiuchi, Jouji(Department of Biomedical Engineering, Toyo University, Saitama, Japan)

Inmammals,stressorsevokeatypicalcardiovascularresponse.Thiscardiovascularresponsetothestressorsismediatedbythesympa-theticnervoussystem.Thesympatheticresponsetothestress ismainlymediatedviathehypothalamicareas,especiallyinthedorso-medialnucleus.However,thedefensivehypothalamicareainthefrogisnotwell-evolvedcomparetothat inmammals.Thefroghasthemidbrainwhereisalsorecognizedasadefenseareaandisamoreprimitivepartofthebrain.Inthepresentstudy,weexaminedwheth-erthefrog(Rana catesbeiana)showsthecardiovasculardefensivere-actionexposedtomechanicalandenvironmentalstressors(pinprick,pinchingand10%NaClsolution)and,ifithas,thecardiovascularre-sponsetothestressisneurogenicornot.Bloodpressure(BP)andheartrate (HR)weremeasuredthroughabranchofabdominalartery inconsciousfrogs.Twotypesofadrenoceptorantagonistswereintrave-nouslyinjected,(anα1receptorantagonist,prazosin(0.3mg/kg)andaβ1antagonist,atenorol (10mg/kg)).Theallstressorscauseddecentpressorandtachycardicresponsesintheanimals.Bolusinjectionsofbothprazosinandatenololsignificantly inhibitedthepressorandtachycardicresponsesevokedbythestressors.Theseresultssuggestthatthemechanicalandenvironmentalstressorscausedtypicaldefen-sivecardiovascularresponsesimilartomammalsandthecardiovas-cularresponsestothestressorsaremediatedviathesympatheticadrenoceptorsintheheartandthebloodvessels.NoCOI.

2P-099Abnormal bradycardia response to fear in rats with heart failureKoba, Satoshi1; Hisatome, Ichiro2; Watanabe, Tatsuo1(1Division of Integrative Physiology, Tottori University Faculty of Medicine, Yonago, Japan, 2Division of Regenerative Medicine and Therapeutics, Tottori University Graduate School of Medical Science, Yonago, Japan)

Thepresentstudyexaminedautonomicresponsestofearinconsciousratswithheart failure (HF)aftermyocardial infarctionandcentralneuralmechanismsfortheresponses.Ratsexposedto5-minwhitenoisesound (90dB)displayedfreezingbehavior (an indexof fear),renalsympathoexcitation,andbradycardia.Thebradycardiawasparasympatheticallymediatedsinceatropine injected intravenouslypreventedtheresponseinnormalrats(N5).InHFrats(N7,fractionalshortening<35%),thebradycardiaresponsetofearwassignificantly(P<0.05)largerthanthatinnormalrats(N9).Ofnote,inHFrats,ar-rhythmiaappearedinassociationwiththereductioninheartrateseenduring fear.Moreover, innormalrats (N7),microinjection intothelateral/ventrolateralperiaqueductalgray (l/vlPAG)ofmuscimol,aGABAAreceptoragonist,significantlysuppressedthebradycardiaresponsetofear.Lastly,inHFrats(N6),microinjectionintothel/vlPAGofTempol,asuperoxidedismutasemimetic,significantlysuppressedthebradycardiaresponsetofear.Takentogether,theseresultssuggestthat fearstimulates the l/vlPAGandparasympatheticallycausesbradycardia inrats,andthatbrainoxidativestress inHFleadstodysfunctionofthe l/vlPAG,therebyexaggeratingparasympatheticeffectsonheartrateandelevatingrisks forcardiovasculareventsduringfear.NoCOI.

2P-100Stimulation of skin osmoreceptor evokes a cardiovas-cular defensive reaction in the frogMori, Rintaro; Sato, Fumitaka; Taguchi, Isamu; Ishihara, Jun; Takahashi, Tomoyuki; Horiuchi, Jouji(Department of Biomedical Engineering, Toyo University, Saitama, Japan)

Ithasbeensuggestedthatthefrogskinhasavariousfunctionslikeakidney(osmoreceptor),atongue(achemicalsensoroftastebuds)oralung.Externalenvironmentalcondition,especiallyosmolalitycanbecrucialforthefrog,sothatalmostofallfrogsliveinafreshwaterenvironment.Thus,changeintheexternalosmolalitymustbestress-fulsituationinthefrogandtheosmoticconditionmayevokeadefen-sivereaction,which isthecauseofstressresponse.Inthepresentstudy,weexaminedthecardiovasculardefensivereactionexposedtoenvironmentalstressors(NaClandsucrosesolutions)inthefrog(Rana catesbeiana).Bloodpressure(BP)andheartrate(HR)weremeasuredthroughabranchofabdominalarteryinconsciousfrogs.Afrogsaline(0.65%NaClsolution)with2cm2absorbentcottononthebackdidnotchangeofBPandHR.However,5,10and15%ofNaClsolutionswiththeabsorbentcottoneliciteddose-dependentincreasesinBPandHR.Theseincreaseswereblockedafterthetreatmentoflocalanestheticagent (2%lidocaine)onthebackskin. Incontrast, isotonicsucrosesolutions(50,108,163%)comparedwithNaClsolutions(5,10,15%,re-spectively)didnotevokeanychangesinBPandHR.Theseresultssuggestthatthefrogskinhasasensorforchangeinosmolalitycausedbyelectrolyteandthatthechangeinosmolalityevokesacardiovas-culardefensivereactioninthefrog.NoCOI.


Poster PresentationsMotor Function (1)

2P-101Changed response to microstimulation of the senso-rimotor cortex in developmental white matter injury model rat.Ueda, Yoshitomo; Misumi, Sachiyo; Ishida, Akimasa; Shimizu, Yuko; Jung, Cha-Gyun; Hida, Hideki(Dept of Neurophysiol & Brain Sci, Nagoya City Univ Grag Sch Med Sci, Nagoya, Japan.)

Developingwhitematterinjury(DWMI)causedbyhypoxia-ischemia(HI)isassociatedwithpermanentneurodevelopmentaldisabilitiessuchasparalysisandcognitivedysfunctioninpreterminfants.WemadeDWMImodelratmadebyrightcommoncarotidarteryocclusionfollowedby6%oxygenfor1hinP3Wistarrat.Ourmodelratshowedthatthearrestedprogressiontomatureoligodendrocyte (OL)withminorneuronaldamage.To investigatemotordeficitsafterHI,weperformedthebehavioral tests;motordeficitscore (MDS),rotarod,griptest,horizontalladdertest,andgaitanalysis.MDSandretentiontimeoftherotarodwerereducedintheDWMIratscomparedwithshamgroup.However,nosignificantdifferencewasobservedingriptest,horizontalladdertest,andgaitanalysis.Wetheninvestigatedtheresponsetostimulusbyintracorticalmicrostimulation(ICMS).Underanesthesia,whenbipolarpulses(0.2ms,0–200µA,333Hz)weregivenintothelayerVsensorimotorcortexusingatungstenelectrode,wecandetecttheevokedtwitchesincontralaterallowerlegjoints,whichcanmakethebrainmapofresponses.Theresponsemapresultsindi-catedthathiprepresentativeareawaslargerinshamgroupratherthanthatofPWMIgroup.Moreover, thealteredareas inPWMImodelarecontributedtoevokethetwitchesoftheotherjoints.Thedatasuggeststhatmotorcoordinationratherthanprimarymotorfunction is impaired inourmodel,andthatsensorimotorcortex isreorganizedinneonatalHI.NoCOI.

2P-102Arrested progression to mature oligodendrocytes with minor neuronal damage in developing white matter in-jury model ratMisumi, Sachiyo; Ueda, Yoshitomo; Shimizu, Yuko; Ishida, Akimasa; Jung, Cha-Gyun; Hida, Hideki(Dept of Neurophysiol & Brain Sci, Nagoya City Univ Grad Sch Med Sci, Nagoya, Japan)

Developingwhitematterinjury(DWMI)causedbyhypoxia-ischemia(HI)isassociatedwithpermanentneurodevelopmentaldisabilitiesinpreterminfants.Inadditiontoselectivelossofoligodendrocytepro-genitorcells(OPC),arrestedprogressiontomatureoligodendrocytes(OL)anddisturbanceofmyelinationarereportedasacauseofpatho-logicalchange inDWMI.Toanalysisneuropathologicalchanges inDWMImodelrat,madebyrightcommoncarotidarteryocclusionfollowedby6%hypoxia for1hour,we investigatedthepatternofcellulardegeneration,countedthenumberofOLlineagecellsandcheckedneuronalinjuryafterthedamage.Activecaspase-3positivecellsweredetectedinboththewhitematter(WM)andthesensory-motorcortexwithin24hoursafterHI.Fluoro-JadeBpositivecellsweredistributedinthedeepcortexlayer.However,active-caspase3/NeuNdouble-positiveapoptoticneuronswerenotdetected inthecortex.DetectionofOLlineagemarkersrevealedthatNG2positivecells increasedintheipsilateralWM(137.2±6.0%ofcontralateralWM,n=4)at2dayafterHIandPDGFRαpositivecellsincreased(108.9±2.74%,n=4)at7dayafterHI.However,matureOLofAPC-positivedecreasedby84±2.45%(n=4)at6monthlater.DatasuggestthatarrestedOLlineageprogressionoccurinneonatalHIinjurywithac-companiedbyminorneuronaldamage.NoCOI.

2P-103Projection pattern of the corticospinal tract, its forma-tion and cervico-lumbar varianceKameda, Hiroshi1; Kamiyama, Tsutomu1; Murabe, Naoyuki1; Fukuda, Satoshi1; Yoshioka, Noboru1; Mizukami, Hiroaki2; Ozawa, Keiya2; Sakurai, Masaki1(1Department of Physiology, Teikyo University School of Medicine, Tokyo, Japan, 2Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical University, Tochigi, Japan)

Thecorticospinal(CS)tractisessentialforvoluntarymovement.De-spitenumerousstudies,whatweknowaboutCStractorganizationanddevelopmentremainslimited.Torevealtotalcorticalareasinner-vatingasinglespinalsegmentofC7whichcontrols the forelimbmovements,weinjectedaretrogradetracerintoC7sothattheyspreadtotheunilateralgraymatteraswidelyaspossible,butnottotheventralmostdorsalcolumn(CStract).Wefoundthatinbothinfantsandadultmice,neuronsdistributedoveranunexpectedlybroadareaoftherostraltwo-thirdsofthecerebralcortexconvergetothissegment.Moreover,thecorticalareasinnervatingC7includeareascontrollingthehindlimbs(L4).However,withagingfromtheinfanttotheadultcelldensitiesgreatlydeclined,mainlyduetoaxonbranchelimination.WholecellrecordingsfromspinalcordcellswithoptogeneticselectivestimulationofCSaxonsandacotransfection-expressionsystemthatenabledlabelingofaxonsandpresynapticstructuresshowedthattheexuberantCSaxonsmakesynapticconnectionswithspinalcellsininfants.ThissuggestsneuronalcircuitsinvolvedinCStracttoC7arelargelyreorganizedduringdevelopment.Incontrast,thecorticalareasinnervatingL4werelimitedtotheconventionalhindlimbarea,andthecelldistributionanddensitydidnotchangeduringdevelopment.NoCOI.


2P-104Development and characterization of a monkey model of internal capsular strokeMurata, Yumi; Higo, Noriyuki(Human Technology Research Institute, AIST, Tsukuba, Japan)

Wehaveinvestigatedthemechanismsoffunctionalrecoveryusingamonkeymodelofcorticallesion,inwhichafocallesionisinducedintheprimarymotorcortex(M1).Inthepresentstudy,asthefirststeptounderstandfunctionalrecoverymechanisms inaclinicallymorerelevantmodel,wemadeafocallesionintheinternalcapsule,anareasusceptibletostroke.AnanatomicalMRIscanwasperformedonJapanesemonkeystoidentifythepartoftheinternalcapsuleinwhichdescendingmotortractsfromthehanddigitareaofM1passthough.Endthelin-1,avasoconstrictorpeptide,wasthen injected intotheidentifiedpartoftheinnercapsule(1.5µL/µg;15tracks,120µl).ThelesionwasevaluatedusingaT2-weightedanatomicalMRIscanafterinjection;theareasofincreasedT2signalwereobservedaroundtheinjectedareafrom3daysto1week,thentheygraduallydisappearedwithin1monthafterlesion.Motordeficitoccurredinthecontralesion-alupperlimb,aswasshownbyadecreaseinthesuccessrateofasmall-objectretrieval task, inwhichmonkeysretrieveasmall foodmorselfromanarrowtube.Recoveryofgrossmovementssuchasreachandpowergripoccurredduringthefirstweekafterlesion,whilelittlerecoveryofdexterousmovementsincludingprecisiongripwasobservedevenat1monthafterlesion.TheresultisincontrasttothatobservedinourM1-lesionmodel,inwhichdrasticrecoveryofdexter-ousmovementswasobservedduringthefirstmonth,suggestingthatthecompensatorymechanismafterinternalcapsulestrokeisdifferentfromthataftercorticallesion.NoCOI.

2P-105Neuronal activity in supplementary motor area of an unrestrained Japanese monkey walking on a treadmillNakajima, Katsumi1; Mori, Futoshi2; Murata, Akira1; Inase, Masahiko1(1Dept. Physiol., Facult. Med., Kinki Univ., Osaka, Japan, 2Lab. Vet. Sys. Sci., Joint Facult. Vet Med., Yamaguchi Univ., Yamaguchi, Japan)

Tofurtherunderstandcorticalmechanismsforcontrollingbipedal(Bp)gaitinhumans,werecordedneuronalactivityinsupplementarymotorarea(SMA)ofanunrestrainedmonkeywalkingeitherquadrupedallyorbipedallyonatreadmill.EMGactivitywasalsorecordedfromtrunkandlimbmuscles.Wefoundthat,duringquadrupedal(Qp)locomotion,manyneurons intrunk/hind-limbregionsofSMAmodulatedtheirdischargetonicallyand/orphasicallyalongwiththestepcycle,whilesomedidnot.WhenthemonkeyconverteditslocomotorpatternfromQptoBp,majorityofthese (task-related)neuronssubstantially in-creasedandmaintainedtheirdischargefrequencytonicallywithorwithoutenhancedphasiccomponent.Amongtherestofrecorded(nontask-related)neurons,somedisplayedburstactivitycloselyrelatedtoturningmovementsofheadtowardtheleftorright,whichwassuper-imposedontheon-goinglocomotormovements.Meanwhile,activityoftrunkandhind-limbmusclesduringQplocomotiondisplayeddiscreteburstswithouttonicactivityineachstep.DuringBplocomotion,theEMGburstwasdrasticallyenhancedinallthesemuscles.Particularly,thetrunkmusclesdisplayedsubstantialtonicactivityinadditiontotheenhancedburst.Theseresultssuggestthat,during locomotion,monkeySMAcontributestothecontrolofmaintaininguprightposturesoastoaccommodatemovementsofhead,trunkandlimbs,andmayprovideinsightintothebasisoffrontalgaitdisordersinhumans.NoCOI.

2P-106Intermittent Grasping a High Repulsion Cushion Grip Increases Skin Temperature and Muscle Activity of Hand, Arm and Face as well as Blood Flow in the Prefrontal CortexNishino, Hitoo1; Jung, Cha-Gyun1; Hida, Hideki1; Shiraki, Motoyuki2; Yamauchi, Masateru3; Sakakibara, Kazumasa4; Urakawa, Susumu5; Nishijo, Hisao5(1Dept. of Neurophysiology and Brain Science, Nagoya City Univ., Nagoya, Japan, 2Whitesuns, Nagoya, Japan, 3CCRC Japan, Nagoya, Japan, 4Sunbarden, Chita, Japan, 5Toyama Univ., Toyama, Japan)

Cerebralstrokeofteninducescontractureofhand,rigidity,expression-lessandspeechdisturbance.Continuousimproper(flexiondominant)afferentstimulifromcontractedhanddisturbthenormalsensorimotorneuralnetworkinthespinalcordandbrain,andwoulddisturbanappropriateenvironmentformotoroutput.Ahighrepulsioncushiongripdesignedbyushasastrongrepulsivepower,thusthestrongeritwasgrasped,thestrongerrepulsivepowerwasinduced.Thecon-tinuousgraspingthegrippromotedapromptrecoveryfromhandcontractureafterstroke.Inthisreportweinvestigatedtheeffectofhandmovement(intermittentgraspandreleaseofthegrip)ontem-peratureandmuscleactivityofhand,armandfaceaswellasonce-rebralbloodflow.Skintemperatureofhand,arm,neckandfacemea-suredbythermographincreasedwithinminutes.Muscleactivitiesinflexorandextensorofarm,masseteraswellasperioralmusclein-creased.Thecerebralbloodflowespeciallyinprefrontalcortexwasincreased.Dataindicatedthatintermittenthandmovementincreasedtheactivitiesinnotonlyhandandarmbutalsothoseofneckandfaceaswellasthebrain.Theseresultswouldbethebaseoftheeffectofrepulsiongripforpromptrecoveryfromaftereffectsofstroke.NoCOI.

2P-107Rewiring of subcortical projections from ventral pre-motor cortex after primary motor cortex lesion in ma-caque monkeysYamamoto, Tatsuya1,2,3; Hayashi, Takuya4; Murata, Yumi2; Onoe, Hirotaka5; Higo, Noriyuki2,6(1Fac Med & Health Sci, Tsukuba Int Univ, Tsuchiura, Japan, 2Human Tech Res Inst, AIST, Tsukuba, Japan, 3JSPS Research Fellow, Tokyo, Japan, 4Func Architechture Imag, RIKEN Ctr Life Sci Tech Unit, Kobe, Japan, 5Imaging Function Group, RIKEN Ctr Life Sci Tech Unit, Kobe, Japan, 6PRESTO, JST, Kawaguchi, Japan)

Neuromotorsystemshaveacapacitytorecovermotorfunctionfol-lowingthelocaldamage.Wepreviouslyreportedthattheipsilesionalventralpremotorcortex(ip-PMv)wasactivatedduringfunctionalre-coveryofhandmovementsintheprimarymotorcortex(M1)lesionedanimals.Inthepresentstudy,westudiedthesubcorticalconnectionsofip-PMvintheM1-lesionedmonkeysaftermotorrecoveryincom-parisonwiththoseintheintactanimals,byusingbiotinylateddextranamine(BDA).Aremarkabledifferencebetweenthelesionedandintactmonkeyswasobservedinthedeepcerebellarnuclei,especiallyintheipsilesionalfastigialnucleus(ip-FN):theBDA-labeledterminalswereobservedinallofthethreeM1-lesionedanimals,butnotineitheroftwointactanimals.Thelabeledterminalsweremainlylocatedinthecaudalandmediumpartof ip-FN,knowntocontain fastigiospinalneurons.Theseresultssuggestthatthenewly-formedprojectionfromip-PMvtoip-FNisinvolvedinfunctionalcompensationafterM1lesion.NoCOI.


2P-108The property of Ia excitation and recurrent inhibition of abdominal motoneurons in the catNiwa, Masatoshi1; Sasaki, Sei-Ichi2(1Department of Occupational Therapy, Faculty of Health Sciences, Kyorin University, Tokyo, Japan, 2Center for Medical Sciences, Ibaraki Prefectural University of Health Sciences, Ibaraki, Japan)

Abdominalmuscleshavedivergent functionssuchasrespiration,posturalcontrol,vomitingandinnervatedfromT6-T13andfromL1-L3incats.Thepresentexperimentswereundertakentoinvestigatehowreflexactionsexertedonthemotoneuronsworkandwhetherrecurrentinhibitorypathwaysarepresentincatabdominalmotoneurons.Intra-cellularrecordingsweremadefromexternaloblique (EO), internaloblique(IO),transversusabdominis(TA)andrectusabdominis(RA)motoneuronsinL1-L2oncatsanesthetizedwithsodiumpentobarbital.1)DorsalrootswereintactandmusclenerveselectricallystimulatedtoactivateIaafferents. Ia-EPSPselicitedwerefound inalmostallmotoneuronesfollowingthestimulationofthehomogeneousnerve.TheyareresponsibleforthemonosynapticEPSPofmotoneuronesoftheirmuscles,althoughthesevaluesarelongerthanthosevaluesofthehindlimbandtheintercostalsmuscles.2)Dorsalrootsweresec-tionedandmusclenerveswereelectricallystimulatedtoactivatemotoraxons.RecurrentIPSPselicitedwerefoundinsomeEOmoto-neurons followingthestimulationofEOnerveswithan intensitysubthresholdactivation foraxonof the impaledmotoneuron.ThepresentresultsprovideevidenceofIaexcitationandrecurrentinhibi-tionofabdominalmotornucleus.Theseneuronalcircuitsmightberelatedtothecontrolofabdominalmusclesduringvariousmotorac-tivities.NoCOI.

2P-109Neuronal activity in the motor thalamus of dopa-mine-intact and Parkinsonian ratsNakamura, Kouichi C.1; Sharott, Andrew2; Mallet, Nicolas2; Magill, Peter J.2(1Dpet Morphological Brain Science, Grad Sch Med, Kyoto Univ, Kyoto, Japan, 2MRC Anatomical Neuropharmacology Unit, University of Oxford, Oxford, United Kingdom)

MovementdifficultiesofParkinson’sdiseaseareoftenassociatedwiththeemergenceofabnormalbeta(13–30Hz)oscillationsincortexandbasalganglia.Themotorthalamus,whichmediatestheinfluencesofbasalgangliaoncorticalinformationprocessing,isparcellatedintotwomajorzonesaccordingtotheirsubcorticalinputs;thebasalganglia-re-cipientzone(BZ)andcerebellar-recipientzone(CZ)arebombardedbyinhibitoryGABAergicinputsfrombasalgangliaandexcitatorygluta-matergicinputsfromcerebellarnuclei,respectively.Toaddressthekeyissueofhowtheactivitiesofneuronsinthemotorthalamusaredisturbed inParkinsonism,werecordedthespontaneousfiringofidentifiedBZandCZneuronsunderanesthesiaindopamine-intactratsaswellasParkinsonianratswithunilateral6-hydroxydopamine (6-OHDA) lesions. IntheParkinsonianrats,wefoundnoevidenceofpathologically-reducedfiringratesineitherzoneofmotorthalamus,contrarytoclassicmodels.However,afterdopamine loss, thetightcouplingoffiringofBZneuronstocorticalslow(~1Hz)oscillationswasphase-shiftedby~100 °.Duringcorticalactivation,manyBZneurons,butnotCZneurons,exhibitedpronouncedbetaoscillations,whichwerenotseenindopamine-intactrats.WeconcludethatthethalamocorticalsubstratesofParkinsonianmovementdifficultiesaremorecloselyrelatedtoabnormalfiringpatterns,asexemplifiedbyexcessivebetaoscillations,thanalteredfiringrates.NoCOI.

2P-110NR2B antagonist ifenprodil improves abnormal fore-limb movement induced by D1 agonist SKF38393 in hemi-parkinsonian ratHabata, Toshiya1; Igarashi, Masakazu2; Akita, Hisanao1; Ogata, Masanori1; Noda, Kazuko1; Saji, Makoto1(1Dept. Physiol., Sch. Allied Health. Sci., Kitasato Univ., Kanagawa, Japan, 2Grad. Sch. Med. Sci., Kitasato Univ., Japan)

Forthe lastdecade, theantiparkinsonianactionofNR2BselectiveNMDAreceptorantagonisthasbeenstudied.However,theameliora-tiveeffectofNR2Bantagonistonmotorsymptomsinanimalmodelofparkinson’sdisease (PD)orpatientswithPDisstillcontroversial.RecentlywehavefoundthatsingleapplicationofNR2Bantagonistifenprodildemonstratesnoanti-parkinsonianeffectinhemi-parkinsonianrats(hemi-PD),whileco-applicationofifenprodilandL-DOPAimprovestheL-DOPA-inducedabnormalforelimbmovementinhemi-PD.Inthisstudy,totestwhethertheameliorativeeffectofNR2BantagonistontheL-DOPA-inducedmotorabnormalityinhemi-PDisresultedfromcooperativeactionofNR2BantagonistwithdopamineD1receptoragonisttomotorsystem,weinvestigatetheeffectofco-applicationofNR2BantagonistifenprodilandD1agonistSKF38393ontheabnormalforelimbmovementinducedbySKF38393inhemi-PDbyusingbehav-ioraltest.WeperformedthecylindertesttoestimateforelimbuseasmotoractivityaftersingleadministrationofSKF38393orco-adminis-trationofifenprodilandSKF38393.Asaresult,theco-administrationof ifenprodilandSKF38393completelyreversedthedyskinesia-likemotorabnormalityinducedbySKF38393.Fromthisresult,wesuggestthatifenprodilimprovestheL-DOPA-inducedabnormalforelimbmove-mentinhemi-PDthroughitscooperativeactionwithD1agonisttomotorsystem.NoCOI.

2P-111Stabilometry test in stroke patients during optokinetic stimulation.Komagata, Junya1; Watanabe, Shiori2; Suzuki, Yutaka3; Kitama, Toshihiro3(1Department of Physical Therapy, Health Science University, Yamanashi, Japan, 2Department of Physical Therapy, Yumura Onsen Hospital, Yamanashi, Japan, 3Center for Life Science Research, University of Yamanashi, Yamanashi, Japan)

Theposturalstabilityofstrokepatientswithhemiplegiawasstudiedbyusingastabilometricplatformwithoptokineticstimulation.Fourpatientswhocouldmaintainasittingposturewithoutassistancewererecruited.Thepatientswereaskedtositquietlyonaforceplatformfixedonastoolinadimroom.Foroptokineticstimulation,apatternofrandomdots,projectedontoascreenplaced100cminfrontofthesubjects,wasmovedcontinuouslyinhorizontal,vertical,ortorsionaldirectionswithavelocityof20deg/s.Asacontrolcondition,thesamepatternwithoutanymovementwaspresented.Ineachcondition,swaypath (SP)andthecenterofpressure (CoP)positionalongtheante-ro-posterior(x)andmedio-lateral(y)axeswereexamined.NosignificantdifferenceswerefoundintheSPvaluesinanystimulusdirectionwhencomparedwithcontroldata.However, theCoPpositionalongthex-axistendedtoshifttotheparalyticsideduringhorizontalortorsion-aloptokineticstimulationwithparalyticdirection,whilenoclearshiftwasobservedduringstimulationwithnonparalyticdirection.Incon-trast,theCoPpositionalongthey-axisdidnotshowclearchangewithanyofthedirections.TheseresultssuggestthattheCoPshiftinducedbyoptokineticstimulationscanbeusedasamodelfortherecoveryofdeviationsintheCoPobservedinpatientswithhemiparesis.NoCOI.


2P-112Characteristics of Dynamic Postural Adjustments during floor inclination using Posturography Tech-nique in RatsSasaki, Takeshi1,4; Nagamine, Takashi2; Matsuyama, Kiyoji3,4(1Dept. of Physical Therapy, Sch. of Health Sci., Sapporo Med. Univ., Sapporo, Japan, 2Dept. of Systems Neurosci., Sch. of Med., Sapporo Med. Univ., Sapporo, Japan, 3Dept. of Occupational Therapy, Sch. of Health Sci., Sapporo Med. Univ., Sapporo, Japan, 4Graduate Sch. of Health Sci., Sapporo Med Univ., Sapporo, Japan)

Inordertounderstandcharacteristicsofdynamicposturalcontrolofanimals,itisimportanttopreciselymeasurechangesoftheircenterofpressure(COP)inresponsetoposturaldisturbances.Inthisstudy,weaimedtoexaminethepropertiesofdynamicposturaladjustmentinrats.WemeasuredtheCOPduringfloorinclinationintheright-leftdirectionsatanglefrom0to30degreesandatanglevelocitiesof1.8,5,10,15deg/secusinganovelposturographytechniquethatwede-veloped.WefurthermeasuredEMGactivitiesofextensormusclesoffore-andhind-limbsinsomeanimals.Theresultsthatweobtainedwereasfollows:1)duringfloorinclinationatanglefrom0to30degreesinright-leftdirections,twosuccessivedynamicCOPchangeswerecommonlyobservedacrossanimals,2)regardlessofinclinationveloc-ities,theinclinationanglesthatdynamicCOPchangesoccurredwereataround8and13degrees,3)duringfloorinclination,animalsshowedtonicEMGactivitiesofextensormuscles,andfurtherexhibitedphasicEMGactivitiescorrespondingtothesedynamicCOPchanges.Fromtheseresults,dynamicCOPchangestogetherwithphasicEMGactiv-itiesarethoughttocorrespondtocompensatoryposturaladjustmentsthattakeplaceinresponsetoposturaldisturbances.NoCOI.

2P-113Thalamo-cortical inputs to the motor cortex during a self-initiated motor taskTanaka, Yasuyo H1,3; Tanaka, Yasuhiro R1,3; Wake, Hiroaki1; Masamizu, Yoshito1,3; Kawaguchi, Yasuo2,3; Matsuzaki, Masanori1,3 (1Division of Brain Circuits, National Institute for Basic Biology, Okazaki, Japan, 2Division of Cerebral Circuitry, National Institute for Physiological Sciences, Okazaki, Japan, 3JST, CREST, Saitama, Japan)

Thalamo-cortical(TC)pathwaysdriveinformationprocessinginthecerebralcortex.Intheprimarymotorcortex(M1),TCinputsintolayer1(L1)arethoughttocarrybasalgangliainformation.However,itremainsunknownhowtheactivityofL1TCprojectionsinM1isrelatedtotheexecutionofavoluntarymovement.Toaddressthisissue,weconductedtwo-photonimagingandoptogeneticstimulationinM1whilemiceperformedaself-initiatedlever-pulltask(Hiraetal.,2013).Weinjectedadeno-associatedvirusthatencodedGCaMP6intothethalamus,andthen,performedtwo-photoncalciumimagingofGCaMP6-expressingTCaxons inM1duringthetaskperformance.TheactivityofL1TCaxonswashigherduringthelever-pullperiodthanduringotherperiods.Next,weperturbedtheactivityofChan-nelrhodopsin-2-expressingL1TCaxonsduringthetaskperformancebyblue-lightilluminationonthecorticalsurfaceofM1.Wefoundthatthetaskperformancegotworseduringthephotostimulationperiod.WealsofoundthatL1TCaxonsinnervatedtheapicaldendritictuftsofL5corticospinalneurons.TheresultssuggestthatL1TCaxonssendacriticalsignaldirectlytoL5corticospinalneuronstoexecutetheleverpull.WearecurrentlyexaminingthetemporalcorrelationbetweenL1TCaxonalactivityandthedendriticactivityofcortico-spinalneuronsduringthelever-pulltask.NoCOI.

2P-114Purkinje cell activity in the cat cerebellar uvula during optokinetic stimulationKitama, Toshihiro1; Komagata, Junya2(1Center for Life Science Research, University of Yamanashi, Yamanashi, Japan, 2Department of Physical Therapy, Health Science University, Yamanashi, Japan)

WehavebeenstudyingtheresponsepatternofPurkinjecells(P-cells)inthecerebellarnodulusanduvuladuringsinusoidalheadrotationinverticalplaneinawakecats.Thepresentstudyexaminedthecomplexspike(CS)andsimplespike(SS)duringoptokineticstimulioftheP-cellsintheuvula.Forvisual-patternrotation,arandomdotpatternwaspresentedonthemonitorthatwasplacedinfrontoftheanimal.Therandom-dotpatternwasrotatedsinusoidallyinhorizontal,vertical,ortorsionalplaneundercontrolbyacomputer.Thestimulusparameterofatafrequencyof0.25Hzwith4degamplitudewasusedforeachstimulusplane.Inaboutfortypercentoftestedcells,clearfiringre-sponsewasobservedinCSactivitiesduringvisual-patternrotationineitherverticalortorsionalplane.NoclearCSresponsewasfoundduringstimulationinthehorizontalplane.On-direction(stimulusdi-rectionthatincreasesfiringrate)ofCSfiringwasupwardforverti-cal-rotation-respondingcellsandextorsionaldirectionfortorsion-rota-tion-respondingcells.ForSSactivities,clearresponsewasnotobservedforallstimulusdirectioninalltestedcells.Theresultssuggestthatthere isadirectionalspecificity fortheCSvisualresponse inthecerebellarregion.NoCOI.

2P-115Reward-timing dependent bidirectional modulation of spontaneous activity during single-cell operant condi-tioning with two-photon calcium imagingHira, Riichiro1; Ohkubo, Fuki1; Masamizu, Yoshito1; Ohkura, Masamichi2; Nakai, Junichi2; Okada, Takashi3; Matsuzaki, Masanori1(1National institute for basic biology, okazaki, Japan, 2Brain Science Institute, Saitama University, Saitama, Japan, 3National Institute of Neuroscience, NCNP, Tokyo, Japan)

Bydeliveryofarewardwhenthefiringrateofaneuronincreases,animalscanlearntheassociationbetweentheactivityoftheneuronandthereward,resultinginspecificincreaseoftheactivity(single-celloperantconditioning,SCOC).Thisabilityisfundamentaltothebrainmachineinterface(BMI)buttheunderlingmechanismsremainunclear.HerewedevelopedSCOCwithtwo-photoncalciumimaging inthemousemotorcortex.Head-fixedmicethatweretransfectedwithanadeno-associatedviruscarryingtheGCaMP7gene inmotorcortexweretrainedfor1–2weekstoperformaself-initiatedlever-pulltask.Aftertraining,weconductedtwo-photoncalciumimagingandmeanfluorescenceinthetargetcellwascontinuouslymeasured.Eachtimeacalciumtransientinthecelloccurred,awaterdropwasdeliveredwithalagtimeof<300ms.Theactivityoftargetcellspecificallyin-creasedwithin15minutes.Thenon-targetcellswhichwereacciden-tallyactivatedbeforeandaftertherewardincreasedanddecreasedtheactivities,respectively.Thisreward-timingdependentbidirection-almodulationoftheactivitywasreproducedbyphotostimulationofcellswithChR2.Theseresultsindicatethatcorticalneuronsindivid-uallyrespondtothetemporaldifferencebetweentheiractivitiesandrewards,whichmaybefundamentalprocessinBMIlearning.NoCOI.


2P-116An analysis of the activity of the arm muscles during the service motion in badminton- A longitudinal study during the period between the first and second years of junior high school Masu, Yujiro; Muramatsu, Ken; Komagata, Jyunya(Department of Physical Therapy, Health Science University, Yamanashi, Japan)

[Objective]Everybadmintongamestartswithaserve,andaplayer’sserviceskillsareconsideredtoinfluenceralliesinagame.Withtheaimofacquiringknowledgeofthedevelopmentofstroketechniques,thepresentstudy,involvingplayerswhostartedplayingbadmintoninthefirstyearofjuniorhighschool,examinedimprovementsintheirservesforoneyear.[Methods]Thesubjectsweresevenbadmintonplayerswhostartedtoplaywhentheywereinthefirstyearofjuniorhighschool-alongitudinalstudyduringtheperiodbetweenthefirstandsecondyearsofjuniorhighschool.Thesubjectswereaskedtoperformshortback-handserves,andtheactivityofthearmmuscleswasas-sessed. [Results]Themean iEMGlevelsof themusculusextensorcarpiulnarisandmusculusflexorcarpiradialisweresignificantlylowerwhenthestudentswereinthesecondthanfirstyear.ThemeaniEMGlevelofthedeltoidwassignificantlyhigherwhentheywereinthesecondthanfirstyear. [Conclusion]Theseresultssuggestthatplayerswhohavecontinuouslypracticedtendtousethedeltoidinsteadofforearmmusclesduringtheservemotion.Changesinmuscleactiv-ityareconsideredtoberelatedto:adecreaseinthetimebetweenthedecisiontohittheshuttlecockandmomenttheracketcontactsit;andimprovementsintheserviceaccuracy.NoCOI.

2P-117Arm preference of ophiuroids during locomotion and its implication for the internal control scheme of arm usageMatsuzaka, Yoshiya1; Sato, Eiki2; Kano, Takeshi2; Sakamoto, Kazuhiro2; Aonuma, Hitoshi3; Ishiguro, Akio2(1Graduate School of Medicine, Tohoku University, 2Research Institute of Electrical Communication, 3Graduate School of Life Science, Hokkaido University)

Ophiuroidsarepentaradiallysymmetricalechinodermsthatlocomoteontheseafloorbythecoordinatedrhythmicmovementsoftheirmul-tijointarms.Howsuchcoordinatedmovementsareachievedisafocusof interest fromtheviewpointofneurobiologyaswellasrobotics,becauseophiuroidslackthecentralnervoussystemthatexertstheintegrativecontroloverthefivearms.Toexploretheunderlyingmechanismofthearmcoordination,westudiedtheirarmusageduringlocomotion.Previousstudiesreportedtwotypesof locomotionofophiuroids,rowingandreverserowing.Intheformer,onearmactsastheleadingarm,beingpointedforwardalongtheaxisoflocomotionwhileinthelatter,anarmispointedbackward.Wefoundthefollow-ings.1)Duringrowing,aparticulararmpreferentiallyactedastheleadingarm.2)Whenthepreferredleadingarmwasanesthetized,itwasreplacedbyoneoftheotherarms.3)Bothintactandanesthetizedindividualspreferentiallyperformedrowing,andfinally4)reverserowingoccurredinfrequentlyevenwhenmultiplearmswereanesthe-tizedsuchthatitwouldhaveallowedmoreefficientusageoftheintactarms.Thesefindings indicatethattheophiuroids’nervoussystemprioritizestheselectionoftheleadingarmoverothersforlocomotion.Anautonomousdecentralizedcontrolmodelwasdevelopedthatac-countsfortheobservedarmusageduringlocomotion.NoCOI.

Poster PresentationsReproduction

2P-118Regulation of sperm hyperactivation by serotoninFujinoki, Masakatsu(Department of Physiology, School of Mediceine, Dokkyo Medical University, Tochigi, Japan)

Onmammalianspermatozoa,capacitationistheessentialprocesstofertiletotheegg.Capacitatedspermatozoaexhibitanacrosomereac-tioninheadandhyperactivationinflagellum.Recently, ithasbeeninvestigatedthatsomehormonesregulatedspermhyperactivationinadosedependentmanner.Inthepresentstudy,Iexaminedwhetherserotonin,whichreleases fromcumulus-oocytecomplex,regulatedhyperactivationusinghamsterspermatozoa.Serotoninsignificantlyenhancedspermhyperactivationinadosedependentmanneralthoughitdidnotaffectspermmotility.Serotoninregulatedspermhyperacti-vationthroughtwotypesofserotoninreceptor.Highconcentration(nM-µM)ofserotoninenhancedspermhyperactivationthrough5HT4receptor.Ontheotherhand,lowconcentration(fM-pM)ofserotoninenhancedspermhyperactivationthrough5HT2receptor.Ingeneral,spermhyperactivationisregulatedthroughcAMPsignalsandCa2+signals.Because5HT2receptorand5HT4receptorareassociatedwithCa2+signalsandcAMPsignalsrespectively,itwasexaminedwhetherserotoninregulatesspermhyperactivationstimulatingCa2+signalsandcAMPsignalsviaeachreceptor.Fromresults,serotoninregulatedspermhyperactivationthroughIP3receptorandPKAwhenitstimu-latedspermatozoavia5HT2receptor.Whenserotoninstimulatedspermatozoavia5HT4receptor, itregulatedspermhyperactivationthroughPKAonly.Inconclusion,serotoninregulatesspermhyperac-tivationinadosedependentmannerthroughCa2+signalsandcAMPsignalsvia5HT2receptorand5HT4receptor.NoCOI.


2P-119Regulation of hyperactivated motility by fluid osmolal-ity in hamster spermatozoaTakei, Gen Leon; Fujinoki, Masakatsu; Seo, Yoshiteru(Department of regulatory physiology, Dokkyo Medical University, Tochigi, Japan)

Mammalianspermatozoacannotfertilizewithovumwithoutcapacita-tion.Generally,capacitatedspermatozoaexhibithyperactivation,aspecializedflagellarmovementwith increasedbendamplitudetopenetratethezonapellucida.Inmammals,osmolalitiesofthefluidsfrommaleinternalgenitalia(seminalvesicle,prostate,andepididymis)arehigher(<420mOsm)thanthatofthefluidsfromfemalereproduc-tivetract(around290mOsm).Thismeansthatmammalianspermato-zoaexperienceosmoticshockinthefemalereproductivetract.How-ever,theimpactofosmoticshockonflagellarmotilityhasnotbeenelucidated.Inthepresentstudy,weinvestigatedtheeffectofphysio-logicalosmoticchangeonmammalianspermmotilityusinghamster.Theosmolalitiesoffluidsfrommaleinternalgenitaliaandvaginainhamsterwerehigher(around390mOsm)thanthatofbloodplasma(around300mOsm).Whenspermatozoawere incubated invariousosmoticconditions(220–390mOsm),osmolalitynearsemenandvaginalfluidsuppressedspermmotilityactivation,andspermmotilitywasactivatedastheextracellularosmolalitydecreased.Moreover, theappearanceofhyperactivatedmotilitywasdelayedattheosmolalitynearsemenandvaginalfluid,andwasacceleratedastheextracellularosmolalitydecreasedtothatofoviductalfluid.Ourdatasuggestthatmammalianspermmotilityisregulatedbyfluidosmolalitytobehy-peractivatedatoviduct,wherespermfertilizewithovum.NoCOI.

2P-120Identification of the interactive regions between dical-cin and gp41; development of compounds that control the fertilization rate in frogs.Miwa, Naofumi; Hanaue, Mayu; Takamatsu, Ken(Dept. Physiol.,Toho Univ.)

Fertilizationisawell-coordinatedandsequentialprocess,beginningwithtaxon-specificsperm-bindingtoegg-coatingenvelope.Werecent-lydiscoveredthatXenopusdicalcin,presentintheegg-coatingenve-lope,remarkablysuppressesfertilizationin vitrothroughbindingtogp41,aglycoproteinoftheeggenvelope.Wehavepreviouslyreport-edthattwoamino-acidregionsofdicalcinwerecandidatesofthenativebindingregionondicalcinforgp41,andthecorrespondingpeptidesdecreasedthefertilizationrate inadose-dependentmanner.Inthepresentstudy,we,oppositely,attemptedtodeterminethebindingregionongp41fordicalcin.Toroughlyestimatetheregion,wegen-eratedpoly-histidine-taggedgp41andtwoZPdomainsofgp41:ZP-NandZP-C,andexaminedtheirbindingtodicalcin.Theresultsshowedthatdicalcinboundtorecombinantgp41andZP-C,butnottoZP-N,suggestingthatZP-Cdomainistheresponsibledomainforthebindingofgp41todicalcin.WenextpreparedasetofdeletionmutantsofZP-Cdomain,andfoundthepotentialdicalcin-bindingregionofgp41,amongwhichweobtainedasynthesizedpeptidethatincreasedthefertiliza-tionrate,indicatingthatthisaminoacidregionisresponsibleforthenativebindingofgp41todicalcin.Thus,ournovelresultselucidatedtheinteractiveregionsbetweendicalcinandgp41,andhelptounder-standmolecularmechanismsoftheactionofdicalcin,andalsoleadtodevelopmentofbioactivepeptidesthatarecapableof‘increasing’thefertilizationrateaswellas‘decreasing’theone.NoCOI.

2P-121New intrauterine probe for measuring uterine electri-cal impedance of mice using four-electrode methodArakawa, Mayu1; Yamamoto, Megumi1; Nakamoto, MIki1; Igi, Chihiro1; Akasaka, Keisuke1; Nishimura, Yukako1; Hosono, Takayoshi1; Nakamura, Hitomi2; Kimura, Tadashi2(1Dept Biomedical Engineering, Grad Sch Osaka Electro-Commun Univ, Shijonawate, Osaka, Japan, 2Dept. Obstet Gynecol, Grad Sch Med, Osaka Univ)

Biologicalelectricalimpedance(EI)measuredusingafour-electrodemethod(FEM)hasbeenwidelyappliedtoassessbodyconstituents.TheFEMinvolvestwopairsofelectrodes,anouterpair (currentelectrodes,CE)andaninnerpair(voltageelectrodes,VE).EIiscalcu-latedbymeasuringthevoltagebetweenVEproducedbyACcurrentspassingbetweenCE. Theuterineendometrium(UE)changes itsconditionsineachestrusphase.TheaimofthisstudywastoclarifythecharacteristicsofUEEIusingmice.EightadultfemaleSlc:ddyfemalemicewereusedinthisstudy.Wecheckedtheestruscycleofmicebythecytologicalevaluationofvaginalsmears.Theintrauterineprobe(IUP)wascomposedoftwoelectrodesandacoreoftungstenwireof0.3mmindiameter.WerolledplatinumwireonthecorewireasCEandVEatfoursiteswithin10.0mmfromthetip.Wemeasuredelectricalimpedanceusinganimpedancecheckerbetween2.5and350kHz,andcalculatedcharacteristicparameterssuchastheimpedanceat0Hz(R0)andinfinityHz(Ri).Underinspiredanesthesia,weexposedtheuterusandinsertedtheIUPintothevaginaandadvanceditstipintotheuterinecavity,andthenmeasuredUEI. R0andRiwerelargerinmetestrusthaninproestrusanddiestrus.TheUEIparam-etersmeasuredbyFEMmightbenovelparametersforevaluatingUEconditions.NoCOI.

2P-122Changes of G-protein-coupled Receptor (GPR) 30 mRNA Levels in the Rat Uterus during Pregnancy, La-bor and the Estrous CycleMurata, Takuya; Ichimaru, Toru; Narita, Kazumi(Department of Integrative Physiology, Faculty of Medical Science, University of Fukui, Fukui, Japan)

Estrogeninducesphysiologicalandmorphologicalchangesassociatedwithalargenumberofgeneexpressionsintheuterus.TheG-proteincoupledreceptor30(GPR30)hasbeenreportedtobeamembrane-as-sociatedestrogenreceptor(ER).Inthisstudy,toinvestigatethereg-ulationofGPR30intheuterus,changesofGPR30mRNAlevelsintheuteruswereexaminedinratsduringpregnancy,laborandtheestrouscycleandinestrogen-treatedovariectomized(OVX)rats.NosignificantchangeinGPR30mRNAlevelswasdetectedduringpregnancyandlabor.Duringtheestrouscycle,GPR30mRNAlevelsinthemorningandafternoononproestrusweresignificantlylowercomparedwiththoseondiestrus,metestrusandestrus.ThisdeclineinGPR30mRNAlevelsonproestruswasabolishedbytheadministrationofERantag-onists, ICI182780andraloxifene,ondiestrus,butnotbytamoxifen.Afterasingleinjectionof17β-estradiol(E2,12.5µg/rat),GPR30mRNAlevelsdecreasedwithin2h,butshowednosignificantchange24haftertreatment.TheinjectionofPPT,anERαagonist,orDPN,anERβagonist,decreasedGPR30mRNAlevels3haftertreatment.TheseresultssuggestthatGPR30mRNAlevels intheratuteruschangeduringtheestrouscycleandthatthesechangesareinducedbyestro-gen,probablyviaERαandERβ.NoCOI.


2P-123Essential amino acids deficient diet suppress estrous cyclicity in ratIchimaru, Toru1; Nagao, Kenji2; Narita, Kazumi1; Bannai, Makoto2; Murata, Takuya1(1Department of Integrative Physiology, Faculty of Medical Sciences, University of Fukui, Fukui, Japan, 2Frontier Research Labs, Institute for Innovation, Ajinomoto Co., Inc., Kanagawa, Japan)

Reproductivefunctionsare influencedbycircumstances,especiallytheirnutritionalstatus.Weinvestigatedwhetherdeficienciesofessen-tialaminoacids(EAAs)couldbeasignalofsystemicundernutritiontosuppressreproductivefunctions.AdultfemaleWistar-ImamichiratsweredailyfedacaloricallyidenticaldietwhichlacksoneofEAAs,threonine (Thr-def), tryptophan (Trp-def), lysine (Lys-def)orvaline(Val-def),andmonitoredtheirestrouscyclicitybyvaginalsmears.AlloftheEAAdeficienttreatmentsdecreasedfoodintake,bodyweight,plasmaglucoseandtriglyceride(TG)comparedtoacontroldiet.ItalsostoppedtheestrouscycleindifferenttimingamongEAAs;thedayswhencontinuousdiestrusstartedwere5.8±1.1inVal-def,9.7±0.7inTrp-def,12.0±0.8inThr-defand18.7±4.5inLys-def.Then,ratsweresubjectedtoapair-feeding(PF)experimentinwhichcontroldietwasrestrictedtoeither100,66or33%ofthespontaneousThr-defintake.BothofthePF-100whichconsumedsamecalorieastheThr-def,andthePF-66ofwhichbodyweightwasequallydecreasedastheThr-def,didnotshowadelayedestrouscyclicity.OnlythePF-33groupshowedacessationoftheestrouscycleinthesametiming(12.8±0.2d)astheThr-def.PlasmaglucoseandTGweresimilartotheThr-definthePF-33,buthigherinothergroups.Theseresultsindicatedthatadefi-ciencyofEAAsuppressesreproductivefunctionsinparticularmanneramongEAAs.NoCOI.

Poster PresentationsDevelopment, Growth, Aging

2P-124Functional decline of alpha4beta2 nicotinic receptors in the aged rat brain Uchida, Sae1; Watanabe, Saori1,2; Misawa, Hidemi2; Kawashima, Koichiro3(1Department of Autonomic Neuroscience, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan, 2Department of Pharmacology, Faculty of Pharmacy, Keio University, 3Department of Molecular Pharmacology, Kitasato University School of Pharmacy)

Ourpreviousstudydemonstratedthatlong-termstimulationofnico-tinicreceptorsenhancescorticalacetylcholine(ACh)releaseinadultratsbutnotinagedrats.Inthepresentstudy,weexaminedwhichsubtypeofnicotinicreceptorscontributetothisenhancementofcor-ticalAChreleaseinadultrats.Adultrats(4–9months)receivedsus-tainedsubcutaneousinfusionofeitheraselectivealpha4beta2nicotin-ic receptor agonist (ABT-418, 10µg/kg/h) or a selective alpha7nicotinicreceptoragonist(GTS-21,20–100µg/kg/h)for14days.Underurethaneanesthesia,wedeterminedAChreleaseintheparietalcortexinducedbyelectricalstimulationofthenucleusbasalisofMeynert(NBM).Thestimulusintensity-dependentAChreleaseinthecortexwasgreater inrats treatedwithalpha4beta2agonist than insa-line-treatedcontrolrats.Ontheotherhand,themagnitudesofcorticalAChreleaseinratstreatedwithalpha7agonistweresimilartothoseincontrolrats. Weconcludethatalpha4beta2nicotinicreceptorsmediateenhancementofcorticalAChreleaseinducedbyNBMacti-vationafterlong-termstimulationofnicotinicreceptorsinadultrats.Thesefindings,togetherwithourpreviousstudies,suggestthereduc-tionofthealpha4beta2typenicotinicreceptorfunctioninagedrats.NoCOI.

2P-125Hypoplasia of the vascular smooth muscle cell in the ATBF1 KO mouseJung, Cha-Gyun1; Kawaguchi, Makoto2; Miura, Yutaka3; Hida, Hideki1; Michikawa, Makoto4(1Dept. of Neurophysiology and Brain Science, Nagoya City Univ., Nagoya, Japan, 2Dept. of Pathology, Niigata Rosai Hospital, Niigata, Japan, 3Dept. of Molecular Neurobiology, Nagoya City Univ., Nagoya, Japan, 4Dept. of Biochemistry, Nagoya City Univ., Nagoya, Japan)

ATBF1(AT-motifbindingfactor1),a404-kDatranscriptionfactorthatcontains4homeodomainsand23zincfingermotifs,involvedintran-scriptionalregulationsanddifferentiationonmanytypesofcellsin-cludingneurons.TogaininsightsintothephysiologicalfunctionsofATBF1,wegeneratedATBF1deficientmicebygenetargeting.ATBF1knockout(KO)micearediedonthedayofbirthbecauseofrespiratoryfailurecausedbytheimpairmentofairspaceformationoflung.BodyweightwasdecreasedinE13andE17ATBF1KOmousecomparedtowildtypemouse,andATBF1KOmouseappearsadys-plasia insomeorgans.ATBF1wasexpressed invascularsmoothmusclecellsbutnotinendothelialcells.Althoughpulmonaryparen-chymasuchasalveolusepithelialcellswasnormal,apulmonaryarterydiameterwassmallerthanthatofWT.Andalsowefoundthatnum-berofsmoothmusclecellwasdecreasedincoronaryarteryofATBF1KOmouse,andthat lossofATBF1leadtothedown-regulationofPDGFR-a,akeyregulatoryfactorofvascularformation,andinducedvascularsmoothmusclecelldefects.Together,thesedataindicatethatATBF1mayanimportantroleinthedevelopmentofvascularsmoothmuscle.NoCOI.


2P-126Erythropoietin facilitates the differentiation of mouse embryonic stem cells into insulin-producing cellsOtsuka, Wakako; Kaitsuka, Taku; Kubo, Takuya; Wei, Fan-Yan; Tomizawa, Kazuhito(Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto. Japan)

Todate,therearefewwaysofthefundamentalcurefordiabetes.Cellreplacementtherapyhasbecomepossiblebyutilizingartificiallygen-eratedpancreaticβ-cellsfromembryonicstemcells(ESCs)andinducedpluripotentstemcells (iPSCs).However,boththeefficiencyofthemethodandpotencyofdifferentiatedcellsareinsufficient.Itisimport-anttodevelopthemoreeffectivemethodofgeneratingapancreaticβ-cellsfortheregenerativemedicine.Inthepresentstudy,wescreenedseveralendogenousfactorsinvolvedinorgandevelopmentandfoundthatthetreatmentwitherythropoietin(Epo)facilitatedthedifferenti-ationofmouseESCsintoinsulin-producingcells.Moreover,Epoin-creasedPdx1geneexpressionatthestageofpancreaticprogenitors.Next,weexaminedwhichdifferentiationstageEpoactedon.Epotreatmentduringearlystageof thedifferentiationsignificantly in-creasedSox17geneexpression,asamarkerofdefinitiveendoderm,anddecreasedtheexpressionofOct4,amarkerofpluripotency.TheseresultssuggestthatthetreatmentwithEpoatanearlystageisusefulfortheefficientgenerationofinsulin-producingcells.EpoisacytokinepromotingtheproductionofredbloodcellsviaJAK-STATsignalingpathway.ThemechanismofSox17 inductionbyEpotreatment isunderinvestigation.NoCOI.

2P-127Cell Functional Differences in Relation to the Distri-bution of Cytokeratins 13, 14 and 18 during Morpho-genesis of the TongueIwasaki, Shin-ichi1; Satoh, Yoshihide1; Aoyagi, Hidekazu2 (1Department of Physiology, The Nippon Dental University School of Life Dentistry at Niigata, Niigata, Japan, 2Advanced Research Center, The Nippon Dental University School of Life Dentistry at Niigata, Niigata, Japan)

Onthemorphogenesisoftherattongue,wecouldrecognizethespe-cificappearanceanddistributionofcytokeratin13(K13),14(K14)and18(K18).K14-specificimmunoreactivitywasfirstdetectedonthelingualepitheliumoffetusesonembryonicday17(E17),atwhichtimethelingualepitheliumwascomposedofafewlayersofcuboidalcells,andK13-specificimmunoreactivityonE19.Thenumberoflayersofcuboi-dalcells inthe lingualepitheliumincreased fromE17toE19.Atpostnatalstages,K13-specificimmunoreactivitybecametobeevidentintheintermediatecellsoftheinterpapillarycellcolumnsandK14-spe-cific immunoreactivitydid inthebasalandsuprabasalcellsofthepapillaryandinterpapillarycellcolumnsaccordingtothedevelopmentoffiliformpapillae.K13-specificimmunoreactivitywasevidentincellsofthedifferentiatingintermediatecells,whileK14-specificimmunore-activitywasdetectedinproliferatingcellsofthebasalandsuprabasallayers.K18-specific immunoreactivitywasdetectable inthesinglelayerofperidermcellsthatcoveredthelingualepitheliumoffetusesonE13andE15. Immunoreactivitywasdistributedthroughoutthecytoplasminperidermcells.OnE17,K18-specificimmunoreactivitywasverydistinctintheperidermcells,whichhadbecomeswollenandelliptical.Peridermcellsareestimatedtohavepeculiarfunctionsinthisprocess.NoCOI.

2P-128The expression of PTEN in the development of mouse cochlear lateral wall Youyi, Dong; Li, Sui; Fuminori, Yamaguchi; Kazuyo, Kamitori; Yuko, Hirata; Akram, Hossain; Chisato, Katagi; Masaaki, Tokuda (The Department of Cell Physiology, Kagawa University Faculty Medicine, Japan)

Phosphataseandtensinhomologdeletedonchromosome10(PTEN)isatumorsuppressorgenethatregulatesvariouscellprocessesin-cludingproliferation,growth,synaptogenesis,neuralandgliomastem/progenitorcellrenewal.Inaddition,PTENcanregulatesensorycellproliferationanddifferentiationofhairbundlesinthemammalianco-chlea. Inthisstudyweuse immunofluorescence,westernblotandRT-PCRtorevealtheexpressionofPTENinthedevelopingcochlearlateralwall,whichiscrucialforregulatingK+homeostasis.RelativelyhighlevelsofPTENareinitiallyexpressedinthemarginalcells(MCs)ofthelateralwallatembryonicday(E)17.5whentheystarttodiffer-entiate.Similarlyhighlevelsaresubsequentlyexpressedindifferenti-atingrootcells(RCs)atpostnatalday(P)3andtheninspiralligamentfibrocytes(SLFs)atpostnatalday(P)10.Inthematurecochlea,PTENexpressionis loworundetectableinMCsandSLFsbutitremainshighinRCsandtheirprocesses.TheexpressionpatternforPTENinthedevelopinglateralwallsuggeststhatitplaysacriticalroleinthedifferentiationofthecellularpathwaysthatregulateK+homeostasisinthecochlea.NoCOI.

Poster PresentationsDigestion, Absorption


2P-129Effects of moxibustion on restraint stress-induced de-layed gastric emptying in conscious ratsTaniguchi, Hiroshi1; Imai, Kenji2; Taniguchi, Sazu2; Ogasawara, Chie1; Hino, Kokoro1; Shinbara, Hisashi1; Sumiya, Eiji1 (1Dept. of Basic Acupuncture and Moxibustion, Meiji Univ. of Integrative Medicine, Kyoto, Japan, 2Dept. of Clinical Acupuncture and Moxibustion, Meiji Univ. of Integrative Medicine, Kyoto, Japan)

Moxibustion(MOX)hasbeenusedtotreatgastricsymptoms.AnimalstudieshavealreadyshownthatacupunctureattheST-36,whichlo-catedontibialisanteriormuscle,haveimprovedstress-induceddelayedgastricemptying(GE).However,themechanismofthebeneficialeffectsofMOXremainsunknown.ThepurposeofthisstudywastoinvestigatewhethertheMOXatST-36improvesrestraintstress-inducedalterationintheGEofconsciousrats.Ratsweregivenofsolidfoodafter24-hfasting.Immediatelyaftertheingestion,ratswereloadedtorestraintstress.Ninetyminutesafterthefeeding,ratswereeuthanizedandgastriccontentwasremovedtocalculateGE. IndirectMOXwasperformedabovethebilateralST-36throughoutthestressloading.Toinvestigatewhether vagal nervewas involved inmediating thestress-inducedalterationsofGE,atropinewasadministered(i.p)justbeforethestartofrestraintstress,andbilateraltruncalvagotomywasperformedin14daysbeforeGEmeasurement.GEinthe90minstudyperiodwassignificantlydelayedbyrestraintstress.ThisdelayedGEwassignificantlyacceleratedbyMOXatST-36,whichhadbeendis-appearedbyatropineadministrationandvagotomy.Restraintstress-in-duceddelayedGEis improvedbyMOXatST-36.Enhancedvagalnerveactivityare involved inmediatingthestimulatoryeffectsofMOXonrestraintstress-induceddelayedGE.NoCOI.

2P-130The different pattern of gastrointestinal motility and blood flow following oral glucose and water ingestionKashima, Hideaki1; Eguchi, Kohei1; Kadowaki, Takako1; Fujihara, Chizuko1; Someya, Nami2; Miura, Akira1; Endo, Masako Yamaoka1; Fukuba, Yoshiyuki1(1Department of Exercise Science and Physiology, Prefectural University of Hiroshima, Hiroshima, Japan, 2Chiba Prefectural University of Health Sciences, Chiba, Japan)

Oralglucose ingestion increasessplanchnicbloodflow(SBF),butwaterdoesnot.WehypothesizedthattheincreasedSBFbyglucoseingestionmaybeelicitedbytheexaggeratedelevationofgastrointes-tinalmotility.Toinvestigatethishypothesis,wemeasuredthegastro-intestinalmotilitiesofstomach(byelectrogastrography;EGG)andsmallintestine(byelectroenterography;EEnG),andthebloodflow(BF)ofsuperiormesentericartery(SMA)asanindexofSBFbeforeandevery15minforonehourafteringesting350-mlofeither8%glucose(Glu)orwater(Wa)in6malesubjects.ThegastrointestinalmotilitywasevaluatedthepowerderivedfromthespectralanalysistoEGGandEEnGrecordingsbeforeandafteringestion.BFofSMAwasmeasuredbypulsed-Dopplerultrasonography.Inaddition,gastricemptying(GE)ratewasevaluatedbyecho-ultrasonographicimages.Comparedtothebaseline,theSBFwassignificantlyincreasednotbyWa,butbyGluingestion.WhereasEGGpowerwasincreasedafteringestionofbothsolutions,EEnGpowerwassignificantlyincreasedmerelybyGluin-gestion.GEratewassignificantlydelayedinGlutoWaingestion.Theseresultssuggestthatglucose ingestionelicitsadifferentpatternofgastrointestinalmotilityfromwateringestion.Especially,theexagger-atedEEnGresponsemaybeafactorwhichleadstoincreasingSBF.NoCOI.

2P-131Characteristics of autonomic nervous response in irri-table bowel syndromeSakuragi, Sokichi; Terada, Miki; Nakajima, Sora(Department of School Health Sciences, Aichi University of Education, Japan)

Autonomicdysregulation inducedbyhighersympatheticnervousactivityandlowerparasympatheticnervousactivityisconsideredtobethemaincharacteristicinmalepatients,andvisceralhypersensi-tivityisconsideredtobethemaincharacteristicinfemalepatientswithIrritablebowelsyndrome(IBS).However,autonomiccharacter-isticinthefemalepatientsisstillcontroversial.Thepurposeofthisstudywastoclarifythecharacteristicsofautonomicresponsetophysical(coldpressortest)andpsychophysiological(strooptest)stim-uliinfemalepatientswithIBS,alongwiththemoodchangeassociat-edwiththestimuli.Moodstateswereestimatedbyaprofileofmoodstates(POMS),andautonomicnervousfunctionwasevaluatedbyaspectralanalysisofheartratevariability(HRV),baroreflexsensitivityandbloodpressure (BP).Repeatedmeasuresanalysisofvariance(ANOVA)revealedsignificantinteractionofgroup(IBSandcontrol)xtimecourseafterthestimuliinHFamplitude,thatis,delayedrecov-eryofHFamplitudeafterthephysicalstimulus,anddelayedanddi-minishedrecoveryafterthepsychophysiologicalstimulus(strooptest)infemalepatientswithIBS.Repeatedmeasuresanalysisofvariance(ANOVA)alsorevealedsignificantmaineffectofgroup(IBSandcon-trol) inbaroreflexsensitivity, that is, thebaroreflexsensitivity isgenerallylowerinfemalepatientswithIBS,thoughthetimecoursesinbothgroupsweresimilar.NoCOI.

2P-132Anion secretion induced by short-chain fatty acids in the human terminal ileumKaraki, Shin-Ichiro; Kuwahara, Atsukazu(Laboratory of Physiology, Graduate School of Nutritional and Environmental Sciences / Institute for Environmental Sciences, University of Shizuoka, Shizuoka, Japan)

Short-chainfattyacids(SCFAs),predominantlyacetate,propionateandbutyrate,arefermentedproductsexistingmorethan100mMinallinthelargeintestinallumenofmammalsincludinghumans.WeandsomeresearchgroupshavepreviouslyreportedthatSCFAsnotonlyareabsorbedasnutrient,butalsostimulateintestinalmucosainducingavarietyofphysiologicalresponses,e.g.transepithelialiontransportintherodents.Howeversofar,therehasbeennoreportoftheSCFA-in-duced iontransport inhumanintestine.ThisreasonseemedtobebecauseSCFAsdidnotevokeiontransportinthehumanlargeintes-tineclearly.Howeverinthepresentstudy,wefoundthattheSCFAsinducedaniontransportinthehumansurgicalspecimensofterminalileumbyusingmucosa-submucosalpreparationsmountedontheUssingchambers.Intheratandguineapigcolon,ithasbeenreport-edthatpropionateevokesanionsecretion,butacetatedoesnot.How-everinthehumanterminalileum,theluminaladditionofacetatealsoconcentration-dependentlyevokedananionsecretionthesameaspropionate.Moreover,theluminalacetate-inducedresponsewasat-tenuateddependentontheconcentrationofpropionatepretreated,andviceversa.TheseresultssuggestthattherefluxofSCFAsfromcecumtoterminalileumpassingthroughtheileocecalvalvemayinduceafluidsecretioninthehumanterminalileum.NoCOI.


2P-133Desacetyl bisacodyl induced ion transport in rat colon mucosaFujita, Takuya1,2; Karaki, Shin-ichiro1; Tateoka, Takashi2; Kuwahara, Atsukazu1(1Laboratory of Physiology, Graduate School of Nutritional and Environmental sciences/Institute for Environmental Sciences, University of Shizuoka, 2Mitsubishi Tanabe Pharma Corporation, Safety Research Laboratories)

Bisacodyl,4,4%-(2-pyridylmethylene)-bis-(phenylacetate),isastimulantlaxativeusedforthetreatmentofconstipation.Bisacodylshowstoinhibitintestinalwaterabsorptionandtoinducenetwatersecretioninrats. Desacetylbisacodyl is theactivemetaboliteofbisacodyl.Previousreportssuggestthatthelaxativeactionofbisacodylisiniti-atedthroughadirectinteractionofluminalsideofthecolon.Howev-er,themechanismofactionofbisacodylhasnotyetbeenelucidated.Aimofthepresentstudyistoinvestigatethemechanismofthede-sacetylbisacodyl inducedsecretion. Methods:Mucosa-submucosaltissuepreparationofratcolonandrectumweremadeofrattissues.Short-circuitcurrent(Isc)andtissueconductance(Gt)weremeasuredasindicesoftransepithelialelectrogeniciontransportandpermeabil-itybyUssingchamber.Results:Mucosaladditionofdesacetylbiscodyl(10-6–10-6M)concentration-dependentlyinducedatransientdecreaseandsubsequentincreaseinIIscinratdistalcolonandrectum.Neuralblockadebytetrodotoxin(10--6M)didnotaffect,butinhibitionofpros-taglandinbythepretreatmentofpiroxicam-5almostcompletelyre-ducedthedesacetylbisacodyl-inducedIIsc increase. Theseresultssuggestthatthesecretoryeffectofdesacetylbisacodyl ispossiblyinteraction inapicalsideofdistalcolonandrectum.COIproperlydeclared.

2P-134Effect of electrical acupuncture on colonic transit measured by a new method using the radiopaque marker in conscious rats.Taniguchi, Sazu1; Kawakami, Koichi1; Taniguchi, Hiroshi2; Kitakoji, Hiroshi1; Imai, Kenji1(1Dept. of Clinical Acupuncture and Moxibustion Meiji Univ. of Integrative Medicine, Kyoto, Japan, 2Dept. of Basic Acupuncture and Moxibustion Meiji Univ. of Integrative Medicine, Kyoto, Japan)

Acupuncturehasbeenusedfortreatingfunctionalgastrointestinal(GI)disorders,includingirritablebowelsyndrome,constipationanddiar-rhea.However,theprecisemechanismofacupunctureonGIfunctionremainsunclear.Weexaminedtheinfluenceofacuterestraintstress(RS)andelectricalacupuncture(EA)oncolonictransit(CT)ofconsciousratsbythemethodusingtheradiopaquemarker.MaleSprague-Daw-leyratswereused.Underpentobarbitalsodiumanesthesia,anin-dwell-ingsilasticcannulawasinsertedintothecaecumandpositionedtoentertheproximalcolon.Fivedaysafterthesurgery,ratswereex-posedtoRSfor90min.TheratstreatedwithEA(10Hz,3V,0.5msec)atST-36,whichlocatedontibialisanteriormuscle,for20minbeforestressloading.Twentymetalradiopaquemarkers,whosediameteris1.5mm,wereadministratedintotheproximalcolonwithsaline.ItwasvisiblethroughouttheGItractviasoftX-rayevery30minuntil120min.CTwassignificantlyincreasedbyRS.Furthermore,nearlyall-ra-diopaquemarkerspassedoutofthecolonuntil60minduringRS.Incontrast,CTwasdelayedbyEAregardlessofstressloading,andtheentireradiopaquemarkersremainedat120mininthecolon.TheseresultsraisethepossibilitythatEAatST-36mayinhibitstressin-duced-acceleratedCT.EAmayimproveonGIdisorders,likeirritablebowelsyndrome.NoCOI.

2P-135Colonic transit measured by a new method using the radiopaque marker in conscious rats.Imai, Kenji1; Imai, Kenji1; Taniguchi, Sazu1; Kawakami, Koichi1; Taniguchi, Hiroshi2; Kitakoji, Hiroshi1(1Dept. of Clinical Acupuncture and Moxibustion Meiji Univ. of Integrative Medicine, Kyoto, Japan , 2Dept. of Basic Acupuncture and Moxibustion Meiji Univ. of Integrative Medicine, Kyoto, Japan)

Colonictransit (CT),whichmeasuredbybeadexpulsiontime, thenumberoffecalpelletoutputorcalculatingthegeometriccenter,isnotproperforevaluatingchronologically.Ontheotherhand,throughthereareindividualdifferencesineffectsofacupunctureandrespons-esofdrugs,itisimportantthatCTrepeatedlymeasuresonindividu-al.Therefore,wedevelopedanewmethodusingtheradiopaquemarkerfortime-courseanalysisofCTofrats.MaleSprague-Dawleyratswereused.Underpentobarbitalsodiumanesthesia,anin-dwellingsilasticcannulawasinsertedintothecaecumandpositionedtoentertheproximalcolon.Fivedaysafterthesurgery,20metalradiopaquemarkers,whosediameteris1.5mm,wereadministratedintotheprox-imalcolonwithsaline(1.0ml).ItwasvisiblethroughouttheGItractviasoftX-rayevery30minuntil120minat2consecutivedays.CTwascalculatedbymaximummigrationlengthoftheradiopaquemark-er.Onthe1stday,maximummigrationlengthwas32.6±15.6mmat60minand62.4±20.4mmat120min.Thenextday,itwas24.8±20.4mmand51.2±31.4mm,respectively.Therewerenotsignificantlydifferencesofmaximummigrationlengthbetween1stand2ndday.Theseresultsshowedthatthenewmethodusingtheradiopaquemarkerwasstableandreliably.ThismethodcanmeasureCTbychronologicallyandmaybeusefultoelucidatethemechanismofco-lonicdisorders,likeIBS.NoCOI.

2P-136Curcumin reduces colonic fermentation after the in-gestion of milkShimouchi, Akito1,2; Nose, Kazutoshi1; Yamaguchi, Makoto2; Taniguchi, Ketaro1; Mizukami, Tomoe1; Jinno, Naoya1; Shirai, Mikyasu1; Ishiguro, Hiroshi2; Kondo, Takaharu3(1Department of Cardiac Physiology, National Cerebral and Cardiovascular Research Center, 2Department of Health and Nutrition, Nagoya University, 3Department of Health Science, Chubu University)

Curcuminisoneofthemainconstituentsofturmeric.Wepreviouslyreportedthatdietarytumericactivatedbowelmotilityandcarbohy-dratecolonicfermentation.NinetypercentsofJapaneseadultaremilkintolerance.Theingestionofmilkcausesmarkedincreaseinbreathhydrogen(H2)bythecolonicfermentation.Thus,wewonderedhowcurcuminaffect thecolonic fermentationafterthemilk ingestion.Subjectsfastedfor12handingestedmilkwithorwithoutcurcumin.BreathH2concentrationswereanalyzedevery15minfor6hbygaschromatographywithasemiconductordetector. IngestionofmilkwithoutcurcuminshowedadelayedandsustainedincreaseofbreathH2insubjectswithmilkintoleranceforupto9hours,whereasinges-tionofmilkwithcurcuminsignificantlydecreasedtheriseinbreathH2inadose-dependentmanner.Weconcludethatcurcumindecreas-esthecolonicfermentationcausedbytheingestionofmilk.NoCOI.


2P-137Anti-fibrotic effects of Daikenchuto (TU-100) associat-ed with intestinal myofibroblast TRPA1 channelSumiyoshi, Miho1; Kurahara, Lin Hai1; Hung, Hsichin1; Aoyagi, Kunihiko2; Inoue, Ryuji1(1Dept. Physiol., Fukuoka Univ. Sch. Med., Fukuoka, Japan, 2Dept. Gastroenterol., Fukuoka Univ. Sch. Med., Fukuoka, Japan)

Daikenchuto(TU-100),atraditionalorientalherbalmedicineusedforpost-operativeileusandconstipation.TU-100,composedofZingiberis Rhizoma,Ginseng RadixandZanthoxyli Fructus,generallyacceleratesgastrointestinalmotility,andincreasesintestinalbloodflowandgas-trointestinalhormonesecretion.However,themechanismofTU-100’sactionsremainsunclear.Intestinalmyofibroblastsplayanimportantrole infibroticstenosis.Previously,wereportedpathophisiological involvementoftransientreceptorpotential(TRP)channelsincolonicmyofibroblasts.Micro-ar-rayassaywithanintestinalmyofibroblastcellline(InMyoFib)detect-edTRPA1athighestexpressionamongTRPfamily.TheTRPA1agonistAITCandanactiveingredientofTU-100[6]-shogaolrespec-tively inducedCa2+ influx inInMyoFib,whichwasantagonizedbyco-treatmentwithaselectiveTRPA1channelblockerHC-030031.Importantly,24-hourincubationwithTU-100100µg/mlenhancedthemRNAandproteinexpressionsofTRPA1inInMyoFibs.WenextexploredapotentialroleofTU-100onfibrosissignaltrans-ductionandcollagensynthesisassociatedwithTGF-β1.TU-100 in-creasedTypeICollagenexpressionandthephosphorylationofSmad2andp38-MAPKatthedownstreamofTGFreceptors.TheseresultssuggestthatTU-100-mediatedmyofibroblasticTRPA1expressionandCa2+influxcouldbeanimportantprocesssuppressingintestinalfibrogenesis,themechanismforwhichmayaccountforthepharmacologicalactionsofTU-100.NoCOI.

2P-138Regional difference in blood flow response to cold pressor testMiyaji, Akane1,2; Ikemura, Tsukasa1,2; Hamada, Yuka1,2; Hayashi, Naoyuki2(1Graduate school of human-environment studies, Kyushu university, 2Graduate school of decision science and technology, Tokyo institute of university)

Toinvestigatewhethervascularresponsestoincreaseinbloodpres-suredifferamongvariousbloodvessels,werecordedrelativechangesofmeanarterialpressure(MAP)andbloodflowvaluesinthemiddlecerebralartery(MCA),retinalarterial(RA),andchoroidalvessels(CV)duringcoldpressortest(CPT)in24healthymales(24.1±0.2yrs).TheCPTwasinducedbyplacinghandincoldwater(5~10°C)for2min.MAPsignificantlyincreasedfromthebaselineby18±2%(P<0.05).BloodflowvaluesinMCA,RA,andCVsignificantlyincreasedby4±2%,9±2%,and16±3%,respectively(P<0.05).Conductanceindex(CI),whichwascalculatedaseachbloodflowvaluedividedbyMAP,inMCAandRAsignificantlydecreasedby10±3%and6±2%,respec-tively(P<0.05).TherewasasignificantcorrelationinrelativechangesofbloodflowvaluesbetweenRAandCV(r=0.55),whilenocorrelationwasobtainedamongothervessels.Therewerenocorrelations inrelativechangesofCIamonganybloodvessels.ItwassuggestedthatvascularresponsestoincreaseinbloodpressureduringCPTdifferamongvessels.NoCOI.

Poster PresentationsEndocrinology

2P-139Morphological and histological properties of MCH neurons in ES cell-derived hypothalamic cultureKodani, Yu1; Nagasaki, Hiroshi1,2; Suga, Hidetaka2,3; Wataya, Takafumi3; Nakashima, Akira1; Kaneko, Yoko S1; Oiso, Yutaka2; Sasai, Yoshiki3; Ota, Akira1(1Dept. Physiol., Fujita Health Univ. Sch. Med., Toyoake, Japan, 2Dept. Endocrinol. & Diabetes, Nagoya Univ., Grad. Sch. Med., Nagoya, Japan, 3RIKEN CDB, Kobe, Japan)

Mouseembryonicstemcell-derivedhypothalamicculture(ES-hypo)isreportedtogeneratepeptidergicneuronsproducingvasopressin,neuropeptideY,oragouti-relatedprotein(Watayaetal.,2008,PNAS).However, itremainsunclearwhetherES-hypois identicalwiththenativehypothalamuswhereavarietyofpeptidergicneuronsareworkinginharmony.Thepresentstudyfocusedonthedevelopmentofmelanin-concentratinghormone(MCH)neuronsinES-hypo.MCHisahypothalamicneuropeptidecontrollingfeedingbehavioranden-ergyhandling.WefoundthatMCH-positiveneurons increased innumberandmaturityinES-hypoduring3rd-5thweeksofinduction,whichissimilartodevelopmentalneurogenesis.TheseMCHneuronsshowedhistologicalcharacterscommontonativeonesin vivo:co-ex-pressionofMCHwithGABAorcocaine-andamphetamine-regulatedtranscript(CART),andreciprocalsynapticconnectionswithorexin-pos-itiveortyrosinehydroxylase-positive (i.e.,dopaminergic)neurons.Moreover,theMCHneuronsexhibitedvariousmorphologiescorre-spondingtothosefoundinthedevelopingstages.Insummary,wehaveidentifiedMCHneuronsco-existwithotherpeptidergicneuronsinES-hypo,andalsotheysharemanycharacterswithnativeMCHneurons.ItissuggestedthatES-hypoprovidesanewexperimentalsystemto investigatethedevelopmentandneurophysiologyofthehypothalamicMCHsystem.NoCOI.


2P-140Adiponectin regulates proopiomelanocortin and neruopeptide Y neurons in the hypothalamic arcuate nucleusSuyama, Shigetomo1; Maekawa, Fumihiko2; Maejima, Yuko1; Kubota, Naoto3; Kadowaki, Takashi3; Yada, Toshihiko1(1Department of Physiology, Jichi Medical University, Tochigi, Japan, 2Center for Environmental Health Sciences, National Institute for Environmental Studies, Tsukuba, Japan, 3Dept. Metabolic Diseases, Graduate Sch. Med, Univ. Tokyo, Tokyo, Japan)

Aim:Adiponectinfacilitatesinsulinsensitivityandregulatesenergyexpenditureintheperipheralorgans,Ithasalsobeensuggestedthatserumadiponectinsecretedfromadipocytesentersandexertseffectsinthebrain.Adiponectinreceptorsareexpressedinseveralregionsofthebrainincludingthehypothalamicparaventricularnucleusandarcuatenucleus (ARC), thecentersregulatingfeedingandenergyexpenditure.Theaimofthisstudywastodeterminetheeffectofadiponectinontheactivityofproopiomelanocortin(POMC)neuronsandneuropeptideY(NPY)neuronsintheARC.Methods:Slicescon-tainingARCwereproducedfromthehypothalamusofPOMC-GFPorNPY-GFPmice.TheeffectsofadiponectinonthemembranepotentialandfiringinPOMCneuronsandNPYneuronsinsliceswererecord-edundercurrentclampmode.Foodintakewasmeasuredfrommicewhichreceivedintracerebroventricularinjectionof150ngadiponectinjustbeforedarkperiod.Results:Bathapplicationofadiponectin(100ng/ml)depolarizedandincreasedfiringinPOMCneuronswhereasitdecreasedfiringinARCNPYneuron.Foodintakewassuppressedbyintracerebroventricularadiponectininjection.Conclusion:AdiponectinactivatesPOMCneuronsandinhibitsNPYneuronsinARC.Theseeffectsmayberelayedtosuppressionoffoodintakebyadiponectin.NoCOI.

2P-141The expression of the oxytocin-monomeric red fluo-rescent protein 1 fusion gene in the hypothalamus and spinal cord of adjuvant-induced arthritic ratsMatsuura, Takanori1; Yoshimura, Mitsuhiro1; Ohkubo, Junichi1; Maruyama, Takashi1; Hashimoto, Hirofumi1; Ishikura, Toru2; Kawasaki, Makoto2; Ohnishi, Hideo2; Ueta, Yoichi1(1Department of Physiology, School of Medicine, University of Occupational and Environmental Health,Kitakyushu,Japan , 2Department of Orthopaedics, U.O.E.H, Japan)

Oxytocin (OXT) isawell-knownneurohypophysialhormonethat issynthesizedintheparaventricular(PVN)andthesupraopticnuclei(SON).SeverallinesofevidencehavesuggestedthatOXTplaysanimportantrole inpainmodulationandanalgesia.However, little isknownabouttheneuronalspinalnetworksresponsibleforOXTeffects.Thepresentstudyexaminedtheeffectsofchronicinflammatory/no-ciceptivestressontheexpressionoftheOXT-monomericredfluores-centprotein1(mRFP1)fusiongeneinthehypothalamusandspinalcord,usingtheadjuvantarthritis (AA)ratmodel.To induceAA,OXT-mRFP1transgenicratswereintracutaneouslyinjectedheat-killedMycobacteriumbutyricum(1mg/rat)inparaffinliquidatthebaseoftheirtails.WeobservedmRFP1fluorescenceinthePVN,theSON,andthedorsalhorninthespainalcordwhenAAwasestablished.TheexpressionofthemRFP1,andtheOXTgenesinthehypothalamuswerealsomeasuredbyin situhybridizationhistochemistry.OXTandmRFP1mRNAlevelsinthePVNandtheSONweresignificantlyin-creasedonday22inAArats.ThemRFP1fluorescencesintheSON,thePVN,andthedorsalhornwereapparentlyincreasedondays15and22inAAratscomparedwithcontrols.TheseresultssuggestthatOXTmayplayanroleinpainmodulationandanalgesiainAArats.NoCOI.

2P-142Roles of secretin-oxytocin system in the control of so-cial recognitionTakayanagi, Yuki; Onaka, Tatsushi(Division of Brain and Neurophysiology, Department of Physiology, Jichi Medical University, Tochigi, Japan)

Ithasbeenknownthatoxytocinreceptorsareimplicatedinautism.Furthermore,mice lackingtheoxytocinoroxytocinreceptorgeneshowautisticphenotypes,suchassocio-behavioraldeficitsincludingsocialrecognitiondeficits.Ontheotherhand,secretin,agastrointes-tinalhormone,hasbeenreportedtobeapossibletreatmentforautism.Micelackingthesecretinorsecretinreceptorgenealsoshowsocio-be-havioraldeficits.Secretinadministrationfacilitatesoxytocinrelease.However,relationshipbetweenoxytocinandsecretinconcerningsocialbehaviorremainstobedetermined.Wehavedemonstratedthatsecretinactivatesoxytocinneuronsofthesupraopticnucleusandfacilitatesoxytocinrelease.Inthepresentstudy,weinvestigatedwhethersecretinfacilitatessocialrecognitionviaac-tivationofoxytocinreceptor.Anintracerebroventricularinjectionofsecretinincreasedthecapabilityofsocialrecognitioninrats,andthefacilitativeeffectofsecretinwasblockedbyanoxytocinreceptoran-tagonist.Furthermore,localapplicationofsecretinintothesupraopticnucleusfacilitatedsocialrecognitionanditsactionwasblockedbyanoxytocinreceptorantagonistinjectedintothemedialamygdala.Alltheseresultssuggestthatsecretinactivatesoxytocinneuronsinthesupraopticnucleus,potentiatesdendriticoxytocinrelease,andfacilitatessocialrecognitionviatheoxytocin/oxytocinreceptorsystem.NoCOI.

2P-143Paired-pulse facilitation in parallel fiber-Purkinje syn-apses were decreased in congenital hypothyroid mice.Amano, Izuki; Takatsuru, Yusuke; Toya, Syutaro; Haijima, Asahi; Koibuchi, Noriyuki(Department of Integrative Physiology, Gunma University Graduate School of Medicine, Gunma, Japan)

Thyroidhormone(TH)playsacriticalroleondevelopmentofbrain.DeficiencyofTHduringdevelopmentinducestheorganizationalchang-esofthecerebellum,causingcerebellarataxia.However,underlingmechanismsofsuchabnormaldevelopmentarepoorlyunderstood.ToclarifytheeffectofTHonthedevelopmentofneuronalcircuit,weusedthedualoxidasematurationfactor (DUOXA)mutationmice,whichshowcongenitalhypothyroidism.Aspreviouslyreported,pro-liferationandmigrationofgranulecellsweredelayedafterP15 inhomozygousmice (Duoxa-/-)usingCresylvioletstaining.However,organizationalchangesofthegranulecellinDuixa-/-micedisappearedbyP25.ThebranchingofdendriteandthesizeofsomainthePurkin-jecellwerenotsignificantlydifferentbetweenDuixa-/-andcontrolmice.Althoughthestructureofthecerebellumwasnotdifferentbe-tweenDuixa-/-andwildtypeatP25,cerebellarataxiawasdetectedinDuixa-/-mice.Toexaminethemechanism,wenextperformedtheneurophysiologicalstudy.Wefoundthatpaired-pulsefacilitation,whichiscommonlydetected inparallelfiber-Purkinjecellsynapses,weredecreasedinDuixa-/-miceespeciallyatP15withouttheobviouschangeofexpressionlevelsofprotein,whichregulateneurotransmitterrelease.Takentogether,weconcludethattheanatomicalcatch-upgrowthofcerebellumcannotnormalizeitsfunctionbecauseofthefunctionofneuronalcircuititselfwascontinuouslyaffectedbyhypothyroidism.NoCOI.


2P-144Comparison of the effect of current therapeutic agents for diabetes in Cdkal1-deficient miceWatanabe, Sayaka; Wei, Fan-Yan; kaitsuka, taku; tomizawa, kazuhito(Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan)

Geneticvariations intheCdk5regulatorassociatedprotein1-like1(CDKAL1)genehavebeenassociatedwithtype2diabetes (T2D).Cdkal1catalyzes2-methylthiomodificationatposition37adenosineincytosolictRNALys(UUU).The2-methylthiomodificationiscriticalfortheaccuratedecodingof lysinecodon.Deficiencyof2-methylthiomodificationinpancreaticbeta-cell-specificcdkal1-deficient(KO)miceshowaberrantproinsulintranslation,andresultedinimpairedinsulinsecretionandglucoseintolerance.GiventhesimilarityofsymptomsbetweenCdkal1KOmiceandT2DpatientscarryingriskCDKAL1variation,weutilizedCdkal1KOmiceasT2Dmodeltoinvestigatethelong-termeffectsofanti-diabeticdrugs.WetreatedCdkal1KOmciewithglibenclamide,glucagonlikepeptide1(GLP1)agonistsincludingexendin-4andliraglutide,andsitagliptin,aninhibitorofGLP-1protease(DDP-4)upto8weeks.Glucosemetabolismandgeneexpressionweretheninvestigated.Long-termapplicationofglibenclamideimpairedtheglucosetoleranceandinsulinsecretioninKOmice.Incontrast,GLP-1agonistsandsitagliptinsignificantlyimprovedtheglucosetoleranceand insulinsecretion.Examinationofgeneexpressionshowedthatbothliraglutideandsitagliptin,butnotglibenclamide,decreasedtheexpressionofunfoldedproteinresponse-relatedgenesaswellasL-Myc,adedifferentiation-relatedgene.OurresultssuggestthattheGLP-1relateddrugswillbebeneficialforT2DpatientscarryingriskCdkal1variation.

2P-145Suppression of AMPK activity in skeletal muscle im-proves metabolic abnormalities of streptozotocin-in-duced diabetes mellitus in mice.Yokota, Shigefumi; Okamoto, Shiki; Minokoshi, Yasuhiko(Div Endocrinol Metab, Natl Inst Physiol Sci)

AMPK(AMP-activatedproteinkinase)isactivatedbydecreasedin-tracellularenergylevel.Herewefoundthatstrepotozotocin(STZ)-in-duceddiabetesactivatesAMPKanditssignalingpathwaysinskeletalmuscleinmice.Surprisingly,inhibitionofAMPKactivityinskeletalmuscle inSTZ-induceddiabetesbypreferentiallyexpressingdomi-nant-negativeAMPK(DN-AMPK)significantlyimprovedSTZ-inducedhyperglycemia,andhighlevelofplasmafreefattyacidsandketonebodies,althoughplasmainsulinlevelwaslow.Moreover,STZ-treatedDN-AMPKmiceimprovedatrophyofwhiteadiposetissue(WAT)andskeletalmuscle,bodyweightlossandincreasedsurvivalrate.Inpar-allelwiththeWATmass,STZ-treatedDN-AMPKrecoveredandin-creasedplasmalevelofleptinandadiponectin,respectively.IL-6(Interleukin-6)andanovelmyokine,irisin,stimulatelipolysisandfatutilizationinWAT.STZ-induceddiabetesincreasedbothIL-6andirisnlevelsinskeletalmuscleandplasma,andthosearereturnedtothecontrol levels inDN-AMPKmice. Infusionof leptinorneutralantibodyforIL-6byosmoticminipumpimprovedthemetabolicchang-esinSTZ-induceddiabetes,similartothoseinSTZ-treatedDN-AMPKmice.Furthermore,chronicinfusionofAMPKinhibitor,compoundC,also improvedthemetabolicabnormities inSTZ-induceddiabetes.TheseresultsthusunveilakeyroleformuscleAMPKinmetabolicabnormities inSTZ-induceddiabetes,and importantroleoforgannetworksinthemetabolicregulation.NoCOI.

2P-146Alteration of thyroid hormone signaling by treadmill training with different intensity in male rat skeletal muscle.Iwasaki, Toshiharu; Lemana, Ronny; Shimokawa, Noriaki; Koibuchi, Noriyuki(Dept Integrative Physiology, Gunma Univ Grad Sch Med, Gunma, Japan)

Thyroidhormone(TH)controlsmetabolicactivityinawiderangeoftissue.Aerobicexercisetrainingfacilitatesoxidativephosphorylationandglycolysisofskeletalmuscle.Thus,westudied ifTHsignalingpathwayisactivatedbyaseveralintensityoftraining.Maleadultratsreceived30min/daytreadmilltrainingwithdifferentexerciseinten-sityfor12days.Bylactatelevels,ratsweredividedintostationarycontrol(SC,0m/min),aerobic(A,15m/min,for30min)andanaerobic(AN,25m/min,30min)traininggroups.WehavepreviouslyreportedthatTSHlevelwassuppressedinbothAandANgroupsduringtheexercise.TRβ1mRNAandproteinlevelswereincreasedinAgroup,butnotinANgroup.Inthepresentstudy,weexaminedwhetherthesensitivitytotriiodothyronine(T3)wasalteredinseveralTH-targetgenes includingMyoD,andetc.Twenty-fourhoursafterthe lasttraining,T3was injected intravenously.Sixhours later,ratsweresacrificedandthesoleusmusclewasdissectedouttoextracttotalRNA.Then,semiquantitativeRT-PCRwascarriedout.Particularly,inductionofNa+K+-ATPaseβ1expressionbyT3wassignificantlyaugmentedinAgroup,butnotinANgroup.Chromatinimmunopre-cipitationassayinL6myoblast-derivedclonalcellsshowedthatTRβ1boundtothenucleotidesequencethatissimilartotypicalTHresponseelementinpromoterregionofNa+K+-ATPaseβ1gene.TheseresultsindicatethataerobictrainingaltersTHsignalingatleastinpartbyincreasingTRβ1expression.NoCOI.

2P-147Distribution of TRPC channels expression and their role in catecholamine secretion of adrenal medullary cells.Harada, Keita; Matsuoka, Hidetada; Inoue, Masumi(Department of Cell and System Physiology, University of Occupational and Environmental Health School of Medicine)

Adrenalmedullary(AM)cellssecretecatecholaminesinresponsetoAChreleasingfromthesplanchnicnerveterminal.ThisACh-mediat-edsignalisthoughttobereceivedmainlybynicotinicAChreceptors,andtheroleofmuscarinicAchreceptorsintheneurotransmissioniscontroversialalthoughmuscarinicreceptorshavebiochemicallyand/orfunctionallybeendetectedinseveralspeciesincludingguinea-pig,ratandhumanAMcells.WehavepreviouslydemonstratedthataninhibitionofTASK1channelwasresponsibleformuscarinicactivationinratAMcells.WhereasanactivationofnonselectivecationchannelsaswellasaninhibitionofTASK1channelswasinvolvedinguinea-picAMcells.TheelectrophysiologicalandpharmacologicalstudiessuggestTRPCchannelsareacandidate.Infact,immunoblottingandimmuno-cytochemistryrevealedexpressionofTRPCs1,4,5,and7inguinea-pigAMcells;TRPCs1and4weremainlylocalizedinthecytoplasmintherestingcellswhereasTRPC5wasinand/orneartheplasmamembrane.Inthisstudy,weexaminedwhetherornotthelocalizationofTRPCsinguinea-pigAMcellsischangeduponthemuscarinicstimulationandTRPC1channelsarecoupledwithTRPC4orTRPC5channels.WealsoutilizedPC12cellstoinvestigatedetailedmechanismsofthetraffickingofTRPCchannelsinthemuscarinicstimulation.NoCOI.


2P-148Ghrelin attenuates incretin effect of GLP-1 in ratsDezaki, Katsuya; Rita, Rauza Sukma; Boldbaatar, Damdindorj; Yada, Toshihiko(Department of Physiology, Jichi Medical University, Tochigi, Japan)

Ghrelin,anacylated28-aminoacidpeptide,reportedlyrestrictsinsulinreleaseinisletβ-cellsandtherebyregulatesglucosehomeostasis.WehavereportedthatghrelinattenuatescAMPsignaling,therebyinhib-itingglucose-inducedincreasesincytosolicCa2+concentration([Ca2+]i)andinsulinreleaseinisletβ-cells.Thisstudyaimedtoclarifywhetherghrelincounteractstheeffectsofglucagon-likepeptide-1 (GLP-1),aphysiologicalincretinhormone,oninsulinrelease,cAMPand[Ca2+]isignalinginisletβ-cells.Inratisolatedisletsunderstaticincubation,glucose (8.3mM)-induced insulinreleasewaspotentiatedbyGLP-1,andthispotentiationwassuppressedbyghrelin.Theglucose (8.3mM)-inducedcAMPproductioninisletswasenhancedbyGLP-1,andthisenhancementwasblockedbyghrelin.Inthepresenceof8.3mMglucose,GLP-1evoked[Ca2+]iincreasesinsingleβ-cellsandtheyweresignificantlyattenuatedbyghrelin.Theresultsindicatethatghrelinattenuatesglucose-dependentinsulinotropicactionsofGLP-1bysup-pressingcAMP-mediated [Ca2+]i signaling in isletβ-cells.Wenextperformedinvivostudiesinrats.InoralglucosetolerancetestsinwhichGLP-1isreleasedandexertsitsincretineffect,intraperitonealadministrationofghrelinmarkedlyattenuatedtheincreaseinplasmainsulinandenhancedthe increase inbloodglucose.ThesefindingsindicatethatghrelinattenuatesincretineffectofGLP-1andthatinter-actionbetweenghrelinandGLP-1playsanimportantroleinphysio-logicalregulationofglucose-inducedinsulinreleaseinisletβ-cells.NoCOI.

2P-149Effects of Ghrelin on tuberomammillary nucleus neu-ronsWakabayashi, Yuji; Kim, Juhyon; Nakajima, Kazuki(Division of Bio-Information Engineering, Faculty of Engineering, University of Toyama, Toyama, Japan.)

Ghrelin,knownasstimulatorofreleaseofgrowthhormone(GH)andfoodintake,isproducedbystomach,otherperipheralorgansandsomebrainregionsandactsonGHsecretagoguereceptors(GHS-Rs)inthesomeperipheralorgansandbrainregions.Recentstudyalsosuggeststhatghrelinparticipatesinregulationofsleep-wakefulness.Tubero-mammillarynucleus(TMN),whichinvolveshistaminergicneuronsthatcontributetomaintainarousalstate,alsoexpressesGHS-Rs.However,directactionofghrelinonTMNneuronsisremainedunclear.Thus,weexaminedelectrophysiologicaleffectsofghrelinonTMNneuronsusingratbrainslicepreparationsandwhole-cellpatchclamprecordingtechnique.ApplicationofghrelindepolarizedTMNneuronsinbothabsenceandpresenceoftetrodotoxin,andthedepolarizationwasin-hibitedbyantagonistforGHS-Rs.Theghrelin-induceddepolarizationwasaccompaniedbyincreaseofmembraneresistance,anddecreasedunderhigh-K+and/orCa2+freeextracellularsolution.TheseresultssuggestthatghrelindepolarizesTMNneuronspostsynapticallyviaGHS-RswithadualionicmechanismincludingadecreaseinK+con-ductanceandanincreaseofCa2+conductance,andmayparticipateintheregulationofsleep-wakefulnessviatheexcitatoryeffectonTMNneurons.NoCOI.

2P-150Mifepristone upregulated adiponectin secretion during 3T3-L1 adipogenesis.Hashimoto, Takeshi; Igarashi, Junsuke; Yamashita, Tetsuo; Kosaka, Hiroaki(Department of Cardiovascular Physiology, Faculty of Medicine, Kagawa University, Japan)

Mifepristone,aputativesteroidreceptorantagonist,clinicallyservesasananti-canceragent,elicitingbothcytostaticandcytotoxiceffectsonmalignantcells.However,themetaboliceffectsoflong-termtreat-mentwithmifepristonehaveremainedunclear.Hereweshowthatmifepristonealsopromotestheabilityofculturedmouse3T3-L1cellsundergoadipogenesiselicitedinducedbyco-treatmentwithDexameth-asone (Dex),1-methyl3-isobutylxanthine (IBMX),andInsulin (Ins).Specifically,weobserved increases incells treatedwithmifepris-tone-Dex-IBMX-InswhencomparedtothosewithDex-IBMX-Insalonein1)theproteinandgeneexpressionlevelsofadipocytemarkeraP2,theadipocyte fattyacidbindingprotein,2) the lipidaccumulation(Bodipy493/503staining),and3)theadiponectinsecretionlevelsintoculturemedium.Intriguingly,mifepristone-inducedgeneexpressionofaP2andadiponectinwasmarkedlyattenuatedbyneutralizingan-tibodytoadiponectin(ANOC9104).Becauseadiponectin,anantidiabet-ichormone,isabletoenhanceadipogenesisinanautocrine/paracrinefashion,weproposethatmifepristonemaystimulatedifferentiationofpreadipocytesintoadipocytespossiblybymodulatingtheexpressionandsecretionlevelsofadiponectin.Theseresultsthereforeidentifyapreviouslyunknownpharmacologicalactionofmifepristoneonfatcellmetabolism.NoCOI.

Poster PresentationsCell Physiology, Molecular Physiology

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2P-151Re-evaluation of regulatory volume decrease of sever-al cell lines by flow cytometric measurements.Hazama, Akihiro; Kobayashi, Daisuke; Otsuki, Lucia(Dept.Cellular & Integrative Physiology, Fukushima Medical University School of Medicine)

Themechanismofregulatoryvolumedecrease(RVD)afterhypotonicstresshasbeenwidelyinvestigated.Theindependentactivationsofvolumeregulatorychloridechannelandpotassiumchannelarecon-sideredtoinducetheKCleffluxfromthecelltogetherwithwater,causingcellvolumedecrease.Althoughthisconceptiswidelyaccept-ed,thespeedofregulatoryvolumedecreasediffersbycelltypes.Inaddition,initialswellingafterthehypotonicstressdiffersmongcellspecies.Tore-evaluatethemechanismofRVD,wemeasuredinitialcellswellingafterhypotonicstressandvolumechangeinHeLacells,HEK293cells,andPC12cells.WeobservedthattheRVDprocessesofHEK293cellsandPC12cellsareslowercomperedwithHeLacells.InitialcellswellingofPC12cellsareslowerthanHeLacellsandHEK293cells.Theseinitialswellingprocessmayrepresentstheexpressionlevelofaquaporinsofplasmamembranewhich increasethewaterpermeabilityofcellmembrane.Furtherstudieswouldbenecessarytoprovethispossibility.NoCOI.

2P-152Cytotoxic anions damaged HeLa cells via volume reg-ulatory chloride channelItagaki, Yuya; Koiwai, Megumi; Eka, Prasedia Sunarwidi; Kobayashi, Daikuke; Hazama, Akihiro(Dept.Cellular & Integrative Physiology, Fukushima Medical University School of Medicine)

Specialanionsdamagesthecellswithunknownmechanism.Wecallthoseanionscytotoxicanions,includingVO4

3-,CrO42-,SeO3

2-,WO42-,etc.

Weexaminedthecytotoxiceffectsoftheseanions.SeO32-wasfound

tohavemostcytotoxiceffectstoHeLacellswithmicro-molarconcen-tration.Thechloridechannelinhibitors,DIDSorNPPB,blockedtheeffectofcytotoxicanions.Whensuchanionswereappliedjustafterhypotonicchallenge,thecytotoxiceffectsweremarkedlyincreased.TheseresultssuggestthatcytotoxicanionsdamagedHeLacellsviavolumeregulatorychloridechannels.Themechanismofcelldamagebytheseanionsafterentranceintothecellsshouldbefurtherexamined.NoCOI.

2P-153AVP neurons possess the cell volume regulation mechanism under hyperosmotic conditionsSato-Numata, Kaori1; Ueta, Yoichi2; Okada, Yasunobu1(1Department of Cell Physiology, National Institute for Physiological Sciences, Okazaki, Japan, 2Department of Physiology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan)

Itisknownthatmanytypesofmammaliancellscanregulatetheircellvolumeaftertransientosmoticcellshrinkageundersustainedhyperosmoticconditionsbythemechanismcalledtheregulatoryvolumeincrease(RVI).Incontrast,itwasreportedthatbrainosmo-sensorymagnocellularneuronsisolatedfromratsupraopticnucleus(SON)cannotexhibitRVIduringhyperosmoticperturbation.Inagree-mentwiththisreport,inthepresentstudy,theRVIeventwasnotobservedinarginine-vasopressin(AVP)neuronsselectivelyisolatedfromtheSONofAVP-enhancedGFPtransgenicrats.Inthepresenceofflufenamicacid(FFA),however,asignificantRVIwasobservedinAVPneuronsundersustainedhyperosmoticstress.TheRVIresponseobservedwithFFAwascompletelyinhibitedbyastilbene-derivativeinhibitorofCl-/HCO3

-anionexchanger(AE),DIDS,oraninhibitorofNa+/H+exchanger(NHE),amiloride.Thus,itisconcludedthatbrainosmosensoryAVPneuronspossesstheRVIabilitywhichismaskedbysomeFFA-sensitivemechanismundernormalconditions.NoCOI.

2P-154Structural and functional analysis of TRPC6 for the interaction with PKG IβHonda, Akira; Inoue, Ryuji(The Department of Physiology, School of Medicine, Fukuoka University, Fukuoka, Japan)

cGMP/cGMP-dependentproteinkinase(PKG)signalingpathwayplaysanimportantroleinvasorelaxation.Inthispathway,PKGplaysakeyroleofloweringtheintracellularCa2+leveland/orCa2+sensitivityofcontractilemachinerybyproteinphosphorylation.However,thedown-streamofPKGisstillpoorlyelucidated.Recently,wereportedthatatransientreceptorpotential (TRP)non-voltage-gatedcationchannel,TRPC6,isanovelPKGsubstrateandnegativelyregulatedviapho-shorylationonitsthreonine69(Takahashiet al. J Physiol,2008).Fur-thermore,usingyeasttwo-hybridsystemtoidentifyresponsiblebind-ingdomainsinbothTRPC6andPKGtypeIproteins,werevealedthattwoPKGsubtypes,bothPKGIαandIβ,interactwithTRPC6N-ter-minaldomain(the87thannualmeetingofthePhysiologicalSocietyofJapan,2010),andthatonlyPKGIβshowedthecGMP-dependentin-teractionwithTRPC6N-terminaldomainandacidicaminoacids,lo-catedintheleucinezipperregion,areresponsibleforthatinteraction(the89thand90thannualmeetingofthePhysiologicalSocietyofJapan,2012and2013).Inthisstudy,todeterminewhichaminoacidsarere-sponsiblefortheinteractionbetweenthePKGIβandTRPC6,seriesofmutationsareintroducedtoTRPC6N-terminaldomain,andproteininteractionbetweenPKGandTRPC6wasexaminedbyyeasttwo-hy-bridassay.Onemutant(K115E)showedthelossofinteractionbetweenPKGIβandTRPC6.Thisresultindicatesthatbasicaminoacidlocat-edintheankyrin-likerepeatdomainofTRPC6N-terminalregionisresponsibleforinteractionwithPKGIβ.NoCOI.


2P-155diDCP-LA-PE stimulates GLUT4 translocation to the cell surfaceTsuchiya, Ayako; Kanno, Takeshi; Nishizaki, Tomoyuki(Division of Bioinformation, Department of Physiology, Hyogo College of Medicine, Hyogo, Japan)

ThepresentstudyinvestigatedtheeffectsofthenewlysynthesizedphospholipidderivativesdiDCP-LA-phosphatidylethanolamine (PE),-phosphatidylserine(PS),-phosphatidylcholine(PC),and-phosphatidy-linositol(PI),whichcontainthelinoleicacidderivativeDCP-LAattheαandβposition,onGLUT4trafficking.diDCP-LA-PE,diDCP-LA-PS,diDCP-LA-PC,anddiDCP-LA-PIactivatedmostofthe investigatedPKCisozymes,butdiDCP-LA-PEalonesignificantlyactivatedPKCλ/ιand -ζ. diDCP-LA-PE,diDCP-LA-PS,anddiDCP-LA-PIexhibitedastronginhibitiononPTP1Bactivity,whilediDCP-LA-PCenhancedtheactivity. diDCP-LA-PEstimulatedGLUT4translocationtothecellsurfaceindifferentiated3T3L1-GLUT4mycadipocytes,withthehigh-estpotentialamongthephospholipidderivatives,andtheeffectwaspreventedbyan inhibitorofPI3K,Akt1/2,orPKCoreachknock-ing-down.TheresultsofthepresentstudyshowthatdiDCP-LA-PEstimulatesGLUT4translocationtothecellsurfacestillintheabsenceofinsulinbyupregulatinginsulinsignalingalonganinsulinreceptor/insulinsubstrate1(IRS-1)/PI3K/Akt(1/2)axisasaresultofsuppress-ingtyrosinedephosphorylationofinsulinreceptorandIRS-1inasso-ciationwithPTP1BinhibitionandbyactivatingPKCλ/ιand-ζ.NoCOI.

2P-156Characteristics of exogenous expression of ROMK K channels with EGFP fused to the different terminus in polarized and non-polarized membranes in cultured M-1 cellsKubokawa, Manabu1; Nakamura, Kazuyoshi1; Mayanagi, Taira2; Sobue, Kenji2(1Department of Physiology, School of Medicine, Iwate Medical University, Yahaba, Japan, 2Institute for Biomedical Science, Iwate Medical University, Yahaba, Japan)

ROMKKchannels intheapicalmembraneofrenalcollectingductplaysakeyrole inpotassiumsecretiontotheurine. Inthisstudy,ROMK1Kchannelgene(KCN1)withEGFPfusedtoN-orC-terminuswasexogenouslytransfectedtoculturedM-1cells.Weusedtwotypesofcultureconditions.Onewassinglecells,andtheotherwasconfluentM-1cellsonmembraneinsetwithpolarizedapicalmembrane.AlthoughtherewasnoappreciabledifferenceinfluorescenceintensityandthelocalizationbetweenthecellswithROMK1fusedtoN-terminalEGFPandtoC-terminalEGFPinbothsingleandconfluentcells,frequencyofchannelcurrentobservationwassignificantlydifferent insinglecells.Namely,channelcurrentisobservedin71.4%ofthecellstrans-fectedROMK1withEGFPfusedtoN-terminus,andin12.2%ofthosefusedtoC-terminus.However,intheconfluentcells,channelcurrentwashighlyobservedintheapicalmembraneofthecellswithROMK1fusedtoeitherterminus(69.2%and66.7%ofEGFPfusedtoN-andC-terminus,respectively).SincethefusionofEGFPtothespecificterminusmaydisrupttheroleoftheterminalfunction,itissuggestedthatC-terminusofROMK1is important forexpressionofchannelactivityinsinglecells,whereasexpressionofchannelactivityinpo-larizedapicalmembraneofM-1cellsrequirednospecificterminalfunctionsofROMK1.NoCOI.

2P-157Calcium holes: a novel PMCA-mediated mechanism for calcium signalingShioya, Takao(Dept. Physiol. Fac. Med. Saga Univ. Saga, Japan)

Calciumion isaversatilesignalingmoleculethatregulatesvariouscellularfunctions.Accordingly,calciummediatesmultiplesignalingprocessesconcurrentlyinasinglecell,owingtoanunveiledcellularmachinerythatencapsulatesmanydifferentcalciumsignalingprocess-es. Here,Iproposetheconceptof“calciumholes”thatprovideanencapsulatednano-environmentforcalciumsignaling.AcalciumholeisaCa2+-deficientnanodomainhavingadiameterofabout100nmthatdevelopsaroundaplasmamembraneCa2+-ATPase(PMCA)molecule.Ca2+-extrusionbyPMCAmaintainsthelocalCa2+-levelinthecalciumholeslowerthanthegloballevel,providingencapsulatedintracellulardomainsthatareinsulatedfromeachother,andalsofromthebulkCa2+-environment.ExperimentalevidenceindicatesthatcardiacNa/Caexchanger(NCX)inwhole-cellclampedmouseheartcellsisregu-latedbycalciumholes.SelectiveinhibitionofPMCAenhancedtheNCXcurrentreflectinganelevatedlocalCa2+-level,withoutachangeinitsreversalpotentialreflectinganunchangedglobalCa2+-level.ThisindicatestheintracellularregulatoryCa2+-bindingdomain(CBD)oftheNCXsensesthelocalCa2+-levelinthecalciumhole,ofwhichthelocalCa2+-leveliselevatedbythePMCAinhibition.Numericalsimulationof localCa2+-diffusionpredictsthediameterofacalciumholetobeabout100nm.Theregulationofothercalciumsignalingmoleculesbycalciumholesandtheirrelationtospecialmembranestructuressuchaslipidraftsarealsodiscussed.NoCOI.

2P-158GPRC6A receptor is involved in amino acid-induced glucagon-like peptide-1 secretion from GLUTag cellsOya, Manami1; Kitaguchi, Tetsuya2; Tsuboi, Takashi1(1Dep. of Life Sci, Grad Sch. of Arts and Sci,Univ. of Tokyo, 2WABIOS, Waseda Univ, Singapore)

Glucagon-likepeptide-1(GLP-1)isapeptidehormonesecretedfromenteroendocrineLcellsinresponsetodietarynutrients.However,theprecisemolecularmechanismsbywhichdietarynutrientsregulateGLP-1secretionfromenteroendocrineLcellsremainsunclear.Inthepresentstudy,weusedenteroendocrineLcell lineGLUTagcells toclarifymolecularmechanismofGLP-1secretion.FirstweperformedRT-PCRanalysisandfoundthatGPRC6A,whichisapu-tativeL-aminoacids-sensingreceptor,isexpressedinGLUTagcells.Livecell imagingandELISAanalysisrevealedthatL-ornithine,apotentstimulusforGPRC6A,inducesGLP-1secretionfromGLUTagcellsinadose-dependentmanner.Toelucidatetheintracellularsig-nalingresponsibleforL-ornithine-inducedGLP-1secretionfromGLU-Tagcells,weexaminedtheeffectofaGPRC6Areceptorantagonist,aphospholipaseCinhibitorandaninositoltriphosphatereceptoran-tagonistonL-ornithine-inducedGLP-1secretion.ApplicationofthoseantagonistssignificantlysuppressedL-ornithine-inducedGLP-1secre-tionfromGLUTagcells.Furthermore,depletionofendogenousGPR-C6AexpressioninhibitedL-ornithine-inducedGLP-1secretion.Takentogether, thesefindingssuggest thatGq-coupledreceptor,GPRC6Areceptor functionsasanaminoacidssensorandregulateL-ornithine-inducedGLP-1secretionfromGLUTagcells.NoCOI.


2P-159Vesicular nucleotide transporter regulates ATP stor-age in secretory lysosome in astrocyteTSUBOI, Takashi1; OYA, Manami1; KITAGUCHI, Tetsuya2 (1Department of Life Science, The University of Tokyo, Tokyo, Japan, 2WABIOS, Waseda Univ, Singapore)

Astrocyteshavediverserolesincludingprotectionofneurons,regu-lationofvesicularsystemandmodulationofsynaptictransmission.Inadditiontorespondingtotransmittersreleasedfromneurons,recentstudieshavesuggestedthatastrocytesthemselvessynthesizeandreleasegliotransmitters.However,theprecisemolecularmechanismthatregulatesATPsecretionfromastrocytesandthesourceofATPinastrocytesremainsunclear.Avesicularnucleotidetransporter(VNUT)/solutecarrier family17,member9 (SLC17A9)hasbeenthoughttoregulateaccumulationofATPintovesicles.Inthepresentstudy,wefoundthatVNUTwasexpressedinbothprimaryculturedcorticalastrocytesandgliomacelllineC6cells,andmainlylocalizedinlysosomerevealedbyimmunohystochemicalanalysis.Livecellim-agingrevealedthatthefluorescentintensityofVNUT-associatedse-cretorylysosomesdidnotchangeafterdepolarizationwasinduced.Thisresult indicatesthattheVNUT-associated lysosomesstay inplasmamembraneafter lysosomalexocytosis. InhibitionofVNUTactivitybyEvansBluedecreasedtheamountofATPaccumulationintosecretorylysosome.Furthermore,depletionofendogenousVNUTexpressionbysmallinterferenceRNAandinhibitionofVNUTactiv-itybyEvansBluedecreasedtheamountofATPreleasefromastro-cytes.Takentogether,thesefindingssuggestthatVNUTplaysakeyroleinATPaccumulationintolysosomeandATPreleaseviasecreto-rylysosomeinastrocytes.NoCOI.

2P-160Transient vacuole formation induced by V-ATPase in-hibitor-a hint of the mechanism of vacuole formationHiruma, Hiromi(Department of Physiology, Kitasato University School of Medicine, Sagamihara, Japan)

Wehavepreviouslyshownthatmembrane-permeableweakbasescausepersistentvacuoleformationandthatindividualvacuolesareformedaroundalysosome.Sincethevacuoleoutsidethelysosomewasweaklyacidicandtheinnerlysosomewasstronglyacidic,wehypoth-esizedthatvacuolesareformedbyextrusionofaccumulatedfreeH+andwaterfromthelysosome.Inthisstudy,inordertoclarifythishypothesisinpart,thevacuoleformationwasinvestigatedaftertreat-mentofSaos-2cellswiththeV-ATPaseinhibitorbafilomycinA1(Baf).Time-lapsemicroscopyrevealedthatcontinuoustreatmentwithBafcausedtheformationofvacuoles15minafterthestartoftreatment,andvacuolesdiminishedafter1h.Theindividualvacuoleswereformedaroundthesphericalorganelles.Theseorganelleswereimmunoreactiveforanti-lysosome-associatedprotein-2antibody.Stainingwithamine-re-activedyeCFDA-SEindicatedthatvacuolescontainednoprotein.ThepHindicatorLysosensorYellow/Bluestainingshowedthatorganellesinsidethevacuoleswerestronglyacidicandvacuoleswereweaklyacidic.Aftervacuolesdiminished,bothorganellesandcytosolwereweaklyacidic.TheseresultssuggestthatBaf-inducedtransientvacu-olesareformedaroundlysosomesandthevacuoleformationiscausedbyextrusionofH+andwaterfromthelysosome.ThenH+inthely-sosomesisdepletedbylong-terminactivationofV-ATPaseandH+inthevacuolesdiffusestocytosol,leadingtotheequalizeddistributionofH+among lysosomes,vacuolesandcytosol,whichparalleledbydisappearanceofvacuoles.NoCOI.

2P-161Involvement of Ras-PI3K-mediated calcium signaling in the regulation of endocytosisFujioka, Yoichiro1; Tsuda, Masumi2; Nanbo, Asuka1; Hattori, Tomoe3; Saski, Junko4; Sasaki, Takehiko4; Miyazaki, Tadaaki3; Ohba, Yusuke(Dep. of Cell Physiol., Hokkaido Univ. Grad. Sch. of Med., 2Dep. of Cancer Pathol., Hokkaido Univ. Grad. Sch. of Med., 3Dep of Bioresources, Hokkaido Univ. Research Centre for Zoonosis Control., 4Dep. of Med. Biol., Akita Univ. Grad. Sch. of Med.)

WehavereportedthatPI3KbindstoactivatedRasintheendosomes,whichisinvolvedintheregulationofclathrin-independentendocytosisandtheincorporationofarangeofsubstances.Inthisstudy,weex-ploredthemechanismbywhichRasisactivatedduringendocytosis.UsingtheFRET-basedbiosensorCameleon,transientincreasesintheintracellularCa2+concentrationwereobservedupon infectionwithinfluenzaviruses,whichareknowntoenterhostcellsviaendocytosis.BecausebufferingofbothintracellularandextracellularCa2+abolishedthevirus-dependentRasactivation,Ca2+ isresponsible fortheRasactivation.WealsorevealedthattheCa2+elevationtriggeredRhoAactivationatthemembrane.Ontheotherhand,expressionofdominantnegativeformofRhoAinhibitedsuchCa2+oscillations,whichhamperedsubsequentvirusentryandinfection.Hence,RhoAandCa2+constituteapositivefeedbackloop,withRhoAnotonlybeingactivatedbyCa2+,butalsoinducingsubsequent[Ca2+]ielevationduringvirusinfection.Moreover,thissignalingcircuitwasalsofoundtoparticipateinuptakeofothersubstances,includingdextranandtransferrin.Takentogeth-er,thevirusesmayexploitsignalingpathwaysreservedforphysio-logicalsubstrateuptake,whichismediatedbyRhoAandcalcium,fortheireffectiveentry.NoCOI.

2P-162Cytosolic chloride ion is a key factor in lysosomal acidification and function of autophagy in human gas-tric cancer cellHosogi, Shigekuni1,3; Marunaka, Yoshinori1,2,3; Niisato, Naomi1,3; Kusuzaki, Katsuyuki1,3; Miyazaki, Hiroaki1,3(1Dept. of Molecular Cell physiology,Kyoto Prefectural University of Medicine, Kyoto, Japan, 2Dept. of Bio-Ionomics, Kyoto Prefectural University of Medicine, Kyoto, Japan, 3Japan Institute for Food Education and Health, Heian Jogakuin (St. Agnes) University)

Thepurposeofthepresentstudywastoclarifyrolesofcytosolicchlorideion(Cl-)inregulationoflysosomalacidification(intra-lysosom-alpH(pHlys))andautophagyfunctioninhumangastriccancercellline(MKN28).TheMKN28cellsculturedunderalowCl-conditionelevat-edpHlysandreducedtheintra-lysosomalCl-concentration([Cl-]lys)viareductionofcytosolicCl-concentration([Cl-]c),showingabnormalaccu-mulationofLC3IIandp62participatinginautophagyfunction(dys-functionofautophagy)accompaniedbyinhibitionofcellproliferationviaG0/G1arrestwithoutinductionofapoptosis.WealsostudiedeffectsofdirectmodificationofH+transportonlysosomalacidificationandautophagy.ApplicationofbafilomycinA1orEIPAelevatedpHlysanddecreased[Cl-]lysassociatedwithinhibitionofcellproliferationviain-ductionofG0/G1arrestsimilartothecultureunderalowCl-condition,howeverunlikelowCl-conditionapplicationofthecompound,bafilo-mycinA1orEIPA, inducedapoptosisassociatedwith increases incaspase3and9withoutlargereductionof[Cl-]ccomparedwithlowCl-condition.Theseobservationssuggestthatthelowered[Cl-]cpri-marilycausesdysfunctionofautophagywithoutapoptosisviadysfunc-tionoflysosomeinducedbydisturbanceofintra-lysosomalacidification.NoCOI.


Poster PresentationsEnvironmental Physiology

2P-163Orexin contributes to feeding and drinking behaviour induced by methamphetamineIkoma, Yoko1; Kuwaki, Tomoyuki1; Ootsuka, Youichirou1,2 (1Department of Physiology Graduate School of Medical and Dental Sciences Kagoshima University, Kagoshima, Japan, 2Centre for Neuroscience, Department of Human Physiology, Flinders University, Adelaide 5001, SA, Australia)

Methamphetamineisanaddictivedrugandknowntohaveanorecticandhyperactiveeffect.Recently,orexinhasbeensuggestedtocon-tributetodrugaddictionandrewarding.Orexinisaneuropeptidelocalizedinhypothalamusandisknowntoplayanimportantroleinmanyphysiologicalsystemsincludingfeedingbehaviour,motivationandmaintainingwakefulness.Therefore,we investigatedwhetherorexincontributedtochangesinfeedinganddrinking(asamakerofdrug-seeking)behaviourinresponsetooraladministrationofmeth-amphetamine.Inorexinknockout(KO)miceandtheirwildtype(WT),weprovidedmethamphetaminecontainingdrinkingwater(0.005%)for21daysandmeasuredfoodintake,drinking,bodyweightandlocomo-toractivityduringthetreatment.Methamphetaminetreatmentde-creasedfoodintakeby25±3%(n=6,P<0.05)duringthefirstthreedaysoftreatmentinWTmice,butnotinKOmice.Methamphetaminein-creasedtheamountofdrinkingby17±4%(n=6,P<0.01)inWTmice,butnotinKOmice.Largeramountofdrinkingindicateanincreaseindrug-seekingbehaviour,suggestingaddictiveeffectofmethamphet-amine.Thesedatasuggestthatorexincontributestomethamphet-amine-inducedanorecticandaddiction.NoCOI.

2P-164Effects of intracerebroventricular administration of colchicine on oxytocin-mRFP1 expression in trans-genic ratsHashimoto, Hirofumi1; Matsuura, Takanori1; Yoshimura, Mitsuhiro1; Ohkubo, Junichi1; Katoh, Akiko1,2; Maruyama, Takashi1; Ueta, Yoichi1(1Department of Physiology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan, 2Department of Otorhynolaryngology, National Kyushu Medical Center)

Colchicineblockstheaxonaltransport,resultinginpeptideaccumula-tion inthecellbody.Recently,wegeneratedatransgenicratthatexpressestheoxytocin (OXT)-monomericredfluorescentprotein1(mRFP1) fusiongene inthehypothalamo-neurohypophysialsystem(HNS).Inthepresentstudy,weinvestigatedtheeffectsofintracere-broventricular(icv)administrationofcolchicineonOXT-mRFP1ex-pression inthesetransgenicrats. Invehicle-treatedrats (controls)OXT-mRFP1fluorescencewasobserved intheHNS, includingthemagnocellularneurosecretorycellsofthesupraopticandtheparaven-tricularnucleiandthenucleuscircularis,andposteriorpituitary(PP).IcvadministrationofcolchicinecausedmarkedincreaseofOXT-mRFP1fluorescenceinthehypothalamicMNCs,whiledecreaseinthePPincomparisonwithcontrolrats.TheOXT-mRFP1fluorescencewasnotobservedintheectopicareaofneithercontrolsnorcolchicines-treatedrats.TheexpectedchangesofOXT-mRFP1fluorescenceintheHNSaftericvadministrationofcolchicineindicatethatOXT-mRFP1fusionproteinmaybetransportedbyaxonalflowandsecretedfromthePPintothesystemiccirculation.TheOXT-mRFP1transgenicratsareusefulanimalmodeltostudydynamicchangesofOXTintheHNS.NoCOI.

2P-165Hypergravity- and microgravity-induced plastic alter-ation of the vestibular system and its countermeasure.Morita, Hironobu1; Abe, Chikara1; Obata, Koji1; Tanaka, Kunihiko2(1Department of Physiology, Gifu University Graduate School of Medicine, Gifu, Japan, 2School of Health Sciences, Gifu University of Medical Science, Seki, Japan)

Thevestibularsystemisknowntobehighlyplastic;thus,itispossiblethatifanimalsorsubjectsareindifferentgravitationalenvironment,functionsofthevestibularsystemarealtered.Toexaminethis,aseriesofexperimentswereperformed.Effectsofhypergravity:Thevestib-ular-mediatedpressorresponseandthevestibular-relatedmotorskill(measuredbyrotarod)wereimpaired,ifratswererearedunder3gconditionfor2weeks.Effectsofmicrogravity:Thevestibular-mediat-edpressorresponse inducedbyhead-uptiltwasexamined intwoastronautsbeforeandafter4-monthstayinInternationalSpaceStation.Itwas16mmHgbeforelaunch,butcompletelyabolishedatthe2nddayoflandingand2weeksafterlanding,butrecoveredby2monthsafterlanding.Countermeasure:Notonlytheeffectofmicrogravitybutalsohypergravityonthevestibularsystemareduetothereducedphasicinputtothevestibularorgans,sinceheadmovement-relatedvestibular inputwasreducedto<20%under3gcondition.Thus,externallyappliedvestibularstimulationmightimprovethevestibularfunction.Toexaminethis,galvanicvestibularstimulation(GVS;±50µAbiphasicsquarewave,1sduration)wasappliedthroughout2-weekhypergravitycondition.GVSsignificantlyimprovedthevestibular-me-diatedpressorresponseandrotarodskill.Theseresultssuggestthat,GVScanbeusedforacountermeasureto improvethevestibularfunctionseeninastronauts.NoCOI.


2P-166Candidate mechanism of skin vasodilation caused by contact with high concentration-CO2-water in the anesthetized ratHashimoto, Masaaki1; Yamamoto, Noriyuki2(1Lab Physiol, Dept Phys Ther, Fac Med Sci, Teikyo Univ Sci, Tokyo, Jpn, 2Dept Health Sci, Fuc Nurs, Jpn Red Cross Hokkaido Coll Nurs, Kitami, Jpn)

Hotspringwatercontaininghighconcentration(>1g/L)carbondiox-ide(CO2-hotspring)haslongbeenappliedtothepatientsofcardiovas-culardiseases.TheskinsurfaceintheCO2-hotspringiscoveredwithsmallbubblesanditscolorturnsredwithinseveralminutesofbathingevenatthewatertemperaturelessthan35°C.Theskinreddeningisreproduciblebyartificiallymadebath-watercontainingcomparableCO2-concentrationtoCO2-hotspring (CO2-water)and iscausedbyvasodilationfollowedbyanincreaseinskinbloodflow.Althoughskinreddeningisnotobvious,theskin-vasodilationisobservedinlabora-toryanimalratsalso.Inthisstudy,weinvestigatedendogenoussub-stanceswhichmightbeinvolvedinthemechanismofskinvasodilationcausedbysoaking inCO2-water.ExperimentwasperformedusingmaleWistarratsunderurethaneanesthesia.Controltap-waterimmer-sionfor30minfollowedbyCO2-waterimmersionfor30min(watertemperatureofabout35°C)wasrepeated2timestoconfirmarepro-ducibilityoftheresponseineachrat.SkinvasodilationduringCO2-wa-terimmersionwasinhibitedbytreatmentofcyclooxygenaseinhibitorindomethacin,butnotbyNOS-inhibitorL-NAME.Amongvasodilatoryprostaglandins(PGE1,PGE2,PGI2),PGE2levelincreasedbyabout30%inthehomogenateoftheskinremovedfromratduringCO2-waterimmersion.LocalPGE2-productionmaybeinvolvedintheskinvaso-dilationobservedunderthisexperimentalcondition.NoCOI.

2P-167The role of TRPV1 that contribute to the thermal ef-fects of immersion to the carbon dioxide-rich waterNishimura, Naoki1; Iwase, Satoshi1; Nishimura, Rumiko1; Uta, Daisuke2; Matsumoto, Takaaki3; Sato, Motohiko1(1Department of Physiology, Aichi Medical University, Nagakute, Japan, 2National Institute for Physiological Sciences, Department of Information Physiology, Okazaki, Japan, 3Chukyo University School of Health and Sport Science, Toyota, Japan)

Carbondioxide-richwater(CO2)bathinghavebeenusedforbalneo-therapyofpatientswithhypertensionorperipheralocclusivearterialdisease.WehavepreviouslyreportedthatCO2bathingexertsthermaleffectsthanfreshwater(FR)bathing.Currently,themodificationoftheactivitiesofskinwarmandcoldreceptorsbyCO2applicationiswidelyaccepted.Asaresult,subjectsfeelaneutralsensationmaybeloweredby2°CduringCO2bathing.Thetransientreceptorpotentialvanilloid1(TRPV1)isnon-selectivecationchannelthatcanbeactivat-edbyheatabove43°Candvariouschemicalagonistsareknownin-cludingsuchascapsaicinorH+.However,themechanismoftheactionofCO2onthermoreceptorhasonlybeenpartiallyclarifiedinhumans.WeexaminedtheroleofTRPV1channelrelatedtoan increase inthermalsensationwhenitwasimmersedintoCO2ofFRat33°Caftercapsaicinapplicationtotheskinoftheforearm.ThermalsensationofimmersionintotheCO2washigherabout30%thanimmersionintotheFR.TheseresultssuggestthattheCO2mighthaveactivatedtheTRPV1.Wealsoreportchanges inthermalsensationof immersionintotheCO2whenitinhibitstheactivityofsodiumchannelsby5%oflidocaineapplicationtotheskin.NoCOI.

2P-168Physiological responses to passive heating in young-er and elderlySaito, Masahisa; Daikoku, Eriko; Shiraiwa, Yuka; Yamaji, Junko; Kubota, Takahiro(Dept. Physiol., Osaka Medical College, Takatsuki, Japan)

Oneolder(aged59years)andtwoyounger(aged22and24years)healthymenwereexposedtoastandardheatstress.Thesesubjectssat inacontrolledenvironmentatambienttemperature35.1±0.3°Candrelativehumidity48.9±7.2%tobereceivedtheheatstressplacingthelowerlegsandfeetinawaterbathat42°C,for60min.Theear-drumtemperature(ET)inboththeolderandyoungermenincreasedduringpassiveheating.ButETatthelast30minofyoungermenwerelowerthanthatoftheolder.Thesestressincreasedthelocalsweatrates(LSR,mg/cm2/min)onthighandchestforboththeolderandtheyoungermen,butinducednodifferencesbetweentwogroupsforLSR.Systolicanddiastolicbloodpressure(mmHg),meanarterialpressure (mmHg),andheartrate (beats/min)changedsimilarlybe-tweentheolderandyoungermenduringpassiveheating.Now,wearetryingtoincreasethenumberofobservations,andtomeasuretheinflammatorycytokines.NoCOI.

2P-169Effects of lumbar warming on dysmenorrheal pain and uterine blood flowNakamoto, Miki; Igi, Chihiro; Nishimura, Yukako; Yamamoto, Megumi; Arakawa, Mayu; Hosono, Takayoshi(Dept Biomedical Engineering, Grad Sch Osaka Electro-Commun Univ, Shijonawate, Osaka, Japan)

Morethan80%ofmenstruatingwomencomplainofdysmenorrhealpain,which ismainlycausedbypain-producingsubstances intheuterineendometrium.Warmingthelowerabdominalorlumbarregionisknown to relievedysmenorrhealpain. Especially, aheat andsteam-generatingsheet(HSG)whichwarmstheattachedskinareatoaround40°Cfor5–8hoursiseffectivefordysmenorrhealpainrelief.However,themechanismofthispainreliefisstillunknown.Theaimofthisstudywastoclarifythemechanismbyinvestigatingpainreliefanduterinebloodflow,whichmayremovepain-producingsubstances.Sevenuniversitystudents(mean:21.7±0.3yrsold)wereincludedinthisstudy.Onthesecondorthirddayofmenstruation,eachsubjectappliedanHSGsheetof54cm2toherlowerlumbarregionfor60min.WemeasuredvelocitiesofuterinebloodflowusingultrasoundDopplerflowmetryevery15min.Wedetectedmaximum(Vmax)andminimum(Vmin)velocitiesandevaluatedtheresistanceindex,RI=(Vmax-Vmin)/Vmax,whichisknowntoreflectperipheralvascularresistance.Thesubjectsalsoself-rateddysmenorrhealpainusingnumericalratingscale(NRS),from0to10,atthesametimeasflowmetry.Fiveofthesubjectsexperiencedpainreliefwithinthe60-minwarming.TheRIalsodecreasedinallsubjects.However,nomarkedcorrelationswereobservedbetweentheRIandNRS. TheremaybenoassociationbetweenpainreliefandauterineRIdecrease.NoCOI.


2P-170Effect of hyperthermia on adult rats after neonatal hy-poxic-ischemic encephalopathy on learning abilityNishimura, Yukako; Hosono, Takayoshi(Department of Biomedical Engineering, Graduate School of Osaka Electro-Communication University, Osaka, Japan)

Amaternalbodytemperaturehigherthan38.5°Cisknowntobeoneofthemostseriousriskfactorsworseningtheprognosis inhumanneonatalhypoxic-ischemicencephalopathy(HIE).However,itseffectsonlearningabilityaftergrowthhavenotbeenclarified.Weinvesti-gatedtheeffectofhyperthermiaafterneonatalHIEonthelearningabilityofanadultratmodel.Theleftcarotidarteryof7-day-oldratswasligatedunderanesthesia.After1-hrecovery,allratswereexposedto8%oxygenatanambienttemperature(Ta)of40°Cfor15min,andthenplacedinachamberwithTaof40°C(n=10;Hgroup).Ashamgroup(n=6;Sgroup)wasalsoestablished.Eleventotwelveweeksaftertheoperation,thelearningabilityoftheratswasassessedusingastep-downpassiveavoidancetest.Beforemeasurement,wedecreasedtherats’bodyweightby15%byfasting.Onthefirstdayofmeasure-ment,ratswereplacedonaninsulatedrubberplatformontoamet-al-gridfloor.Oncetheratssteppedontothefloor,theyexperienceddiscomfortviaanelectricalshocktothe foot.Weperformedthisprocedureuntiltheratsnolongersteppeddownontothefloor.Theintervalbetweenratsmovingfromtherubbertometalfloorwasmeasuredonceadayforthenextthreedays.Onthelastdayofmea-surement,althoughtheSgroupratsstayedontherubberfloorfor595sec,theHgroupratssteppedontothefloorafteranintervalof181sec.ThisindicatesahigherhyperthermiainlaborwithHIEmayresultinarestrictedlearningabilityaftergrowth.NoCOI.

2P-171Comparison of average changes in brain level blood pressure between SHR and WKY rats during acceler-ation stressMaruyama, Satoshi1; Nishida, Yasuhiro2(1Aeromedical Laboratory, JASDF, Tokyo, Japan, 2Department of Physiology, Natl Def Med Col, Saitama, Japan)

Gzstressgreatlyaffectsnormalbloodcirculation inthebrainandcauses lossofconsciousness. IncreasedaorticvalvebloodpressureimpartsaprotectiveeffectagainstGzstress.Hypertensioninvolveshigheraorticvalvebloodpressure,butbloodpressureregulationisdifferent fromnormotension.Wehypothesizedthathypertension isnotanadvantageousfactoragainstsustainedGzandestimatedtheeffectofnifedipineonbloodpressureduringGzloadinginhypertensiverats.Weusedmalespontaneouslyhypertensiverats(SHR)andWistarKyoto(WKY)rats.TheSHRwererandomlyallocatedtothenontreat-ment(nSHR)andtreatment(tSHR)groups.ThetSHRwereadminis-tered0.1%nifedipinedietfor2weeks.Bloodpressureatthelevelofthebrain (APLB)andheartrate (HR)werecontinuouslymeasuredduringGz loadingusingacentrifugeforsmallanimals.ThemeanAPLBwassignificantlyhigherinthenSHRandtSHRthantheWKYratsbeforecentrifugation,andthemeanAPLBwaslowerinthetSHRthanthenSHR.During+5Gzloading,averagechangesinAPLBwassignificantlyhigher inthenSHRthan inthetSHRandWKYrats;however,nosignificantdifferencewasobservedbetweenthesechang-esinthetSHRandWKYrats.NosignificantdifferencewasobservedinHRbetweenbothgroups.TheseresultssuggestthatAPLBdecreas-eseasierinthehypertensiveratthaninthenormotensiveratduringGzloading,andthisisimprovedbycareforhypertension.HypertensionisnotanadvantageousfactoragainstGzloading.NoCOI.

2P-172Changes in cerebral and muscle oxygenation during normoxic and hypoxic exercise.Masuda, Atsuko1; Yokoi, Mari1; Sakakibara, Yoshikazu2; Masuyama, Shigeru2(1Medical Education Center, Faculty of Health Science, Ryotokuji University, Chiba, Japan, 2Department of psychological informatics, Kanazawa Institute of Technology, Kanazawa, Japan, 3Travellers Medical Center, Tokyo Medical University Hospital, Tokyo, Japan)

Thepurposeofthisstudywastoexaminetheinfluenceofacutehy-poxiaandexerciseonoxygenationresponsesinbrainandexercisemuscle.Tenmalejudoathletesperformedanincrementaltreadmillexercisefor6minuteswhilebreathingroomair(normoxia)andhy-poxicgas(FIO2=0.112).Near-infraredspectroscopy(NIRS)wasusedtomonitor concentration changes of oxy- and deoxyhemoglobin(d[O2Hb],d[HHb]) inthebothright frontalcerebralcortexand leftvastuslateralismuscle.ChangeintotalHb(d[THb]=d[O2Hb]+d[HHb])andwasusedasanindexofthechangeinregionalbloodvolume.Bothduringnormoxicandhypoxicexercise,d[O2Hb]andd[THb]incerebralandmuscle,afterinitialdip,increasedsteadilyafterwards.Incrementind[O2Hb]inbothtissuesatnormoxiawasgreaterthanthatduringhypoxiaandasignificantdifferencewasdetectedearlier inmuscletissuethanincerebraltissues.Ontheotherhand,incrementind[THb]inbothtissuesathypoxiawasgreaterthanthatatnormoxiaandasignificantdifferencewasdetectedearlierinmuscletissuethancere-braltissue.Thesefindingswouldsuggestthatdysoxygenationinbrainaswellasmusclewascompensatedbyrelativelypreservedbloodflowandthatbrainmaloxygenation inbraintissuemightbeoneoftheexerciselimitingfactorsduringhypoxiccondition.NoCOI.

2P-173In utero exposure to hydroxylated polychlorinated bi-phenyl induces hyperactivity in adult male mouse off-springHaijima, Asahi; Shimokawa, Noriaki; Takatsuru, Yusuke; Amano, Izuki; Lesmana, Ronny; Koibuchi, Noriyuki(Dept. Integrative Physiol., Gunma Univ. Grad. Sch. Med., Gunma, Japan)

Polychlorinatedbiphenyls(PCBs)arerecognizedaspersistentenvi-ronmentalpollutantsthatcanaffectneurobehavioraldevelopment.Here,wedeterminedwhetherin uteroorlactationalexposuretohy-droxylated-PCB106(OH-PCB106;4-hydroxy-2’,3,3’,4’,5’-pentachlorobi-phenyl)affectsthespontaneouslocomotoractivityinadultmalemouseoffspring.Forin uteroexposure,pregnantC57BL/6Jmicereceivedcornoilvehicle,0.05or0.5mg/kgb.w.ofOH-PCB106viagavageeveryseconddayfromgestationalday10to18.Forlactationalexpo-sure,damsreceivedcornoilvehicle,0.05or0.5mg/kgb.w.ofOH-PCB106viagavageeveryseconddayfrompostnatalday3to13.Aftermalemiceoffspringreachedadulthood,theirspontaneouslocomotoractivityinthehomecageandopenfieldwereevaluated.PrenatallyOH-PCB106-exposedmiceshowedsignificantlyincreasedspontaneouslocomotoractivitycomparedtothecontrolgroupbothinthehomecageandopenfield.Conversely,lactationalexposurestoOH-PCB106hadnosignificanteffectsonthespontaneouslocomotoractivityundersuchconditions.TheseresultssuggestthattheneurotoxiceffectsofOH-PCB106exposuremaybedifferentdependingontheperiodofexposure,andin uteroOH-PCB106exposurecaninducehyperactiv-ityinadultmalemouseoffspring.NoCOI.


2P-174Effects of nursing on PER2::LUC rhythm in mouse mammary tissue and endocrine organsYoshikawa, Tomoko1,2; Jia, Shu Sheng2,3; Honma, Sato2; Honma, Ken-ichi2(1Photonic Bioimaging Section, Hokkaido University Graduate School of Medicine, Sapporo, Japan, 2Department of Chronomedicine, Hokkaido University Graduate School of Medicine, Sapporo, Japan, 3Department of Breast Surgery, The Third Affiliated Hospital of Harbin Medical University, Harbin, China)

Mammaliancircadiansystemconsistsofmasterandperipheralcirca-dianclocks.Themasterclockislocatedinthehypothalamicsuprachi-asmaticnucleusandtheperipheralclocksarefoundinmanyorgansthroughoutthebody.Currentunderstandingisthatthemasterclockentrainstheperipheralclocks.Theentrainingsignalscontainneuralandhumeralsignals,bodytemperature,activityandpossiblymanyothers.Fornursingfemales,sucklingbehaviorofpupsmayactasthetimecueforthecircadianclocksinendocrineorgansrelatedtonurs-ing.Inthepresentstudy,weexaminedtheeffectsofnursingoncir-cadianrhythminthemammarytissue,pituitaryglandandovarybyusingPeriod2::Luciferaseknockinmouse.Nursingdamsweredeprivedfromtheirpupsfor6hoursduringthefirsthalfofthedayfromlac-tatingday1(L1)toL7.ThetissuesampleswerecollectedfromnursingdamsonL7,andbioluminescencewasrecordedfromtheculturedtissuestoanalyzePER2::LUCrhythm.Innon-deprivedcontrolmam-marytissue,anteriorandposteriorpituitariesshowedthepeakphaseoftherhythmatearlynight,whiletheovaryhadthepeaksomewhatearlier.Inthedeprivedgroupthephaseoftheanteriorpituitarywassignificantlyadvanced.Thisresultsuggeststhatnursingpatternisatimecueforthecircadianclockintheanteriorpituitary.NoCOI.

2P-175Effects of daily exercise and mental stress on forearm endothelial function in male and female university stu-dentsChen, Xin; Fujiwara, Yurika; Yamamoto, Sachiho; Tanaka, Aoi; Kumano, Yuri; Takamata, Akira; Morimoto, Keiko(Dept. Environmental Health, Facult. Human Life and Environmental Sci., Nara Women's Univ., Nara, Japan)

Mentalstressorphysicalinactivityisariskfactorforatherosclerosis.Theunderlyingmechanismsarenotclearlyknown,butendothelialfunctioncouldbeinvolved.WeexaminedtheeffectsofdailyexerciseandmentalstressonendothelialfunctionofbrachialarteryusingechoandDopplerultrasoundinmaleandfemaleuniversitystudents.Younghealthysubjectsweresubjectedtomentalstressevokedbythemod-ifiedSTROOPColor-WordTest(CWT)in10min.Theyunderwentreactivehyperemia(RH)forflow-mediateddilation(FMD)andmaximalincreaseinbloodflow(%)assessmentsbeforeandat5minaftertheCWT.Inaddition,5-minhand-gripexercise(HGEX)wasperformedfortheestimationofFMDresponsetoHGEX-inducedincreaseinshearstress.ThebasallevelofFMDinRHwashigherintheexercisegroupcomparedwiththesedentarygroupinmen,butnotinwomen.TheCWTinducedaslightvasoconstrictionandareductionofbloodflowinbrachialartery.However,therewasnosignificantdifferenceamongmenandwomen,oramongtheexerciseandsedentarygroups.FMDinRHwasincreasedat5minafterCWTcomparedwiththebasallevelonlyintheexercisegroupofmen,butnotinanothergroups.Inaddition,FMDinHGEXwashigherintheexercisegroupcomparedwiththesedentarygroupinmen.Thesefindingssuggestthatdailyexercisehadbeneficialeffectsonforearmendothelialfunctionatthebasalconditionandrecoveryphasefrommentalstressinmen.NoCOI.

2P-176Central ketone bodies regulate sleep homeostasis in miceChikahisa, Sachiko; Shimizu, Noriyuki; Shiuchi, Tetsuya; Sei, Hiroyoshi(Department of Integrative Physiology, Institute of Health Biosciences, The University of Tokushima Graduate School)

Theketonebodiesaregeneratedfromthebreakdownoffattyacids,andtheyaremajormetabolicfuelsforthebrainunderconditionsoflowglucoseavailability.Ketogenesisisknowntobemodulatedbytheactivityofperoxisomeproliferatoractivatedreceptoralpha(PPARα).Inourpreviousstudy,treatmentwithaPPARactivatorhasinducedamarkedincreaseinplasmaacetoacetate(AcAc)anddecreasedβ-hy-droxybutyrate(BHB)inmice,accompaniedbyincreasedslow-waveactivity(SWA)duringnon-rapideyemovement(NREM)sleep.Inthepresentstudy,wehaveinvestigatedtheroleofketonebodiesinthesleepregulation.Six-hoursleepdeprivationincreasedplasmaketonebodies.Moreover,sleepdeprivation increasedmRNAexpressionofketogenesissuchasPPARαand3-hydroxy-3-methyl-glutarate-CoAsynthase2 (Hmgcs2) inthebrainanddecreasedketolyticenzymessuchassuccinyl-CoA:3-oxoacidCoAtransferase(Scot).Additionaly,central injectionofAcAc,butnotBHB,markedly increasedSWAduringNREMsleepandsuppressedglutamaterelease.Theseresultssuggestthatcentralmetabolismofketonebodies (especiallyAcAc)playaroleintheregulationofsleephomeostasis.NoCOI.

2P-177Physiological responses when viewing aquarium fish-es and their changing movement activitiesIchinose, Mitsuyuki; Maeda, Takuya; Matuura, Tetsuyu(Faculty of Engineering, Iwate University, Morioka, Japan)

Inordertoinvestigatephysiologicaleffectsofviewingaquariumfish-es,wemeasuredEEGandheartrate.Subjectswereyounghealthy4men.Akindongoldfish,Ryukinwaskeptinplasticaquarium(29×30×34cm),whichwasplaced1minfrontofasubjectinanexperimentalroom.Toinducebehaviorchangeoffishes,baitwasdroppedtwotimes2.5minuteinterval.ComparingtoEEGduringwatchingawhiteboard,amplitudesofαandβbandweresuppressedduringviewingaquariumfishes.Whenbaitwasdroppedintotheaquarium,goldfishestookthebaitandactivityoftheirmovementwasincreased.Whenthefeedingmotionwasactivated,amplitudesofαandβbandweresuppressedmuchmoreatthattime.Thesuppressionsuggestedthatneuralac-tivityofFzwasreducedbywatchingfishesandmoreeffectivelywatchingactivemotion.Slopeofpowerspectraldensity(PSD)ofEEG,whichshowedaveragedneuralactivitiesbetweenδtoβband, in-creasednegatively inFzandO2of twosubjectsduringviewingaquariumfishes.Thismeantthatviewingaquariumfishesreducedactivityoffrontalandoccipitalneuralactivity.Inothertwosubjects,areductioninslopeofPSDonFzwasalsosuppressed,suggestingthatfrontalactivitywasnotstimulatedrelativelycomparingtoO2,asarepresentativeoftheoccipitalarea,whichcorrespondedtothevisu-alcortex.Thesedatasuggestedthatviewingaquariumfishesrestedgeneralneuralactivityofsubjects,namely leadingtorelaxation infeelings, thoughneuralactivity invisualsensorysystemshouldbeactivatedbecauseofwatchinggoldfishes.NoCOI.


2P-178Chronic exposure of extremely low-frequency magnet-ic field stimulates aldosterone secretion in H295R cellKadoriku, Fumiya1,2; Kawata, Shiyori1,2; Kitamura, Mitsuo1; Kitaoka, Kazuyoshi1(1Department of Physiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan, 2Student Lab, The University of Tokushima, Faculty of Medicine, Tokushima, Japan)

Anextremelylow-frequencymagneticfield(ELF-MF)isgeneratedbypowerlinesandhouseholdelectricaldevices.Manystudieshavein-vestigatedwhetherELF-MFhasbiologicaleffectusingcellline,animalmodel,humansubject,andepidemiologicalstudy,buttheeffectsremaincontroversial.WepreviouslyreportedthatchronicELF-MFexposureisincreasedplasmacorticosteroneandaldosteronewithoutenhance-mentofhypothalamic-pituitary-adrenalaxis inmice.Also invitro,corticosteroneandaldosteronesecretionwasincreasedsignificantlyby24hELF-MFexposureinmouseadrenalcortex-derivedY-1cell.ThesepreviousstudiessuggestthepossibilitythatchronicELF-MFexposureaffectstheadrenalstroidgenesisalso inhumans. Inthepresentstudy,wecarriedoutchronic(6h,12h,24h,and48h)ELF-MFexposure(1.5mTintensity)tohumanadrenalcortex-derivedH295Rcellandestimatedtheeffectforadrenalsteroidbiosynthesis.Astheresults,12and24hELF-MFexposuresignificantlystimulatesaldoste-ronesecretioninH295Rcell.Ontheotherhand,thesecretionofcor-tisoldidnotaffectsignificantly.TheresultssuggestthepossibilitythatchronicELF-MFexposurestimulatessteroidgenesisinzonaglomer-ulosa,notzonafasciculateofadrenalglandinhumans.NoCOI.

2P-179Exploration of the mechanism for extremely low-fre-quency magnetic field-induced adrenal steroids up-regulation in Y-1 cellKawata, Shiyori1,2; Kadoriku, Fumiya1,2; Kitamura, Mitsuo1; Kitaoka, Kazuyoshi1(1Department of Physiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan, 2Student Lab, The University of Tokushima Faculty of Medicine, Tokushima, Japan)

Extremely low-frequencymagneticfield (ELF-MF) isgeneratedbypowerlinesandhouseholdelectricaldevices.Manystudieshavein-vestigatedbiologicaleffectofELF-MFbyusinghumansubjectsandexperimentalanimals.However,theeffectremainscontroversial.WepreviouslyreportedthatchronicELF-MFexposurewas increasedplasmacorticosteronelevelanddepression-likebehaviorwithouten-hancementofhypothalamic-pituitary-adrenalaxisinmice.In vitro,thesynthesisofcorticosteronewasalsoincreasedinmouseadrenalcor-tex-derivedY-1cellby24hoursELF-MFexposure.However, themechanismisunknownatpresent.SomereportsindicatethatELF-MFaffectsvariouscellularprocessesviareactiveoxygenspecies(ROS)production.Inthisstudy,wemeasuredintracellularROSinELF-MFtreated(1.5mTintensity)Y-1celltorevealhowtheup-regulationofcorticosteronesynthesiswaselicited.Astheresults,24hoursELF-MFtreatedY-1cellsshowedthedecreaseofintracellularROScomparedwithsham-treatedcontrol.OurevidencesuggeststhepossibilitythattheROS-scavengingmechanismsareinvolvedintheup-regulationofadrenalsteroidssynthesis.NoCOI.

2P-180Changes of systemic arterial pressure,common carot-id arterial flow and heart rate after onset of 90 de-grees head-up tilt in anesthetized young ratsNishimura, Hironobu1,2; Yamasaki, Masao2; Ito, Yasuhiro2; Nomura, Hiroko3(1Graduate Student of Master Course, Depat. of Physiol. and Med., Graduate School of Health Sciences, Fujita Health Univ., 2Dept. of Physiol. and Med., Graduate School of Health Sciences, Fujita Health Univ., 3Dept. of Physiol. and Pathophysiol., Graduate School of Health Sciences, Fujita Health Univ.)

Wepreviouslyreportedthe initialchangesofthesystemicarterialbloodpressure(BP),thebloodflowinthecommoncarotidartery(BF)andtheheartrate (HR)afteronsetof90°head-uptilt (HUT)fromsupineposition.Toelucidatethese initialchangesandtheroleofbaroreflexduringHUTfor30mininanesthetizedyoungrats(urethane,1.0–1.5g/kg),wemeasuredtheBP,BFandHRbyanalog-digitalre-cordingsystem(MP-36;Biopac,USA).Underanesthesia,weinsertedacatheterintoarightcommoncarotidarterytowardtheheartforBPmeasurement,attachedaprobeofultrasoundflowmeter(T206;Tran-sonic,USA)toanartery forBF,andplacedECGelectrodesonasubcutaneousofachestforHR.AfteronsetofHUT,BPandBFim-mediatelydecreasedto86.4±9.4mmHgand2.96±0.45ml/minat2.7±0.7sec,respectively,comparedwiththevaluesundersupinebeforeHUT(102±10.1mmHg,3.82±0.65ml/min;n=6,p<0.01).BothoftracesinBPandBFturnedtothevaluesbeforeHUT,andtheaverageofHRslightlyincreased.TheseresultsindicatedthattheinitialchangesinBPandBFproducedbythehydrostaticpressuregradientduetoHUTweresimilartothose intheadultrats.FromtheviewpointofHRchangeineachanimal,itwasunclearthatthebaroreflexworkactive-ly.NoCOI.

2P-181Responses of serotonin release in the central nucleus of the amygdala to cutaneous pinching of rats- Contri-bution of type 1 and 2 corticotropin releasing recep-tors in the dorsal raphe nucleusTokunaga, Ryota1; Takagi, Noriaki1; Shimoju, Rie2,3; Shibata, Hideshi4; Yamaguchi, Rie4; Kurosawa, Mieko1,2(1Physical Ther., Health & Welfare Sci., Intl. Univ. Grad. Sch. Health & Welfare, Tochigi, Japan., 2Center Med. Sci., Intl. Univ. Health & Welfare, Otawara, Japan, 3Dept. Physical Ther., Intl. Univ. Health & Welfare, Otawara, Japan, 4Lab. Veterinary Anatomy, Fac. Agriculture, Tokyo University of Agriculture and Technology)

Wehaveshownthatresponsesofserotonin (5-HT)release inthecentralnucleusoftheamygdala (CeA)tocutaneouspinchingwereabolishedafteradministrationofanon-selectivecorticotropinreleasingfactor(CRF)receptorantagonist,α-helical-CRF(9-41),intothedorsalraphenucleus (dRN) inanesthetizedrats. Inthepresentstudyweaimedtodefinethecontributionofbothtype1andtype2CRFreceptorsinthedRNtotheresponses,withuseofselectivetype1andtype2CRFreceptorantagonists.Acoaxialmicrodialysisprobewasstereo-taxicallyimplantedintheCeAandperfusedwithmodifiedRinger’ssolutionataspeedof1µl/min inanesthetizedrats.Pinchingwasappliedtothebackfor10min.AftervehicleinjectionintothedRN,pinchingincreasedthe5HTrelease.Theresponsesof5-HTtopinchingwereabolishedafterinjectionofantisauvagine-30(ASV-30),atype2CRFreceptorantagonist,whilethosewerenotinfluencedbyinjectionofantalarmin,atype1CRFreceptorantagonist.Theseresultssuggestthatresponsesof5HTreleaseintheCeAtocutaneouspinchingaremediatedviathetype2CRFreceptorsinthedRN.NoCOI.


Poster PresentationsDrug Actions

2P-182Evaluation of muscarinic actions of the antipsychotic clozapine and its metabolite N-desmethylclozapine by using cultured hippocampal neuronsSugawara, Yuto; Ohno-Shosaku, Takako(Fac. Health Sci. Kanazawa Univ., Kanazawa, Japan)

Clozapineisauniqueantipsychoticdrug,andhasbeenusedfortreat-ment-resistantschizophrenicpatients.Clozapineanditsactivemetab-olite,N-desmethylclozapine (NDMC), interactwithvarioustypesofneurotransmitterreceptors.RecentbiochemicalstudiessuggestedthatNDMCexhibitsM1muscarinicagonistaction,whichisuniqueandnotsharedbyanyotherantipsychotics.However,directelectrophysiolog-icalevidenceforthemuscarinicactionofNDMConintactneuronshasbeenscanty.Inratculturedhippocampalneurons,wepreviouslyre-portedthatapplicationofamuscarinicagonist,oxotremorine-M(oxo-M),inducedsuppressionofthebasaloutwardK+currentat-40mVinaPLC-dependentmanner.Inthepresentstudy,weexaminedagonist-an-tagonistpropertiesofNDMCandclozapineusingthismuscarinicre-sponse.Theoxo-M-inducedsuppressionofK+currentwassensitivetoboth0.1µMpirenzepine(M1antagonist)and3nMDAU5884(M3antagonist), indicating involvementofM1/M3muscarinicreceptors.NDMCsuppressedtheK+current,whichwasreversedbythemus-carinicantagonistatropine.Clozapinewasmuchlesseffectiveinsup-pressingthecurrent.Next,antagonistactionswereexaminedbyusingoxo-M.Theeffectofoxo-MwasreversedpartiallybyNDMC,andmorestronglybyclozapine.CoapplicationofNDMCandclozapineexhibitedweakagonistandstrongantagonistactions.TheseresultsindicatethatNDMCalonecanactasmuscarinicagonist,butitsagonistactionissuppressedbytheantagonisticactionofclozapinewhenthebothco-exist.NoCOI.

2P-183Mesenchymal stem cells ameliorate central and pe-ripheral neural pathologies in mice with spinocerebel-lar ataxia type 1Nakamura, Kazuhiro; Hirai, Hirokazu(Department of Neurophysiol, Gunma University Graduate School of Medicine, Maebashi, Gunma, japan)

Spinocerebellarataxiatype1(SCA1)isadevastatingneurodegenera-tivedisordercausedbytheexpansionofapolyglutaminetractintheataxin-1protein.Wefoundthat intrathecal injectionofonly3x103MSCssignificantlymitigatedthecerebellarandspinalcordpathologiesseeninSCA1-transgenic(SCA1-Tg)mice.AlthoughthePurkinjecells(PCs)of24-week-oldnon-treatedSCA1-Tgmicedisplayedamulti-lay-erarrangementanddendriticatrophy,MSCs-injectedSCA1-Tgmicedisplayedmono-layerPCsandwell-developeddendrites.Furthermore,MSCsinjectionpartiallyrestoreddelayednerveconductionvelocityinspinalmotorneurons inSCA1-knock inmice.Finally,behavioraltestsdemonstratedthat intrathecal injectionofMSCsnormalizeddeficitsinmotorcoordinationinSCA1-Tgmice,whichwasreplicatedbyanintrathecalandfollowingintravenousinjectionsofMSCs-condi-tionedmedium.Thus,MSCs likelyexerttheirtrophiceffects inaparacrinemannerandfuturestudiesdeterminingthetrophicfactorswouldbeworthatrytodevelopatreatmentforSCA1patients.NoCOI.

2P-184Lansiumamide B and SB-204900 isolated from Clau-sena lansium inhibit histamine and TNF-α release from RBL-2H3 cellsMatsui, Takuya1; Shiono, Hiroyuki1; Ito, Chihiro2; Itoigawa, Masataka3; Masubuchi, Satoru1(1Department of Physiology, Aichi Medical University, Aichi, Japan, 2Faculty of Pharmacy, Meijo University, Aichi, Japan, 3Faculty of Human Wellness, Tokai Gakuen University, Aichi, Japan)

AimsMastcellsplayacentralroleinallergicandchronicinflammation.ExtractsfromClausena lansium(Lour.)Skeels(Rutaceae)possessmanypharmacologicaleffectsincludinganti-inflammatory,anti-oxidant,an-ti-cancer,andanti-trichomonalactivities.Inaddition,theleavesandfruitareusedinChinesefolkmedicine.Wehaveisolatedandidentifiedfourknowncinnamamidesfromthisplant:lansiumamideC,lansamideI,lansiumamideB,andSB-204900.However,thebiologicalactivitiesofthesecompoundsarenotyetunderstood.Thepurposeofthisstudyistoclarifythepharmacologicaleffectsofthesecompoundsonmastcells.MethodsWemeasuredinflammatorymoleculesinA23187-stim-ulatedratbasophilic leukemiacells (RBL-2H3) treatedwiththesecompoundsusingHPLC,ELISA,and immunoblottingmethods. Inaddition,somesignalingmoleculeswereinvestigatedbyimmunoblot-ting.ResultsLansamideI,lansiumamideB,andSB-204900significant-lydecreasedhistaminerelease.Furthermore, lansiumamideB-andSB-204900-treatedcellsalsoreducedtheproteinand/ormRNAlevelsofTNF-α.AlthoughIL-6proteinexpressiononlansiumamideB-andSB-204900-treatedcellswerenotdetected,theircompoundstendedtoreduce itsmRNAlevels.SB-204900markedlysuppressedthephos-phorylationofp38MAPK.ConclusionOurfindingssuggestthatlan-siumamideBandSB-204900attenuatemastcell-inducedinflammation.NoCOI.


2P-185Triptolide suppresses the adjuvant-arthritis in rats.Saito, Hiroyuki; Ishikawa, Shintaro; Asano, Kazuhito; Gomi, Norihiro; Hisamitsu, Tadashi(Department of Physiology, School of Medicine, Showa University, Tokyo, Japan)

VariousextractsoftheChineseherbalmedicineTripterygiumwilfor-diiHook.f.(TWHF)havebeenreportedtobetherapeuticallyeffectiveforrheumatoidarthritis(RA)inChina,buttheiractionmechanismhasnotbeenunderstood.Althoughtriptolide,aditerpenoidtriepoxidefromTWHF,suppressestheinflammatorycytokineproductioninvitro,theinfluenceoftriptolideonimmunesystemisnotfullyunderstoodwell.Thepresentstudy,therefore,wasdesignedtoexaminetheeffectsoftriptolideoncytokinesproductioninvitroandinvivobyusingadjuvantarthritisrats.Thefirstpartof theseexperimentswasdesignedtoexaminetheeffectsoftriptolideonarthritisrats.AdjuvantarthritiswasinducedinmaleWistarratsbyasinglesubcutaneousinjectionof0.1mLcompleteFreund’sadjuvantintohindpaw.Arthritisratswereinjectedintraperitoneallywithvariousdosesoftriptolideonceadayfor1week,startingoneweekafteradjuvantinjection.Theminimumconcentrationoftriptolide,whichcausedsignificantsuppressionofpawswelling,was0.1mg/kg.Thesecondpartoftheseexperimentswascarriedouttomeasuretheabilityofcytokineproduction,andtran-scriptionfactoraboutthecytokineproductionbylienallymphocytesfromthearthritisratswhichweretreatedwithtriptolide.TriptolidecausedsuppressionofIL-1βproduction in lymphocyteswhichwasactivatedbyConcanavalinA.ThesedatasuggestthatthetherapeuticeffectsofTWHFinRAareinpartduetothenovelchondroprotectiveeffectsoftriptolideviasuppressionofIL-1βproduction.NoCOI.

2P-186Effect of glucosamine on MMP production of the fi-broblast cells derived from the osteoarthritis kneeGomi, N.; Asano, K.; Saito, H.; Yoshida, N.; Suga, H.; Hisamitsu, T. (Dept. Physiol, Sch. Med, Showa Unv. , Tokyo, Japan)

Osteoarthritisoftheknee(kneeOA)isoneoftheimportantdiseasesinthefieldoforthopedics.RecentlyitwasreportedthatglucosaminemightbeeffectiveforkneeOA.Extracellularmatrixsplittingenzyme(MMP)whichisproducedbyinflammatorycell,synoviacellandcar-tilagecellhastheimportantroleforalterationofarticularcartilage.Thisstudywasexaminedtheeffectofglucosaminehydrochloride(GH)onMMPproductioninthekneeOAfibroblasts.ThesubjectswerefivecaseskneeOApatientswhovisitedfortotaljointreplacementsurgeryatShowaUniversityhospital.Intheexperiment,theseparatedfibro-blastsfromarticularsynovialtissuecollectedatthetimeofsurgerywereused.ThefirstpartoftheseexperimentswasdesignedtoobtaintheoptimalconcentrationofTNF-αonthefibroblasts.Inthesecondpartoftheseexperiments,MMPmRNAexpressioninthepresenceofGHwasmeasuredusingRT-PCRmethod,andTIMPproduction,TNF-α-dependentMMPandtranscriptionfactor inthepresenceofGHweremeasuredusingELISAmethods.AstheresultsofstimulationwithTNF-α(2.5ng/ml)fibroblasts,MMPintheculturesupernatant(MMP-2,MMP-3andMMP-13)levelsweresignificantlyincreased.GH(1.0mg/ml)inhibitedTNF-αdependentMMPproductionoffibroblastcells,andhadnoeffectonTIMPproduction.ThenGHdecreasedNF-κBproductionandMMPmRNAexpressioninTNF-αdependent.GHshowedtheproductionofMMPinhibitoryeffectonarticularcartilagedegeneration.ItwassuggestedthatGHintakewasusefulforQOLimprovementofthekneeOApatients.NoCOI.

2P-187Effects of Herbal Medicines in Rats with Chronic Constriction Injury ~ Comparative Study of Yokukan-san and KamishoyosanSuga, Hiroki; Sunagawa, Masataka; Ikemoto, Hideshi; Gomi, Norihiro; Saito, Hiroyuki; Hisamitsu, Tadashi(Dept.Physiol.,Sch.Med.,Showa Univ.,Tokyo,Japan)

Chronicpaincausesstressandpsychiatricsymptoms,suchasanxietyanddepression,;therefore,itisnecessarytoprovidementalhealthcareinassociationwithpainmanagement.Kamishoyosan(KSS)isusedtotreatneuropsychiatricsymptoms.Recently,yokukansan(YKS),whichcontainsaformulasimilartoKSS,hasbeenreportedtobeeffectiveagainstpaindisorders,suchasheadachesandchronicpain.Inthisstudy,theeffectsofthesemedicinesonneuropathicpainandstresscausedbypainwereinvestigatedusingChronicConstrictionInjury(CCI)modelrats.TheCCImodelratswerepreparedaccordingtothemodelofBennett.Twoweeksafterasurgery,adecreaseintheme-chanicalwithdrawalthresholdwasconfirmedintheCCIratsusingthevonFreyTest;thedrugshadbeenadministeredforthistwo-weekperiod.Fourweekspost-operatively,thelevelsofplasmacorticosterone(Corti)andchromograninA(CgA),markersofmentalstress,signifi-cantlyincreasedintheCCIrats.Moreover,thepainthresholdsignifi-cantlydecreasedandtheactivationofspinalastrocytes,whichareinvolvedintheexpressionofchronicpain,wasobserved.TheincreaseinthelevelsofCortiandCgAwassignificantlysuppressedbytheadministrationofYKSandKSS.However,theactivationofastrocytewascontrolledandthedecreaseinthepainthresholdwasreducedfollowingtheadministrationofYKSonly.WeconcludethattreatmentwithYKSeffectivelyreducesbothneuropathicpainandthestresscausedbypain.NoCOI.

2P-188Suppressive effect of Hochuekkito on lung metastasis of B16 melanoma cells in miceYoshida, Yuri; Tamaki, Misako; Ishikawa, Shintaro; Yoshida, Norio; Hisamitsu, Tadashi(The Department of Physiology, School of Medicine, Showa University, Tokyo, Japan)

Hochuekkito(HET)iswellknowntobeoneofKampo(Japaneseherb-al)medicineconsistedoftencomponentherbs,andusedforthesup-plementaltherapyofcancerpatientswithremarkablesuccess.How-ever,theprecisemechanismsbywhichHETcouldfavorablymodifytheclinicalconditionsofcancerpatientsarenotwelldefined.Thepresentstudy, therefore,wasundertakentoexaminethepossibletherapeuticmechanismsofHEToncancerusingexperimentalmousemodel.Inthefirstpartofexpeiment,HETwaswellmixedwithrodentchowatconcentrationsofeither0.1%,1.0%or3.0%,andadministeredorallyadlibitum,whichwasstartedoneweekbeforetumorcellinjec-tionandcontinuedthroughouttheexperiment.OraladministrationofHETatconcentration1.0%and3.0%intoC57BL/6malemicesignifi-cantlyinhibitedtumormetastasisinlungs,whichwasinducedbytheintravenousinjectionof1×105B16melanomacell.Thesecondpartofexperimentwastomeasurethecytokineproductionabilityoflienaland lung lymphocytes fromthefirstexperimentmice.HETcausedincreaseofINF-γproductioninlymphocyteswhichwasactivatedbylipopolysaccharide (LPS).TheseresultsstronglysuggestthatoraladministrationofHETcaused increase intheproductionofIFN-γ,whichisresponsiblefortheactivationofbothNKcellandNKTcell,inthelungsandresultsininhibitionofB16melanomacellmetastasisinthelungs.NoCOI.


2P-189Search of Hyugatouki (Angelica Furcijuga) extract fraction which stimulated melanogenesisFujiwara, Hiroshi1; Atsumi, Toshiyuki2; Ishikawa, Shintaro1; Sato, Takao1; Watanabe, Daishi1; Takashima, Masashi1; Sunagawa, Masakata1; Kakiuchi, Nobuko2; Hisamitsu, Tadashi1 (1Department of Physiology, School of Medicine Showa University, Tokyo, Japan, 2Kyushu University of Health and Welfare, School of Pharmaceutical Sciences, Department of Pharmaceutical Sciences, Miyazaki, Japan)

PurposeAngelicafurcijuga(AF)isanendemicspeciesandperennialherbofJapaneseparsleydepartmentthatgrowswildintheSouthKyushuislandinJapan.Itisfacedwithextinctionbutitsusefulnessisattractedattentionbysuccessoforganicgrow.Wehaveeverreport-edthattheAFextractsfromlobeandstem,stimulatedmelanogenesisinmouseB16MelanomaCell(B16cell)andmousehair,andeffectofmelanogensisinfractionofwaterandethyl-acetatelayer.Inthepresentstudy,ontheeffectsoftheeachextractsolventfractionoftheAF’sextractsfromlobeandstem,melanogenesiswereexaminedusingtheB16cell.MethodsTheB16cellwereculturedwiththeeachfractionofAF’sextracts,anddrugsusceptibilitytestwasconducted.AndthenthemelanincontentandtyrosinaseactivityweremeasuredintheB16cell,theeffecttotheabilityofmelaninproductionwaschecked.ResultsandDiscussionWeobserveddifferenteffectsofeachfractiononmela-nogenesisandtyrosinaseactivity.ThisstudysuggeststhecertainfractionofAF’sextractscontainsthemelaninproductionpromotingsubstances.NoCOI.

2P-190Influence of Palmatine for bone metabolism in OVX mouseTamaki, Misako1,2; Ogawa, Yui1,2; Ishikawa, Shintaro1; Hisamitsu, Tadashi1(1Dept. Physiol, Sch. Med, Showa Unv., Yokyo, Japan, 2Sch. Pharm. Showa Unv., Yokyo, Japan)

Receptoractivatorofnuclearfactor-κBligand(RANKL)orosteopro-tegerin(OPG)wasfoundtobeakeyosteoclastrelationmoleculethatregulatesitscognatereceptor,RANK,onosteoclastprecursorcells.Thepresentstudywasdesignedtoexamineaninhibitoryeffectofthepalmatine,anisoquinolinealkaloidoriginallyisolatedfromCoptis chin-ensis,onanosteoclastdifferentiationinvivobyusingovariectomized(OVX)mice.Thefirstpartofexperimentsweredesignedtoexaminebyhistologicalapproachofshank-proximalobtainedfromOVXmicetreatedwithpalmatine.Theyweredividedintofourgroupsof5miceeach:thesham-operated(Sham),OVX,OVX-palmatineintakegroups(1mg/kg,10mg/kg),randomly.OVXmicewereservedbysondeforc-iblywithvariousdosesofpalmatineonceadayfor12week,startingtwo-weeksafterOVX-operation.ThesecondpartofexperimentswasexaminedthecytokinelevelofserumfromOVXmicetreatedwithpalmatine.ThethirdpartofexperimentswasexaminedtheinfluenceofPalmatinefortheosteoblast-likecell(MC3T3-E1)invitro.Palmatinecausedsignificantsuppressionoftheosteoclastnumberontissue.Inthemicetreatedbypalmatine,RANKLdecreased,andOPGincreased.Theseresultssuggestthatpalmatineattenuatesosteoclastdifferenti-ationthroughinhibitionofRANKLexpression,andactivationofOPGexpressionfromtheosteoblast.Therefore,palmatinemightbeacan-didateforananti-resorptiveagentofdisorderssuchasosteoporosis.NoCOI.

2P-191Antinociceptive Effect of Yokukansan, a Traditional Herbal Medicine, in Rats with Morphine ToleranceIkemoto, Hideshi1; Sunagawa, Masataka1; Takemoto, Mariko1,2; Suga, Hiroki1; Katahira, Haruto1; Kitamura, Atsuko1; Fukushima, Masaya1; Okada, Mayumi2; Hisamitsu, Naoko1; Hisamitsu, Tadashi1(1Department of Physiology, School of Medicine, Showa University, Tokyo, Japan, 2Department of Anesthesiology, School of Medicine, Showa University)

Morphineisoneofthemostcommonlyuseddrugstotreatsevereandchronicpain;however,itschronicuseresultsinantinociceptivetoler-anceaswellasaddiction.Ithasbeenreportedthatglialcellsmaybeinvolvedinthedevelopmentofmorphinetolerance.Inaddition,wepreviouslyreportedthatanherbalmedicine,Yokukansan(YKS),con-trolsactivatedmicrogliainratswithchronicinflammatorypain.Inthepresentstudy,theeffectsofYKSinratswithmorphinetolerancewereinvestigated.MaleWistarrats(7weeksofage)wereinjectedwithmorphinehydro-chloride(10mg/kg)dailyforsevendays.Thehotplatetest (47.5°C)wasusedtoassessthethermalnociceptivethreshold.Thethresholdbegantodecreaseonday3followingtheadministrationofmorphine,andtheantinociceptiveeffectcompletelydisappearedwithinfourdays.However,theadministrationofYKS(manufacturedbyTsumura&Co.)significantlyinhibitedthisdecrease.Moreover,glialcells(microg-liaandastrocytes)wereobservedintheposteriorhornofspinalcordimmunohistochemically.Thegliacellsweresignificantlyactivatedinthemorphine-treatedrats;however,thisactivationwasreducedbytheadministrationofYKS.OurfindingsrevealthatYKShasaneffectinreducingmorphinetol-erancebycontrollingtheactivationsofmicrogliaandastrocytes.NoCOI.

2P-192Spectrum of dose-response relationship for radical scavenging activity of edaravoneTokumaru, Osamu; Shuto, Yachiko; Ogata, Kazue; yokoi, isao(Department of Neurophysiology, Oita University Faculty of Medicine, Yufu, Japan)

Edaravoneisapowerfulfreeradicalscavengerandhasbeenclinical-lyusedforthetreatmentofcerebralinfarction.Butdataconcerningitsspectrumofdose-responserelationshipagainstmultiplefreeradicalsarestillsparseas farastheauthorssurveyed.Thusweevaluateddirectscavengingactivityofedaravoneagainstmultiplefreeradicalsandestimateddose-responsecurvesandEC50’s.Antioxidantactivityonperoxidationinbrainhomogenatewasalsoevaluatedbythiobarbi-turicacidreactivesubstances(TBARS)assay.SixkindsoffreeradicalsweremeasuredbyESR;O-centeredradicals:HO·,O2·-,ascorbatefreeradicals(AFR),t-BuOO·-;N-centeredradicals:NO,DPPHradicals.Freeradicalswerequantifiedbyspin-trappingmethodusingDMPOorCYPMPOandpeakheightofspinadductsrelativetointernalstandardMn2+werecalculated.Edaravonescavengedallfreeradicalsexamined;EC50’sareasfollows;HO·0.4mM,O2·-4mM,t-BuOO·-0.3mM,NO40µM,DPPHradicals5µM(p<0.001).EdaravonesignificantlyinhibitedAFRat5mM(p<0.01),butEC50wasnotsignificant(p=0.098).Anti-oxidantactivityofedaravoneonperoxidation inbrainhomogenatewassignificantagainstHO·andAFRwithEC50’sof60µMand100µ,respectively.Itisconcludedthatedaravonehadabroadspectrumofradicalscavengingactivity.Although itsEC50againsteachradicalvaried,itsantioxidativeactivityis,atleast,partiallyattributabletoitsdirectfreeradicalscavengingactivityagainstmultiplefreeradicals.NoCOI.


2P-193The newly synthesized naftopidil analogue HUHS1015 induces apoptosis in malignant mesothelioma cells by activating caspase-4Kanno, Takeshi; Nishizaki, Tomoyuki(Division of Bioinformation, Department of Physiology, Hyogo College of Medicine, Nishinomiya, Japan)

Theaimofthepresentstudywastoassesstheantitumoreffectofnewlysynthesizednaftopidilanaloguesonhumanmalignantmesothe-liomacelllinesNCI-H28,NCI-H2052,NCI-H2452,andMSTO-211Hcells.Wenewlysynthesized21naftopidilanalogues,andofthem1-[2-(2-me-thoxyphenylamino)ethylamino]-3-(naphthalene-1-yloxy)propan-2-ol(HUHS1015)mosteffectivelyreducedcellviabilityforalltheinvesti-gatedmalignantmesotheliomacell lines inaconcentration (1-100µM)-dependentmanner.TreatmentwithHUHS1015(100µM)for12hmarkedly increasedTUNEL-positivecells inall the investigatedmalignantmesotheliomacelllines,andHUHS1015activatedcaspase-3and-4,withoutactivatingcaspase-8and-9.Theresultsofthepresentstudy,thus,indicatethatHUHS1015inducesapoptosisinmalignantmesotheliomacells,possiblybyactivatingcaspase-4andtheeffectorcaspase-3.ThisraisesthepossibilitythatHUHS1015couldbedevel-opedasapromisinganticancerdrugfortreatmentofmalignantme-sothelioma.NoCOI.

2P-194Spinal neuronal responses elicited by intradermal pruritogen injection in hairless miceUeda, Yuhki1,2; Ishii, Ritsuko2; Uta, Daisuke1; Imoto, Keiji1,3; Furue, Hidemasa1,3(Department of Information Physiology, National Institute for Physiological Sciences, 2Maruho Co., Ltd. Kyoto R&D Center, Kyoto, Japan, 3School of Life Science, The Graduate University for Advanced Studies)

Antipruriticeffectofdrugshasbeenevaluatedbyusingpruritogen-in-ducedscratchingbehavior.Inordertobetterunderstandthedetailmechanismsofantipruriticdrug,weexaminedspinalneuronalrespons-eselicitedbyintradermalinjectionofserotonin,andthetimecoursewascomparedwiththatobtainedfromscratchingbehavior.HR-1hairlessmiceweredeeplyanesthetizedwithurethane.Afterdorsalsurfaceofthelumbarspinalcordwasexposed,extracellularrecordingsweremadefromspinaldorsalhornneurons.Forbehavioralexperi-ments, thenumberofscratchingbehaviorwascountedfor30minafterinjectionofserotonin.Serotonininjectedintheipsilateralpawskin increasedthefrequencyofactionpotentialselicited inspinalneurons,andtheactionwaslastedformorethan20min.Thenumberofscratchingbehaviorwasalsosignificantlyincreasedbyserotoninskininjection,andthetimecoursewasquitesimilartothatofsero-tonin-inducedspinalresponses.Theseresultssuggestthatspinallong-lastingresponseselicitedbyintradermalserotonininjectionmayberelatedtoitch.NoCOI.

2P-195Reduction of TRPV1-mediated pain sensation by TMEM16A inhibitionTakayama, Yasunori1; Uta, Daisuke2; Tominaga, Makoto1,3(1Division of Cell Signaling, Okazaki Institute for Integrative Bioscience, Aichi, Japan, 2Division of Neural Signaling, National Institute for Physiological Sciences, 3Department of Physiological Sciences, The Graduated University of Advanced Studies)

Acalcium-permeablechannelTRPV1isimportantforthepainsensa-tionbecauseTRPV1isactivatedbypungentcompounds includingcapsaicin.Acalcium-activatedchloridechannelTMEM16Aisco-ex-pressedwithTRPV1inDRGneurons.ThissuggeststhatTMEM16AcouldbeactivatedbycalciuminfluxthoughTRPV1activation.TME-M16Aactivationinducesdepolarizationbecausearestingpotentialislowerthantheequilibriumpotentialofchloride inDRGneurons.Therefore,wehypothesizedthatTMEM16Ainhibitoroperatestoreducepain.ToconfirmtheTRPV1-TMEM16Afunctionalinteraction,weperformedwhole-cellpatch-clamprecordings inHEK293Tcells.Thecapsaicin-inducedcurrentswere larger inthecellsexpressingTRPV1andTMEM16Athan inthecellsexpressingTRPV1alone.Moreover,thisinteractiondependedoncalciuminfluxthroughTRPV1activation.Next,weinvestigatedtheeffectofaTMEM16Ablocker(A01)inDRGneurons.A01reducedthecapsaicin-inducedcurrentsbyapproximately50%.ThesefindingssuggestthatTRPV1-mediat-ednociceptioncouldbeinhibitedbyTMEM16Ablocking.Therefore,weinvestigatedcapsaicin-inducedpain-relatedbehaviorsinmice.TimeoflickingandbitingwasshorterwithaconcomitantadministrationofA01withcapsaicinthaninadministrationofcapsaicinalone.TheseresultssuggestthatTMEM16Ainhibitionwouldbeaneffectivetreat-mentofacuteperipheralpaininvolvingTRPV.NoCOI.

2P-196Effects of melatonin administration on motor function recovery after internal capsule hemorrhageIshida, Akimasa; Ueda, Yoshitomo; Masuda, Tadashi; Misumi, Sachiyo; Jung, Cha-gyun; Hida, Hideki(Department of Neurophysiology and Brain Sciences, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan)

Intracerebralhemorrhage(ICH)causesseveredisability.FreeradicalproducedfrombloodmetabolitesisinvolvedinthebraindamageafterICH.Toinvestigatewhethermelatonin(ML),aradicalscavenger,couldreducebraindamagebyICH,ratswereinjectedcollagenase(15Units,1.4µl)intotheinternalcapsulewithML(15mg/kg)thatwasorallyadministratedfor7days.Motorfunctionwasassessedbymotordefi-citscoretest.Survivingcorticospinalneuronswereevaluatedbyretrogradetracerfluorogold(FG)intocervicalcord.Immunostainingof8-hydroxy-2’-deoxyguanisine(8-OHdG)wascarriedout forDNAdamageassessment.Measurementsofmotormapinipsilateralmotorcortexweredonebyintracorticalmicrostimulation(ICMS),inwhichbipolarpulses(0.2ms,0–200µA,333Hz)weregiveninlayerVsenso-rimotorcortextoevoketwitches incontralateral lower leg joints.Behavioral testsrevealedthatMLsignificantlyamelioratedmotordysfunction.Inaddition,moreFG-labeledneuronswereshowninthemotorcortexinML-treatedrats.ICMSrevealedthatMLresultsinlowerthresholdforevokingforelimbmovementthancontrol.Less8-OHdGpositivecellsweredetected inMLinperi-hematomaarea.DatasuggestedthatoralMLadministrationtoICHmodelratsreduc-esthedamageofperi-hematomaareaincludingcorticospinalpathwaybyscavengingradicals,resultinginbetterfunctionalrecovery.NoCOI.


2P-197Long-term feeding of rare sugar D-psicose preserved pancreas beta cells and thus prevented diabetes de-velopment in T2DM model OLETF rats Hossain, Akram1; Yamaguchi, Fuminori1; Sui, Li1; Hirata, Yuko1; Kamitori, Kazuyo1; Dong, Youyi1; Tsukamoto, Ikuko1; Iida, Tetsuo2; Tokuda, Masaaki1(1Department of Cell Physiology, Kagawa University, Kagawa, Japan, 2 Research Institute, Matsutani Chemical Industry Co., Ltd., Hyogo, Japan)

Prevalenceofglobalobesity,mostlyduetoexcesscalorieintakeandinsufficientphysicalactivitiesleadingtooneofitscomplications,type2diabetesmellitus(T2DM)hasbeenincreasingwithalarminghealthproblems.Thiscircumstancedemandshealthscreeningfromearlylifewiththeintakeofage-adjustedbalancedfoodinobese-tendencysub-jects.Weintroduceazero-caloriefoodadditive,D-psicose,asweetraresugarproducedinKagawaUniversityRareSugarResearchCentre.Long-termfeedingofmorethan60weeksof5%D-psicoseindrinkingwaterhasbeenproventocontrolbodyfataccumulationandthuspreventedexcessbodyweightincreaseincomparisontonon-treatedcontrolrats.D-psicoseimprovedinsulinresistancethroughconstantmaintenanceofbloodsugar levelswiththepreservationof insulinproducingpancreasbetacells.Subsequently,serumlevelsofpro-in-flammatoryandanti-inflammatoryadipocytokineswerealsocontrolledwellbyD-psicosedrink.ThesefindingsdemonstratethatD-psicosemightbeapromisinganti-obeseandanti-diabeticagent.NoCOI.

Poster PresentationsMembrane Transport

2P-198Slowing of Mg2+ influx by TRPM7 inhibitors in rat ven-tricular myocytesTashiro, Michiko; Inoue, Hana; Tai, Shinobu; Konishi, Masato(Dept. Physiol.,Tokyo Med. Univ., Tokyo, Japan)

Adultratventricularmyocyteswereisolatedenzymatically.Intracel-lularfreeMg2+concentration([Mg2+]i)wasestimatedusingafluorescentindicatorfuraptra(mag-fura-2).IncubationofthecellsinaMg2+-deplet-ingsolution (high-K+,divalent-free)causedadecrease in [Mg2+]iby0.4–0.6mM.The lowered [Mg2+]iwasrecoveredbyperfusionwithCa2+-freeTyrode’ssolution.Thetimecourseoftherecoverycouldbewellfittedbyasingleexponentialfunction,thefirstderivativeofwhichattime0wasanalyzedasaninitialMg2+influxrate.AdministrationofTRPM7inhibitors,2-Aminoethoxydiphenylborate(2-APB,100µM)andNS8593(10µM)suppressedtheinitialrateofMg2+influx,respec-tively,to43±10%andto12±8.6%.ThesecompoundsalsoinhibitedtherateofNi2+ influxestimatedbyquenchingof furaptrafluorescenceduringtheinitial10min.Thehalfinhibitoryconcentrations(IC50)of2-APBforMg2+-andNi2+-influxrateswerecomparable:17µMand20µM,respectively.TheIC50valuesofNS8593(2.0µMforMg2+influxand4.4µMforNi2+influx)werealsocomparable.WemeasuredMICcurrents (IMIC) likelyviaTRPM7channelsactivatedbyremovalofintracellularandextracellulardivalentcationsunderthewhole-cellpatch-clampconfiguration.IMICat -120mVwas inhibitedby2-APBwithIC50of50µM,andwasreducedto50±12%by10µMNS8593.Thus,Mg2+influxwassignificantlyslowedbyTRPM7inhibitors,whichsupportourpreviousconclusion[JPhyNoCOI.siolSci63:S273,2013]thatTRPM7/MICchannelsserveasamajorphysiologicalpathwayofMg2+influxinratventricularmyocytes.NoCOI.

2P-199Activation of the Na+-H+ exchanger by Ca2+ and cAMP signals during bicarbonate secretion from rat salivary ductsUeno, Kaori1; Hirono, Chikara2; Kitagawa, Michinori2; Sugita, Makoto2; Shiba, Yoshiki2(1Dept. Physiol. & Oral Physiol., Grad. Sch. Biomed. Sci., Hiroshima Univ., 2Dept. Physiol & Oral Physiol., Inst. of Biomed. & Health Sci., Hiroshima Univ., Hiroshima, Japan)

TocharacterizetheNa+-H+exchanger(NHE)activationduringbicar-bonatesecretionfromratsalivaryducts,wemeasuredintracellularpHchangesinducedbyCa2+andcAMPsignals.Ratparotidglandswereremoved,mincedand incubatedwithcollagenasetoseparateaciniandducts.TheintralobularductsegmentswereloadedwiththepH-sensitivefluorescencedye,BCECF.TheintracellularpHchangesweremeasuredwiththeARGUS-HiSCAsystem.Applicationofcar-bachol(CCh)didnotaffecttheintracellularpH,whileforskolin+IBMX(F+I)decreasedthepH.CChrestoredthepHlevelthathadbeenre-ducedbyF+I.InthepresenceoftheNHEinhibitor,5-(N,N-dimethyl)amiloride(DMA),CChandF+IdecreasedtheintracellularpHcom-paredwiththeireffectsintheabsenceofDMA,indicatingthattheNHEwasactivatedduringbicarbonatesecretion inducedbythestimulations.TherateofF+I-induceddecreaseinpHwasgreaterthanthatofCCh-induceddecreaseinthepresenceofDMA,suggestingthattherateofH+generationbyF+IishigherthanthatbyCCh.WhenbicarbonateandH+wereatanequilibriumstateundertheinhibitionofthecarbonicanhydrasebymethazolamide,additionofCChincreasedthepH,whileF+Ididnot.ResultsobtainedsuggestthatCChstrong-lyactivatestheNHEduetothehighpHsetpoint.F+ImayactivatethegenerationofbicarbonateandH+bythecarbonicanhydraseratherthanH+extrusionbytheNHE.NoCOI.


2P-200Adenosine A2B receptor regulates CFTR in pancreatic duct cellsHayashi, Mikio1; Inagaki, Akihiro2; Matsuda, Hiroko1(1Department of Physiology, Kansai Medical University, Hirakata, Japan, 2Department of Physiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan)

Introduction:PancreaticacinisecreteATPandnucleotide-modifyingenzymesthatincludeCD39andCD73.Adenosine,theendproductofATP,stimulatedcysticfibrosistransmembraneconductanceregulator(CFTR)inpancreaticductcells.However,theidentityofadenosinereceptorshasnotbeenextensively investigated. Objectives:Thepresentstudyaimedto identify functionaladenosinereceptors inpancreaticductcells.Methods:Themolecularbasisofadenosinere-ceptorswasrevealedbyRT-PCRanalysisandimmunostaining.Wemeasuredequivalentshort-circuitcurrent(Isc)inhumanadenocarci-nomacellline(Capan-1)monolayer.Results:Capan-1cellsexpressedadenosineA2Breceptor(Adora2b)asreportedpreviously.AdenosineA2BreceptorlocalizedinluminalmembraneofpancreaticductsandCapan-1monolayer.TheluminaladditionofadenosinesignificantlyincreasedIscinCapan-1monolayer.Theeffectwasconsistentwithanionsecretioninepithelia.PSB603,anadenosineA2Breceptoran-tagonist,inhibitedthisincrease.BAY60-6583,anadenosineA2Bre-ceptoragonist, increasedIsc,whichwas inhibitedbyCFTRinh-172.Conclusion:TheseresultsindicatedthattheadenosineA2BreceptorregulatesCFTRinCapan-1cells. Furthermore, theadenosineA2Breceptormaybeinvolvedinaniontransportinpancreaticducts.NoCOI.

2P-201Optimization of a mathematical model of HCO3

- se-cretion by pancreatic duct cellYamaguchi, Makoto1; Yamamoto, Akiko1; Steward, Martin2; Sohma, Yoshiro3; Ko, Shigeru4; Ishiguro, Hiroshi1(1Human Nutrition, Nagoya Univ Grad Sch Med, 2Life Sciences, Univ Manchester, 3Dept Pharmacol, Keio Univ Sch Med, 4Systems Medicine, Keio Univ Sch Med)

Pancreaticductcellproducesisotonicfluidsecretioncontaining~140mMHCO3

-.Wehavebeenconstructingamathematicalmodelofpan-creaticductcellthatvariousiontransporters,channels,andpumpsareallocatedinthebasolateralandapicalmembranesbyusingMAT-LAB/Simulink.Inthepresentstudy,wehavetriedtooptimizethepermeabilityofseveral transporters/channels/pumpsatonetimeusinganalgorithm“fminsearch”whichisbasedontheNelder-Meadmethod.ThepermeabilityvaluestobeoptimizedincludedthoseofNa+-K+pump,K+channel,NBC11Na+-2HCO3

-cotransporter,andAE21Cl--1HCO3

-exchanger inthebasolateralmembrane,andofCFTRanionchannel(PHCO3-/PCl-wassetat0.4)andSLC26A61Cl--2HCO3

-ex-changerintheapicalmembrane.Thevalueswereoptimizedtorepro-ducethepublishedexperimentaldataof interlobularducts isolatedfromguinea-pigpancreas.Thedataincluded(1)intracellularpH,[Cl-],andmembranepotential intherestingandcAMP-stimulatedductsluminally-perfusedwithlowHCO3

-(25mMHCO3--125mMCl-)orhigh

HCO3-(125mMHCO3

-–25mMCl-)solutionand(2)themaximalrateoffluidsecretion(3.5nl/min/mm2epithelium)andfluid[HCO3

-](140mM)intotheclosedducts.Thestandarderrorsofexperimentaldataweresetasacceptablevariationrangesforoptimization.ThepermeabilityvaluesoftransportersweresuccessfullyoptimizedtoreproduceHCO3

-secretionandintracellularparameterswithinacceptableranges.NoCOI.

2P-202Short-chain fatty acid might increase intracellular Ca2+ concentration in rat colonic surface and crypt cellsInagaki, Akihiro(Department of Physiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan)

Short-chainfattyacids(SCFAs)suchasacetate,propionate,andbu-tyratearesynthesizedfromdietarycarbohydratebycolonicbacteriafermentation.TheseSCFAsareconsideredthattheymaycontributenotonlyenergysourceorpreventionofcancerbutalsoregulatingiontransportbyaffectingSCFAreceptor,GPR43.ThisreceptorcouplesG-proteintypeGq(andGi),thuswhenSCFAsbindthisreceptor,intra-cellularCa2+([Ca2+]i)mightchange.However,theseeffectsonlyidentifiedinculturecells.Thus,Iinvestigatedthatwhetherbutyratecanincrease[Ca2+]iinratcolonicsurfaceandcryptcellswithFura-2.Asaresultthatbutyrateshowedelevationof[Ca2+]iincolonicsurfaceandcryptcells.Thisbutyrate-induced[Ca2+]ielevationwasnotinhibitedbyat-ropine,whichshowedinhibitiontheeffectsofacetylcholineoncoloniccryptcellsbutnotsurfacecells.Toevaluatethecontributionofbu-tyrateabsorptionviaNa+-coupledmonocarboxylatecotransporter1(SMCT1)andH+-coupledmonocarboxylatetransporter1 (MCT1), Iinvestigatedtheeffectofibuprofen,SMCT1inhibitor,orα-cyano-4-hy-droxycinnamicacid (α-CHC), MCT1 inhibitoronbutyrate-induced[Ca2+]i increase.Although ibuprofendidn’tshowanyeffects,α-CHCshowedcomparableinhibitiononcoloniccrypt,butnotonsurfacecells.Theseresultsindicatedthatbutyratemightincrease[Ca2+]iwithoutaffectingintracellularbutyratewhichabsorbedviaSMCT1orMCT1.NoCOI.

2P-203Short-chain fatty acid inhibits cAMP-activated short-circuit current in rat distal colonInoue, Rentarou; Inagaki, Akihiro(Department of Physiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan)

Introduction:Short-chainfattyacids(SCFAs)areproducedincolonofomnivore, fromdietaryfiberfermentedbyentericbacteria.ThemainSCFAsincolonareacetate,butyrate,andpropionateandtheyarethoughttobeabsorbedviacolonicepitheliainhibitingcolorectalcancerorprovidedasnutrientsforepithelia.SCFAarealsothoughtthatitreducesCl-secretionandfollowingintestinalfluidflow.Thisregulationmightbe inducedviaSCFAreceptorGPR43oncolonicepithelia,however,ithasnotbeenclearyet.ThuswefocusedonGPR43whichcouplestoGiprotein.Method:WetestedhowSCFAaffectscAMP-activatedCl-secretion,whichmaybeviacysticfibrosistrans-membraneconductanceregulator(CFTR)withincreasingintracellularcAMPconditionwithcolorectalmucosalonUssingChambertomea-sure short-circuit current (ISC). We also tested the effect of4-chloro-α-(1-methylethyl)-N-2-thiazolylbenzeneacetamide(4-CMTB),anagonistofGPR43.Result:ApicaladditionofSCFAwith10mMreducedcAMP-activatedISC.Afterthisprocess,ISCwasnotchangewithaddingCFTRinh-172,aninhibitorofCFTRonapicalside.Ontheotherhand,furtheradditionof5-nitro-2-(3-phenylpropylamino)benzoicacid(NPPB),anonspecificCl-channelinhibitor,showedadditionalreductionofISCandmadeitturntoinitialstate.4-CMTBalsoreducedISCinsimilarwayofSCFA.Conclusion:TheseresultsindicatedthatSCFAsreducedcAMP-activatedCl-secretionbydecliningcAMPviaGPR43and itmayregulateintestinalfluidsecretion.NoCOI.


2P-204LAT1 Is a Critical Transporter of Essential Amino Ac-ids for Immune Reactions in Activated Human T Cells.Anzai, Naohiko; Hayashi, Keitarou; Jutabha, Promsuk(Department of Pharmacology and Toxicology, Dokkyo Medical University School of Medicine, Tochigi, Japan)

SufficientnutrientsupplyisimportanttosupportimmunereactioninTcells.SinceourresultsdemonstratethataspecificinhibitorofL-typeaminoacidtransporter1(LAT1)suppressestheactivationofTcells,LAT1isseemedtobecrucialforimmunefunctionofTcells.PurifiedhumanperipheralbloodTcellswereactivatedwithanti-CD3andanti-CD28antibodyinthepresenceorabsenceofJPH203,aspecificinhibitorofLAT1,andLAT1expression,[14C]-labeledL-leucineuptakeorcytokineproductionwereanalyzed.Theup-regulationofDNA-dam-age-inducibletranscript3(DDIT3)inLAT1-treatedactivatedTcellswasconfirmedbyrealtimePCR.Thereportergenesforwhichex-pressionispromotedbyNF-κBorNFATwereco-transfectedwithaDDIT3expressionvectorintoJurkatTcells,andtheactivityofeachtranscriptionfactorwasdeterminedbyareporterassay.LAT1ex-pressionwasdramaticallyincreasedbyanti-CD3andanti-CD28stim-ulationinhumanTcells,butthisexpressionwasinhibitedbyNF-κBorAP-1inhibitor.Leucineuptakeandcytokineproductioninactivat-edTcellswereinhibitedbyJPH203.DDIT3expressionwasfacilitat-edbyJPH203treatment inactivatedTcells.DDIT3 inhibitedthefunctionofNF-κBandNFAT.TheseresultssuggestthatinductionofLAT1expressionmediatedbyNF-κBandAP-1is indispensabletoachievemaximumincorporationofessentialaminoacidsfornormalimmunereactioninhumanTcells.NoCOI.

2P-205Mechanisms of regulation of clathrin heavy chain CHC22 assemblySakamoto, Kazuho1; Camus, Stephane M2; Torres, Jorge A2; Brodsky, Frances M2(1Department of Pharmacology, Fukushima Medical University School of Medicine, 2University of California San Francisco, CA, USA)

ClathrinheavychainCHC22isrequiredforformationoftheGLUT4storagecompartment(GSC).CHC22controlsproteinsortingfromlateendosomestothetrans-Golginetwork.ItisalsoassociatedwithGLUT-4mobilizationinhumanskeletalmuscle.Wehavecomparedthebio-chemicalpropertiesofCHC22tothoseofCHC17todefinespecies-re-strictedcharacteristicsofthemembranetrafficpathwaythatleadstohumanGSCformation.Cellstarvationcausedreduction inmem-brane-associatedCHC22alongwithitsinteractingadaptormolecules.StimulationofAMPKactivitybyAICARphenocopiedthiseffect,buttreatmentofcellswithrapamycin,anothermTORantagonistdidnot.ThusCHC22membranedissociationresultsfromAMPKactivation,apathwaythatincreasesGLUT4availability.Notablyhowever,insulintreatmentdidnotaffectCHC22membraneassociation.CHC22 ismissingthebindingsequencefortheuncoatingATPaseHsc70,whichinteractswithauxilintouncoatCHC17,andwasnotdisassembledbythisproteincomplex.TheseobservationssuggestthatinsulinsignalingaffectsGLUT4releaseafteritissortedinaCHC22pathway,butthattheCHC22-dependentGLUT4sortingmightbeaffectedbyexercise.Thus,wehypothesizethatduetoitsmembraneassociationpropertiesCHC22contributestoformingahumanGSCthatismorestablethanthemurineGSC,andconsequentlymorepronetoinsulinresistance.NoCOI.

2P-206Estimation of activity of electro-neutral ion transporter by establishing mathematical model of Cl- secretion in epithelial cells based on measurement of transcel-lular ion transport and membrane ion conductanceSasamoto, Kouhei1; Niisato, Naomi2,4; Marunaka, Yoshinori2,3,4

(1Undergraduate Student (4th-year), Kyoto Pref. Univ. Med., Kyoto, Japan, 2Dept. of Molecular Cell Physiology, Kyoto Pref. Univ. Med., 3Dept. of Bio-Ionomics, Kyoto Pref. Univ. Med., 4Japan Institute for Food Education and Health, St. Agnes' Univ.)

Epithelialcellsplaykeyrolesinpreventionofourbodyfromenviron-mentalchangesbyregulatingtransepithelial transportof ionsandwater;e.g., the liquidcoveringepithelialapicalmembranesurfaceproducedbyCl--secretion-drivenwatermovementplaysanessentialroleinprotectionofourbodyfrombacterialandviralinfection.Tran-sepithelial (transcellular)Cl-secretion iscomposedoftwosteps; i.e.,entryandreleasingsteps.Inthisstudy,weestablishedmathematicalmodelsdescribingtranscellularCl-secretionbasedonfollowingthreepathways.(1)theentrystepofCl-intotheintracellularspaceacrossthebasolat-eralmembraneviaelectro-neutraliontransporter(2) thesecretionstepofCl- fromthe intracellularspaceacrosstheapicalmembraneviaCl-channel(3)therecyclestepofCl-acrossthebasolateralmembraneviaCl-chan-nelInadditiontomeasurementsofthetranscellularCl-secretionbyelec-trophysiologicaltechnique(short-circuitcurrent),wemeasuredapical/basolateralCl-conductanceinUssingchamberusingCl-channelblock-er(NPPB).Thepresentstudyenablesustomeasuretheactivityofelectro-neutral iontransporter,NKCC,byelectrophysiologicaltech-niques,providinguswithdeepunderstandingintheresearchfieldofiontransport.NoCOI.

2P-207Regulation of the Cl- extrusion activity in osteoclasts via farnesyl diphosphate synthaseKajiya, Hiroshi; Ohgi, Kimiko; Okamoto, Fujio; Okabe, Koji(Department of Physiological Science and Molecular Biology, Fukuoka Dental College, Fukuoka, Japan)

Farnesyldiphosphatesynthase(FDPS),animportantenzymeinme-valonicacidmetabolismcatalyzestheproductionofgeranylpyrophos-phateandfarnesylpyrophosphate,resultinginincreaseprenylationinsmallGTPproteins.Nitrogen-containingbisphosphonates(NBPs)havewellknowntoinhibitFDPS,leadingtoinhibitboneresorptioninmatureosteoclasts.However,littleisknownwhetherFDPSregulatedirectlythehydrochronicacidtransportersinmatureosteoclasts.WefoundthattheFDPSwerecombinedwithcytoplasmicdomainofClC-7Cl-transporters.TheaimofpresentstudywastoinvestigatetheexpressionandfunctionalroleofFDPSontheextrusionactivityofClC-7Cl-transportersinosteoclasts.FDPSwereexpressedinosteoclastprecursorsandslightlyupregulatedbyRANKLduringosteoclasto-genesis.FDPSwascolocalizedwithClC-7Cl-transportersinmouseosteoclasts.Extracellularacidification inducedoutwardlyrectifyingCl-currentsanddecreasedintracellularCl-concentration([Cl-]i)associ-atedwith inClC-7Cl- transporters inosteoclasts.ZoledronicacidaNBPsuppressedacid-inducedCl-currentsand [Cl-]i reduction inadose-dependentmanner.Incontrast,theinhibitoryactionofzoledron-icacidwasrescuedbyadditionofgeranylgeranylacid,aderivativeofmevalonicacidmetabolism.TheresultssuggestthatFDPSmaycon-tributetoregulatetheboneresorptionactivity,especiallytheClC-7Cl-transporteractivityinmatureosteoclastsbecauseNBPsdirectlysuppressedtheactivityofCl-transporters.NoCOI.


Poster PresentationsOral Physiology (2) / Heart, Circulation

(3) / Muscle Physiology (2)

2P-208Involvemnet of thermosensitive TRP channel in rapid wound healing in oral epithelia.Aijima, Reona1,2,3,4; Aijima, Reona1,2,3,4; Wang, Bing1; Takao, Tomoka1; Mihara, Hiroshi5; Kashio, Makiko5; Ohsaki, Yasuyoshi1; Zhang, Jingqi1; Tominaga, Makoto5; Kido, Mizuho1(1Dept. of Molecular Cell Biology and Oral Anatomy, Grad. Sch. of Dental Sci., Kyushu Univ., Maidashi, Japan; , 2Dept. of Oral and Maxillofacial Surgery, Fac. of Med., Saga Univ., Nabeshima, Japan, 3Div. of Histology and Neuroanatomy, Fac. of Med., Saga Univ., Nabeshima, Japan, 4JSPS Research Fellow, 5Div. Cell Signal., Okazaki Inst. Integr., Okazaki, Japan)

Theoralcavityprovidesanentrancetothealimentarytractandservesasaprotectivebarrieragainstadrasticvariationofstimulifromfoodanddrink.Becauseoftheirlocation,oralmucosaissusceptibletoin-jury.However,woundinoralmucosaisknowntohealfasterthanskinwith littlescar formation.Hereweshowedthattransientreceptorpotentialvanilloid3(TRPV3),athermosensitivecationchannelacti-vatedbywarmtemperatures(>33°C),isfunctionallyexpressedinoralepithelia.WeexploredthephysiologicalsignificanceofTRPV3,andfounddelayedclosureofwoundaftertoothextractioninTRPV3-defi-cient(TRPV3KO)mice.Furthermore,TRPV3expressionwasup-reg-ulatedduringthewoundhealingprocess.WealsofoundthatTRPV3activationincreasedthenumberofproliferatingcellsinprimarycul-turedoralepithelialcellsfromwild-typebutitisnotfoundinthecellsfromTRPV3KO.Interestingly,thenumberofproliferatingcellsinoralepitheliawasalsomarkedlyreducedinTRPV3KOmice.TheseresultssuggestTRPV3inoralepitheliapromotetheproliferationoforalepi-thelialcellsandcontributerapidwoundrepair.NoCOI.

2P-209Functions and localization of a calcium sensor NCS-1 as a mediator of the nuclear calcium regulation in car-diomyocytesNakao, Shu; Nakamura-Nishitani, Tomoe. Y; Wakabayashi, Shigeo(Dept. of Mol. Physiol., Natl. Cer. Cardiovasc. Ctr., Osaka, Japan)

Incardiomyocytes(CMs),cytoplasmiccalcium(Ca2+) isessentialforexcitation-contractioncoupling,whereasnuclearCa2+hasbeenimpli-cated ingeneexpression leadingtohypertrophy.Severalstudiesdemonstratethatakeymoleculefortheregulationofnuclearcalciumlevels([Ca2+]n)isinositol1,4,5-trisphosphatereceptor(IP3R),anintracel-lularCa2+releasechannel.However,precisemechanismofcontrolling[Ca2+]n inCMsremainsunclear.WerecentlyreportedthataCa2+sensorprotein,neuronalcalciumsensor-1(NCS-1),interactswithIP3Rsintheheartandregulatescardiachypertrophy.ToclarifywhetherNCS-1togetherwithIP3Rsare involved inregulationof [Ca2+]n,weexaminedtheeffectsofreceptorstimulationwhichactivatesIP3RsonnuclearCa2+transients,subcellularlocalizationandexpressionpatternsofNCS-1aswellasIP3Rs,andinteractionbetweenNCS-1andIP3RsusingwildtypeandNcs1knockout(KO)mouseCMs.Wefoundthat1)receptorstimulation-inducedelevationof[Ca2+]nwaslowerinKOCMs.2)NCS-1wascolocalizedwithIP3Rsintheperinuclearregion.3)ReceptorstimulationincreasedtheexpressionofNCS-1inthenucleuswhereIP3Rsweremainlyexpressedaswellasinthecytoplasmandmembrane fractions,and4)strengthenedthe interactionbetweenNCS-1andIP3R.TheseresultssuggestthatNCS-1contributestotheregulationof[Ca2+]nthroughdirectmodulationofIP3RactivityintheperinuclearregionofCMs.NoCOI.

2P-210Dysregulation of Cav1.2 Ca2+ Channel by Calpasta-tin was Involved in Myocardial Hypoxic-Ischemic Inju-ryHu, Huiyuan; Sun, Deri; Zhao, Yan; Zhao, Meimi; Liu, Shuang; Guo, Feng; Feng, Rui; Sun, Xuefei; Gao, Qinghua; Hao, Li-Ying (Department of Pharmaceutical Toxicology, School of Pharmacy, China Medical University, Shenyang, China)

OurpreviousstudyrevealedthatcalpastatindomainL(CSL)couldreprimeactivityofCav1.2Ca2+channelfromrun-downincell-freepatches.Recently,itwasreportedthatCSLcanbindtointracellularC-terminalregionofCav1.2channelandregulateitsactivity.However,thepathophysiologicalroleofCav1.2-channel-regulating functionofcalpastatinwaslargelyunknown.Inthisstudy,weinvestigatedthechangesinexpressionandfunctionofCav1.2channelsassociatedwithcalpastatininbothinvitroH9c2hypoxiamodelandinvivomyocar-dial infarction (MI)modelofrats.WefoundthatbothmRNAandproteinlevelsofCav1.2andcalpastatindecreasedatthelatestageofhypoxic-ischemic(H-I)injury.Atthesametime,influxofCa2+inducedbyKClwasattenuatedinH9c2cellsconfrontedtohypoxia.Inaddition,colocalizationofCav1.2andcalpastatinonthecytoplasmicmembranewasobservedinsham-operatedrats,butitwasonlydetectedincyto-solinratsencounteredMI.KnockdownofcalpastatinwithsiRNAinH9c2cellsresulted inbothdecreasedCav1.2 levelsanddepressedCav1.2channelactivity.Takentogether,theseresults indicatethatdysregulationofCav1.2Ca2+channelbycalpastatinwasinvolvedinmyocardialH-Iinjury,whichprovideinsightsthatproperregulationofCav1.2Ca2+channelbycalpastatinwasneededinmaintainingthenormalcardiacfunction.NoCOI.


2P-211Spin-spin relaxation of 1H NMR signals from myofibril suspension of rabbit skeletal muscle with or without ADPOhno, Tetsuo1; Kimura, Masako2; Yamaguchi, Maki1; Takemori, Shigeru1(1The Department of physiology, The Jikei University school of Medicine, Tokyo, Japan, 2Department of Integrative Physiology, Kagawa Nutrition University)

Thedynamicchangesofwatermoleculesstructuresurroundingcon-tractileproteinsmightplayanimportantroleincross-bridgecyclingduringcontraction.Thespin-spinrelaxationprocessof1H-NMRsignalsfromsuspensionofmyofibrilspreparedfromrabbitpsoasmusclecouldbewellrepresentedbythesummationofseveralexponentialsindicat-ingthatwatermolecules inthesuspensioncouldbeconvenientlygroupedintoseveralcomponentsbasedontherelaxationtimeconstant(T2).Theslowesttwocomponentsdominatedoverfasterrelaxationcomponentsatthemyofibrilconcentrationrangestudied.Withincreaseintheconcentrationofmyofibrils,watercomponentthatrelaxedwithT2around0.15sprogressivelyreplacedtheslowestcomponentofT2>0.4s.Anequivolumicpointforthesetwocomponentswasfoundat12mg/mland20mg/mlmyofibrilconcentrationat20°CintheabsenceandpresenceofMgATPrespectively. IntheabsenceofMgATPmyofibrilaffectswatermoleculeswithin500nmfromitssurface,andreleasesmanywatermoleculesinthepresenceofATP.InthepresenceofADPmyofibrilsmayreleasemanywatermolecules.Thisreleasemightplayanimportantroleincross-bridgecyclingduringcontraction.NoCOI.


3P-001Development of light-induced insulin secretion sys-tem using channelrhodopsinMizutami, Takahiro1; Kaitsuka, Taku1; Luo, Huan2; Wei, Fan-Yan1; Song, Wen-Jie2; Tomizawa, Kazuhito1(1Dept. Molecular Physiology, Facult. Life Sci., Kumamoto Univ., Kumamoto, Japan, 2Dept. Sensory and Cognitive Physiology, Facult. Life Sci., Kumamoto Univ., Kumamoto, Japan)

Channelrhodopsin-2(ChR2)isamembraneproteinthathasafunctiontotransportcationintothecellbythestimulationofbluelight.CellsaredepolarizedwhenNa+flowsviaChR2,andactivationofthischan-nelisstrictlyregulatedbylightstimulation.Inthisstudy,wetriedtodevelopthe light-induced insulinsecretionsystemusingChR2.WeconstructedChR2-expressinglentivirusvectorfusedtoGaussialucif-erase,whichgeneratesluminescenceof475nmbluelightregionviacatalyzingcoelenterazine(CTZ).Therefore,weutilizethisluminescenceforChR2activation.Lentivirusesweretransducedintomouseislets,andChR2expressionlevelswereanalyzedbyWesternblottinganal-ysis.Thechannelactivitywasalsomeasuredbyvoltage-clamprecord-ing.Wenextquantifiedinsulinsecretion,andthecellsshowedmod-eratelevelofsecretedinsulinbytheadditionofCTZ.However,thislevelislessthanthatbyhighglucosestimulation.Itmightbeduetolowlevelsofproteinexpressionandmembranetrafficking.FurtherimprovementisneededforsufficientandcontrollableinsulinsecretionviaChR2.Artificiallygeneratedpancreaticβ-cellsfromiPScellsaredesiredforregenerativemedicineofdiabetes.However,potencyofdifferentiatedcellsisshowntobeinsufficient,asresponsebyglucosestimulationisextremelylow.ItispossiblethatoursystemusingChR2maybeausefultoolfortheinductionofinsulinsecretioninthesecells.NoCOI.

3P-002Reproduction of cyclic GMP-dependent Channel Cur-rent under various light intensity by Retinal Rod Pho-totransduction ModelHosoki, Yukari1; Koike, Chieko2; Takeda, Yukari1; Amano, Akira1 (1Department of Life Sciences, Ritsumeikan University, 2Department of Pharmaceutial Sciences, Ritsumeikan University)

Thephototransductionsysteminretinalphotoreceptorcellsconvertslightsignaltoanelectricalsignal.Inthissystem,visualpigmentsac-tivatedbytheincidentlightstimulatetransducinandtheactivatedtransducinraisethecyclicGMP(cGMP)decompositionactivityofphosphodiesterase.ThecGMP-dependentchannelsinretinalphotore-ceptorcellmembranetendtobeclosedbythereducedcGMPconcen-tration. Inaddition, thecGMPconcentrationrecoversthroughthecalcium-dependentguanylatecyclaseactivity.Inthissystem,asinglevisualpigmentmayactivatehundredsoftransducin,amplifyingtheincomingsignal.Efficacyofthesignalamplificationsystemisreportedtobe light intensity-dependent.Inexperiments,positivecorrelationbetweenlightintensityandactivatedtransducinhasbeenobservedata lowstimulationrange,whereasactivatedtransducinstartstodecreaseatveryhighlightstimulation.Inthephysiologicalcondition,succeedingcGMP-dependentchannelcurrentshowsnegativecorrela-tionwithactivatedtransducin.Sincenoneoftheproposedphototrans-ductionmodelcouldreproducethecomplexcharacteristicsof thesignaltransductionsystemofretinalphotoreceptorcell,weproposeamodelwhichcanreproducetherodcurrentofcGMP-dependentchan-nelsundervariouslightintensity.Themodelincludesthelightinten-sity-dependentamplificationofincomingsignalsinrods.Thepresentmodelcouldsuccessfullyreproducetheexperimentaldata.NoCOI.


3P-003Bi-stability of chimeric channelrhodopsinsHososhima, Shoko1,2,3; Ishizuka, Toru1,2; Yawo, Hiromu1,2(1Molecular and Cellular Neuroscience Laboratory, Department of Developmental Biology and Neuroscience, Tohoku University Graduate School of Life Sciences, Sendai, Japan, 2CREST, JST, Tokyo, Japan, 3JSPS, Tokyo, Japan)

[Introduction]Channelrhodopsin1and2(ChR1andChR2)arelight-gat-edcationchannels,eachofwhichhasaseven-passtransmembraneproteinwithacovalentlyboundretinal.Lightabsorptionisfollowedbythephotoisomerizationoftheall-transretinaltoa13-cisconfigura-tionandsubsequentconformationalchangesofthemolecule,whichallowthechannelstructuretobecomepermeabletocations.Recentlyithasbeenexperimentallyevidencedthatthecation-permeablestatesisstabilizedbytheintroductionofpointmutationsinC128orD156ofChR2.Thecounterpartsoftheseaminoacidresidues,C167andD195lieclosetoall-transretinalandformretinal-bindingpocketundercrystallographicstudyofC1C2/channelrhodopsin-widereceiver(ChR-WR),achimericChR.Herewetestedthehypothesisthattheseresiduesarealsoinvolvedinthestabilizationofcation-permeablestateofC1C2/ChRWR. [Method]Theexpressionplasmidsweretransientlytrans-fectedinND7/23cellsusingEffecteneTransfectionReagent(Qiagen).Thephotocurrentwasmeasuredunderconventionalwhole-cellpatchclamp.[ResultsandDiscussion]ThemutationC167orD195positionmarkedlyprolongedtheOFFphaseofthephotocurrentofC1C2/ChRWRorChRFR,anotherchimericChR.Theresultssuggestthattheinteractionbetweenthesepositionsand13-cisretinalisconservedagainsthelixexchangebetweenChR1and2.NoCOI.


3P-004Activation of peripheral mGluR5 contributes thermal and mechanical hyperalgesia via TRPA1 and TRPV1Honda, Kuniya1,2; Iwata, Koichi1(1Department of Physiology, Nihon University School of Dentistry, Tokyo, Japan, 2Department of Oral and Maxillofacial Surgery, Nihon University School of Dentistry, Tokyo, Japan)

Objective:Peripheral tissue injurycausesglutamaterelease fromkeratinocytes,resulting inthermalhyperalgesia.Wehavereportedthatperipheralglutamate injection inducesthermalhyperalgesia.However,itisstillnotunderstoodthemechanismsunderlyinghyper-algesiafollowingperipheralglutamateinjection.Theaimistoclarifythe involvementofperipheralTRPA1,TRPV1andPKCεingluta-mate-inducedthermalandmechanicalhyperalgesia.Methods:Weanalyzednocifensivebehaviorstoheat,coldandmechanicalstimulationfollowingantagonistsadministration1weekaftercontinuoussubcuta-neousinjectionofglutamateintothefacialskin.Next,weexaminedtheexpressionofTRPA1- andmGluR5-immunoreactive (IR) andTRPV1-andmGluR5-IRTGneuronsinnervatedtofacialskin.Moreover,weperformedaneuronalrecordingfromtheTGneuronsfollowingglutamateinjection.Results:Head-withdrawalthresholdtocold,heatandmechanicalstimulation1weekafterglutamate injectionweresignificantlydecreasedcomparedtovehicle-injectedrats,andthedecreasedhead-withdrawalthresholdwassignificantlyrecoveredbyantagonistsadministration.TRPA1-andmGluR5-IRneuronsandTRPV1-andmGluR5-IRneuronswereobservedintheTG.NeuronalactivityinTGneuronswassignificantlyincreasedfollowingglutamatetreat-ment.Conclusions:PresentfindingssuggestthatTRPA1orTRPV1activationthroughmGluR5signalingviaPKCεis involved in facialthermalandmechanicalhyperalgesia.NoCOI.

3P-005Analysis of physiological functions of transient recep-tor potential vanilloid 2 (TRPV2) in adult primary sen-sory neurons using a tissue specific conditional knockout mouse.Katanosaka, Kimiaki1; Takatsu, Satomi2; Mizumura, Kazue3; Naruse, Keiji2; Katanosaka, Yuki2(1Dept. Neurosci. II, Res. Inst. Environ. Med., Nagoya Univ., Aichi, Japan, 2Dept Cardiovasc. Physiol., Grad. Sch. Med., Dent. and Pharmac. Sci., Okayama, Japan, 3Dept. Phys. Ther., Coll. Life Health Sci., Chubu Univ., Aichi, Japan)

TRPV2,isaheat-sensitiveionchannelexpressedinasubsetoftheprimarysensoryneurons(PSN)inadultanimals.Thischannelhasbeenproposedtobeaheattransducer(>52°C)innociceptiveneurons,andrecentlyrevealedtocontributetostretch-dependenteventsindevel-opingperipheralneuronsandothernon-neuronalcells.However,aroleinadultanimalsremainsunclear.Tounderstandthe functionsofTRPV2intheadultPSN,wehaveestablishedaconditionalknockout(cKO)mouselackingTRPV2inPSN,usingtheCre/loxPrecombinationsystem.Usingseveraltypesofbehavioralassaymethods,wefoundthatTPRV2-cKOmicehadimpairedmechanicalnociception,butnor-malheatnociception.Next,weexaminedtheCa2+responseofacutelydissociatedPSNwithFura-2.Intheneuronsfromthenormallitter-mates,about20%ofthecellsshowedstretch-inducedCa2+increaseand12.9%ofthecellswereprobenecid(aTRPV2activator)-sensitive.However,intheneuronsisolatedfromTRPV2-cKOmice,theprobe-necid-sensitivecellsandthestretch-sensitivecells,especiallywithrelativelyhighmechanicalthreshold,werelargelydiminished.TheseresultssuggestthatTRPV2playsanimportantroleinthehigh-thresh-oldstretch-dependentexcitationofthePSNandmechanicalnociceptioninadultanimals.NoCOI.


3P-006Skilled motor training increases synaptic delivery of AMPA receptors at layers II/III synapses in primary motor cortexKida, Hiroyuki1; Tsuda, Yasumasa1; Yamamoto, Yui2; Owada, Yuji2; Mitsushima, Dai1(1Systems Neuroscience Yamaguchi University Graduate School of Medicine Ube, Yamaguchi, 2Organ anatomy Yamaguchi University Graduate School of Med, Ube, Yamaguchi)

SynapticplasticityviaAMPAreceptortraffickingatsynapseterminalsisassociatedwithmemoryandlearning.Toinvestigatetheneuronalmechanismofmotorlearning,weperformedarotorrodtestandmadeacutebrainslicetoanalyzelayersII/IIIneuronsinthemotorcortexusingpatchclampmethod.Motorskillconsistentlyimprovedwithin2daysoftraininginallanimals(n=12).Incurrentclampanalysis,motorlearningsignificantlydecreasedfiringthresholdandincreasedfiringprobability,sincetherestingmembranepotentialsignificantlyincreasedaftermotorlearning(MannWhitneytest,p<0.05).Involt-ageclampanalysis,motorlearningsignificantlyincreasedthefrequen-cyandamplitudeofminiatureEPSC(mEPSC)butnotthoseofminia-tureIPSC(mIPSC).Inaddition,motorlearningproducedasignificantcorrelationintheamplitudebetweenmEPSCandmIPSC(ρ=0.28,p<0.05).TheaverageAMPA/NMDAratioatglutamatergicsynapsesdidnotchangesignificantly,probablyduetoanincreaseofNMDAcurrent.Further,paired-pulseanalysisshowedthatmotor learningdecreasedpaired-pulseresponses(ANOVAandFisher’stest,p<0.05),suggestinganincreaseinpresynapticglutamaterelease.HistologicalanalysiswasalsoperformedtoexamineAMPAreceptorsinspines.TheseresultssuggestthatmotorlearningderivedAMPAreceptordeliveryatexcitatorysynapse in layersII/IIIneuronsofprimarymotorcortex.NoCOI.

3P-007Taste-related aversive behaviors evoked by microin-jection of GABAA receptors agonist into rat ventral pallidumInui, Tadashi; Shimura, Tsuyoshi(Division of Behavioral Physiology, Department of Behavioral Sciences, Graduate School of Human Sciences, Osaka University, Suita, Osaka, Japan)

Previouslyweshowedthataversivetastereactivityresponsestoaconditionedstimulus(CS)intasteaversionlearningweredecreasedbytheblockadeofGABAAreceptorsintheventralpallidum(VP).Inaddition,theintraoralinfusionofaCSelevatedGABAeffluxintheVP.TheseresultssuggestthatVPGABAmediatestheexpressionofaversiveresponses.Inthepresentstudy,weinvestigatedwhetherthestimulationofVPGABAAreceptors inducestaste-relatedaversiveresponses.RatsreceivedmicroinjectionsoftheGABAAreceptorago-nistmuscimol(0,10,100ng/100nl)intotheVP,thentheirbehaviorswerevideotaped.Lowdose(10ng)ofmuscimolevokedingestivebe-haviorssuchasrhythmicmouthmovementsandfloor licking. Incontrast,higherdose(100ng)elicitedalargenumberofchinrubbingandforelimbtreading,whichareknownasaversiveresponsesinthetastereactivityresponsetest.Todeterminethebrainregionsinvolvedintheaversiveresponsestohigherdoseofmuscimol,weexaminedFos-likeimmunoreactivity(FLI)asamarkerofneuralactivationinmultiplebrainsites.Thehigherdoseofmuscimol inducedgreaternumberofFLIinthelateralhabenula,lateralhypothalamus,suprama-mmillarynucleus,andventraltegmentalarea.Itislikelythatthein-creasedactivationofVPGABAAreceptorsaffectedthesebrainregionstocausethealteration inbehavioralexpressionfromingestivetoaversive.NoCOI.

3P-008Role of presynaptic P2X3 receptor in excitatory inputs onto GABAergic inhibitory interneurons in Substantia Geratinosa of rat spinal cordKoga, Keisuke1,2; Tsuda, Makoto2; Inoue, Hidekazu2; Imoto, Keiji1,3; Furue, Hidemasa1,3(1Department of Information Physiology, National Institute for Physiological Sciences, Higashiyama, Myodaiji, Okazaki, Japan, 2Department of Molecular and System Pharmacology, Graduate School of Pharmaceutical Sciences, Kyushu University Fukuoka, Japan, 3School of Life Science, The Graduate University for Advanced Studies (SOKENDAI), Higashiyama, Myodaiji, Okazaki, Aichi, Japan)

Neurons inthespinaldorsalhornprocesssensory informationandtransmit it toseveralbrainregionswhichareresponsible forpainperception.SpinalGABAergicinterneuronsontheotherhandinthesuperficialdorsalhornhaveanimportantroleonmodulationofsen-soryinformation.However,whichkindofafferentfibersinteractwithGABAergicinterneuronsispoorlyunderstood.Here,weexaminedtheeffectofα,β-methyleneATP,aP2X3agonist,onsynapticresponseselicitedinGABAergicneuronsusingwhole-cellpatchclamprecordingsfromspinalcordslicesofvesicularGABAtransporter(VGAT)-venusrats.α,β-methyleneATPincreasedthefrequencyandamplitudeofspontaneousEPSCinvenus-labeledneurons.Inthepresenceoftetro-dotoxin,α,β-methyleneATPalso increasedthefrequencybutnotamplitudeofminiatureEPSC.TheseresultssuggestthatactivationofspinalpresynapticP2X3facilitatesexcitatorysynapticresponsesinGABAergicinterneurons.TheP2X3-mediatedactivationofGABAer-gicinterneuronsmayhaveanimportantroleonspinalmodulationofitchorpaininformation.NoCOI.

3P-009P2X7 receptor in microglia contributes tongue cancer pain in ratsTamagawa, Takaaki1; Honda, Kuniya2; Shinoda, Masamichi2; Yonehara, Yoshiyuki1; Iwata, Koichi2(1Department of Oral and Maxillofacial Surgery, Nihon University School of Dentistry, Tokyo, Japan, 2Department of Physiology, Nihon University School of Dentistry, Tokyo, Japan)

[Introduction]Although,P2X7receptorisimplicatedinbothneuropath-icandinflammatorypain,P2X7receptormechanisminpathologicalpainisunclear.Thus,wedevelopedaratmodeloftonguecancerpain,and investigatedthe involvementofP2X7receptor inthetonguecancerpain.[Materials&Methods]Squamouscellcarcinoma(SCC)cellssubcutaneouslywereinoculatedintothetongue.ThemechanicalorheatstimulusintensityevokingneckEMGactivitywasdefinedasthehead-withdrawalreflexthreshold(HWT)underlightanesthetization.Ondays3,4and14afterSCCcell-inoculation,themicroglialactivationandP2X7receptorexpressionwereimmunohistochemicallyexaminedintrigeminalsubnucleuscaudalis(Vc).WealsoexaminedtheHWTinSCCcells-inoculatedratswithsuccessive intrathecalselectiveP2X7receptorantagonist(A-438079)administration.[Results]TheHWTsig-nificantlydecreasedfollowingSCCcell-inoculation intothetongue.However, therewerenosignificantchanges intheHWTtoheatstimulationinSCCcell-inoculatedrats.Ondays3,4and14,microgliawasactivatedandP2X7receptorlocalizedinactivatedmicrogliainVc.IntrathecalA-438079administrationsuppressedthedecrementofHWTandtheactivationofmicrogliainSCCcell-inoculatedrats.[Conclusion]ThesefindingssuggestthatmicroglialactivationinVcviaP2X7sig-nalingisinvolvedintonguemechanicalallodyniafollowingSCCcellsinoculationintothetongue.NoCOI.


3P-010Identification of cutaneous low-threshold mechanore-ceptors related to inhibition of cardiovascular re-sponses to noxious somatic stimuliWatanabe, Nobuhiro; Hotta, Harumi(Dept. Auton. Neurosci., Tokyo Metropol. Inst. Gerontol.)

Wehavereportedthattouch inhibitedcardiovascularresponsestonoxioussomaticstimuli.Sucheffectsweredependentonthetextureofsurfaceofthetouchingobjects.Weaimedto identifycutaneousmechanoreceptorsthatinhibitsomatically-inducedcardiovascularre-sponses.Singleorafewmultipleunitaryafferentactivitieswerere-cordedfromsaphenousnerveinanesthetizedrats.Touchwasappliedtotheinnerthighfor10minusing1)asoftelastomerbrush(microcone),whichinhibitedsomatically-inducedcardiovascularresponses,and2)asoftelastomerflatdisc(flatdisc),whichdidnotinfluence.Anincre-mentoffiringrateduringeachtypeoftouchwascomparedforeachunit,andadifferencebylessthan10%wasconsideredasnodifference.Atotalof33slowlyadaptingunitswereobtained(Aβ,AδandCfibers;13,12and8units,respectively).Mechanicalthresholdofallunitswaslessthan0.4g.OfAβunits,firingratewasgreaterduringmicroconein5unitsandduringflatdiscin5units,andnotdifferentin3units.OfAδunits,firingratewasgreaterduringmicroconein11unitsandduringflatdiscin1unit.OfCunits,firingratewasgreaterduringmicroconein6unitsandduringflatdiscin2units.MeanincrementsofAβ,AδandCunitsduringmicroconeandflatdiscwere0.55vs.0.80Hz,0.57vs.0.26Hz,and0.31vs.0.17Hz,respectively.Thepresentstudysuggeststhatexcitationofcutaneouslow-thresholdmechanore-ceptiveAδandCunitsmayresponsibleforinhibitionofsomatically-in-ducedcardiovascularresponses.NoCOI.

3P-011Cholinesterase inhibitor improves contrast sensitivity in freely behaving ratsSoma, Shogo1; Suematsu, Naofumi2; Shimegi, Satoshi1,2(1Graduate School of Medicine, Osaka University, Toyonaka, Osaka, Japan, 2Graduate School of Frontier Biosciences, Osaka University, Toyonaka, Osaka, Japan)

Acetylcholine (ACh) isknowntomodulateneuronalactivity intherodentprimaryvisualcortex(V1).WerecentlyexaminedeffectsofamicroionophoreticallyandtopicallyadministeredAChinV1ofanes-thetizedrats,findingthatAChfacilitatedorsuppressedvisualrespons-estovaryingstimuluscontrastsbymultiplyingthecontrolresponses,i.e.responsegaincontrol.TheseACheffectsshowedalaminarbias,wheretheresponsesuppressionandfacilitationprevailedinlayers2/3andlayer5,respectively.Infacilitatedcells,AChimprovedthesignal-to-noise(S/N)ratio,whileinsuppressedcellsitenhancedtheF1/F0ratiowithoutanyconcurrentreductionintheS/Nratio.TheseeffectsonS/NandF1/F0ratioswereobservedinregular-spikingcells,butnotinfast-spikingcells.OurfindingssuggestthatAChpromotesthesignalingofgrating-phase informationfromsupragranularcells tohigher-orderareasbythesuppressivemodulation,andenhancesfeed-backsignalswithahighS/Nratiofrominfragranularcellstosubcor-ticalareasbythefacilitatorymodulation.ToexaminewhethersuchfineregulationofvisualinformationprocessingbyAChcontributestotheimprovementofvisualperformanceinbehavinganimals,wetrainedratstodetectvisualstimulusinatwo-alternativeforced-choicetaskcombinedwithastaircasemethod,findingthatdonepezil,acholines-teraseinhibitor,improvedthecontrastsensitivitydependingonthestimulusconditions.NoCOI.

3P-012Extracellular matrix proteoglycan plays a pivotal role in mechanical sensitization by low pH of thin muscle afferent fibersMizumura, Kazue; Hotta, Norio; Kubo, Asako(Coll. Life Health Sci., Chubu Univ. Japan)

Exerciseleadtotissueacidosis,whichinducesacutemusclepainandmechanicalhyperalgesia.Correspondingtothis,enhancedthin-fiberafferentresponsestomechanicalstimulationhavebeenrecordedinvitroatlowpH.Recentlywehavedemonstratedusingthewholecellpatch-clampmethodthatthelowpH-inducedmechanicalsensitizationinratcultureddorsalrootganglionneuronswereinhibitedbychon-droitinsulfateandchondroitinaseABC,andproposedanovelmecha-nismforsensitizationthatinvolvesextracellularproteoglycan,versican(Kuboetal,J.Physiol.2012).Wethereforeexaminedwhetherthismechanismworksalsointissuelevel.Werecordedsinglefiberactiv-itiesinmuscle-nervepreparationsinvitroobtainedfromSprague-Daw-leyrat.AfteridentifyingsingleAδ-orC-fiberafferents,mechanicalstimulus(from0to196mNin10s)wasappliedtothereceptivefieldonthemuscle.TheresponsemagnitudewassignificantlyincreasedbypH6.2,andthemechanical thresholdwassignificantly lowered.Injectionof5-µlof0.3%chondroitinsulfateneartothereceptivefieldsignificantlyreversedtheloweredthresholdandincreasedresponsemagnitude(n=26).InjectionofpH7.4controlsolutiondidnothaveanysignificanteffects (n=15). Itwasnotpossibletoexaminewhethersensitizedafferentshadversican,butthemajorityofthemareconsid-eredtohaveversicanbasedonthepreviousreportthatover70%ofthinmuscleafferentshaveit.Ourpresentresultsupportsthenovelmechanismforsensitizationthatinvolvesextracellularproteoglycan,versican.COIproperlydeclared.

3P-013Effect of forelimb stimulation on the hindlimb stimula-tion-induced propagating excitation in the rat senso-rimotor cortex detected by optical recording systemHama, Noriyuki; Kawai, Minako; Ito, Shin-Ichi; Hirota, Akihiko(Dept of Physiol, Shimane Univ. Sch of Medicine, Izumo, Japan)

Wehavedevelopedanopticalrecordingsystemusingavoltage-sen-sitivedye.Usingthissystem,wehavereportedthattheneuralexci-tationinducedbyasomaticstimulationspreadsoverthesensorimotorcortex likeapropagatingwave initiated fromthesomatotopicallycorrespondingsite.Inthisstudy,toanalyzetheinteractionbetweenthesepropagatingwaves,weexaminedtheinfluenceofforelimbstim-ulationonthehindlimbstimulation-inducedresponse.Thesensorimo-torcortexincludingthehind-andfore-limbregionswasexposedandstainedwithavoltagesensitivedye(RH-414).Anelectricalforelimbstimulationwasgivenpriortohindlimbstimulation.Theinter-stimulusintervalswere0,20,50,100,200or300msec.Thepropagatingpatternwasanalyzedonthebasisofisochronemaps.Whentheintervalwas0or20msec,propagatingwavesranintoeachothersomewhereinbetweenanddidnotappeartoexceedthecollision line.Whentheintervalwaslongerthan50msec,thepropagatingwaveoftheforelimbresponseshouldhavepassedtheinitiationsiteofthehindlimbresponsebeforeitoccurred.Fortheintervalslessthan100msec,thehindlimbresponse,ifany,wasoverriddenbytheforelimbresponse.Forlongerintervals,i.e.,200or300msec,hindlimbresponsewasobservedbuttheextentofthepropagationareadecreased.Theseresultssuggestthatthesomaticevokedwave,onthewayofpropagation,suppressestheoccurrenceandreducesthepropagationofanotherone.NoCOI.


3P-014Activity is required not for initial formation but for later reorganization of neuronal functions in visual cortexHagihara, Kenta M1; Tagawa, Yoshiaki2,3; Yoshida, Takashi1,3; Murakami, Tomonari1; Ohki, Kenichi1,3(Department of Molecular Physiology, Graduate School of Medical Sciences, Kyushu University, 2Department of Biophysics, Kyoto University Graduate School of Science, 3CREST)

Visualfunctionsofcorticalneuronsareestablishedbyactivity-inde-pendentand-dependentmechanisms.However,thepreciserolesofneuronalactivityinthedevelopmentoftwoprominentvisualfunctions,orientationanddirectionselectivity,arestilldebated.Hereweshowthat inthemouseprimaryvisualcortex (V1), thedevelopmentoforientationanddirectionselectivityproceedsintwosteps,whichdifferintheiractivitydependency.Theresponsivenessandtuningsharpnessofneuronsrapidlymaturewithinonedayofeyeopening,butthereisastrongbiasinthedistributionsofpreferredorientationsanddirec-tions.Wefoundthatthis initialbiasreducesmoreslowlythanthematurationoftuningsharpness.Tostudytherolesofneuronalactiv-ityintheseprocesses,wegeneticallysuppressedtheactivityofneuronsinV1duringdevelopmentonly.Wefoundthatneuronswithsuppressedactivityacquiredresponsivenessandtuningsharpnessnormally,butthebiasreductionwasblocked.Thus,therapidmaturationofrespon-sivenessandtuningsharpnessislargelyactivity-independent,where-asthelaterprocessofbiasreductionisactivity-dependent.Thisresultsuggeststhattheinitialneuronalfunctionsarespecifiedbyactivity-in-dependentmechanisms,suchasgeneticfactors,buttheadultfunctionsarereorganizedbyactivity-dependentmechanismstorepresentmorediverseinformation.NoCOI.

3P-015Characterization and synaptic connections of a new class of neurons in layer II of the cerebral cortexLuo, Huan; Hasegawa, Kayoko; Song, Wen-Jie(Dept of Sensory and Cognitive Physiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan)

Itisnowestablishedthatpyramidalneuronsareexcitatoryneuronsofthecerebralcortex.Whethernon-pyramidalexcitatoryneuronsexistincorticallayersotherthanlayerIV,however,remainstobeelucidated.Herewecombinedmultiplewhole-cellrecordingsandhistologicalmethodstoinvestigatethemorphologicalandfunctionalpropertiesofagroupof layerIIneurons locatedattheborderoflayerIandlayerII.WerefertotheseneuronsaslayerIIroofneurons(L2RN).AllL2RNswereregular-spikingneurons,havinghighlyho-mogenousintrinsicmembraneproperties.ManyL2RNswerenon-py-ramidalinshape.Inpaired-recordings,L2RNs,aspresynapticneurons,alwaysexcitedpostsynapticneuronswithlatencieslessthan1.5msec.L2RNsexcitedlayerIfast-spikingneuronsandreceivedinputsfromotherlayerII/IIIandlayerVneurons.Takentogether,ourresultssuggestthataportionofL2RNsarenon-pyramidalexcitatoryneurons,integratinginputsfrombothdeeplayersandupperlayers.NoCOI.

3P-016Two-photon imaging of lateral inhibition among neu-ronal ensemble in the superficial layer of the superior colliculusKASAI, Masatoshi1; Isa, Tadashi 1,2(1Department of Developmental Physiology, National Institute for Physiological Sciences, Okazaki, Japan, 2Department of Life Sciences, The Graduate University for Advanced Studies, Hayama, japan)

Thesuperiorcolliculus(SC)isabrainstemcenterwhichplaysakeyroleinmediatingthesignalforsensory-motortranslation.Thesuper-ficiallayeroftheSC(sSC)isdirectlyinnervatedbytheoptictractandvisualspaceisrepresentedintheretinotopiccoordinates.Inthevisu-alpathway,firingactivityofneuronsinresponsetothestimulipre-sentedintheirresponsefieldareofteninhibitedbystimulipresentedoutsidetheirreceptivefield.Thiseffectisknownas“lateralinhibition”.Ithasbeendebatedwhetherhorizontal inhibitoryconnectionsareresponsibleforthelateralinhibitioninthesSC;however,thewayofitsneuralimplementationremainselusive,especiallyattheneuronalpopulation level.Therefore,weappliedanin vivotwo-photonCa2+imagingtosSCinanesthetizedmice,andtriedtoelucidateneuronalpopulationactivitiesandthosemicro-circuitmechanisms.First,weexaminedstimulussizetuningpropertiesinthesSC.Thelargerthestimulussize,thelesstheneuralactivityoftheneuronswhichwerelocatedneartheresponsecenter.Second,weconfirmedeitherexcit-atoryorinhibitoryeffectsbytwo-pointstimuluspresentation,whichdependedonthedistancebetweenthetwostimuli.Thesmallersep-arationcausedmutuallypotentiatingresponses.Ontheotherhand,mutualinhibitionwasobservedwhenthetwostimuliwerepresentedwithlargeseparation(>9deginthevisualspace).NoCOI.

3P-017Involvement of rostral ventromedial medulla (RVM) cells in cortex stimulus-induced anti-nociceptive ef-fects in normal and chronic constriction injury ratsKitazawa, Hiromasa1; Kitamura, Taiko1; Yamada, Jinzo2(1Dept of Histology&Neuroanatomy, Tokyo Medical University, Tokyo, Japan, 2Dept of Psychiatry, Kashiwazaki Kosei Hospital)

Motorcortexstimuliprovideanti-nociceptiveeffectsinchroniccon-strictioninjury(CCI)ratsbyspinalcordinhibition.Ithasalsobeenshownthateveninnormalratsspinalcordneuronsreducetheirre-sponsestonociceptivestimulationduringmotorcortexstimuli,buttheprecisemechanismofthisspinalcord inhibitionremainstobeunknown.Inthepresentstudy,wetestedtherostralventromedialmedulla (RVM) involvement inthismotorcortexstimulus-inducedspinalcordinhibitioninnormalratsandinCCIratsmadebyunilat-eralsciaticnerveligation.SingleunitactivityoftheRVMcellwasrecordedwithtungstenmicroelectrodeunderpentobarbitalanesthesia.Priortocorticalstimuli,theRVMcellswereclassifiedintothreegroups,ON-,OFF-,andNeutralcells,basedontheirresponsestonociceptivepinchstimulationappliedatthehindpaw.Corticalstimuluscurrentintensitywasranged30–110µA.WefoundthatinnormalratsRVMcellactivitychangedduringcortexstimuli,andinCCIratsthechangeoftheactivityprolonged.Theseresultssuggestthatanti-nociceptiveeffectsofmotorcortexstimuliarederivedfromtheRVMactivity.NoCOI.


3P-018In vivo analysis of sensory synaptic responses evoked in parasympathetic preganglionic neurons in the rat spinal cord.Hakozaki, Atsushi1,2,3; Imoto, Keiji1,2; Hayashi, Yukio3; Sasaki, Eiji3; Kawatani, Masahito4; Furue, Hidemasa1,2(1Dept Information Physiol, NIPS, Aichi, Japan, 2Sch Life Sci, SOKENDAI, Aichi, Japan, 3TAIHO pharm Co LTD, Ibaraki, Japan, 4Dept Neurophysiol, Akita Univ Grad Sch Med, Akita, Japan)

Coordinatedmovementofthebladder,urethraandexternalurethralsphincterinthelowerurinarytract(LUT)isessentialforthebladderfillingandvoiding.Parasympatheticpreganglionicneurons(PGN)inthelumbosacralspinalcordplayanimportantroleof inregulatingdifferentpelvicorganfunctionincludingmicturitionanddefecation.AlthoughpharmacologicalandbehavioralapproacheshavebeenwellutilizedtostudythespinalcontrolofLUTfunctions, thecellularmechanism,however,isstillunclear.Inthisstudy,wedevelopedanin vivoextracellularrecordingandwhole-cellpatch-clamprecordingtechniquestoinvestigatehowthespinalcordcontrolstheLUTfunc-tionatthesynapticlevel.ThefiringfrequencyofspinaldorsalhornneuronsinthePGNwassynchronouslychangedwiththeintravesicalpressure.TheelectrophysiologicalpropertiesofPGNinL6spinalcordsliceswithanattacheddorsalrootwerealsostudied.MonosynapticEPSCsmediatedthroughAδandCfiberwereelicitedinPGN.Andalsotheseneuronswereexpressedacholineacetyltransferase.Thenewlydevelopedin vivorecordingtechniquesinadditiontothelum-bosacralslice-patchrecordingareusefulforelucidatingthedetailedmechanismforspinalcontrolofLUTfunction.NoCOI.

3P-019Effect of CCL-1 on synaptic transmission in the spinal superficial dorsal hornAkimoto, Nozomi1; Uta, Daisuke1; Honda, Kenji4; Takano, Yukio4; Imoto, Keiji1,3; Noda, Mami2; Furue, Hidemasa1,3(1Department of Information Physiology, Division of Neural Signaling, National Institute for Physiological Sciences, Okazaki, Japan, 2Laboratory of Pathophysiology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan, 3School of Life Science, The Graduate University for Advanced Studies, SOKENDAI, Okazaki, Japan, 4Department of Physiology and Pharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka, Japan)

Cytokinesarewell-knowntohaveeffectsonneurotransmissioninthespinalcord.However, thedetailmechanismhowchemokine (C-Cmotif) ligand1(CCL-1),awell-characterizedchemokinesecretedbyactivatedTcells,modulatesspinalneurotransmissionremainsunclear.HereweexaminedactionofCCL-1onsynaptictransmissioninthespinalsuperficialdorsalhornbyusingslicepatch-clamprecordings.Pain-relatedbehavioraltests,vonFreyfilamenttest,Randall-Selittotestandtailflicktest,showedthatCCL-1inducedmechanicalallody-niaandhyperalgesia,butnotthermalhyperalgesiaafterintrathecalinjectionofCCL-1recombinant.CCR-8,thereceptorforCCL-1,wasdetected inthesuperficialdorsalhornneurons.BathapplicationofCCL-1(50ng/ml)enhancedthefrequencyofspontaneousandminiatureexcitatorypostsynapticcurrentselicited insuperficialdorsalhornneuronsataholdingpotentialof -70mV.Ourresults indicatethatCCL-1increasesglutamatereleasefrompresynapticterminalinthesuperficialdorsalhorn.ThissynapticfacilitationmayaccountforCCL-1-inducedmechanicalallodyniaandhyperalgesia.NoCOI.

3P-020Facilitation of spinal inhibitory synaptic transmission by optoactivation of the locus coeruleus in the brain stemFurue, Hidemasa1,2; Anthony, Pickering E3; Imoto, Keiji1,2 (1Department of Information Physiology, National Institute for Physiological Sciences, Japan, 2School ofLife Sciences, The Graduate University for Advanced Studies, Japan, 3Department of Pharmacology and Physiology, Bristol University, UK)

Locuscoeruleus(LC)neuronsinthebrainstemsendnoradrenergicprojectionsthroughouttheneuroaxisandareimplicatedinthecontrolofmanyhomeostatic functionssuchasarousal,cardio-respiratorycontrol.Inaddition,theLCisalsoamajorsourceofnoradrenergicprojectionstothespinalsuperficialdorsalhornwhichplayasignificantroleinpainmodulation.Underurethane-anesthesia,invivowhole-cellcurrent-andvoltage-clamprecordingsweremadefromspinalsuper-ficialdorsalhornneurons.Bathapplicationofnoradrenalinetothesurfaceofthespinalcordelicitedanoutwardcurrentandfacilitatedinhibitorypostsynapticcurrents.OptoactivationoftheLCneuronsexpressingChR2,however,elicitedabarrageofIPSCsinspinalsuper-ficialdorsalhornneuronswithoutelicitinganypostsynapticslowoutwardcurrents.Slicepatch-clamprecordings fromGABAergicneuronsinthespinalcordshowedthatnoradrenalineexcitesGAB-Aergicneuronsmediatedthroughalpha1receptors.TheseresultssuggestthatactivationofpontospinalnoradrenergicsystemfacilitatesspinalGABAergicneuronstoinhibitnociceptiveinformation.NoCOI.

3P-021Spinal cord imaging during post-ischemic numbness in miceWatanabe, Tatsunori1,2; Komagata, Seiji1; Tsukano, Hiroaki1; Hishida, Ryuichi1; Kohno, Tatsuro2; Baba, Hiroshi2; Shibuki, Katsuei1(1Dept Neurophysiol, Brain Res Inst, Niigata Univ, Niigata, Japan, 2Dept Anesthesiol, Sch Med, Niigata Univ, Niigata, Japan)

Weexperiencenumbnessaftertransient ischemiaofextremities.However,themechanismsareunknown.Inourpreviousstudy,con-ductionblockofperipheralsensorynervesinducedabnormalsensiti-zationofthespinalneuralcircuits.Therefore,itispossiblethatsimilarspinalcordmechanismsmayberesponsibleforpost-ischemicnumb-ness.Totestthishypothesisdirectly,weinvestigatedspinalcordre-sponsesduringpost-ischemicnumbnessusingflavoproteinfluorescenceimaginginanesthetizedmice.Vibratorystimuliappliedtothehindpawproducedfluorescenceresponsesintheipsilateralspinalcord.Vibra-torystimulationappliedtoeachdigitofthehindpawproducedclearlyseparatedandorderedspinalcordresponses.Thetransientischemiawasproducedbyapplicationofhighpressureintoarubbercuffaroundthehindpawfor30min.Theischemiaeliminatedthespinalresponseselicitedbyvibratorystimuliappliedtothehindpaw.However, theresponsesappearedagain,andwerepotentiatedafterremovaloftheischemiacomparedwiththoserecordedbeforethe ischemia. It isknownthattypeIImetabolicglutamatereceptor(mGluR)isresponsi-bleforthechangesinspinalneuralcircuitsafterconductionblockofperipheralnerves.ApplicationofLY354740,amGluRagonist,ontothesurfaceofthespinalcordsuppressedthepost-ischemicpotentiationofspinalresponses.Theseresultsclearly indicatethepresenceofspinalmechanismsunderlyingpost-ischemicnumbness.NoCOI.


3P-022Somatosensory cortical responses after crossing nerve transfer in miceManiwa, Keiichi1,3; Yamashita, Haruyoshi1,3; Tsukano, Hiroaki1; Hishida, Ryuichi1; Shibata, Minoru2; Endo, Naoto3; Shibuki, Katsuei1(1Department of Neurophysiology, Brain Research Institute, Niigata University, Niigata, Japan, 2Division of Plastic Surgery, Niigata University, Niigata, Japan, 3Division of Orthopedic Surgery, Niigata University, Niigata, Japan)

Weinvestigatedcorticalchangesaftercrossingnervetransferofbrachialplexususingflavoproteinfluorescenceimaginginmice.Thedistalcutendsoftheleftmedianandulnarnerveswereconnectedtothecentralcutendsoftherightmedianandulnarnerveswithasci-aticnervegraftat8weeksold.Eightweeksafterthisoperation,S1responseselicitedbyvibratoryleftforepawstimulationwerevisualized.Incontrolmice,directresponses(DRs)mediatedviathalamicinputwasobservedinthecontralateralS1.Weakindirectresponses(IRs)werealsoobservedintheipsilateralS1.Inmicewithcrossingnervetransfer,DRswereobservedintheipsilateralS1,andclearIRswereobservedinthecontralateralS1.TherelativeamplitudesofIRsnor-malizedbythoseofDRsinmicewithcrossingnervetransferweresignificantlylargerthanthoseincontrolmice.ItisexpectedthatDRswereinitiatedbythalamicinputstolayer4,whileIRswereinitiatedbycallosalinputstolayer2/3.Incontrolmice,layerspecificflavopro-teinfluorescenceresponseswereinvestigatedusingamacroconfocalmicroscope.TheDRsinlayer4wereslightlylargerinamplitudeandfasterinlatencycomparedwiththoseinlayer2/3.Forinvestigatingcorticalplasticityaftercrossingnervetransfer,wearegoingtoperformsimilaranalysesonIRsinmicewithcrossingnervetransfer.NoCOI.

3P-023Firing pattern of spinal dorsal horn neurons receiving pruriceptive afferents in the adult rat spinal cordUta, Daisuke1,3; Ando, Tsugunobu2; Kuraishi, Yasushi2; Imoto, Keiji1,3; Furue, Hidemasa1,3(1Division of Neural Signaling, National Institute for Physiological Sciences, Aichi, Japan, 2Department of Applied Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan, 3Department of Physiological Sciences, The Graduate University for Advanced Studies, Hayama, Japan)

Itching isacommonsymptomindermatologicdiseases.Wehavepreviouslyshownbyusingan invivopatch-clamptechniquethatpruriticsynapticresponsesareevoked inspinaldorsalhorn (SDH)neuronsofrats.Topicallyapplicationof5-HTtotheskinincreasedthefrequencyofthelargeamplitudeofspontaneousEPSCsinabout30%oftheneuronsrecorded.Inthisstudy,weexaminedthefiringpatternsoftheSDHneuronsreceiving5-HT-sensitiveafferentfibers.Incur-rent-clampmode,currentpulseswereappliedtoSDHneuronstoelicitactionpotentials.SDHneuronstestedwereclassifiedintofivetypesbasedontheirfiringpatterns.Most(about70%)ofthe5-HT-re-sponsiveSDHneuronswerethedelayedandsustainedrepetitivefiringtypes,although5-HT-unresponsiveneuronswerealsofoundinthesetwotypesofSDHneurons.Therelationshipbetweenthemor-phologicalclassofSDHneuronsandtheirfiringpatternshasprevi-ouslyshownthatverticalcellsexhibitthedelayedorsustainedfiringtype. Incombinationwiththepreviousstudies, thepresentresultssuggestthatpruriceptive5-HT-sensitiveafferentfibersevokeexcit-atorysynapticresponsesmainlyontodelayedfiringandsustainedrepetitivefiringtypeneuronswhicharemorphologicallyclassifiedastheverticalcell.NoCOI.

3P-024Involvement of lysophosphatidic acid-evoked TRPA1 and TRPV1 activation in peripheral itch sensation of miceKittaka, Hiroki1,2; Uchida, Kunitoshi1,2; Fukuta, Naomi1; Saito, Claire1; Tominaga, Makoto1,2(1Department of Physiological Sciences, School of Life Sciences, the Graduate University for Advanced Studies (SOKENDAI), Okazaki, Japan, 2Division of Cell Signaling, Okazaki Institute for Integrative Bioscience (National Institute for Physiological Sciences), National Institutes of Natural Sciences, Okazaki, Japan)

Lysophospahtidicacid(LPA)isabioactivephospholipidreportedasamainmediatorofcholestaticitch,orpruritus,whileitisalsoreportedtoinduceacutepain.WhetherLPAinducesitch,painorbothintheperipheryisunclear.AlthoughhowLPAinducespainhasbeeninves-tigatedindetail,thecellularandmolecularmechanismofLPA-induceditchremainsunclear.Transientreceptorpotentialankyrin1(TRPA1)andvanilloid1(TRPV1)channelsarecalcium-permeablenon-selectivecationchannelsreportedtocontributetotheitchtransductioninpe-ripheralsensoryneurons.WeinvestigatedtheeffectsofLPAontheintracellularCa2+concentrations ([Ca2+]i) inthedorsalrootganglionneuronsobtainedfromwild-type,TRPA1KO,TRPV1KOorTRPA1/TRPV1doubleKOmice,andfoundthatLPAinduced[Ca2+]iincreasesthroughbothTRPV1andTRPA1activation.WealsoobservedLPA-in-ducedTRPA1-mediatedsinglechannelopenings inHEK293TcellsexpressingmouseTRPA1.Furthermore,weobservedLPA-inducedscratchingbehaviorsinacheek-injectionmodel,whichweresignifi-cantlyreducedinTRPA1KO,TRPV1KOandTRPA1/TRPV1doubleKOmice.Thus,weconcludedthatbothTRPA1andTRPV1arein-volvedintheLPA-induceditchsensationinmice.NoCOI.

3P-025Esophageal peristalsis is regulated by capsaicin-sen-sitive intrinsic neural circuit in ratsShiina, Takahiko; Shima, Takeshi; Naitou, Kiyotada; Nakamori, Hiroyuki; Shimizu, Yasutake(Department of Basic Veterinary Science, Laboratory of Physiology, The United Graduate School of Veterinary Sciences, Gifu University, Gifu, Japan)

Thepurposeofthisstudywastoclarifytheroleofalocalneuralreflexconsistingofcapsaicin-sensitiveprimaryafferentneuronsandintrinsicneuronsinesophagealperistalsis.Ratswereanesthetized,andesoph-agealintraluminalpressureandpropelledintraluminalliquidvolumewererecorded.Intheexperimentalsystem,anintraluminalpressurestimulusevokedperiodicchanges in intraluminalpressureof theesophagus,whichwereconsistentlyaccompaniedby intraluminalliquidpropulsion.Bilateralvagotomyabolishedchangesinintralumi-nalpressureaswellasliquidpropulsion.Theseresultsindicatethatthenovelmethodisappropriateforinducingperistalsisintheesoph-aguscomposedofstriatedmuscles.Then,byusingthemethod,weexaminedfunctionalrolesofthelocalreflexinesophagealperistalsis.Theesophagusofcapsaicin-treatedratsshowedamulti-phasicriseinintraluminalpressure,whichmayduetonon-coordinatedcontractionsofesophagealmuscles,whereasamono-phasicresponsewasobservedintheintactratesophagus.Inaddition,destructionofcapsaicin-sen-sitiveneuronsincreasedthepropelledliquidvolumeandloweredthepressurethresholdforinitiatingperistalsis.Theseresultssuggestthatthe localneuralreflexconsistingofcapsaicin-sensitiveneuronsandintrinsicneuronscontributestocoordinationofperistalsisandsup-pressesmechanosensoryfunctionofvagalafferentsintheesophagus.NoCOI.


3P-026Reduction of pain threshold induced by joint immobi-lization modulates the negative affective component of painNoda, Kazuko; Ogata, Masanori; Akita, Hisanao; Ishibashi, Hitoshi (Division of Physiology, School of Allied Health Sciences, Kitasato University, Sagamihara, Japan)

Immobilizationofjointbycastiscommonlyusedforrestingtheinjuredjoint.Howeverthereductionofpainthresholdformechanicalstimuliisofteninducedbycastimmobilizationofthehindlimb.Inthisstudyusingrats,weexaminedwhetherthehypersensitivityinducedbycastimmobilizationaltersthepain-inducedplaceaversionasanaspectoftheaffectivecomponentofpain.Toexaminetheeffectsofcastimmo-bilizationonpainbehaviorsinrats,onehindlimbwasimmobilizedfor2weekswithacastandobservationofpainbehaviorwasconductedaftercastremovalfor3weeks.Awiremeshcastwaswrappedaroundtheonehindlimbtokeeptheanklejointalmoststraight.ThepainthresholdwasmeasuredbyusingcalibratedvonFreyfilamenttestbeforeandaftercastimmobilization.Thejointimmobilizationelicitedthereductionofpainthresholdwhichcontinuedalmostover10dayperiodafteracastremoval.Thenegativeaffectivecomponentofpainwasassessed inratswithcasttreatmentbyusingtheconditionedplaceaversioninducedwiththeintraplantarinjectionofformalin.Theformalin-inducedconditionedplaceaversionwasdecreasedintheratswithacasttreatment.Theseresultssuggestthatthereductionofpainthresholdbyjointimmobilizationfailstoformtheassociatelearningbetweenthepainfulstimulation-inducedaversiveemotionandthepain-conditionedenvironment.NoCOI.

3P-027Effect of thermal therapy on cutaneous pain threshold and muscular pain threshold in healthy subjectsNasu, Teruaki1; Umeki, Sayaka2; Omi, Kaori3; Terui, Naohito1 (1Faculty of Health Sciences, Mejiro Univ, Saitama, JAPAN, 2Department of Rehabilitation Ageo Kousei Hospital, Saitama, JAPAN, 3Department of Rehacare, Funabashi Municipal Rehabilitation Hospital, Chiba JAPAN)

Therearemanypatientssufferingfromchronicmusculo-skeletalpainsuchasthelowbackpain.Intheclinicaltreatmentformuscularpain,thermaltherapysuchashotpack(HP)isfrequentlyusedasoneofthephysical therapies.Althoughsomehypothesesareproposed inanalgesicmechanism,evidenceofeffectsofHPispoor.Furthermore,usuallytreatmenttimeisused20min,buttheeffectivetreatmenttimeisuncertain.Therefore,weexaminedeffectsofHPonthemechanicalnociceptivethresholdsofmusclesandskin.Effectsofdifferenttreat-menttimewerealsoexamined.HPwasappliedontherightlowerlegofhealthysubjects(n=9).UnilateralapplicationofHPfor20minin-creasedmuscularpainthresholdsofbothsides.Theseeffectslastedformorethan20minafterHP.HPapplicationfor5minincreasedmuscularpainthresholdoftheipsilateralsideonlyfor10min.Unliketheeffectsofmuscularpainthreshold,HPapplicationfor20mindidnotaffectcutaneouspainthresholdinbothsides.ThatHPapplicationtoonesidefor20minincreasedmuscularpainthresholdonbothsides,butthattherewasnoeffectsoncutaneouspainthresholdsuggestthatthedescendingpain inhibitorysystemmayactmorestronglytomuscularnociceptionthancutaneousnociceptioninhumans.HPfor5minisnotenoughtodevelopfullactivationofthedescendingpaininhibitorysystem.NoCOI.

3P-028Involvement of MeCP2 in heat sensitivity and inflam-matory pain in tongueSuzuki, Azumi1; Shinoda, Masamichi2; Shirakawa, Tetsuo1; Iwata, Koichi2(1Department of Pediatric Dentistry, Nihon Univ, School of Dentistry, Tokyo, Japan, 2Department of Physiology, Nihon Univ.,School of Dentistry, Tokyo, Japan)

[Introduction]RegulationofgeneexpressioncanbeachievedthroughDNACpGmethylationwhich induceschromatinremodelingandgenesilencingthroughatranscriptionalrepressorcomplexcomprisingmethyl-CpG-bind-ingprotein2(MeCP2).Thoughitiswellknownthatnoxioussensitivitydecreased inRettsyndromepatientswhohavemutations inMeCP2 gene,themechanismremainsunclear.Inthepresentstudy,wedeter-minedifMeCP2wasassociatedwithalteredheatsensitivityofthetonguefollowingcompleteFreund’sadjuvant(CFA)injectionintothetongue.[Methods]CFAsolutionwasinjectedsubcutaneouslyintothetongueinwildtypeandmecp2knockoutfemalemice.Heatstimulation(35–55°C,1°C/sec,cutoff:60°C)wasalsoappliedtothetonguebyusingacontactheatprobeunderlightanesthetization.ThethresholdtemperatureforevokingEMGactivitybyheatstimulationtothetonguewasdefinedastheheathead-withdrawalreflexthreshold(HHWT).[Results]TheHHWTinmecp2knockoutmiceishigherthanthatinwildtype.TheHHWTsignificantlydecreased followingCFAinjection intothetongueinwildtypemice.However,therewerenosignificantchangesintheHHWTfollowingCFAinjectionintothetongueinmecp2knock-outmice.[Conclusion]ThepresentfindingsprovidethefirstevidencethatMeCP2isinvolvedinheatsensitivityandheathyperalgesiafollowingtongueinflammation.NoCOI.

3P-029The effects of music listening on the pain thresholdTomida, Mihoko; Iwasaki, Takami; Ando, Hiroshi; Fujii, Nobumi; Furuta, Tumugu; Asanuma, Naokazu(Department of Oral Physiology, Matsumoto Dental University, Shiojiri, Japan)

Painplaysacrucialroleintransmittinghazardsignalstothebody,butitcausesdiscomfort.Variousstudieshaveshownthatpainper-ceptionisreducedwhenweareconcentratingonsomethingsuchassports.Inthisstudy,weexaminedtheeffectsofmusiclistening(pop-ularmusicorballads)aswellasseveraloralfunctions(biting,chewingandtasting)onpainperception.Forty-fivesubjectsweregivenstim-ulationbyCO2laserontheirankleswitheachcondition.Subjectsas-sessedtheintensionofpainlevelusingvisualanalogscale(VAS)bythemselves.Thethresholdsofpainonoralareas(tongue,buccalmu-cosa,andjawgingiva)wereinvestigatedbypainvisionPS-2100whilethesubjectswerelisteningtopopularorclassicalmusic,orballads.Furthermore,thebloodoxygenationlevel-dependentBOLDsignalsinthecingulatecortexwereanalyzedusingfunctionalmagneticresonanceimaging(fMRI),whentensubjectsweregivenelectricalstimulationof80µAontheirankleswhilelisteningtomusic.IntheVASlevels,wefoundsignificantpainreductionwhenthesubjectswerelisteningtomusic(Shaffe’stest,p<0.001),whereasnosignificantpainreductionwasseenwiththeoralfunctions.Thethresholdsofpainonoralareasweresignificantlyhigherwhenthesubjectswerelisteningtoballadsorclassicalmusicthanthosewithoutmusic(Wilcoxontest,p<0.05).InthefMRIstudy, listeningtomusicattenuatedBOLDsignals inthecingulatecortexin5subjects.Thesefindingssuggestthatlisteningtomusiciscapableofreducingpainperception.NoCOI.


3P-030Repeated forced swim stress enhances CFA-evoked thermal hyperalgesia and affects the expressions of pCREB and c-Fos in the insular cortexImbe, Hiroki; Kimura, Akihisa; Donishi, Tomohiro; Kaneoke, Yoshiki (Department of Physiology, Wakayama Medical University, Wakayama, Japan)

Exposuretostressorscausessubstantialeffectsontheperceptionandresponsetopain.Inseveralanimalmodels,chronicstressproduceslastinghyperalgesia.Theinsular(IC)andanteriorcingulatecortices(ACC)aretheregionsexhibitingmostreliablepain-relatedactivity.AndtheICandACCplayanimportantroleinpainmodulationviadescendingpainmodulatorysystem.InthepresentstudyweexaminedtheexpressionofpCREBandc-FosintheICandACCafterforcedswimstressandCFAinjectiontoclarifychanges inthecerebralcorticesthataffecttheactivityofdescendingpainmodulatorysystemintheratswithstress-inducedhyperalgesia.Forcedswimstress(day1,10min;days2–3,20min)inducedanincreaseintheexpressionofpCREBandc-FosintheanteriorIC(AIC)andACC.CFAinjectionintothehindpawafterthe forcedswimstressshowssignificantlyenhancedthermalhyperalgesiaandinducedadecreaseintheexpres-sionofc-FosinintheAICandtheposteriorIC(PIC).Quantitativeimageanalysisshowedthatthenumbersofc-Fos-IRneuronintheleftAICandPICweresignificantlylowerintheforcedswimstress+CFAgroup(LAIC,95.9±6.8;LPIC,181.9±23.1)thanthoseinthenaivegroup(LAIC,151.1±19.3,p<0.05;LPIC,274.2±37.3,p<0.05).ThesefindingssuggestaneuroplasticchangeintheICafterforcedswimstress,whichmaybeinvolvedintheenhancementofCFA-inducedthermalhyper-algesiathroughdysfunctionofdescendingpainmodulatorysystem.NoCOI.

3P-031Lowered Threshold for Self-Motion Perception to Gal-vanic Vestibular Stimulation (GVS) in Patients with Weather-Related PainAono, Shuichi1; Sakurai, Hiroki1,2; Saito, Aiko1; Toda, Mayumi1; Ushida, Takahiro2; Sato, Jun1,2(1Center for Animal Research and Education, Nagoya University, Nagoya, Japan, 2Multidisciplinary Pain Center, Aichi Medical University, Nagakute, Japan)

Severalclinicalstudieshavedemonstratedaconsistentrelationshipbetweenchangesinbarometricpressureandpainintensityinpatientswithchronicpain.Wehavedemonstratedthatthebarometricpressuresensorlocatedintheinnerear(vestibule)contributestothemechanismofweather-relatedpain.However,ithasnotbeenknownwhetherthevestibuleofpatientswithweather-relatedpainismoresensitivethanthatofhealthysubjects.Thepresentstudy,therefore,aimedtoinves-tigatethethresholdforself-motionperceptiontoGVSinpatientswithweather-relatedpain.Sixteenhealthysubjects(age22–77yrs,mean38yrs),fourteenpatientswithweather-relatedpain (age31–73yrs,mean52yrs),andeightpatientswithweather-independentpain(age25–73yrs,mean53yrs)wereincludedinthisstudy.SinusoidalGVSwasdeliveredbinaurallyviasurfaceelectrodesappliedoverthemastoidsatstimulusfrequen-cies0.5Hz.Thesubjectbecomesslightlydizzy(self-motionperception)withincreasingcurrent,andtheperceptionthresholdwasmeasured.Averagethresholdofsubjectswithweather-relatedpain(1.0±0.2mA,mean±SE)wassignificantlylowerthanthatofweather-independentpaingroup(2.2±0.3mA)andhealthycontrolgroup(3.1±0.4mA).Theseresultsshowedthatpatientswithweatherrelated-painaresensitivetovestibularinputs.NoCOI.

3P-032Thermal preference test in rat orofacial formalin mod-elSugimoto, Mariko; Miyazawa, Yuta; Takahashi, Yukari; Kato, Fusao (Dept Neurosci, Jikei Univ. Sch. Med., Tokyo, Japan)

Theaffective/emotionalcomponentofpainhelpsanimalstochangetheirbehavioralprogramstoavoidpotentiallyaversiveandharmfulsituations inthe future.Suchfunctionofpaindependsoncentralplasticityandmakesitastrongdriverofnegativeemotionthatformsbasisforpainsufferinginchronicpainpatients.However,incontrasttothehumansubjectsthatreportthestateofpain“also”inaverbalform,objectiveevaluationofpainaffectinanimalshasbeendifficult,comparedtothatofnocifensivebehaviors,suchasthepawwithdrawandreflexogenicescape,widelyuseduntiltoday.Recently,evaluationofplaceaversionorpreferenceusingthenociceptiveoranalgesicexperiencesasunconditionedstimuliforPavlovianlearningisusedtoaddressthisissue,assumingthatthedegreeoflearningrepresentsthedegreeofpainsuffering.Todevelopamethodformoreobjectiveevaluationofspontaneouspaininanimals,weemployedtwo-tempera-turepreferenceparadigm.Ratswereallowedtomovefreelybetweentwoequivalentplatesofdifferenttemperaturesthatwerenear-aversiverangeforsomeperiodswithin30minofobservation.Weoptimizedtheprogrammedchangesinplatetemperaturessothattheratsspon-taneouslymovebackandforthbetweenthetwoplatesinahighlyreproduciblemannerandevaluatedtheeffectsofchronificationprocessofinflammatorypain(6to24hoursafterorofacialformalininjection)onthetimeandfrequencyspentincolderplate.Theresultssuggestthatthechronifiedinflammatorypainaffectsthetemperature-depen-dentdecision-makingprocess.NoCOI.

3P-033Efficacy of electro-acupuncture for paclitaxel-induced peripheral neuropathyIshiakwa, S.; Sunagawa, M.; Murata, K.; Tawaratsumida, Y.; Hisamitsu, T.(Dept. Physiol, Sch. Med, Showa Unv., Yokyo, Japan)

Paclitaxelisamicrotubule-bindingcompoundthatiswidelyusedasachemotherapeuticinthetreatmentofcommoncancers,includingbreastandovariancancer.Howeverpaclitaxelhasneurotoxiceffect,andpaclitaxelexposurefrequentlycausesperipheralneuropathyatasideeffect.TheSDratswererandomlydividedintothreegroups;thePa-clitaxelgroup(PTX),thePaclitaxelandacupuncturegroup(PTX-A),andthecontrolgroup.Allratswereinjectedintraperitoneally(i.p.)onfouralternatedays(days1,3,5,and7)withvehicle(saline)or2.0mg/kgpaclitaxel.Electro-acupuncturewhichcausedslightmuscletwitchwasappliedtotheZuSanliacupoint(S36)inthelimbsoneveryotherday(rightside,1Hz,20min.,3–5V).Behavioralassayswerecarriedoutbytheheat-hyperalgesiatestandmechano-allodyniatestofthehindpaws(sciaticnerveterritory).Allratsweresacrificedonday28,andthelumbosacralvertebralcolumn,andthesegmentofthesciaticnervefrommid-thighwerecollectedforlightmicroscopyexamination.PTXgroupproducedsignificantmechanicalallodyniainboththehindpaws.HoweverthePTX-Agroupdidnotshowthedecreaseofthemechan-ical threshold.Additionally, thedifferenceofheat-hyperalgesiaandaxonalβ-tubulinwasnotshownbetweenthePTXgroupandthePTX-Agroup.Inconclusion,ourstudysuggeststhataacupuncturestimulationrelievedtopaclitaxel-inducedpainfulperipheralneuropathy.ItwassuggestedthatsatellitecellsintheDRGmightplayaroleinthedevelopmentofpaclitaxel-inducedpainfulperipheralneuropathy.NoCOI.


3P-034Neural correlates of a target position represented in a background coordinate: an f-MRI studyUchimura, Motoaki1,2,5; Nakano, Tamami1,2,3; Morito, Yusuke3; Ando, Hiroshi3,4; Kitazawa, Shigeru1,2,3(1Dynamic brain laboratory, Grad School of Frontier Biosciences, Osaka University, 2Physiology 1, Grad School of Medicine, Juntendo University, 3Center for Information and Neural Networks (CiNet), National Institute of Information and Communications Technology, and Osaka University, 4Multisensory Cognition and Computation Laboratory National Institute of Information and Communications Technology, 5JSPS)

Ourbrainrepresentsatargetpositionintermsofourbodyparts,likeinretinalorcraniotopiccoordinates.However,recentstudieshaveshownthatatargetpositioncanberepresentedintermsofaframeinthebackground.Inthisstudy,weaimedtodissociateneuralcor-relatesofthebackgroundcoordinatesfromtheretinalandcraniotopiccoordinatesbyusinganf-MRIadaptationtechnique.Atargetwaspresentedatdifferent locationsonascreen, incombinationwitharectangularframethatwasalsopresentedatdifferentlocations,whileparticipantslookedatafixationcross.Whenatargetwaspresentedatthesamelocationinthebackgroundcoordinate,significantdecreas-esoff-MRIsignals(duetoadaptation)wereobservedinthebilateralprecuneus,therightdorsalpremotorcortex,therighttransverseoc-cipitalsulcus,andtherightmiddletemporalcortex.Ontheotherhand,noregionshowedadaptation,whenatargetwasrepeatedlypresentedintermsoftheretinalorcraniotopiccoordinates.Theseresultsclear-lyshowthatneuralcorrelatesofthebackgroundcoordinateisdistinctfromthoseoftheretinalandcraniotopiccoordinates,andreside inseveralareasincludingtheprecuneus.NoCOI.

3P-035Possible plasticity of the Stevens' power lawNishi, Eri1; Tsuchiya, Saki1; Nishikawa, Yuri1; Tokumaru, Osamu2; Eshima, Nobuoki3; Harada, Chizuru4; Yokoi, Isao2(1Nursing student, School of Nursing, Oita University Faculty of Medicine, Yufu, Japan, 2Depertment of Neurophysiology, Oita University Faculty of Medicine, Yufu, Japan, 3Depertment of Statistics, Oita University Faculty of Medicine, Yufu, Japan, 4Depertment of Fundamental Nursing Sciences, Oita University Faculty of Medicine, Yufu, Japan)

ObjectiveThesubjectivemagnitudeofsensationgrowsasapowerfunctionofthestimulusintensity,knownastheStevens’powerlaw(1957).Thepowerlawwasstudiedforvisualandauditorysensationsasapracti-cumfornursingstudents.MethodsSubjectswereinstructedtoassignnumbersproportionaltotheap-parentmagnitudeoflight(brightness)andsound(loudness)andtheapparentfrequencyofsound(tone)stimulipresented(n=50;Match).Thesubjectswere instructedtoadjustbrightness,soundandtoneuntilitseemedtomatchthenumbergiven(n=45;invMatch).Inversematchingwasstudiedafter“trainingsession”usingstimulatorswithexponents>2(n=7;invMatch/Tr).ResultsInMatch,exponentsforbrightness,loudnessandtonewereestimatedtobe0.25,0.31and0.69,respectively(p<0.001).IninvMatch,exponentswere0.91,0.71and0.82,respectively(p<0.001).ExponentsininvMatch/Trwere1.18,1.14and0.89(p<0.001).TheexponentforloudnesswassignificantlylargerininvMatch/TrthanininvMatch(p=0.013).DiscussionThethreemodalitiesobeyedtheStevens’powerlawinMatch.Theyalso followedthepower lawin invMatch,but it is interestingthatexponentssignificantlydifferedaftertraining.Theseresultsmayin-dicatepossibleplasticityofthepowerfunction.NoCOI.

3P-036Effect of dopamine on excitatory synaptic transmis-sion in the rat spinal parasympathetic preganglionic nucleiIkeda, Azusa1; Imoto, Keiji1,2; Furue, Hidemasa1,2(1Dept. Information. Physiol. NIPS, Okazaki, Japan, 2Sch. Life. Sci. SOKENDAI, Okazaki, Japan)

DopamineisknowntomodulatesynaptictransmissionoverwideareasoftheCNS.Dopaminergicneuronsinnervatetheiraxonsalsointothelumbosacralspinalcord,andarethoughttocontrolactivityofviscer-alorgans.Inthisstudy,weexaminedhowdopamineactsonexcitato-rysynapticresponsesevokedinparasympatheticpreganglionicnucleineurons in theadultrat lumbosacralspinalcord.Whole-cellcur-rent-clampandvoltage-clamprecordingsweremadefromparasym-patheticpreganglionicnucleineuronsinspinalcordslices,anddopa-minewasbath-applied for1–2min.Parasympatheticpreganglionicnucleineuronshadarestingmembranepotentialmorenegativethan-60mV.Undervoltage-clampconditionsataholdingpotentialof-70mV,parasympatheticpreganglionicnucleineuronsexhibitedspontaneousexcitatorypostsynapticcurrents.Dopaminedose-dependentlyde-creasedthefrequencyofspontaneousexcitatorypostsynapticcurrentsinmostoftheparasympatheticpreganglionicnucleineuronstested.Thepresentresultssuggestthat inhibitoryactionofdopamineonspinalexcitatorytransmissionmayhaveanimportantroleonpara-sympatheticcontrolofvisceralorgansbydopamine.NoCOI.

3P-037Effects of Vasopressin on reciprocal synaptic trans-mission between mitral and granule cells in the mouse accessory olfactory bulbTaniguchi, Mutsuo; Namba, Toshiharu; Kaba, Hideto(Dept. Physiol., Kochi Medical School, Kochi University, Kochi, Japan)

Centralvasopressinfacilitatessocialrecognitionandmodulatesnumer-ouscomplexsocialbehaviorsinmammals.Recentanalysisoftrans-genicratsengineeredwithanenhancedgreenfluorescentproteinreporterforvasopressinsynthesisidentifiednewpopulationofvaso-pressinneuronsintheaccessoryolfactorybulb(AOB).TheAOBhasbeendemonstratedtobeacriticalsiteformating-inducedmaterec-ognition(olfactorymemory)infemalemice.Abehavioralstudyrevealedthat inactivationofthevasopressin1breceptorgene impairedtheolfactoryblocktopregnancy.Theeffectofvasopressin,however,onthesynaptictransmissionbetweendendrites intheAOBoffemalemiceislargelyunknown.Toaddressthisissue,weexaminedtheeffectofvasopressinonthereciprocaltransmissionbetweenmitralandgranulecellsbystimulat-ingamitralcellandrecordingtheevokedIPSCsfromthesamecellwiththepatch-clamptechniqueinwhole-cellconfiguration.AOBslic-eswerepreparedfrom23-to36-day-oldBalb/cfemalemice.ToevokeIPSCs,adepolarizingvoltagestepfrom-70to0mVwasappliedtoamitralcell.InMg2+-freesolution,vasopressinsignificantlyblockedtheIPSCs.ThesuppressiveeffectofvassopressinontheIPSCswasdiminishedbyanantagonistforvasopressinreceptors(AVRs),[β-mercapto-β,β-cyclopen-tamethylenepropionyl-O-me-Tyr,Arg]vassopressin.Thepresentre-sultssuggestthatAVRsareinvolvedinreciprocaltransmissionbe-tweenmitralcellsandgranulecellsinthemouseAOB.NoCOI.


3P-038A role for vasopressin in synaptic plasticity in the ac-cessory olfactory bulb of male miceNamba, Toshiharu; Taniguchi, Mutsuo; Murata, Yoshihiro; Okutani, Fumino; Kaba, Hideto(Department of Physiology, Kochi Medical School, Nankoku, Kochi, Japan)

Theaccessoryolfactorybulb(AOB)hasbeendemonstratedtobeacriticalsiteformating-inducedmaterecognitioninfemalemice.How-ever,arolefortheAOBofmalemiceinsocialrecognitionislargelyunknown.ToinvestigatethefunctionalimplicationsofthemaleAOB,weexaminedtheeffectofvasopressinonsynapticplasticityofgluta-matergictransmissionfrommitraltogranulecellsinAOBslicesfrommalemice.Thestrengthofsynaptictransmissionfrommitraltogran-ulecellscanbeanalyzedbylateralolfactorytract(LOT)-evokedfieldpotentialsinparasagittalslices.WemeasuredthemaximalinitialslopeoffieldEPSPsofgranulecellstomonitorthestrengthofglutamater-gictransmissionfrommitraltogranulecells.Whensub-thresholdLOTstimulationthatonlyproducedshort-termpotentiationwaspairedwithvasopressin,robustLTPwasinduced.Next,weexaminedtheeffectofvasopressinonthereciprocal transmissionbetweenmitralandgranulecellsbyusingwhole-cellpatch-clamptechniques.VasopressinsuppressedreciprocalsynapticcurrentstriggeredbyendogenousglutamatereleasefrommitralcellsintheAOB.ThisstudysuggeststhatvasopressinfacilitatesLTPinductionatthemitral-to-granulecellsynapseinthemaleAOBbyreducingdendrodendriticinhibition.NoCOI.

3P-039Functional MRI during odorant induced olfactory stim-ulation revealed brain activation with 7 Tesla MRIFukami, Hideyuki1; Horie, Sawa1,2; Higuchi, Satomi3; Sasaki, Makoto3; Sahara, Yoshinori1(1Dpt. of Physiol., Iwate Med. Univ., Sch. Dent., Iwate, Japan, 2Dept. Tumor Biol., Inst. for Biomed. Sci., Iwate Med. Univ., Iwate, Japan, 3Div. Ultrahigh Field MRI, Inst. for Biomed. Sci., Iwate Med. Univ., Iwate, Japan)

Piriformcortexformsthemainpartoftheprimaryolfactorycortex,yetitsprecisefunctionalrolewithinthebrainremainsunclear.Previ-ously,functionalmagneticresonanceimaging(fMRI)hasbeenusedtoassessodor-inducedpiriformcortexactivationinhumans,howevertheresultshavebeeninconsistent.Twomainfactorswhichareattributedtothisinconsistencyare:1)BOLD-signalchangesevokedbyolfactorystimuliarecomparativelysmall,and2)brainregionsinvolvedinolfac-tiontendtobesusceptibletospatialdistortionandsignallosswithintheimage.InordertoincreaseBOLD-signalstrength,wehypothesizedthattheuseofultrahighfield(7Tesla)MRImaybebeneficial.Inthisstudy,weoptimizedanecho-planarimagingpulsesequenceforfMRIusinga7TMRI(GE)systemtominimizesusceptibilityartefacts,andtoinvestigateodorantinducedolfactorycortexBOLDsignalactivation.Odorantstimulation(isovalericacid,peppermintandcoffeeodor)in-ducedactivationsinthepiriformcortex,amygdala,orbitofrontalcortexandhippocampus.Weobserveddifferences inthespatialextentofBOLDsignalactivationforeachodorant,withsomespatialoverlapwithinthepiriformcortex.Theseresultssuggestthatthepiriformcortexplayan importantrole forodordiscrimination,andthattheodorqualitymaybemappedinthepiriformcortex.NoCOI.

3P-040Inhibitory action of 6-methylthiohexyl isothiocyanate on nasal inflammatory responses induced by tolu-ene2, 4-diisocyanate in ratsAsano, Kazuhito; Ishikawa, Shintarou; Yoshida, Atsumasa; Hisamitsu, Tadashi(Department of Physiology, School of Medicine, Showa University, Tokyo, Japan)

Theinfluenceof6-methylthiohexylisothiocyanate(6-MTITC)extract-edfromWasabia japonicaonallergicrhinitis inducedbytoluene2,4-diisocyanate (TDI)wereexamined inF344rats.F344malerats(4-weeksofage)weresensitizedbyintranasalinstillationof10%TDIonceadayfor5days.Theseratswereexposedtovariousconcentra-tionsof6-MTITCfor4hours/dayfor5consecutivedays,whichwasstarted10daysafterfinalTDIinstillation.Nasalsymptoms,sneezesandnasalrubsfor10minutes,inducedby10%TDInasalchallengewereevaluated24hoursafter6-MTITCexposure.6-MTITCat0.1%butnot0.05%and0.01%couldattenuatetheappearanceofnasalsymptomsanddecreasedthenumberofbothsneezesandnasalrubs.WethenexaminedtheconcentrationsofsubstanceP(SP),calcitoningene-relatedpeptide(CGRP)andnervegrowthfactor(NGF)innasallavagefluidsobtainedfromTDI-sensitizedand6-MTITC-inhaledF344rats60minutesafterTDIchallenge.ExposureofTDI-sensitizedratsto0.1%6-MTITCcouldsuppresstheincreaseinSP,CGRPandNGFlevelsinnasallavagefluids,whichwereincreasebyTDIchallenge.Theseresultsclearlyshowedthatexposureto6-MTITCcouldatten-uatethedevelopmentofallergicnasalsymptomsthroughthesuppres-sionoftheproductionofneuropeptides,suchasSP,CGRPandNGF.It isalsosuggestthataromatherapywith6-MTITCwillbeagoodcandidateforthetreatmentofallergicrhinitis.NoCOI.

3P-041Involvement of actin polymerization in long-term po-tentiation at synapses in the mouse accessory olfac-tory bulbMurata, Yoshihiro; Kaba, Hideto(Department of Physiology, Kochi Medical School, Nankoku, Kochi, Japan)

Microcircuitsinthemouseaccessoryolfactorybulb(AOB)includetheprominentreciprocaldendrodendriticsynapsebetweenmitralcellprojectionneuronsandgranulecellinterneurons.Long-termpotenti-ation(LTP)attheAOBsynapsesisexpectedtounderliepheromonalmemoriesinfemalemice.TheformationofLTPisthoughttoinvolvethereinforcementofsynapticconnectionsthatdependsonproteinsynthesis,andthechangesinthestructureofsynapticconnectionsthatresult fromactincytoskeletonreorganization.Inthepreviousmeetings,wereportedthatLTPattheAOBsynapsesrequiressyn-thesisofproteins,oneofwhichwouldbeatypicalproteinkinaseC.HereweexaminedwhetherLTPattheAOBsynapsesalsorequiresactinpolymerization.UsingAOBslices,wemeasuredfieldEPSPde-rivedfromgranulecellstoexaminetheeffectsofsomereagentsthatmodifyactinpolymerizationontheLTP.Bathapplicationofanactinpolymerizationinhibitor,cytochalacinD,blockedtheLTPinducedbythetetanicstimulation(100Hz×4),buthadnoeffectsontheshort-termpotentiation.Underbathapplicationofanactinpolymerizationactivator,jasplakinolide,LTPisinducedbythetetanicstimulation(100Hz×2)thatelicitsonlyshort-termpotentiation.Theresultsarecon-sistentwiththehypothesisthatactinpolymerizationisrequiredfortheformationofLTPattheAOBsynapse.NoCOI.


3P-042Hard-diet and soft-diet feeding change neurogenesis in the subventricular zone and olfactory functionsKashiwayanagi, Makoto1; Utsugi, Chizuru1,2; Miyazono, Sadaharu1; Osada, Kazumi3; Sasajima, Hitoshi1; Noguchi, Tomohiro1; Matsuda, Mitsuyoshi2(1Department of Sensory Physiology, Asahikawa Medical University, Asahikawa, Japan, 2Department of Oral and Maxillofacial Surgery, Asahikawa Medical University, Asahikawa, Japan, 3Department of Oral Biology, School of Dentistry, Health Sciences, University of Hokkaido, Tohbetu, Japan)

Thesubventricularzone (SVZ)generatesan immensenumberofneurons,whichmigratetotheolfactorybulb(OB),evenduringadult-hood. Recentstudieshaveshownthatareductionofmasticationimpairsbothneurogenesis inthehippocampusandbrainfunctions.IngestionofharddietinducedhighernumberofexpressionofFos-ircellsattheprincipalsensorytrigeminalnucleus(Pr5)offemalemicethanthoseofsoftdietornodietdid,suggestingthatsoft-dietfeedingmimicimpairedmastication.After1month,thedensityofBromode-oxyuridine-immunoreactivecells,newlygeneratedcells, intheSVZwas lower inthesoft-diet-fedmicethan inthehard-diet-fedmice.Avoidancebehaviorstobutyricacidwerealsoreducedbythesoft-di-etfeeding.Wethenexploredtheeffectsofthehard-dietfeedingonolfactoryfunctionsandneurogenesisintheSVZofmiceimpairedbysoft-dietfeeding.At3monthsofhard-dietfeeding,avoidancebehaviortobutyricacidandresponsestoodorswererecovered,aswasneuro-genesisintheSVZ.ThepresentresultssuggestthatfeedingwithaharddietimprovesneurogenesisintheSVZ,whichinturnenhancesolfactoryfunctionattheOB.NoCOI.

3P-043Orexin neurons are involved in antinociceptive effect of olfactory inputTashiro, Shogo1,2; Kashiwadani, Hideki1; Kanmura, Yuichi2; Kuwaki, Tomoyuki1(1Dept. Physiology, Grad. Sch. Med. and Dent. Sci., Kagoshima Univ., 2Dept. Anesthesiology, Grad. Sch. Med. and Dent. Sci., Kagoshima Univ.)

Someodormoleculeswerereportedtohaveantinociceptiveeffects.Howevertheantinociceptiveeffectmediatedbytheolfactorysystemandthecentralnervoussystemhasnotyetexamined.Inthisstudy,wedemonstratedthatodorexposureofanodormolecule(odorantX)inducedantinociceptiveeffectandtheeffectwasmediatedbyolfacto-rysystem.Furthermore,wedemonstratedtheinvolvementofhypo-thalamicorexinneurons,oneof thekeyneurons involved inpainprocessing, intheantinociceptiveeffectofolfactory input.Weper-formedhot-platetestsandformalintestsforassessmentofnociceptiveresponseandexaminedtheeffectofodorantXexposure.WhentheodorantXwasexposedtowildtypemice,thelatencytonociceptiveresponsesinhot-platetestssignificantlyincreasedandthenociceptiveresponsestimesinformalintestssignificantlydecreased.ThesedataindicatedthattheexposureofodorantXinducedantinociceptiveeffect.Additionally,weperformedtheformalintestswitholfactorybulbec-tomymice.Theantinociceptiveeffectbyodorexposurewasdisap-peared, indicatingthattheantinociceptiveeffectwasmediatedbyolfactoryinputevokedbytheodorantXvapor.Next,informalintestwiththemicewhoseorexinneuronsweregeneticallyablated, theantinociceptiveeffectbyodorexposurewasdisappeared.Inconclusion,wefoundthathypothalamicorexinneuronswereinvolvedinthean-tinociceptiveeffectofolfactoryinputevokedbytheodorantXvapor.NoCOI.

3P-044The effective period of anti-inflammatory treatment in the traumatic olfactory dysfunctionTamari, Kengo1; Kobayashi, Masayoshi2; Takeuchi, Kazuhiko2 (1Center for Medical and Nursing Education, Mie University Faculty of Medicine, 2Otorhinolaryngology-Head and Neck Surgery, Mie University Graduate School of Medicine)

Previousstudieshavereportedthatrecoveryintheolfactorysystemdependsontheseverityofinjuryandthattreatmentwithanti-inflam-matorydrugassteroidiseffectiveinimprovingrecoveryduringanacutephaseofhead injury. It isunknownwhenweshouldstartsteroidforimprovingolfactorydysfunction.Weinvestigatedtheeffec-tiveperiodofanti-inflammatorytreatmentusingmiceperformedol-factorynervetransection(NTx).Subcutaneousinjectionofdexameth-azonesodiumphosphate(DXM)forconsecutive5dayswasstarted7,14,28and42daysaftertheNTx.Histologicalassessmentofnerverecoveryintheolfactorybulbwasmadeat5,14and42daysafterinjuryusingX-galstainingandimmunohistochemicalstainingofglialfibrillaryacidicprotein (GFAP)andCD68.AnimalsthatDXMwasinjected7daysaftertheNTxhadlessinjury-associatedtissuewithfewerastrocytesandmacrophagesandbetternerverecoverycom-paredtocontrolmice. However,thosethathad14daysor longerintervalsbetweentheNTxandDXMinjectiondidnotshowsignificantdifference.Ananti-inflammatorytreatmentusingsteroidsforolfac-torydysfunctionbyheadtraumamaybeeffectiveuntil7daysbutineffective14daysorlongerafterheadinjury.Thesefindingssuggestthatdifferenttherapeuticstrategyfrominhibitionofinflammationmaybeneededfortraumaticolfactorydysfunctioninachronicphase.NoCOI.

3P-045Aversive olfactory learning is prevented by intrabulbar infusion of tunicamycinTong, Jia; Okutani, Fumino; Kaba, Hideto(Department of Physiology, Kochi Medical School, Nankoku, Japan)

Theendoplasmicreticulum(ER) isanorganelle inwhichsecretoryandtransmembraneproteinsarefoldedorprocessed,andissuscepti-bletovariousirritants,suchastunicamycin,thatprovoketheaccu-mulationofunfoldedproteinresponseand induceERstress inthelumen,whichislinkedtoneuronaldeathinvariousneurodegenerativediseases,includingParkinson’sdisease,amyotrophiclateralsclerosis,Alzheimer’sdisease,andmanyothers.Youngratscan learntheirmother’sodorandapproachherwithoutvision.Theydothisinpartbylearningtheirmother’sodorasaconditionedstimulusthatispairedwithanunconditionedsomatosensorystimulusgivenbymaternalcare.Toestablishaversiveolfactorylearning,anartificialodorcanbepairedwithfoot-shockduringtraining.Tunicamycin(0.2,0.25,0.5and1.0mM)infusedintothebilateralolfactorybulbsduringodor-shocktrainingonpostnatalday11dose-dependentlyimpairedaversiveolfactorylearningtestedonthenextdaywithoutaffectingmemoryretentiononehourafterthetraining.Theseresultssuggestthattunicamycin-inducedERstresscausestheselectiveimpairmentofthelong-termmemorypro-cessofaversiveolfactorylearninginyoungrats.NoCOI.


3P-046Evaluation of olfactory function in the elderly using a card-kit of odor identification test for JapaneseOkutani, Fumino1; Ito, Hiroaki2; Kaba, Hideto1; Hyodo, Masamitsu2 (1Department of Physiology, Kochi Medical School, Nankoku, Japan, 2Department of Otolaryngology, Kochi Medical School, Nankoku, Japan)

Wehavealreadyexaminedolfactoryfunctionofmorethan500healthyvolunteerswithOpenEssence.Itisacard-kitofodoridentificationtestforJapanesewhichconsistsof12odorantswith6alternatives.Thenumberofcorrectanswersisdefinedasthescoreonthistest.Elderlypeopleareknowntodecreaseolfactoryfunctiongradually.Ourresultsalsorevealedthatwomenover70yearsoldandmenover65yearsoldshowsignificantlylowerscoresonOpenEssencecomparedwithyoungpeople.Thenweanalyzedthedataindetailfromthisstudyinordertorevealthepropertiesofolfactoryfunctionofelderlypeople.Allparticipantswerediagnosedas“non-dementia.”Weobtainedtheresultsasfollows:[1]Elderlywomenshowgradualolfactoryimpairmentasaging.Especiallyover85yearsoldOpenEssencescoresdeclinesignificantlycomparedwithyoungerpeople.[2]Over70yearsoldmenshowlowscoresonOpenEssence.Even if theygetoldscoresnolongerchangesignificantly.[3]Althoughinelderlywomenthenumberof“Detectablebutnotrecognizable”answersisincreasingasaging,thenumberof“Nosmelldetected”answersdoesnotchange.Itsuggeststhatodordetectionattheperipheralolfactoryorganismaintainedinseniorwomen.[4]Inwomenself-evaluationofolfactoryfunctionshowgoodcorrelationwithscoresonOpenEssence.Itseemsthatwomenarecapableofestimatingtheirolfactoryfunctioncorrectly.NoCOI.

3P-047The odor of Osmanthus fragrans modulates feeding behaviorTsuji, Tadataka1,2; Inui, Tadashi3; Yamamoto, Takashi4(1The First Department of Oral and Maxillofacial Surgery, Graduate School of Dentistry, Osaka University, Osaka, Japan, 2Department of Oral and Maxillofacial Surgery, Saiseikai Matsusaka General Hospital, Mie, Japan, 3Division of Behavioral Physiology, Department of Behavioral Sciences, Graduate School of Human Sciences, Osaka University, Osaka, Japan, 4Department of Health and Nutrition, Faculty of Health Science, Kio University, Nara, Japan)

Odorshavebeenshowntoexertaninfluenceonvariousphysiologicalandbehavioralactivities.HereweshowthattheneuraltransmissionbyOsmanthus fragrans (OSM)decreasedthemRNAexpressionoforexigenicneuropeptides,suchasagouti-relatedprotein,neuropeptideY,melanin-concentratinghormoneandprepro-orexin,whileincreasedanorexigenicneuropeptides,suchascocaine-andamphetamine-regu-latedtranscriptandproopiomelanocortininrats.ThelatencytoeatingandthefeedingtimeforafixedamountofpelletswereextendedintheOSMrats.EMGrecordingsfromboththedigastricandmassetermusclesshowedtwodistinctpatternsofburstscorrespondingtothegnawingandchewingphases.DuringtheOSModorexposure, themagnitudeoftheburstsbecamesmaller ingnawingphase inbothmasseteranddigastricmuscles,theburstdurationbecamelongerinbothphasesinmassetermuscle.Consequently,theburstfrequencyinbothphases inmassetermusclewasdecreased,consistentwithsluggishmasticatorymovements.ThisstudysuggeststhattheOSModordecreasesfoodintakeaccompaniedbychangingeatingpattern,whichcontrastsmarkedlywiththefacilitatoryfeedingpatterninratintracerebroventricularly-injectedwithorexins.NoCOI.

3P-048Influence of aging on visual field for color visionYada, Takeshi1,2; Tokumaru, Osamu1; Yokoi, Isao1(1Dept. Neurophysiol., Oita Univ. Facult. Med., Oita, Japan, 2Dept. Occup. Ther., Toka Med. Tech. College, Oita, Japan)

BackgroundDecayofsightwithaginghasbeenstudied;declineinlightsensitivitywithage(Spryetal.,2001),decreaseinretinalnervefiberlayerthickness(DaPozzoetal.,2006)andthenumberofopticnervefibers(Frisenetal.,1991).Butknowledgeabouttheeffectsofagingonvisualfield(VF)issparse.ItiswellknownthatVFvariesamongcolor,butthereisnostudyoneffectsofagingonVFforcolorvisionasfarastheauthorsknow.TheauthorsinvestigatedinfluenceofagingonVFforcolorvisioninelderly.MethodsThirteenhealthyelderly (≥70yrs)andninehealthyadults (<70yrs)participatedasunpaidvolunteersubjects.VFsweremeasuredbyaperimeterwithφ8mmtargets(100–300lux).VFsfordetection(VFdet)anddiscrim-ination (VFdis)offivecolors (white,red,green,blue,yellow)werecompared.ResultsNosignificantdifferencewasobservedinVFdisforeachcolorbetweentheelderlyandthecontrolgroups.ThereweresignificantdifferencesinVFdisamongfivecolorsinsubjectsunder70yrs(p<0.01),VFsbeingblue>white>yellow>red>green.ButthereweresmallerdifferencesinVFdisobservedamongcolorsintheelderlyover70yrs.SignificantlynarrowerVFdetwasobservedintheelderlythan inthecontrol.ConclusionsThepresentstudyshowedVFdetmightbenarrowerintheelderlythanthecontrol,indicatingdecayinperipheralvisionbyaging.VFdisintheelderlydidnotdifferfromthose inthecontrolgroup,whichmight indicatenodecay incolorvisionprocessintheelderly.NoCOI.

3P-049The integration of gustatory and olfactory information in the agranular insular cortex Mizoguchi, Naoko1; Kobayashi, Masayuki2; Muramoto, Kazuyo1(1Division of Physiology Department of Human Development & Fostering, Meikai University School of Dentistry, Saitama, Japan, 2Department of Pharmacology, Nihon University School of Dentistry, Tokyo, Japan)

Gustatoryinformationprocessingcouldbemodulatedbyolfaction.Notonlygustatorybutalsoolfactoryareprocessedintheinsularcortex(IC).However,littleinformationisavailablehowthesesensoryinputsareconvergedintheIC.Thepresentstudy,aimedtoexaminethespatiotemporaldynamicsofexcitatorypropagationinducedbysimul-taneousstimulation(5trainofpulsesat50Hz)of(1)theparvicellularpartofventroposteromedialthalamicnucleus(VPMpc)andolfactorybulb(OB),or(2)thelateralolfactorytract(LOT)andchordatympaninerve (CT). Weperformed invivooptical imagingwithavoltagesensitivedye.TheVPMpcstimulationevokedexcitatorypropagationinthedysgran-ularIC(DI).Ontheotherhand,theventralOBstimulationevokedexcitatorypropagationthatspreadinthepiriformcortex(PC)andtheagranularregion(AI),whichlocateadjacenttotheDI.TheexcitatoryregionsbystimulatingtheOB/VPMpcweresegregatedexceptfortheAI.TheexcitatorysignalsintheAIshowedanadditiveeffectofOB/VPMpcstimulation.OurdataraisethepossibilitythatapartofintegrationofgustatoryandodorsensewerecarriedoutinAI.ThesimileradditiveexcitationintheAIwasobservedbythesimultaneousstimulationoftheLOT/CT.TheseresultssuggestthattheAImayplayaroleinintegratingthegustatoryandolfactoryinformation.NoCOI.


3P-050Gustatory innervation in taste bud is stable for the rapid reconnection to the ever-renewing taste recep-tor cellsNakashima, Noriyuki; Ishii, Takashiro M; Ohmori, Harunori(Dept. Physiol., Facult. Med, Kyoto Univ., Japan)

Tasteperceptionprovidesflavorsinfoodintakeandeventuallyaffectsourmoods.Itsimpairmentbringsabouttheprofounddeteriorationonqualityoflife.Tastereceptorcells(TRCs)inlingualtastebudsrespondtovariouschemicalsandcomprisethreemajorsubtypes;typeI-(mild-lysalt-sensitive),typeII-(umami-,bitter-orsweet-sensitive)andtypeIII-(sour-sensitive)cells.Remarkably,TRCscontinuouslyrenewandthustheconsistency intasteperceptionshouldrequirethestablemaintenanceofneuralconnectionsonceestablished.Thelossofinputusuallyatrophiesthenetworkstructures,butwhathappensifeverrenewingTRCsareablatedforalongperiod?Hereweeliminatedthepre-synaptictypeIIIcells inthemousetastebudsbyconditionalchemo-geneticcellablationtechniquecalledTRECK(diphtheriaTox-inReceptor-mediatedCellKnockout).TRECKresulted inthesoledeteriorationofsoursensitivityinbehavioraltestandnerverecordingasexpected.WhentheTRECKwassuspended,therecoveryofthesoursensitivityoccurredratherpromptlywithin2daysonaverage.HistologicalinvestigationrevealedthatthegustatorynerveterminalspersistednormallyinsidethetastebudevenduringtheTRECKab-lation.Thus,theseresultssuggestthatthegustatoryinnervationinthetastebudsshouldbemaintainedstablyandindependentlyfromthepre-synaptictypeIIIcells.NoCOI.

3P-051Zinc-induced currents in the rod cells of frog taste discOkada, Yukio1; Miyazaki, Toshihiro2; Fujiyama, Rie1; Toda, Kazuo1 (1Integrative Sensory Physiology, Graduate School of Biomedical Sciences, Nagasaki University, Japan, 2Cell Biology, Graduate School of Biomedical Sciences, Nagasaki University, Japan)

TRPM5isactivatedbytheriseofintracellularcalciumconcentrationandplaysanimportantroleintastetransductionmechanisminmam-mals.Frogshavesufferedalossofthegene.However,therodcellsinfrogtastediscdisplaytheappearanceofaninwardcurrentinresponsetotheriseofintracellularcalciumconcentration.Inthepresentstudy,weinvestigatedtheeffectofintracellularzinconthemembraneprop-ertiesoftherodcells.When100µMzincwasdialyzedwithcesiuminwhole-cellconfiguration,therodcellsdisplayedtheincreaseofinwardcurrent(-19.4±5.6pA/pFat-50mV,n=6)andthemembranepoten-tialdepolarizedfrom-36±5mVto-2±2mV(n=6).TheEC50ofzincforelicitingtheinwardcurrentwas24µM,whiletheEC50ofcalciumwas2.5µM.Gadolinium(30µM)inhibitedtheinwardcurrentsalmostcompletely.Intracellularalkalization(pH8.27)withTAPSalsoinducedlargeinwardcurrentsof-53±16pA/pFat-50mV(n=6)intherodcells.IthasbeenreportedthatTRPA1channelscouldbeactivatedbyintracellularzinc,calciumandalkalization.TheresultssuggestthatTRPA1channelsexpressinthefrogrodcells.NoCOI.

Poster PresentationsCNS Function

3P-052Human auditory steady state responses to fine struc-ture periodicityKeceli, Sumru; Kakigi, Ryusuke(Dept. of Integrative Physiology, National Institute for Physiological Sciences)

Auditorysteadystateresponses(ASSR)areoscillatorybrainrespons-esthatarephase-lockedtothetemporalenvelopeofperiodicsoundssuchasclicktrainsoramplitudemodulated(AM)whitenoises/tones.Theenvelopesofthesesoundsarecreatedbytheirwell-definedonandoffgatingpatternsandmostprominentoscillatoryresponsesareobtainedfor40Hzperiodicity.Wepreviouslyobservedthatincaseof40HzAMwhitenoises,whenweusedthesamewhitenoisesegmentfortheon-periodsofthesignal,theevoked40HzASSRamplitudesweresignificantly increased.Basedonthisfindingwe investigatedwhetheritwouldbepossibletoobtainASSRinresponsetoacontin-uousperiodicsoundthathasaflattemporalenvelope.Wehavecre-atedspecialshortnoisesegmentsthathavethelowestpossibleampli-tudevariationsintimedomainandconcatenatedthesesegmentstoprepareperiodicsoundswith40-and80Hzrepetitionrates.Usingthesenovelsoundswerecordedauditoryevokedcorticalresponsesin15healthyadultsusingmagnetoencephalographyandperformedthefollowinganalyses:1)dipoleanalysisofthemagneticfielddatatodefinetheauditorycortexasregionofinterest,2)spatialfilteringofthesensordatatoobtainsingle-trialsourcewaveforms,and3)time-fre-quencyanalysisofsingle-trials.Ourdatashowedthatcomparedtonon-periodiccontrolnoisesounds,40-and80Hzperiodicnoisesevokedsignificantoscillatoryactivity intheauditorycortex.Ourfindingssuggestthatinadditiontotheperiodicenvelope,repetitivefinestruc-turealsoplaysaroleinASSRgeneration.NoCOI.


3P-053Cortical top-down input evokes dendritic spike in so-matosensory L5 neuronsMurayama, Masanori1; Sato, Masaaki1; Ohkura, Masamichi2; Matsubara, Chie1; Nakai, Junichi2; Hayashi, Yasunori1; Manita, Satoshi1(BSI, Riken, Japan, 2Brain Science Institute, Saitama Univ., Japan)

Ourpreviousstudyusingcurrentsourcedensityanalysiswithalinearprobe inprimarysomatosensorycortex (S1)duringdirectcorticalmicrostimulation(DCMS)ofsecondarymotorcortex(M2)suggestedthatM2top-downinputarrivesincorticallayers(L)6and2/3,ratherthaninL5.However,thiswasinconsistentwithmulti-unitdatathatshowedthehighestfiringactivityinL5.Oneexplanationforthisin-consistencycouldbethatM2synapticinputtoL2/3causedlocalCa2+spikes inL5apicaldendritesthat ledtotheir increasedfiring.TosubstantiatewhetherM2top-downinputevokesdendriticCa2+activ-ity inL5neurons,weperformed2-photon imagingandmeasureddendriticCa2+activityinthepresenceandabsenceofCNQXduringDCMSofM2inanesthetizedmice.CorticalapplicationofCNQXsig-nificantlyreducedtheaveragedresponsetotop-downinput inthewholefieldofview.Inindividualdendrites,CNQXhadmuchlargereffectsondendriteswithalargerinitialdendriticresponse.DendriticCa2+activitycausedbybackpropagatingactionpotentials (BPAPs),butnotbydendriticspiking,shouldnotbeaffectedbyglutamatergicblockers.Moreover,dendriticspikingisknowntoinducelargerfluo-rescencechangesindistaldendritescomparedtoBPAPs.ThesedataarethereforeconsistentwiththesmalldendriticCa2+responsesweobservedinS1arisingfromBPAPs,andthelargeresponsesfromlocal,dendriticCa2+spikesevokedspecificallybytheobservedtop-downprogrammedcoincidentinputsfromM2.NoCOI.

3P-054Cortical Top-Down Input Regulates Conscious Per-ceptionHomma, Chihiro1; Suzuki, Takayuki1; Kamoshida, Atsushi1,3; Mainta, Satoshi1; Matsumoto, Takashi1; Yamada, Kazuyuki1; Odagawa, Maya1; Matsubara, Chie1; Inutsuka, Ayumu2; Yamanaka, Akihiro2; Murayama, Masanori1(1Brain Science Institute RIKEN, Saitama, Japan, 2Research Institute of Environmental Medicine, Nagoya Univ., Aichi, Japan, 3National Instruments Japan Corp., Tokyo, Japan)

Ourpreviousstudydemonstratedthatasecondarymotorcortex(M2)projectionregulatesdendriticspikingandsomaticfiringinsomatosen-sorycortex (S1) layer5pyramidalneurons.Totestwhetherthisprojection influencesconsciousperception,weusedanoptogeneticapproachtospecificallyinhibitthetop-downM2toS1projectionduringasomatosensory-associatedtaskinbehavingmice.WeinjectedAAV-GFPorArchTintoM2,andilluminatedM2projectionfibersinS1.Inthespontaneousplacepreferencetest,whichusestactilecues,miceexpressingGFPaloneorArchTwithoutLED-illuminationalsoexhib-itedastrongtexturepreference.However,uponinactivationoftheM2toS1projectionviaLED-illumination,ArchTmicelostthetexturepreference.Wealsoappliedoptogenetic inactivationtomicehighlytrainedinatactilediscriminationtask.OptogeneticinactivationoftheM2-S1projectionsignificantlydegradedaccuratediscriminationinthistask.Moreover,weconductedseveralgeneralbehavioraltasks,includ-inganopenfieldtest,gaitanalysis,etc.,butnosignificantdifferenceswereobservedbetweenilluminationconditions.Together,theseresultssuggestthatM2top-downinputsmodulateS1activity,andspecifical-lyaltersensoryperceptioninbehavinganimals.NoCOI.

3P-055Identification of indirect top-down pathways in the sensory-motor circuit through the thalamusAtsumi, Yusuke1,2; Odagawa, Maya1; Ota, Keisuke1; Murayama, Masanori(1BSI, RIKEN, Saitama, Japan, 2Dept. Biotechnological, Tokyo Institute of Technology)

Top-downinputfromahighercorticalareatowardasensoryareaisessential forsensoryperceptionandbehavioralexecution.Recentphysiologicalstudieshaveshownthatmousesensory-motorcircuitsconsistoflong-rangerecurrenthorizontalprojectionsbetweenprima-rysomatosensorycortex(S1)andsecondarymotorcortex(M2),theso-calleddirectpathway.However,otherstudieshaveshownthateachcorticalregionformsthalamicconnections,calledindirectpathways.Little isknownaboutthephysiological functionsof these indirectpathwaysduringsensoryperception.Here,wefocusedontheS1-M2circuitthroughthethalamusandinvestigateditsfunctionsduringM2top-downinputtoS1.Inordertoanatomicallyfindtheindirectpathway,welabeledM2fibersbyinjectingadeno-associatedvirus-greenfluo-rescentproteinintoM2asananterogradetracerandlabeledthalam-icsomatabyinjectingcholeratoxinsubunit-BintoS1asaretrogradetracer.WefoundthatlabeledM2axonsandneuronsprojectingtoS1coexistedintheventrallateralnucleus(VL),whichispartofthemotorthalamus.ThisanatomicalresultsuggestedthatVLrelaysM2top-downsignalstoS1.Totestthishypothesis,weextracellularlyrecordedneuralfiringactivityfromVL,S1,andM2duringhindpawstimulationthatcausedM2feedbackinputtoS1.Wewillpresenttheseanatomicalandphysiologicalresultsanddiscussthe functionsof the indirectpathwayinsensoryperception.NoCOI.

3P-056Non-noxious skin stimulation activates the nucleus basalis of Meynert and promotes NGF secretion in the parietal cortex via nicotinic ACh receptors in anesthetized rats.Hotta, H.1; Watanabe, N.1; Piche, M.2; Hara, S.1; Uchida, S.1; Yokawa, T.3(1Dept. Auton. Neurosci., Tokyo Metropol. Inst. Gerontol., Tokyo, Japan, 2Dept. Chiropractic, Univ. Quebec Trois-Rivieres, Canada, 3BioView, Tokyo, Japan)

Wehavepreviouslydemonstratedthatstimulationofthebasalfore-brainnucleus(thenucleusbasalisofMeynert;NBM)increasesextra-cellularnervegrowthfactor(NGF)secretionintheparietalcortexbytheactivationofnicotinicAChreceptors(nAChRs)[NeurosciRes63:122,2009].Hereweexaminedwhethernon-noxiousskinstimulationactivatesNBM,resulting inthepromotionofNGFsecretion inthecortex.Weusedanesthetizedandartificiallyventilatedrats.Innocuousskinstimulationwasdeliveredtothe lefthindlimbwithasoft-hairbrush.Firstly,extracellularNGFintherightparietalcortexwascol-lectedandmeasuredbymicrodialysismethodsandanELISA.Brush-ingstimulation,repeatedatafrequencyof1Hzfor100min,producedanincreaseinextracellularNGFlevelintheparietalcortex.There-sponseofNGFwasnotobservedinratspretreatedwithanicotiniccholinergicblockingagentmecamylamine(20mg/kg,i.v.).Secondly,bymeansof functionalMRI,weexaminedthebloodoxygen leveldependent(BOLD)signalevokedbybrushing(at3Hzfor1mintothelefthindlimb)intheNBM.BOLDsignalintherightNBMwashigherduringbrushingcomparedwithbaseline.Theseresultsindicatethatnon-noxiousmechanicalskinstimulationcanpromoteNGFsecretionintheparietalcortexbyactivatingnAChRs,possiblythroughexcitationofcholinergicnervesfromtheNBM.NoCOI.


3P-057Single neuronal activity in the rhesus monkey orbitof-rontal cortex related to reward value processing during the decision-making schedule taskSetogawa, Tsuyoshi1,2; Mizuhiki, Takashi1,3; Akizawa, Fumika1; Kuboki, Ryosuke1; Matsumoto, Narihisa4; Shidara, Munetaka1,3 (1Grad. Sch. of Comprehensive Hu man Sci., Univ. of Tsukuba, Tsukuba, Ibaraki, Japan, 2JSPS Res. Fellow (DC2), 3Faculty of Medicine, University of Tsu kuba, Tsukuba, Japan, 4Human Tech. Res. Inst., AIST, Tsukuba, Japan)

Whenwemakeachoice fromthealternatives,weconsidertheirvaluesandworkloads.Tounderstandtheneuronalmechanismofsuchadecision-makingprocess,wedevelopedadecision-makingscheduletaskandrecordedsingleunitactivity frommonkeyorbitofrontalcortex(OFC)whichhasbeenreportedtobeoneoftheimportantbrainareas forthereward-guidedbehavior.Themonkeywastrainedtoperformarewardscheduletaskwhichconsistsof1,2or4trialsofvisualdiscriminationtoearn1,2or4dropsofliquidreward.Afterlearningthistask, thedecision-makingscheduletask inwhichtwokindsofchoicetarget(CT)weresequentiallypresentedwasintroduced.TheCTbrightnessandlengthindicatedrewardamountandrequirednumberoftrials,respectively.Then,thesetwoCTsweresimultane-ouslyreappeared(choicephase).Themonkeywasrequiredtochooseoneofthem,andthenthechosenrewardschedulestarted.Werecord-edfrom137neuronsintheOFCandanalyzedtheneuronalactivityduringthesecondCTperiod.Someneuronalactivitieswerecorrelat-edwithdifferentialvalueofthetwoCTs(7.3%).Other7.3%ofneuronsshowedlarger/smallerresponseswhenthetwoCTvalueswereclose.TheseresultssuggestthatOFCneuronsplayanimportantroleinthedecision-makingbyrewardvalueinformationprocessing.NoCOI.

3P-058Neuronal activity in the monkey frontal cortex during a novel shape-manipulation taskYagi, Kohei1; Sakamoto, Kazuhiro2; Kawaguchi, Norihiko1; Mushiake, Hajime1,3(1Tohoku University School of Medicine, Sendai, Japan, 2Research Institute of Electrical Communication, Tohoku University, 3CREST)

Humansmanipulatetoolsormachinesbyusingasequenceofstepsoractionstoachievecertaingoals.Themonkey lateralprefrontalcortex(lPFC)andpremotorarea(PM)arethoughttoplayimportantrolesinplanningthestepstoattainabehavioralgoal.Toinvestigatetheinvolvementoftheseareasinplanninggoal-directedactions,weintroducedanovelshape-manipulationtaskthatrequiredstep-by-stepmovementswithamanipulanda.Thegoalofthetaskwastofitthetestshapetothesampleshapebymanipulatingangleandsizebyrotating,expanding,andcontractingthetestshape.Aftertrainingmonkeystoperformthistask,werecordedneuronalactivityinthelPFCandPMusing linear-arraymulti-contactelectrodeswhiletheanimalswereperformingthetask,andexaminedhowthe frontalneuronactivityreflectedbehavioralplanningunderdifferenttaskconditions.This research is supported by the MEXT of Japan(#24120703),FRISofTohokuUniv.andCREST.NoCOI.

3P-059Neural response predicting social stimuli in monkey striatumKuraoka, Koji; Inase, Masahiko(Department of Physiology, Kinki University Faculty of Medicine, Osaka, Japan)

Striatalneuronswhichreceivestrongprojectionofmidbraindopamineneuronshavebeenreportedtoshowpredictiveresponseofrewards.Thestriatum,especiallyintheventralpart,alsoreceivesneuralpro-jectionfromthebrainareasthatprocesssocialinformationsuchastheamygdala.Thus,itispossiblethatsocialinformationalsoaffectstheactivityofthestriatalneurons.Inthisstudy,toelucidatethein-volvementofthestriatuminsocialinformationprocessing,werecord-edtheactivityofthemonkeyventralstriatalneuronswhileanarbitrarygeometricpatternwasassociatedwithoneofthesocialimagesbelong-ingtoseveralcategoriessuchastheoppositesexorafaceshowingnegativeemotion.Theprecedentgeometricpatternsandthefollowingsocialimageswereseparatedbyabriefdelayperiod.49of77neuronsrecordedfromtheventralstriatumofamacaquemonkeyshowedsignificantresponsesinrelationtoatleastoneofthetaskeventsofgeometricpatternpresentation,delayperiodandsocialimagepresen-tation.Toestimatehowwell individualneuronsdiscriminatesocialstimuluscategories,wecomputedthetemporalchangeofROCvaluein49responsiveneuronsbycomparingneuralactivitybetweensocial-lypreferableandunpreferablestimuli.ResultsshowedthatROCvalueincreasedwhilethegeometricpatternsthatpredictedtheso-ciallypreferablestimuliwerepresented.Theseresultsindicatethatthemonkeyventralstriatalneuronsare involved inpredictionofpreferablesocialinformation.NoCOI.

3P-060Real-time change in the firing rate of hippocampal CA1 neurons before, during, and after the exposure to a specific episodeTaniguchi, Hiroki; Isikawa, Junko; Mitsusima, Dai(Department of Systems Neuroscience, Yamaguchi University Graduate School of Medicine, Ube, Japan)

Peoplesremembertheepisodesoftheirfirstloveorsexualrelationship.Tomonitortheprocessofepisodicmemory,werecordedneuronalactivityofhippocampalCA1neurons in freelymovingyoungmaleratsbefore,during,andafterthefirstencounterwithyoungfemalerats.Spontaneousbehaviorandsoundsweresimultaneouslyrecordedusingadigital-videorecordingsystem.Priortotheexperiment,weimplantedmulti-unitrecordingelectrodesintothepyramidalcelllayerofCA1inyoungmales,unisexuallyhousedafterweaning.Onthedayofexperiment,westartedtorecordthespontaneousfiringofCA1neuronsinhabituatedhomecage,butthefiringratewaslow.Tenminaftertherecording,youngfemaleratwasputintothehomecagefor10min.Maleratsshowedaninterestinfemalerats,andfrequencyofthefiringwasincreasedsimultaneously.Afewminutesafterthefemaleratwastakenout,spontaneoushighfrequencyfiring(~100Hz)wassuddenlyobservedforseconds.Minutesafterthehigh-frequencyfiring,theratsbegantoshowripple-likefiring(~50msec)frequently.Thisobservationshowsareal-timechangeinthefiringrateofhippocampalCA1neuronsbefore,during,andafteraspecificepisode.NoCOI.


3P-061The endogenous opioids related with antinociceptive effects induced by electrical stimulation of the amyg-dalaNakamura, Takami; Tomida, Mihoko; Fujii, Nobumi; Ando, Hiroshi; Asanuma, Naokazu(Department of Oral Physiology, School of Dentistry, Matsumoto Dental University, Shiojiri, Japan)

Previousstudiesdemonstratedthatbriefelectricalstimulationoftheamygdaladepressedneuraldischarge inthecingulateareaswhennoxiousstimulationwasappliedtoperipheraltissues.Toclarifywheth-erendogenousopioidsare involved inantinociception inducedbyelectricalstimulationoftheamygdala,weinvestigatedimmunohisto-logicallythenumberofc-Fosexpressionandthedistributionandthequantitativechangesofendogenousopioids(β-endorphin,enkephalin,dynorphinA)secretedinthebrainwhenelectricalstimulationwasappliedtotheamygdalawithorwithoutnoxiousstimulationtotheperipheraltissue.Malewistarratswereusedforthisstudy.Asthenoxiousstimulation,formalinsolution(0.1ml)wasinjectedintotherighthindpaw.Electricalstimulation(2µA,100Hz,15sec)wasappliedtothecentralnucleusoftherightamygdala.C-Fosexpressionwasincreasedintheipsilateralanteriorcingulatecortex(ACC),whichsuggestedthatelectricalstimulationintotheamygdalaactivatedACCneurons.How-ever,onlyafewendogenousopioidswereobservedintheACC.Ontheotherhand,theamountofdynorphinAintheperiaqueductalgray(PAG)wasincreasedbyelectricalstimulationoftheamygdala.TheresultssuggestthattheincreaseofdynorphinAinthePAGinducedbyelectricalstimulationoftheamygdalaactivatesthedescendingantinociceptivesystem,anddepressesthenociceptiveresponseintheACCindirectly.NoCOI.

3P-062Higher expression of cocaine and amphetamine regu-lated transcript (CART) peptide in the amygdala rath-er than the medial prefrontal cortex in ADHD model ratShimizu, Yuko; Yokoyama, Yoshihiro; Misumi, Sachiyo; Ishida, Akimasa; Jung, Cha-Gyun; Hida, Hideki(Department of Neurophysiology & Brain Science, Nagoya City University Graduate School of medical Sciences, Nagoya, Japan)

Spontaneouslyhypertensiverat(SHR)arethemostusedanimalmod-elofattentiondeficithyperactivitydisorder.Previouslywefoundthatenvironmentalenrichment (EE) for5weeksfrompostnatalday25(P25)toP60decreasedhyperactivityandanxiety-likebehaviorinSHRandthatmRNAofcocaineandamphetamineregulatedtranscript(CART),knownto influence locomotoractivity,anxietyandstress,increasedinmedialprefrontalcortex(mPFC)andamygdala(Amy).ToconfirmtheproteinlevelandlocalizationofCARTinmPFCandAmy,Westernblotand immunohistochemistrywerecarriedout.Westernblotwasperformedin16%tricine-gelusingpreproCARTpolyclonalantibody.Theantibody(1:800,Proteintech)recognizedthreebands(14,7.7,5.3kDa)inthearcuatenucleusasthepositivecontrol.AntibodydidnotdetectanybandsinmPFCbutseveralbandsinAmy,indicatingthattheamountofCARTpeptidesinmPFCisunderthedetectionlevel.CARTlocalizationwasinvestigatedusingtheantibody(1:10,000,PhoenixPharmaceuticalsInc).AfewCARTpositivecellswasdetectedinthemPFConlytheratsgrowninEE.Strongimmu-noreactivityforCARTwasobservedintheAmy,especiallycentralnucleusofAmy(CeA).DatasuggestthattheamountofCARTpeptideislargerinAmycomparedtomPFC,probablyrelatingtoanxiety-likebehaviorinSHR.NoCOI.

3P-063The functional role of the prefrontal cortex and baso-lateral nucleus of amygdala in value comparison be-tween reward and punishmentIshikawa, Junko; Ishikawa, Akinori; Mitsushima, Dai(Department of Systems Neuroscience, Yamaguchi University Graduate School of Medicine.)

Comparisonbetweenrewardvalueandpunishmentvalueareessentialtodecidewhethertoexecutetheactionornot, ifoutcomesoftheactionarebothrewardandpunishment.Asamechanismofcompar-ison,wehypothesizedthatprefrontalcortex (PFC)andbasolateralnucleusofamygdala(BLA)playaroleinvalueevaluationsincebothareasregulaterewardseekingandpunishmentavoidancebehaviors.Inthepresentstudy,weinvestigatedwhethertheventral-medialPFC(v-mPFC),v-lateralPFC(v-lPFC)andBLAcouldhavefunctionalroleinthevalue-comparisontask,inwhichthefood-restrictedratsreceivedonepelletandelectricfootshocksimultaneouslyafterlever-pressingduringtonepresentation.Inthistask,thecurrent intensityof footshockbecamestrongereachtimetheratspressedthelever10times,andtheratsallowedtochosewhethertopresstheleverornot.Weevaluatedmaximumcurrent intensity (MCI) inwhich leverpressprobabilitywasmorethan25%.Inactivationofthev-mPFCorBLAbylocaltreatmentwithGABAAandGABABagonistselevatedMCI,whereasv-lPFCinactivationwas loweredMCI.Ontheotherhand,inactivationofthesethreeregionsdidnotaffecttheprobabilityofleverpressforonlyreward.Theseresultssuggestthatthev-mPFCandBLAmaycontributetooverestimatepunishment,whilev-lPFCmaycontributetounderestimatepunishmentinvalue-comparisontask.Inconclusion,thev-mPFC,v-lPFCandBLAprovideafunctionalroleinvalueevaluationundertheconflictingsituation.NoCOI.

3P-064Synaptic potentiation in the nociceptive amygdala in orofacial inflammatory pain model of ratsMiyazawa, Yuta1; Takahashi, Yukari1; Watabe, AyakoM1,2; Kato, Fusao1,2(1Department Neuroscience, Jikei University School of Medicine, Tokyo, Japan, 2Nagoya University, Nagoya, Japan)

Alargemajorityofthespinalcordnociceptiveprojectionneuronssendaxonstothelateralparabrachialnucleus(LPB)andthentothecapsu-larpartofthecentralamygdala(CeC),asubnuclusdubbedas“noci-ceptiveamygdala”.Thisnon-thalamocorticalpathwayiscentralinthenociception-emotion linkandundergoesrobustplasticity invariouspainmodels (Ikedaetal.,2007;Nakaoetal.,2012). Interestingly, insub-acuteinflammatorypainmodelssuchasarthritis(Jietal.2009),thepotentiationalwaysoccursintherightCeCregardlessofthesideofinflammationincontrasttothechronicnerveinjurymodels,inwhichthepotentiationoccursintheCeCcontralateraltotheinjury.Anatom-ically,theLPBreceivesinputsalsofromthespinaltrigeminalnucleus,whichsendsbilateralprojectionstotheCeCwithipsilateralpredom-inance,unlikethedorsalhornneurons(Lietal.,2006).ToidentifytheLPB-CeCplasticityinthetrigeminalpainmodel,weinjected5%for-malinintotheleftupperlipofadultWistarrats,whichshowedrobustnocifensivebehaviorsfor1hourafterinjection,andrecordedLPB-CeCtransmission6hourslaterandfoundthattheLPB-CeCtransmissionwaspotentiatedonlyintherightCeC.Thisstudyisthefirsttodescribethesynapticpotentiation inthetrigemino-parabrachio-amygdaloidpathwayandsuggeststhatthelateralityofLPB-CeCsynaptictrans-missionisdeterminednotbythesideofnociceptionbutratherbythetypeofpainmodels.SupprtedbyKakenhi.NoCOI.


3P-065Synaptic plasticity and electrophysiological proper-ties of layers II/III neurons in primary motor cortex at the early stages of motor learningTsuda, Yasumasa; Kida, Hiroyuki; Mitsushima, Dai(Systems Neuroscience Yamaguchi University Graduate School of Med, Ube, Yamaguchi, Japan)

Rotorrodtestiswidelyusedfortheassessmentofmotorcoordinationandlearning.Rodentscanreachaplateauscoreafter2consecutivedaysofthetraining.Afterthetraining,wefoundanincreaseofbothAMPAandNMDAcurrentinthemotorcortex,followedbyplasticchangesinvolvingstructuralreorganizationinspinearchitecture(Yangetal.,2009).However,theeffectsofmotortrainingonelectrophysio-logicalpropertiesoflayersII/IIIneuronsarestillknown.Onthe1stdayoftraining,motorskillwassignificantlyimprovedwithin10trialsinallanimals(n=8).Then,wepreparedacutebrainslicesofprimarymotorcortextoanalyzetheelectrophysiologicalpropertiesandthesynapticplasticityusingwholecellpatchclampmethods.Involtageclampanalysis,thetrainedratsshowedsignificantlyhigherAMPA/NMDAratiothanuntrainedcontrolrats,butthe increasewasnotobserved inrats2daysafterthetraining (Fisher’stest,P<0.05).Further,motortrainingdidnotaffectpaired-pulseresponses,suggest-ingthatAMPAreceptor-mediatedpostsynapticplasticityratherthanpresynapticglutamatereleaseisinvolvedinthemotorlearning.Theseresultssuggestapossibleneuralmechanismatearlystageofmotorlearning.NoCOI.

3P-066The enhancement of the reward prediction error sig-nal in the midbrain dopamine neuron by the cost paid for the rewardTanaka, Shingo1; John, O'Doherty P2; Sakagami, Masamichi1(1Brain Science Institute, Tamgawa University, Tokyo, Japan, 2CALTECH, CA, USA)

“Oneofthegreatestjoysinlifeisanicecoldbeerafteraharddayofwork.”Asthisphraseindicates,thevalueofarewardcanbemodu-latedbytheeffortorthecostput into itsachievement.However,relativelyfewstudieshaveshowntheneuralbasisoftheeffectcausedbythepaidcostonthevalueofthereward.Whencalculatingthevaluesofrewards,theactivityofdopamineneurons,whichrepresenttherewardpredictionerror, isusedtoupdatethevalue.Here,weexaminedwhethertheactivityofthedopamineneuronsinresponsetorewardpredictivecueswasincreasedbythecostprecedingtothesecues.Twomacaquemonkeysperformedasaccadetask.Afterfixationonafixationpoint, thesubjectswererequiredtomakeasaccadetoaconditioncueandthenatargetappeared. Inthehighor lowcostcondition,longorshortfixationtothetargetwasrequired,respective-ly.Afterfixationonthetarget,thesubjectsmadeasaccadetotherewardcue.Whilethesubjectsperformedthesaccadetask,theactiv-itiesofdopamineneuronswererecordedfromtheSNcinthemidbrain.Theneuronalresponsetothelow-costcuewaslargerthanthattothehigh-costcue.Incontrasttotheresponsestotheconditioncues,theresponsestotherewardcueafterthehigh-costconditionwerelargerthanthosetotherewardcueafterthelow-costcondition.Wesuggestthatinformationaboutthecostistransmittedtothedopamineneuronsandthepaidcostincreasestherewardpredictionerrorsignalinthedopamineneurons.NoCOI.

3P-067Memory-guided navigation behavior of mice in a virtu-al linear trackMizuta, Kotaro1; Sato, Masaaki1,2; Kawano, Masako1; Sekine, Yukiko1; Ohkura, Masamichi3; Nakai, Junichi3; Hayashi, Yasunori1,3(1BSI, RIKEN, Saitama, Japan, 2JST PRESTO, Saitama, Japan, 3Saitama University, Saitama, Japan)

Althoughvirtualreality(VR)hasincreasinglybeenusedtoinvestigatenavigation-relatedneuralactivityinhead-fixedmice,itisstillunclearwhetherthemiceusetheirspatialmemorywhentheynavigatewith-inaVRenvironment.Toaddressthis issue,wehaveestablishedanewVRspatialmemorytask.Inthistask,micebegintorunfromoneendofavirtuallineartracktoseekforrewardsdispensedatatargetzoneinthemiddleofthetrack.Thetrainingprotocolconsistsofthefollowingtwophases.(1)Non-delayedtask:micearerewardedimme-diatelywhenevertheyenterandpassthroughthetargetzone. (2)Delayedtask:micehavetonavigatetothesametargetvisuallyandstaytherefor1-2sectoreceiverewards.Inthenon-delayedphaseofthetraining,micedemonstratedsteadyincreaseindistancetraveled,runningspeedandthenumberofrewards. Inthedelayedphase,however,thenumberofrewardsdroppedbutgraduallyrecoveredastheylearnedtostopandstayinthetargetinmosttrials.WetrainedtransgenicmicethatexpresstheG-CaMP7fluorescentcalciumsensorproteinsinhippocampalCA1pyramidalneuronssimilarlyandimagedtheneuronalactivitiesduringthebehaviorbyin vivotwo-photonmicroscopy.Ourpreliminaryanalysissuggeststhatapopulationofneuronswithvirtualplace-specificactivitycanbeopticallyidentified.ThisVRtaskthusprovidesanexperimentalparadigmtostudyplas-ticityofhippocampalmemorycircuitswithimagingandelectrophys-iology.NoCOI.

3P-068Short-term changes in behavioral efficiency and brain activity associated with response inhibtionHirose, Satoshi1,2; Jimura, Koji1,2,3; Kunimatsu, Akira4; Abe, Osamu4; Ohtomo, Kuni4; Miyashita, Yasushi1; Konishi, Seiki1,2 (1Department of Physiology, The University of Tokyo School of Medicine, Tokyo, Japan, 2Department of Physiology, Juntendo University School of Medicine, Tokyo, Japan, 3Precision and Intelligence Laboratory, Tokyo Institute of Technology, Tokyo, Japan, 4Department of Radiology, The University of Tokyo School of Medicine, Tokyo, Japan)

Behavioralperformance is improvedthroughlearning,andthis im-provementmayentaildevelopmentalneuralchangesevenwithinoneexperimentalsession.However,previousstudiesofresponseinhibitionhaveoverlookedsuchshort-termchangesinperformance,whichmayhavehelpedininvestigatingneuralmechanismsofresponseinhibition.InthepresentfMRIstudy,wemeasuredbrainactivitychangeswith-inashortrange(~1hour)offMRIruns,andtrackedsignalincrease/decreaseinbroadsetsofbrainregions.Twosetsofbrainregionswereidentifiedthatshowedincreaseordecreaseofbrainactivityfollowingperformanceimprovement:theinferiorfrontalcortex(IFC),thepre-supplementarymotorarea (preSMA), thecaudatenucleusandthecerebellumshowed increaseofbrainactivity,whereasthemedialprefrontalcortexandtheanteriorcingulatecortexshowedthebrainactivitydecrease.GiventhepivotalrolesoftheIFCandthepreSMAinresponseinhibition,theseresultssuggestthatthecaudateandthecerebellumalsoplayanimportantroleinresponseinhibitionincoor-dinationwiththesecerebralregions.NoCOI.


3P-069Therapy of stress-induced depressive-like state of mouse by tail vein injection of adipose stem cell-con-ditioned mediumInoue, Akio; Nishimoto, Takaaki; Jin, Yu; Tamura, Takashi; Kanno, Takeshi; Nishizaki, Tomoyuki(Dept. Physiol., Hyogo College of Med.)

Adiposestemcells(ASC)havetheabilitytodifferentiateintomultiplecelllineagesandusedforclinicalapplicationagainstvariousdiseases.Recently,thetherapeuticeffectsofstemcellsareconsiderednotbythetransplantedcells,butbysecretedgrowthfactors.Inthepresentstudy,weexaminedthetherapeuticeffectsofASC-conditionedmedi-um(ASC-CM)onstress-induceddepression-likestateofmouse.Thedepressive-likestateofmaleC57BK/6Jmousewasproducedbyap-plicationofrepeatedrestraintstress(3hoursfor3days).Whentreat-edmicewereexaminedbytailsuspensiontestorforcedswimtest,theybecameimmobilestatemorerapidlythannon-treatedmice.Thedepression-likestate inmicecontinuedmorethantwomonths.WepreparedASCfromfattissueoffemaleC57BK/6Jmouse,andculturedinDMEMwith10%FBS.ASC-CMwasobtainedbyculturingadiposestemcellsinPBScontaining1mMCaCl2for24hours.Themediumwascollectedandfiltered.WeinjectedASC-CM(0.4ml)intotailveinofstressedmice,andmeasuredtheirresponseontailsuspensiontestorforcedswimmingtest.Theimmobilityperiodwasreducedgradu-allyandrecoveredtothelevelofcontrolmiceby3weeks.Wefoundthattheconditionedmediumofmusclestemcells(satellitecells)alsohadtherapeuticeffect.However,nochangewasobservedwhenbufferwasinjectedintostressedmice.Thisresultshowsthatstem-cellcon-ditionedmediumiseffectivefortherapyofbraindiseaseevenbyveininjection.NoCOI.

3P-070Identification and characterization of a top-down cir-cuit in the mouse somatosensory systemSuzuki, Takayuki1; Odagawa, Maya1; Matsubara, Chie1; Yanagawa, Yuichio2; Murayama, Masanori1(1Brain Science Institute RIKEN, Saitama, Japan, 2Gunma University Graduate School of Medicine, Gunma, Japan)

Despitethe fundamentalroleof top-downcontrol inbehavior, thestructureandoperationoftheseneuralcircuitsarepoorlyunderstood.Here,wereporttheidentificationandcharacterizationofatop-downcircuitinthemousesomatosensorysystem.Usingwide-fieldcorticalvoltage-sensitivedye(cVSD)imaging,wedetectedthatprimaryso-matosensory(S1)andsecondarymotor(M2)cortexwereactivatedinsuccessionduringhindpawstimulation.Intheawakestate,hindpawstimulationevokedasignificantlylargercVSDresponseinM2,butnotinS1andotherareas,relativetotheanesthetizedstate.ApplicationofTTXtoS1significantlydecreasedcVSDactivityinM2,whileap-plicationtoM2significantlydecreasedalatecomponentofcVSDac-tivityinS1duringhindpawstimulation.WenextexaminedthedetailedanatomicalconnectivitybetweenS1andM2.Toanterogradelylabelaxons,aviral tracer (AAV-CAG-GFP)was injected intoeacharea,whiletheretrogradetracercholeratoxinBsubunitconjugatedtoAlexa555wasusedtolabelthesomataofprojectionneurons.TheaxonalinnervationpatternoffibersfromM2toS1showedtargetingtolayer1(L1)anddeepcorticallayerswhileavoidingthemiddlelayers,suggestiveoftop-downconnectivity.ReversetracingexperimentsalsorevealedapredominanceofprojectingneuronsfromL2/3,L5aandL6ofS1toM2,inabottom-upconnectivitypattern.ThesephysiologicalandanatomicalresultsindicateareververatingcircuitbetweenS1andM2.NoCOI.

3P-071Cortical top-down programmed input to primary sen-sory area in miceMatsumoto, Takashi1; Ota, Keisuke1; Suzuki, Takayuki1; Manita, Satoshi1; Odagawa, Maya1; Matsubara, Chie1; Inutsuka, Ayumu2; Yamanaka, Akihiro2; Murayama, Masanori1(1Brain Science Institute, Riken, Saitama, Japan, 2Research Institute of Environmental Medicine, Nagoya University)

Wehavefoundthatprimarysomatosensorycortex(S1)andsecondarymotorcortex(M2)formareverberatingcircuitvialong-rangerecip-rocalconnections,indicatingthatM2canexecutetop-downcontrolofsensoryperception.However,thestructureandinformationflowofthistop-downinputtotheS1columnispoorlyunderstood.Here,wereportthe identificationandcharacterizationofatop-downcontrolcircuitinthemouseforebrainsomatosensorysystem.Duringhindpawstimulation,S1andM2wereactivatedinsuccession.M2thenprovid-edfeedback inputtoS1, initiatingdendriticspikingand increasedfiringoflayer5neurons.Thedendriticspikeandthelayer5-selectiveoutputwerealsoobservedbydirectcorticalmicrostimulationofM2,andblockedbyTTXapplicationtoM2.Byperformingcurrentsourcedensityanalysis,wefoundthatM2synapticinputsselectivelycontact-edS1bottomandtoplayers,consistentwithanatomicaltracingstud-ies.ThisfindingindicatesthatM2top-downinputsinducefiringintheS1columninaprogrammedtemporalsequenceoflayer-specificactivity.Wehavedescribedthiscoordinated inputpatterntothelowerandupperlayersofS1as“programmedcoincidentinput”(PCI),becauseitreliesontheorderedrecruitmentofactivityfromlowertohigher layers,coordinatedwithdirect inputtothedistaldendritestimedtomosteffectivelyproducedendriticspikes.NoCOI.

3P-072Gene expression of the transcription factors in the monkey cerebral cortex during postnatal developmentOishi, Takao1; Higaki, Sayuri2; Sato, Akira3; Kondo, Shinji4; Kojima, Toshio5(1Primate Res Inst, Kyoto Univ, Inuyama, Japan, 2Biobank Omics Unit, Natl Cntr Geriatr Gerontol, Obu, Japan, 3Lab Mol Neurosci, Tokyo Univ Sci, 4Transdiscipl Res Integr Cntr, Natl Inst Polar Res, Tokyo, Japan, 5Res Cntr Physical Fitness, Sports Health, Toyohashi Univ Tech, Toyohashi, Japan)

TranscriptionfactorsrecognizespecificDNAsequencesandregulategeneexpressions.Inthecentralnervoussystem,theyareinvolvedinthecontrolofmultiplebiologicalphenomenaincludingcellulardiffer-entiation,regionaldifferentiation,andmaintenanceofregionalspecia-tion.Therefore,weanalyzedgeneexpressionpatternsoftranscriptionfactorsinthecerebralcortexofmacaquemonkeys,whichhaswelldifferentiatedcorticalareas,at3stages inpostnataldevelopment(postnatalday70,1yearandatadult).Developmentalchangeinthegeneexpressionwascomparedbetweensensorimotorareas(M1andS1)andtheassociationareas(PFC,TE,andPE)byusingDNAmi-croarraysystem(Agilent,4×44K).Amongtranscriptionfactorgeneswhoseexpressionwasdevelopmentallyregulated,expressionpatternswerecategorizedinto10patternsfromlogicallypossible14patterns.Expressionlevelofsometranscriptionfactorgeneswasinaccordancewiththeextentofmyelinationeitherpositivelyornegatively.Expres-sionlevelofsomeothertranscriptionfactorgeneswasdevelopmen-tallyup-regulatedandalwayshigherintheassociationareas.Thesearea-specificanddevelopment-specificexpressionofspecifictranscrip-tionfactormayberelatedtoarea-specificstructureandfunction.NoCOI.


3P-073Human Brain Areas Activated by Attention-related Stimuli in Relation to Genetic VariationsYamada, Kazuhiro1,2; Fujii, Kosuke2; Kuroki, Chihiro1,3; Akiyoshi, Jotaro4; Kawano, Yoshihisa2(1Dept. of Neurophysiol., Univ. of Oita Facult. of Med., Yufu, Japan, 2Kawano Neurosurg. Hosp., Oita, Japan, 3Suwanomori Hosp., Oita, Japan, 4Dept. of Psychiat., Univ. of Oita Facult. of Med., Yufu, Japan)

Brainactivitiesare“intermediate”phenotypesbetweengenesandbehavior.Toassessthe intermediatephenotype inhumans,brainimagingbyfMRIismostsuited.WeemployedANT(attention-networkstest)thathasbeenappliedtofMRIbrainimaging(FanJ.etal.,2005).ThetaskinANTistorequestparticipantstoreportthedirectionofanarrowprojectedonascreen.Threeattention-relatedprocessescanberesolved,i.e.,1)alertingbypresentingacue,2)orientingbyspatialcues,and3)conflictresolvingbysimultaneouslypresentingflankerarrowsheadingtowardstheotherend.Unlikethepredictionsmadesofar,mostoftheactivatedareasweredetectedinduplicateintwoorallofthethreeprocesses(p<0.05,FWEcorrected).Theseactivatedareaswerelefthigher-ordervisualandinferiorparietalcortices(oc-cipitoparietalprojection),lefthigher-ordervisualandadjacenttempo-ralassociationcortices(occipitotemporalprojection)andrightSMA.Exceptionwasthatrightoccipitotemporalprojectionareaandrightanteriorcingulatecortexwereactivatedonlyintheconflictresolving.Weareinvestigatingvariations(SNPs)incandidategenestofindthegeneticpolymorphismintheactivationpatterndetected.Inthiswaywemaybeableto investigatetherelationshipbetweengenesandbehavior,andtheattention-relatedneuralsubstrateandfunctioninhumanbrain.NoCOI.

3P-074Neural correlates of personality traits: A resting-state fMRI studyDonishi, Tomohiro1; Terada, Masaki2; Kaneoke, Yoshiki1(1Department of System Neurophysiology, Wakayama Medical University, Wakayama, Japan, 2Wakayama-Minami Radiology Clinic, Wakayama, Japan)

Neuralcorrelatesofpersonalitytraitsstillremaincontroversialal-thoughanumberofbrainregionshavebeensuggested.Afurtherunderstandingofitsneuralbasiswouldalsoprovidebetterinsightintopersonalitydisorders.Therefore,theaimofthisstudywastoexaminethecorrelationofpersonalityestimates (obtainedfromCloninger’sTemperamentandCharacterInventory,TCI)withglobalfunctionalconnectivityasmeasuredbyfMRI(“regionalglobalconnectivity,”rGC).Weuseda3-TeslaMRI(Philips)toobtainstructuralandresting-statefunctionalimagesfromhealthymalesubjects(N=89,18–24yearsold).AfterpreprocessingofBOLDsignalsthroughSPM8andMATLAB,cross-correlationcoefficientsofeachvoxel(6×6×6mm)withallothervoxelswerecalculatedandaveragedtodeterminerGCineachvoxel.SignificantpositivecorrelationsbetweenmostofTCIscores(NoveltySeeking,RewardDependence,Persistence,Self-DirectednessandSelf-Transcendence)andrGCwererevealed (p<0.05,correctedformulti-comparisonwithMonteCarlosimulation)atdistinctgraymatterregions(includingmiddle/inferiorfrontalgyri,orbitofrontalcortexandcerebellarhemisphere),whilesignificantnegativecorrelationswererevealedforCooperativenessscoreatprecuneus.Theresultssuggestthattheseregions,eachwithdistinctfunction,areincludedinthebrainnetworkrelatedtopersonalityformation.NoCOI.

3P-075Neonatal dopamine depletion results in a decrease of anxiety-related behaviors in the adulthoodOgata, Masanori; Noda, Kazuko; Akita, Hisanao; Toda, Satoshi; Ishibashi, Hitoshi(Div. of Physiology, Sch. of Allied Health Sci., Kitasato University)

Dopamineneuronsoriginatinginthemidbrainisimplicatedinsever-alimportantfunctions,includingmotorcontrol,attentionandemotion.Theeffectsofdopaminedepletionontheneuralfunctionsprobablydependonthelesioneddevelopmentalperiod.Asanexample,dopaminedepletionintheadulthoodcausesanakineticmotoractivity,andthatintheneonatecausesamotorhyperactivity.Althoughsomereportshaveshownanincreaseofanxiety-relatedresponsecausedbydopa-minedepletionintheadulthood,effectsofneonataldopaminedepletiononanxiety-relatedresponse intheadulthoodarestillunclear.Toclarifycharacteristicsofbehavioralresponsetoanxiogenicstimulationintheadulthoodofratwithneonataldopaminedepletion,weperformedthebehavioralanalyseswithanopenfield(OF)test,alight/darkbox(L/DB)testandanelevatedplusmaze(EPM)testusingtheratsthatreceivedintra-ventricularinjectionof6-hydroxydopamine5daysafterbirth. Intheadulthood,theratswithneonataldopaminedepletionshowedasignificantincreaseindistancetraveledandtimespentincenterareaintheOFtest,andasignificantincreaseinthenumberofopenarmentriesandheaddipsintheEPMtest.Thereisnosig-nificanteffectofneonataldopaminedepletionontheanxiety-relatedbehaviorintheL/DBtest.Theseresultsindicatealackofanxiety-re-latedbehaviorinpart,andsuggestthatdopaminesystemhasacrucialroleinthedevelopmentofinformationprocessingsystemforbehav-ioralresponsetoanxiogenicstimulation.NoCOI.

3P-076The development of attentional prioritization of own face during adolescence.Doi, Hirokazu; Shinohara, Kazuyuki(Graduate School of Biomedical Sciences, Nagasaki University)

Adolescenceisthedevelopmentalstagecharacterizedbytheabruptchangeofphysicalandpsychologicalattributestriggeredpartlybythegonadalhormonesecretion.Humandevelopmentresearchersarguethatasuddensurgeoftheinterestinownbody-imageisobservedduringthisstage.However,atthispoint,suchclaimisbasedlargelyontheanecdotalorobservationalevidences,andfewexperimentalstudieshaveverifiedthiscontention.Theprimaryaimofthepresentstudyistoobjectivelyquantifytheinterestinownbodyimage,andtrackthedevelopmentalcourseofitduringadolescence.Tothisend,wemeasuredthevisuo-spatialattentiondirectedtowardsownface,familiarfriend’sfaceandunfamiliarstranger’sfaceinearly-andmid-dle-adolescentparticipantsusingeye-tracker.Theresultshaveshownthattheparticipantsintheearlyadolescencespendlongerdurationonfixatingtofriend’sthanownface.Thoseinthemiddleadolescencehaveexhibitedthereversepatternofattentionalprioritization.Thatis,theyspendlongeronlookingatownfacethanonthefriend’sface.Therewasnodiscernibledifferenceinthefixationdurationonthestranger’s facebetweenearly-andmiddle-adolescentparticipants.Theseresults indicatethatthepatternofattentionalprioritizationqualitativelychangesduringtheadolescence,andthatthischangeischaracterizedmainlybytheincreasedattentivenesstowardsownfaceconsistentlywiththeexistingviewthatthereisasurgeofself-con-sciousnessduringadolescence.NoCOI.


3P-077Age-related human brain changes detected by MRI T1W/T2W ratioIshida, Takuya1; Iwatani, Jun2; Shinosaki, Kazuhiro2; Donishi, Tomohiro1; Terada, Masaki3; Kaneoke, Yoshiki1(1Department of System Neurophysiology,Wakayama Medical University,Wakayama,Japan, 2Department of Neuropsychiatry, Wakayama Medical University,Wakayama,Japan, 3Wakayama-Minami Radiology Clinic,Wakayama,Japan)

MRIT1w/T2wratiosignalintensity(calculatedbyT1weightedimagesignal intensitydividedbythatofT2weighted image)cancelsthereceivercoilbiasandincreasesthecontrastrelatedtomyelincontent.Weexaminedtheage-relatedT1w/T2wsignalchangetoevaluatetheusefulnessoftheT1w/T2wratioimageforstudyingthenormalhumanbrain functionsandvariousbraindiseases.NormalizedT1w/T2wratioimageswerecreatedfor33healthysubjects(23–60yearsold).Ateachvoxel,correlationbetweenthesignalintensityandtheagewerecalculated.Significantnegativecorrelationswerefound(p<0.05,correctedformulti-comparisonwithMonteCarlosimulation)atcorpuscallosum,pyramidaltract,coronaradiata,andleftopticradiationinwhitematterandatinsular,basalganglia,cingulategyrus,parieto-oc-cipitalcortexinthegraymatter,whichcorrespondstotheage-relatedmyelinreduction.Significantpositivecorrelationswerefoundatrightinferiorfrontal,leftinsularwhitematter,andrightexternalcapsuleandthesignalintensitypeakwasaroundtheageof45,whichcorre-spondstothe latedevelopmentof themyelination intheseareas.Age-relatedsignalintensitychangeinT1w/T2wratioimageindicatesthepossibilityofquantitativeanalysisofhumannormalbrainandpathologyofvariousbraindiseasesincludingdevelopmentaldisordersanddegenerativenervousdiseases.NoCOI.

3P-078Neuronal responses to pure-tone and complex sound stimuli in the putamen, globus pallidus and amygdala of awake catsZhong, Renjia; Qin, Lin; Ma, Hanlu(Department of Physioplogy, Interdisciplinary Graduate School of Medicine and Engineering (Faculty of Medicine), University of Yamanashi, Yamanashi, Japan)

Thebasalgangliaandamygdalahavebeenknowntobehighlyinvolvedingoal-directedbehaviorsandemotionalfunctions.Todate,littleisknownabouthowthesensorysignalsareprocessedinthesenonau-ditoryneuralloci.Here,weinvestigatedtheacousticresponseprop-ertiesofneuronsinputamen(PU),globuspallidus(GP),lateralamyg-dala(LA)andcentralamygdala(CA)oftheawakecats.Wefoundthat1)PUneuronswellrespondedtothepure-tonestimuli. Theyhadshorterresponselatencies,lowerintensitythresholdsandlargerre-sponsemagnitudes.Theresponsivenesstopure-tonesgraduallyde-creasedintheorderofPU,GP,LAandCA.2)Theneuralresponsive-nesstocomplexsounds(vocalizationsofcon-speciesandotherspeciesandnonlivingenvironmentalsounds)alsodeterioratedintheorderofPU,GP,LAandCA,whiletheselectivityforthecomplexsoundsin-creased(responsetoasmallfractionofthestimuli).3)Theproportionofneuronspreferredtocomplexsoundsratherthanpure-toneswaslargerinLAandCAthaninPUandGP.Theratiobetweenthere-sponsemagnitudesofcomplexsoundsandpure-toneswashigherinCA.OurresultssuggestthatPUandGPmightreceivemoredirectauditoryinputs,whichcanprovideprecisesensoryreferencesmeetingtherequirementstomakearapidmotorresponse.Onthecontrary,LAandCAmightprocessmoreindirectauditorysignalstofulfilltheemotionaland/orcognitivefunctions.NoCOI.

3P-079An experimental model of an emotional response that induced by auditory stimulation, which are linked to the experience-stimulation couplingShutoh, Fumihiro1,2; Sugimoto, Koji2; Yamashita, Kenya2; Hisano, Setsuji1,2(1Fac. Medicine, Univ. Tsukuba, Tsukuba, Japan, 2Kansei, Behav. and Brain Sci, Grad. Sch. Compreh. Hum. Sci., Univ. Tsukuba, Tsukuba, Japan)

Perceptionofenvironmentalsensorystimulithroughsoundsinducesseveralkindsofemotions,despitetheunderlyingbiologicalmechanismisstillunclear.Toclarifythemechanism,wehaveattemptedtoestab-lishusefulexperimentalmodels.Inananimalmodelstudy,weexposedmicetothesesoundswithinasoundproofbox.Serotoninconcentrationinthefrontalforebrainwasshowntobehigherinthesound-exposedmicethaninmicekeptinsilence.Auditoryenvironmentalsurroundingscaninfluenceemotionalresponsethroughserotonergicneuronsystem.However,hearingasoundoftenelicitsanemotionthatisinducedbynotinstinctbutexperience.Inlightofthissoundeffect,wetriedtoexaminewhetherornotthehearingexperienceactuallyevokesemo-tionalresponseusinganexperimentalmousemodel, inwhichmicewereexposedtoenvironmentalsoundstimulationcoupledtodifferenthousingconditions.Weexposedmicetoanartificialsoundstimulationundereachof thepleasantandtheunpleasanthousingconditions.Aftermicewerespentineachofthetwoconditionsforseveraldays,weanalyzedsomephysiologicalparametersofmicewhileexposingtoasequenceofsoundstimulations.Amongtheparameters,hartbeatratewassignificantlydecreasedbypresentingasoundcoupledtothepleasanthousingcondition.Thisstudyprovidesanmodeltounderstandneuronalmechanismsofemotioninducedbyauditoryperception.NoCOI.

3P-080Enhanced sociality by umami intake during the period of development is mediated by vagus nerve afferent in ADHD model ratYokoyama, Yoshihiro1,2; Simizu, Yuko1; Ueda, Yoshitomo1; Misumi, Sachiyo1; Isida, Akimasa1; Jung, Cha-Gyun1; Yokoi, Motoo2; Hida, Hideki1(1Dept Neurophysiology & Brain Sci, Nagoya City Univ Grad Sch Med Sci, Nagoya 467-8601, Japan, 2Department of Maxillofacial Surgery,Nagoya City University Graduate School of Medical Science)

Spontaneouslyhypertensiverat(SHR)arethemostusedanimalmod-elofattentiondeficithyperactivitydisorderthatischaracterizedbyhyperactivity,impulsivityandinattention.WefoundthatoralintakeofmonosodiumL-glutamate(MSG),atastesubstanceforumami,for5weeksfrompostnatalday25(P25)toP60alteredsocialbehaviorinSHRthatwasgrowninsingleisolatedcondition.HoworalMSGintakeduringtheperiodofdevelopmentinducedenhancedsocialityinadult-hoodisunknown.Toinvestigatetheeffectofbrain-gutcommunicationviavagusnerveontheMSGeffect,SHRinisolationenvironmentre-ceivedvagotomyatP25followedbyMSG(0.6%solution)intakefor5weeks,andthenbehavioraltests(openfieldtest:OFT,socialtest:ST)werecarriedout.InOFT,nodifferencewasobservedinthevagoto-my-groupascomparedtosham-operatedMSG-treatedgroup.Howev-er,inST,sniffingtimeandridingnumberinthevagotomy-groupin-creasedbacktosamelevelofnon-MSGtreatedgroup.DatasuggestthatMSGeffectonsocialbehaviorduringtheperiodofdevelopmentwasmediatedbytheafferentofvagusnervefromthegastrointestinaltractreceptor.NoCOI.


3P-081Neural activities to frequency-modulated sounds in the frequency bands of the primary auditory cortex of guinea pigs observed by optical recordingHosokawa, Yutaka1; Kubota, Michinori2; Sugimoto, Shunji3; Horikawa, Junsei3(1Dept. of System Physiol., Grad. Sch. Univ.of Ryukyus, Okinawa, Japan, 2Med. Res. Inst., Tokyo Medical and Dental Univ. Tokyo, Japan , 3Grad. Sch. of Comp. Sci. and Eng., Toyohashi Univ. of Technology, Toyohashi, Japan)

Neuralactivitiestofrequency-modulated(FM)soundswithdifferentFMsweepratesintheprimaryauditoryfield(AI)oftheleftandrightauditorycorticesoftheguineapigwere investigatedusingopticalimagingwithavoltage-sensitivedye(RH795).Eightguineapigswereanesthetizedwithketamine(80mg/kg)andxylazine(40mg/kg).Activ-itypatternstotheFMsounds(upwardanddownwardlinearlysweptfrequency:0.5–16.5kHzin16–400msdurationorFMsweeprate0.04–1kHz/ms)at75dBSPLandthetones(0.5,16kHz)wererecordedfromtheAI.ThepeaksoftheactivitytoFMsoundsandtonesweremeasuredinthedorsalandventralregionsofthe0.5,1,2,4,8,16-kHzfrequencybands(FB).Thepeakstothe0.5-kHztoneinthe0.5-kHzbanddidnotchangemuchinamplitudewithrespecttotheduration(withsmallmaximaat40and640ms)andthose intheotherFBsshowedthesimilardurationfunctionwithsmalleramplitudes.ThepeakstotheFMsoundsinthe8-and16-kHzbandsshowedthedura-tionfunctionwiththemaximalamplitudeat16msanddecreasedamplitudesbeyond64ms.Thistendencyinthedurationfunctionwassalient inthedorsalbutnottheventralregionofthesebandsandneitherintheotherlowerFBs.WediscussthefunctionaldifferenceofthefrequencyprocessinginAIofguineapigs.NoCOI.

3P-082Temporal information processing of visual and audito-ry cues in monkey prefrontal cortex during a duration discrimination taskChiba, Atsushi; Oshio, Ken-ichi; Inase, Masahiko(Dept. Physiol. Kinki Univ. Facult. Med. Osaka-Sayama, Japan)

Weexaminedneuronalactivity inmonkeyPFCduringadurationdiscriminationtaskwithvisual(Vis,agreensquare)andauditory(Aud,2000Hztone)cues.Inthetask,twocues(firstcue,C1andsecondcue,C2)werepresentedconsecutivelyfordifferentdurationrangingfrom0.2to1.8sec.Eachcuewasfollowedby1secdelayperiod.Subjectswererequiredtochoosethelongerpresentedcueaftertheseconddelay(D2)period.Therearefourkindsofcuemodalitycombinations;Vis-Vis,Vis-Aud,Aud-VisandAud-Aud.Durationoftwocues,orderofcueduration (long-short,LSorshort-long,SL),andcuemodalitycombinationwerepseudo-randomized.Outof860PFCneuronsexam-ined,64and139neuronsrespondedtotheC1andC2,respectively.Morethan80%ofC1responseneuronsweremodality-specific,re-spondedtoeitherthevisualorauditoryC1,while14%oftheneuronswerebimodal.Morethan70%ofC2responseneuronswerealsomo-dality-specific,while25%oftheneuronswerebimodal.Outof192D2responseneurons,57neuronsexhibitedgreaterD2activityintheLStrialsthantheSLtrials,while85neuronsshowedgreateractivityintheSLtrialsthantheLStrials.MorethanhalfoftheseD2responseneuronsrespondedsimilarlyafterthevisualandauditoryC2anddidsoamongthefourcuemodalitycombinations.TheseresultssuggestthatthePFCintegrates interval informationondifferentmodesofsensorystimuliandcontributestotemporaldiscriminationbetweenthesestimuliafterthetwocuepresentations.NoCOI.

3P-083Sound-shape association memory tested using a M-maze in miceYamagishi, Tatsuya1,2; Tsukano, Hiroaki1; Baba, Hironori2; Honma, Yusuke1,2; Ohsima, Shinsuke2; Kubota, Yamato2; Takahashi, Kuniyuki2; Takahashi, Sugata2; Shibuki, Katsuei1(1Dept Neurophysiol, Brain Res Inst, Niigata Univ, 2Dept of Otolaryngology, Faculty of Medicine, Niigata Univ)

Wecanrecallaparticularshapefromasoundstimulus intimatelyassociatedwiththeshape.Toinvestigatethissound-shapeassociationmemoryinmice,wedevelopedaM-mazeequippedwithascreenandaspeaker.First,acombinationofsoundAandshapeA,orsoundBandshapeBwaspresentedtowater-deprivedmice.Afterthat,shapeAandshapeBwerepresentedatthetwoinletsoftheM-mazebranch-es.Ifthemicechoosethebranchwiththesameshapepresentedbefore,theycouldobtainasmallamountofwater.Thistrialwasrepeated20timespereachday.Micelearnedtoselectthecorrectshapesafterabout20sessions.Wefurthertestedwhetherthemicecouldselecttheshapesbasedonthesoundcuesonly,andweconfirmedthatthetrainedmiceselectedthecorrectshapessuccessfully,indicatingthatmicehavesound-shapeassociationmemory.Afterthetraining,weanesthetizedthemice,andcorticalresponsestothesoundstimuliwithorwithoutpresentationoftheshapeswererecordedintheauditorycortexusingflavoproteinfluorescenceimaging.Theresponsestothesound-shapestimuliwerelargerthanthoseelicitedbythesoundsonly.Theareasventraltotheauditorycortexwerealsoweaklyactivatedbythesound-shapestimuli.Suchpositive interactionbetweenthesoundsandshapeswasnotobservedincontrolmice.Theseresultssuggesttheauditorycortexandtheventralareasmayplayanimport-antroleinsound-shapeassociationmemory.NoCOI.

3P-084Visual responses for one’s own hand during hand ma-nipulation movement of hand manipulation-related and mirror neurons.Maeda, Kazutaka1,2; Ishida, Hiroaki3; Nakajima, Katsumi1; Inase, Masahiko1; Murata, Akira1(1Department of Physiology, Graduate School of Medicine, Kinki University, Osaka, Japan, 2JSPS Res. Fellow, Tokyo, Japan, 3Italian Inst. of Technology, Brain Ctr. for Motor and Social Cognition, Parma, Italy)

Duringmotorbehavior,ourbrainrepresents“bodyschema”,thatisspatiotemporaldynamicorganizationofthebody,basedoninteractionsbetweenmotoroutputsignalandmultisensoryfeedback.Thehandmanipulation-relatedormirrorneuronsininferiorparietalareaAIP/PFGofthemacaquemonkeythatareheavilyinvolvedinvisualandmotorintegration,maycorrelateone’sownbodyschema.Theaimofthisstudywasto investigatewhethertheseneuronsencodevisualfeedbackduringself-generatedhandaction.WeexaminedactivityofsingleneuronsfromAIP/PFGoftwomonkeysduringexecutionofgraspingactionviewingvideomonitorthatpresentedtheironlinemovements,andduringfixatingvideoclipsof theirowngraspingactionfilmedfromthemonkey’spointofviewandthenexperimenter’sgraspingaction.Werecorded54handmanipulation-relatedneurons(including33mirrorneurons)thatrespondedtovideoclipsofone’sownand/orexperimenter’shandmovement.Ofthese,25neurons(including13mirrorneurons)alsorespondedtothevideoclipsofone’sownhandmovementwithouttheimageofthetargetobject.Theseresultssuggestthatsomeofhandmanipulation-relatedormirrorneuronsintheAIP/PFGvisuallyrespondedtoone’sownhandkine-matics,representingthebodyschemeratherthanrepresentingthegoalofaction.NoCOI.


3P-085Relationship between crossed hand illusion and au-tism spectrum quotient scoreWada, Makoto1,2; Suzuki, Mayuko3; Agarie, Hiromi3; Takaki, Akiko4; Kansaku, Kenji2(1Dev Disorders Sect, Dept of Rehab for Brain Func, Res Inst of NRCD, Tokorozawa, Japan, 2Sys Neurosci Sect, Dept of Rehab for Brain Func, Res Inst of NRCD, 3Info Center for Persons with Dev Disorders, NRCD, 4Chichibu Gakuen, Rehab Serv Bureau, NRCD)

Neurotypicialindividualsexperienceasubjectivereversaloftemporalorderjudgmentswhenthetwohandsarestimulatedwhiletheyarecrossed (Yamamoto&Kitazawa,2001)and itmaybecausedbyaconflictbetweenanegocentricandanallocentricstance(Shoreetal.,2002).Recently,wefoundthatthecrossedhandillusionwassignifi-cantlysmallerinautisticthaninneurotypicalchildren(Wadaetal.,2013).Inthisstudy,weinvestigatedarelationshipbetweenthecrossedhandillusionandAutismSpectrumQuotient(AQ)scores.Youngboyswhojoinaself-helpgroupformilddevelopmentaldisordersandalsoattendaregularschoolclassparticipatedinthestudy(n=12,16.3±0.4y.o.).Theywererequiredtojudgetemporalorderoftwotactilestimulithatweredeliveredtotheirbothringfingerswiththeirarmscrossed.Aftertheorder-judgmentprobabilitiesthattherighthandwasstimulatedearlierwerefittedbythedoubleflipmodel(Yamamo-to&Kitazawa,2001),wefoundthatbothright-to-leftandleft-to-rightflipprobabilitieswerenegativelycorrelatedwithAQscore(R=-0.77,P<0.01;R=-0.70,R<0.05)duringthearmcrossedcondition.PresentresultssuggestthattheyoungboyswhohavehigherAQscoresgenerallyshowedsmalleramountsofthecrossedhandillusion,whichwasconsistentwithourpreviousstudythatusedautisticchildren(Wadaetal.,2013).NoCOI.

3P-086Prenatal low-dose bisphenol A enhances behavioral alterations induced by predator odorFujimoto, Tetsuya1; Kubo, Kazuhiko2; Aou, Shuji3; Nishikawa, Yasuo1(1Dept. Physiol., Osaka Dent. Univ., Hirakata, Japan, 2Dept. Otorhinolaryngol., Grad. Sch. Med. Sci, Kyushu Univ., Fukuoka, Japan, 3Dept. Brain Sci. Eng., Kyushu Inst. Technol., Kitakyushu, Japan)

BisphenolA(BPA)isoneofthe‘environmentalendocrinedisrupters’(EEDs).Pastourstudyshowedthatpre-andpostnataladministrationsoflow-levelBPAinduceddepression-likebehaviorinrats.Wefocusedonthestressvulnerabilitywhichwasconsiderablyrelatedtothede-pression.Inordertoinvestigatetherelationshipbetweenthedepres-sive-responseandthestressvulnerability,wedesignedonebehavior-al experiment using the predator odor as a stressor.Wemadecross-formplasticchamber, inwhich,predatorodor (foxodor)waslocatedattwooppositecorners.Ratswerelocatedatthecenterposi-tion,andevaluatedthelocomotoractivityandtheavoidanceresponse.Inthisstudy,pregnantWistarratswereexposedtolow-doseBPAduring7daysjustbeforebirth.Themaleandfemaleoffspringwereevaluatedattheageof10weeks.BothcontrolandBPAgroupsshowedthereduced locomotoractivityunderthepredatorodor,especiallyBPAgroupwasremarkable.Theodor-avoidanceresponsewassignif-icantonlyinBPArats.BPAexposureratswereobviouslysensitivetothepredatorodor.ItsuggestedthatprenatalBPAexposureratshadabackgroundregardingthevulnerabilitytostresssuchasthepredatorodor.Thatmayhavesomerelationshipstotheenhancementofdepression-likebehaviorintheforcedswimmingtest.NoCOI.

3P-087Recordings of neuronal activity in the pig brain relat-ed to the snout functionSaito, Toshiyuki1,2(1Department of Animal Medical Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kyoto, Japan, 2Laboratory of Neurobiology, National Institute of Agrobiological Sciences, Tsukuba, Japan)

Thepig’ssnoutisnotonlythemainorganrelatedtothesenseofsmell,butalsoischieftactileorganaswellaschiefeffector,spadeasthehumanhand.Inthisstudy,weexaminedneuronalactivityinthepigbrain,relatedtothesnoutfunction.Inthefreely-movingcondition,localfieldpotentials (LFP)weremeasuredbyuseof thewirelesstechnique.Largenegativewaves intheLFPwererecorded inthehippocampus,priortopushingaswitchbythesnouttogetarewardduringoperantlearningtasks.Undergeneralanesthesia,evokedpo-tentialswererecordedinthehippocampusaswellasintheposteriorpartoftherostralregionbycontralateralelectricalstimulationonthesurfaceoftheupperpartinthesnout.Thehippocampusinthepigmaybeareceptivefieldtotheafferentinputfromthesnoutandmay,inpart,beinvolvedinmotorcontrolforthesnoutaswellaslearningandmemory.Furtherinvestigationsneedtorevealneuronalmecha-nismsgeneratingthelargenegativewavesinthepighippocampusassociatedwithpushingtheswitchduringtheoperantconditioningtasks.NoCOI.

Poster PresentationsNutrition, Metabolism,

Thermoregulation


3P-088Evening-based feeding habit impaires insulin sensitiv-ity via central AgRPShiuchi, Tetsuya1; Otsuka, Airi1,3; Shimizu, Noriyuki1; Chikahisa, Sachiko1; Sei, Hiroyoshi1(1Dept. Integrative Physiology, Inst. Health Biosciences, The Univ. Tokushima Graduate School, Tokushima, Japan, 2PRESTO, JST, Saitama Japan, 3Dept. Food Science, Inst. Health Biosciences, The Univ. Tokushima Graduate School, Tokushima, Japan)

Disturbanceoffeedingrhythmhasbeenunderstoodasariskfactorfordevelopmentofinsulinresistance,however,itisnotelucidatedthedetailedmechanism.Inthisstudy,3groupsofC57BL/6Jmiceweregivenlabchowsfreelyduringdarkphase(ZT12–24,Controlgroup),first4-hourindarkphase(ZT12–16;Morninggroup),orlast4-hourindarkphase(ZT20–24,Eveninggroup)for8weeks.MiceinEveninggroupshowedimpairedwholebodyinsulinsensitivitybyinsulintol-erancetestdespitemiceinthegroupingestedthesmallerfoodintakethanthatofControlgroup,whilemice inMorninggroupshowednormalinsulinsensitivity.Weobservedhighertriglyceride(TG)con-tent,increasedgeneexpressionoffattyacidsynthase(FAS)andim-pairedinsulinsignalsinskeletalmuscleinEveninggroupcomparedtoothergroup.Theseeffectswerenotobservedinliver.Ontheotherhand,mRNAexpressionofagouti-relatedprotein(AgRP)wasincreasedinhypothalamusinEveninggroup.Weobservedthatacuteand/orchronicICV-injectionofAgRPincreasedFASexpressionandTGcontentinskeletalmuscle.Moreover,inhibitionofcentralAgRPex-pressionbyantisenseoligo improved insulinresistance inEveninggroup.Theseresults indicatethatfeedingrhythmlikeas ingestiononlyintheeveningimpairsinsulinsensitivitybecauseofTGaccumu-lationinskeletalmusclemediatedbyhypothalamicAgRP.NoCOI.

3P-089Hindbrain responsiveness to anorexinergic hormones is reduced in animals showing binge-like overcon-sumptionYamaguchi, Erina; Yasoshima, Yasunobu; Shimura, Tsuyoshi(Div Behav Physiol, Dept Behav Sci, Grad Sch Human Sci, Osaka Univ, Suita, Japan)

Previousstudiessuggestthatthefeedbackinhibitionoffoodintakebypostprandialvisceralstimulationisbluntedinanimalmodelsshow-ingbinge-likeoverconsumption.However,itisunclearwhethertheresponsetogutanorexinergicpeptidesisreducedintheseanimals.Inthepresentstudy,wefocusedonneuralresponsesinthenucleusoftractussolitarius(NTS)andparabrachialnucleus(PBN)toanintraper-itonealinjectionofpeptideYY(PYY),oneofgutpeptidehormones,inC57BL/6Jmiceusingc-fos immunoreactivityasaneuralactivationmarker.Miceweregivenlimitedaccesstosucrosefor4hour/daywith(trainedgroup)orwithout(controlgroup)foodrestriction.Trainedmiceshowedbinge-likesugaroverconsumption.Incontrolanimals,manyc-fos-positivecellswereseenintheNTSandPBNinresponsetothePYYinjection,especiallyintheareareceivinggastrointestinalbutnottaste-related information. Incontrast, intrainedmice, thenumberofc-fos-positivecellsactivatedbytheperipheralPYYinjectionwassmallerinbothnucleicomparedwithcontrolmice.Thepresentresultssuggest thatbinge-likesugaroverconsumptionattenuateshindbrainresponsivenesstogutanorexinergicpeptide.NoCOI.

3P-090Effect of the number of chewing on diet-induced ther-mogenesis and postprandial appetiteHamada, Yuka1; Kashima, Hideaki2; Hayashi, Naoyuki1(1Graduate School of Decision Science and Technology, Tokyo Institute of Technology, Tokyo, Japan, 2School of Health Sciences, Prefectural University of Hiroshima, Hiroshima, Japan)

Thepresentstudywastoexaminetheeffectofthenumberofchew-ingondiet-inducedthermogenesis(DIT)andsubjectiveappetiteaftereating.Afterbaselinemeasurementsintheovernightfastingstate,tenhealthynormal-weightsubjectschewed300-kcalsolidfoodaslongandmanytimesastheycouldbeforeswallowingintheStrial,whiletheyswallowedthesamebutfracturedfoodasfastastheycouldintheRtrial.DITwascalculatedfromoxygenuptakeandbodymass.Subjec-tivescoreofappetitewasassessedbyvisualanalogscale(noappetite:0-appetite:100)every15min.DITandappetitescorewererecordeduntil90minaftereating.DurationandthenumberofchewingweresignificantlygreaterintheStrialthanintheRtrial(497±45vs.103±11s,702±108vs.137±15times,respectively,p<0.05).DITwassignificant-lygreaterintheStrialthanintheRtrial(11±2vs.1±1kcal/90min,p<0.05).Appetitescoredecreasedsignificantlyfromrestingbaselinelevel;thiswasshowninimmediatelyaftermealandcontinueduntil30minintheStrial,while,solelyat75minaftermealintheRtrial.AppetitescorewassignificantlylowerintheStrialthantheRtrialuntil90minaftermeal(44±7vs.61±5,immediatelyaftermeal;50±6vs.67±5,90min;respectively,p<0.05).TheseresultssuggestthatfewernumberofchewingafoodinhibitstheincreaseinDITandthedecreaseinappetite,implyinganassociationbetweeneatingrapidlyandoverweight.NoCOI.

3P-091Effect of systemic ghrelin administration on tail-hiding behavior in a cold exposure in male ratsUchida, Yuki1; Osako, Yoji 1; Nagashima, Kei2,3,4; Yuri, Kazunari1 (1Department of Neurobiology and Anatomy, Kochi Medical School, Kochi University, Kochi, Japan, 2Laboratory of Integrative Physiology (Body Temperature and Fluid Laboratory), Faculty of Human Sciences, Waseda University, Saitama, Japan, 3Sport Sciences for the Promotion of Active Life, Waseda University, Saitama, Japan, 4Institute of Applied Brain Sciences, Waseda University, Saitama, Japan)

INTRODUCTIONRatsplacetheirtailsunderneaththeirbodytrunksinthecold(tail-hidingbehavior).Inacold(15°C),bodytemperature(Tb) in42-h fastedratsdecreasedwhenatail-hidingbehaviorwaspreventedexperimentally(Uchidaetal.,2012).Thus,tail-hidingbe-haviorisathermoregulatorybehaviorinfastedrats.Theaimofthepresentstudywastoelucidatewhetherghrelinthatisanincreasinghormonebyfastingisinvolvedintail-hidingbehaviorinratsduringacoldexposure.METHODSMaleWistarratsweredividedinto‘fed’,‘42-hfasting’andghrelingroups.Theratsreceivedani.p.salineor30µgghrelininjection,thenexposedto27°Cor15°Cfor2-hwithcontin-uousTbandtail-hidingbehaviormeasurements.RESULTSAt15°C,Tbdecreasedonly inthefastedgroup. Thedurationoftail-hidingbehaviorincreasedinthefastedandghrelingroupsthanthefedgroupat15°C. Theonsetoftail-hidingbehavioradvanced intheghrelingroupthanthefedandfastedgroupsat15°C.CONCLUSIONTheseresultssuggestedthatghrelinmightmodulateatail-hidingbehaviorduringacoldexposureinrats.NoCOI.


3P-092Skin warm perception is enhanced by regular endur-ance trainingTakeda, Ryosuke1; Okazaki, Kazunobu1,2; Imai, Daiki1,2; Suzuki, Akina1; Yamashina, Yoshihiro1; Naghavi, Nooshin1; Yokoyama, Hisayo1,2; Miyagawa, Toshiaki1,2(1Department of Environmental Physiology for Exercise, Osaka City University Graduate School of Medicine, Osaka, Japan, 2Research Center for Urban Health and Sports, Osaka City University, Osaka, Japan)

Increase inskinandesophagealtemperature (TskandTes) inducesautonomicandbehavioralthermoregulatoryresponseswiththeper-ceptionofbothtemperatures.Ithasbeenreportedthatregularen-durancetrainingenhancesautonomicthermoregulatoryresponsesandmaymodifywholebodythermalperception.Weassessedwhetherskinthermalperceptionwasalsomodifiedwithregularendurancetraining.Methods:Tentrained(<3daysoftraining/week)andsevenuntrainedhealthyyoungmenunderwentmeasurementsofnoticeableincrease/decrease(±0.1°C/sec)ofskintemperature(warm/coldthresh-old)atchestbyusingathermode(6.25cm2)andofsubjectivewholebodythermalsensation(visualanaloguescale)innormothermia(NT,Tes;36.6±0.2°C)andhyperthermia(HT,Tes;37.3±0.1°C,lowerlegsim-mersionin42°Cwater).TesandTskweremeasuredcontinuously.Results:Inbothgroups,coldthresholdwasdecreasedandsubjectivewholebodythermalsensationwereincreasedwithTesandTskinHTcom-paredwithNT(all,P<0.05),whilewarmthresholdremainedunchanged.InNTandHT,warmthresholdwaslower(moresensitive)intrainedthanuntrainedmen(P<0.05),althoughcoldthresholdandsubjectivewholebodythermalsensationweresimilarinbothgroups.Conclusions:Skinwarmperceptionbutnotskincoldperceptionorwholebodythermalsensationismoresensitiveintrainedthanuntrainedmen.NoCOI.

3P-093The effect of the head or neck cooling on body core temperature and thermal pleasantness in humans.Matsuda, Mayumi1; Wada, Satoshi1; Marui, Shuri1; Sato, Nobuo1,4; Nagashima, Kei1,2,3(1Body Temp. Fluid Lab., Fac. Human Sci., Waseda Univ., Tokorozawa, Japan, 2 IABS, Waseda Univ., Tokorozawa, Japan, 3GCOE Active Life, Waseda Univ., Tokorozawa, Japan , 4Dept. Anesthesiology, Tokyo Women's Med. Univ., Tokyo, Japan)

AimSomeanimalshavemechanismsthatselectivelycoolthebrainduringhyperthermia.However, itremainscontroversial ifhumanshavethemechanism.Inthepresentstudy,weinvestigatedtheeffectoftheheadorneckcoolingontympanictemperatureasanindicatorofbraintemperature.MethodEighthealthymalesubjectsperformedfourlocalcoolingtrials(facialfanning,coolingoftheforeheadorneck,andthecontrol).Subjectssatonachair,puttingonawater-perfusionsuittomaintainskintemperatureat33°C(normothermiccondition)or38 °C (hyperthermiccondition).Tympanic,esophageal,andskintemperatureswerecontinuouslymonitored.After10-minbaselineperiod,localcoolingwasconductedfor15min.Temperaturesensationandthermalpleasantnesswerereportedbythesubjects.ResultsDuringtheheadorneckcooling,whilefacialskintemperaturewassignificantlydecreased,therewasnosignificantdifferencebetweentympanicandesophagealtemperaturesinnormothemiccondition.Onlyinhyperthermiccondition,asignificantdifferencebetweentympanicandesophageal temperatureswasobserved.After facial fanning,tympanictemperaturewaslowerthanesophagealtemperature.Dis-cussionTheseresultsmaysuggestthatfacialfanningcoolsthebraininhyperthermiccondition.However,localcoolingoftheforeheadorneckmaynoteffectontheselectivebraincooling.NoCOI.

3P-094Changes in behavioral thermoregulation in β-amy-loid-infused ratsMatsuzaki, Kentaro; Katakura, Masanori; Hara, Toshiko; Hashimoto, Michio; Shido, Osamu(Shimane Univ. Sch. Med. Shimane, Japan)

Weinvestigatedthebehavioralthermoregulationofβ-amyloid(Aβ)-in-fusedratsbymeasuringtheirselectedambienttemperatures(Ts).MaleWistarratswereanesthetizedwithpentobarbitalsodium(50mg/kg,i.p.)andimplantedintheintraperitonealcavitywithatemperaturetransmitter.Asolventof35%acetonitrileand0.1%trifluoroaceticacidwasusedasthevehicle forAβpeptide (4.9-5.5nmol).Theosmoticpumpcontained234±13.9µlofAβsolutionwassubcutaneouslyinsert-edintothebackandwascannulatedintotheleftcerebralventricle.Moreover,0.5µgofAlCl3wasinjectedintotherightcerebralventricle.Vehicle-infusedratswereusedascontrolrats (CN).After2weeks,ratswereplaced inathermalgradientandtheir intra-abdominaltemperature(Tab)andTsweremeasuredfor3days.Afterthebehav-ioraltest,ratsweresubjectedtoheattolerancetest, i.e. theywereexposedtotheambienttemperatureof36°Cfor3h.Then,ratswereanesthetizedandbrainwasremovedforimmunohistochemicalanaly-sis.Althoughtherewereclearday-nightvariationsofTsandTabinCN,the levelsofTsandTab intheAβ-infusedratswerealtered.However,magnitudesofrisesinTabofCNandAβ-infusedratsduringheattolerancedidnotchange.Immunohistochemicalanalysisshowedthattheexpressionlevelofc-Fosproteininthehypothalamus,acen-terofautonomicthermoregulation,didnotdifferbetweenCNandAβ-infusedrats.TheseresultssuggestthatAβ-infusioninthelateralventriclechangesbehavioralthermoregulationmaybewithoutaffect-ingautonomicthermoregulationinrats.NoCOI.

3P-095Heat acclimation affects circulating levels of PGE2 COX-2 and orexinLee, Beom Jeong; Shin, Oh Young; Min, Ki Young; Yang, Mo Hun (Department of Physiology, College of Medicine, Soonchunhyang University, Cheonan, Republic of Korea)

WeexaminedserumlevelsofprostaglandinE2(PGE2),cyclooxygenase(COX)-2andorexinbeforeandafterheatacclimation(HA)totestthehypothesisthatdecreasedbasalbodytemperatureduetoHAcorrelatewithcirculatinglevelsofthesekeythermoregulatorymolecules.Ninehealthyhumanmalevolunteerswererecruited(age,21.9+/-2.7years).Thesubjectswereexposedtohalf-bodyimmersioninhotwater(42+/-0.5oC)atthesametimeofday(2–5pm)onalternatedaysfor60days.TheHAprotocol included10boutsof30min immersion.Allexperimentswereperformedinanautomatedclimatechamber(tem-perature,26.0+/-0.5oC;relativehumidity,60+/-3.0%;airvelocity,1m/sec).Tympanicandskintemperaturesweremeasured,andmeanbodytemperaturewascalculated.Thedifferenceinbodyweightwasusedtoestimatetotalsweatloss.SerumlevelsofPGE2,COX-2andorexinwereanalyzedbeforeandafterHA.Bodytemperaturede-creasedsignificantlyafterHA,whereassweatvolumeincreasedsig-nificantly.SerumPGE2,COX-2andorexinconcentrationsdecreasedsignificantlycomparedtothoseatpre-acclimation.OurdatasuggestthatdecreasedbasalbodytemperatureafterHAisassociatedwithdecreasesinthermoregulatorymolecules,suchasPGE2,COX-2andorexin.KeywordsprostaglandinE2,cyclooxygenase-2,orexin,heatacclimation,bodytemperature.NoCOI.


3P-096Increase in circulating levels of free fatty acids during passive heat loadingWook, Tae Kim; Jeong, Lee Beom; Yang, Mo Hun; Min, Ki Young (Department of Physiology, College of Medicine, Soonchunhyang University, Cheonan, Republic of Korea)

Thepurposeofthisstudywastodeterminewhetherheatacclimation(HA)iscloselyrelatedtosweatingfunctionandcirculatinglevelsoffreefattyacids(FFA)duringpassiveheatloading(PHL,lowerbodyimmersioninhotwater,42+/-0.5C,30min)inhuman.Thesubjectswere17maleuniversitystudents(age,22.14+/-3.27yr;height,173.52+/-4.10cm;weight,70.40+/-5.64kg;bodyfat,19.84+/-3.28%;musclemass,22.49+/-4.19kg).Tomimicheatacclimation,repeatedlowerbodyimmersioninhotwater(42+/-0.5C,30min/day)wasperformed6timeseveryotherdayfor2weeks.Theresultsshowedthatthesweatfunctionimproved,andwaistcircumferencesignificantdecreasedafterHA.ThelevelofFFAwassignificantlyalteredafterHAinPost-PHLandthereweresignificantcorrelationsbetweenbodytemperatureandFFA,bothpre-andpost-HAtreatmentinPost-PHL(r2=0.595,r2=0.716,respectively).Thethresholdofbodytemperatureforlipoly-siswasdecreasedbyHA.Inconclusion,HAimprovedlipolysisanddecreasedwaistcircumferenceduringPHL.Keywords:Heatacclima-tion,Freefattyacids,Sweatfunction,Bodytemperature,Lipolysis.NoCOI.

3P-097Activation of G protein-coupled receptor 40, one of free fatty acid receptors, induces the enzymes of polyunsaturated fatty acids synthesis and facilitates differentiation of cultured neural stem cellsKatakura, Masanori; Hashimoto, Michio; Okui, Toshiyuki; Matsuzaki, Kentaro; Shido, Osamu(Department of Environmental Physiology, Shimane University Faculty of Medicine, Izumo, Japan)

Polyunsaturatedfattyacids(PUFAs),especiallydocosahexaenoicacid(DHA)andarachidonicacid,areessentialforthegrowthandfunction-aldevelopmentofthebrain.Moreover,decreasingPUFAlevelsanddecreasingmRNAlevelsofenzymesforPUFAsynthesiswereob-served inpostmortemhumanbraintissuesofAlzheimer’sdisease,depressivedisorder,andschizophrenia.Therefore,enhancementofPUFAsynthesisinthebrainisanimportanttargettopreventandtreatthesediseases.Thepresentstudyexaminedeffectsofactivationoffreefattyacid-activatedGprotein-coupledreceptor(GPR)40,whichisoneoffreefattyacidreceptors,ontheenzymesofPUFAssynthesisintheculturedratfetalneuralstemcells(NSCs).GPR40activationbyGPR40agonistGW9508increasedthelevelsofTuj-1(aneuronalmark-er),GFAP(anastrocytemarker)mRNAexpressionaswellasthepercentageofTuj-1-andGFAP-positivecells;suggestingthatGPR40activationenhancedneuronalandglialdifferentiation.GW9508 in-creasedthemRNAlevelsofstearoyl-CoAdesaturase,delta-5anddelta-6desaturases,andfattyacidelongase-5viasterolregulatoryelement-bindingprotein(SREBP)1ctranscriptionalactivation.SREBP1cisknownasamainregulatorofPUFAsynthesis.TheseresultssuggestthatGPR40 isrelatedtoSREBP1c-mediatedactivationofPUFAsynthesisandenhancesdifferentiationofNSCs.NoCOI.

3P-098Orexin involves lipid metabolism in liverMochizuki, Ayako; Nakayama, Kiyomi; Nakamura, Shiro; Inoue, Tomio(Department of Oral Physiology, Showa University School of Dentistry, Tokyo, Japan)

Orexinisahypothalamicneuropeptidethatregulatesmotivatedbe-haviors,includingfeedingandawake-sleepcycle,and,isalsoinvolvedinenergyhomeostasis.Orexinknockout (OxKO)micesignificantlygainedmoreweightthanwild-type(WT)mice,althoughfoodintakewasnotsignificantlydifferentbetweenbothgenotypes,suggestingthatOxKOmicemighthavelowerratesofmetabolismcomparedwithWTmice.However,itisnotclearwhetherorexinaffectsmetabolicratesofmiceornot.Thuswecomparedmetabolism,focusedonlipidmetabolisminthe liver,betweenOxKOand littermateWTmice.LiversintheOxKOmiceweresignificantlylargerthanthoseofWTmiceandwereyellowishinappearance.OilredOstainingrevealedhigherlipidaccumulationintheliversoftheOxKOmicecomparedwithWTmice.ThetotalcholesterolinperipheralbloodwashigherthanWTmice(WT:32.8±15.1mg/dL,n=12;OxKO:156.5±29.2mg/dL,n=12,p<0.05),althoughnosignificantdifferencesinfreefattyacidandserumtriglyceridewereobservedbetweenOxKOandWTmice.Moreover,mRNAlevelsofthehepaticlipogenicgenes,PPAR-γ,CD36,andfattyacidsynthaseinOxKOmicewerehigherthanthatinWTmice.Theseresultssuggestthatorexinislikelytocontributetoreg-ulationoflipidmetabolismintheliver.NoCOI.

3P-099The effect of meal duration and timing of high fat diet on energy metabolic rhythm of mice Haraguchi, Atsushi; Sasaki, Hiroyuki; Tahara, Yu; Shibata, Shigenobu(Laboratory of Physiology and Pharmacology, Schools of Advanced Science and Engineering, Waseda University, Tokyo, Japan.)

Itisknownthattheovereatingatdinnerand/oratnightsnacktime,orbreakfastskippingleadtobecomeobesityinhumans.Therefore,mealtimingandmealfrequencymayaffectbodyweightandbodyfatgain,andwillbeassociatedwithobesity.Inourpreviousexperiment,wegavehighfatdiet (HFD)ornormaldiet (ND)tomiceatactiveperiodoratinactiveperiodin2mealsperdayschedule,andamountoffoodwasregulatedbyfeedingdurationtime(2,4,and8hrsperhalfday).Asaresult,thebodyweightandbodyfatwerehigherin8hrsgroupthanthosein4hrsgroup.Although2hrsgroupkeptsmallfoodintakethan4hrsgroup,thebodyweightandbodyfatwerehigherin2hrsgroupthan4hrsgroup.Moreover,wemeasuredperipheralclockphase in liverusingInVivoImagingSystem(IVIS).MicefedwithHFDatinactiveperiodshowedtheadvanceofliverclockascomparedwiththosefedwithHFDatactiveperiod.Incurrentexperiment,weaskedwhetherabovefeedingconditionsmayaffecttheenergymeta-bolicrhythm.Metabolicrhythmwasmeasuredbymetabolicmeasur-ingsystemafterwebredmicewitheachprotocolfor3or4weeks.Astheresult,micefedwithHFDatinactiveperiodconsumedlessenergyfromfatincomparisontothosefedwithHFDatactiveperiod.ThisresultsuggestedthatfeedingofHFDatinactiveperiodcouldnotconsumemuchenergyfromfat.NoCOI.


3P-100Seasonal differences in thermoregulation, blood pres-sure and melatonin secretion in obese subjectsSato, Maki; Kanikowska, Dominika; Iwase, Satoshi; Shimizu, Yuuki; Nishimura, Naoki; Inukai, Yoko; Sugenoya, Junichi; Sato, Motohiko(Department of Physiology, Aichi Medical University, Aichi, Japan)

Obesityhasbeenincreasingintheworldduringthepastseveralde-cades.Obesepeoplehaveanincreasedincidencefordevelopingcar-diovascular,renal,andhormonaldiseasesandsleepdisorders.Wehaverecentlystudiedthedifferencesofthermoregulation,melatoninsecre-tionduringsleepand24hoursbloodpressure inobesesubjects inJapanintwoseasons,summervs.winter.Fiveobese(BMI,32±5kg/m2)and5non-obese(BMI,23±3kg/m2)menparticipatedinthisex-perimentatlatitude35°10′Nandlongitude136°57.9′E.Theaverageenvironmental temperaturewas29±1°C insummerand3±1°C inwinter.Tympanictemperatureandsweatrateweremeasuredduringlegwaterimmersionat42°Cfor30min.Bloodpressurewasmeasuredoverthecourseof24hoursinsummerandwinter.Salivasamplesformelatoninwerecollectedat11pm,2amand6am.Therelationshipbetweentympanictemperatureandsweatratewassignificantlydif-ferentbetweenobeseandnon-obesesubjectsinbothseasons,therebeingaloweredsweatrateforanycoretemperatureinobesesubjects.Melatoninconcentrationsduringsleepinobesesubjectsweresignifi-cantlylowerthanthoseinnon-obesesubjectsinthewinter.Systolicanddiastolicbloodpressuresinobesemenweresignificantlyhigherinwinterthaninsummer.Thethermoregulatoryresponses,melatoninsecretionduringsleepandbloodpressurecontrolwereattenuatedinwinterseasoninobese.NoCOI.

3P-101Search of the suitable protein content and kinds of amino acid which entrains mouse peripheral clocksIkeda, Yuko; Ohtsu, Teiji; Hirao, Mizuho; Sasaki, Hiroyuki; Tsubosaka, Miku; Shibata, Shigenobu(Laboratory of Physiology and Pharmacology, School of Advanced Science and Engineering, Waseda Univ.)

Itiswellknownthatinsulinsecretionmaybeimportantstepfortheentrainmentofperipheralclockbyrestrictedfeeding(RF)(Hiraoetal.,2009;Tharaetal.,2011),andwefoundthatfasterdigestiblecarbo-hydratecausedbigphase-shiftofperipheralclock.(Itokawaetal.,2013).Howeverwehavenotyetfoundtheratioofproteininfoodandalsorolesofaminoacidsonperipheralclockentrainment.Inthisresearch,weprepared100%caseinonly(100C),50%casein+50%β-cornstarch(50C),and100%β-cornstarchonly(0C)groups,andinsomeexperimentswepreparedchangingratioofcaseinandβ-cornstarch(3C,6C,20C,40C,86C)inAIN-93Mformuladiet.Micewerefedwitheachfoodatdaytimefortwodays.Wemeasuredphase-advanceofliver,kidneyand,submandibulargland(SubGla)clockusinginvivoimagingsystem(IVIS).Inliverandkidney,valuesofphase-advancewerenotdependentonproteincontentbutontotalcalorie.Ontheotherhand,inSubGlaphase-advancewaspositivelydependentoncontentofcarbohydrate,suggestingthatSubGlaisresponsivetocarbohydrate,andliverandkidneyarewellresponsivetoprotein.Inparalleltoaboveexperiment,wescreenedwhetheroraladministrationofeachaminoacid(5mM/kg)in20differentaminoacidscausedphase-shiftofperipheralclocksusingIVIS.Wefoundcysteineandhistidineentrainperipheralclock,suggestingthatcaseinsubstitutedbycysteineand/orhistitidine isinteresting.NoCOI.

3P-102The relevance between estrogen decrease and cold constitutionNigo, Ryosuke1; Nishimori, Atsuko2; Matsuoka, Tomomi2; Yamato, Takako1,2; Aomine, Masahiro1,2(1Grad. Sch. of Health & Nutri. Sci., Nakamura Gakuen Univ., Fukuoka, Japan, 2Fac. of Nutri. Sci., Nakamura Gakuen Univ., Fukuoka, Japan)

Menopausalwomenfrequentlysufferfromuncomfortablecoldconsti-tution,whichisatleastinpartcausedbyperipheralcirculatoryim-perfectionresultedfromvasoconstrictionduetothedeclineofestrogensecretion.Inthisstudy,weexaminedthebloodflow(shoulder,waistandfemur)andtheskintemperature (shoulder, femurandplanta)beforeandaftercold-load(1°C,1hr)ovariectomizedrats(OVX)asamenopause-modelrat,andcomparedthefindingswiththoseobtainedfromsham-operatedrats (Sham)andnon-treatedrats (Control) . Inaddition,wealsostudiedtheeffectsofβ-estradioladministrationonthebloodflowandtheskintemperatureintheseratgroups.Ovariec-tomyproducedasignificantdecreaseinthebloodflowofshoulder,waistandfemurbeforethecold-load,butnottheskintemperature.Therecoveryfromthecold-loadwasdelayedintheskintemperatureoffemurinOVX.Theadministrationofβ-estradiolledtoasignificantincreaseinthebloodflowandtendedtofastentherecoveryoftheskintemperatureafterthecold-load.Thesefindingssuggestthattheestrogenreductionduetoovariectomymaydeterioratethevascularfunction,resultinginthedecreaseofthebloodflow.Thefactthatthedecreaseofthebloodflowdidnotaffecttheskintemperature,butaffecttherecoveryprocessoftheskintemperaturefollowingthecold-loadsuggeststhatthedecreaseofestrogeninmenopausalwomenmaycausetheonsetofcoldconstitution.NoCOI.

3P-103The noradrenergic vasoconstrictor response in leg skin in young women complaining of unusual cold-nessYamazaki, Fumio(School of Health Sciences, University of Occupational and Environmental Health, Kitakyushu, Japan)

InJapan,weoftenencounterwomencomplainingofphysicalcoldness,especiallyintheacralportionofthelowerextremities,evenundernormaltemperatureconditions.Inthisstudy,weexaminedthesym-patheticcutaneousvasoconstrictorresponseinthelowerextremitiesintenyoungwomencomplainingofunusualcoldness(Cgroup)andnineyoungwomennotsufferingfromcoldness(Ngroup).Inprotocol1,theroomtemperaturewasdecreasedfrom29.5°Cto23.5°Cforin-creasingnoradrenergicsympatheticactivityintheskin.Inprotocol2,cutaneousvasoconstrictorresponsesto iontophoreticapplicationofnorepinephrine(NE)wereexaminedat29.5°C.Inbothprotocols,la-ser-Dopplerflow(LDF)wasmonitoredfromthecalfanddorsalfoot.Cutaneousvascularconductance(CVC)wascalculatedastheratioofLDFtobloodpressure.Inprotocol1,theslopesofrelationshipbetweenmeanskintemperatureandCVCinthecalfduringmild-coldexposuredidnotdifferinbothgroups,whiletheslopeindorsalfootwassteep-er(P<0.05)intheCgroupthanintheNgroup.Inprotocol2,theCVCinthecalfsimilarlydecreasedinbothgroupsduringNEiontophoresis,butthereductionofCVCinthedorsalfootwasgreater(P<0.05)intheCgroup.Thus,theCgroupshowedagreatercutaneousvasoconstric-torreflexresponseintheperipheralregioninthelowerextremitiestocold.Itwassuggestedthattheaugmentedsensitivityofthecuta-neousvasoconstrictorresponseintheCgroupwasduetothealterednoradrenergicsensitivityofthelegs.NoCOI.


3P-104Hypoxia-induced hypothermia mediated by the GAB-Aergic transmission in the rostral ventromedial medul-la in anesthetized ratsOsaka, Toshimasa (National Institute of Health and Nutrition)

Hypoxiaevokesaregulateddecreaseinthebodycoretemperature(Tc)inavarietyofanimals.ThisresponseisbeneficialforanimalsbecausealowerTcincreasestheaffinityofhemoglobinforoxygenandreducestheoxygendemandofthetissue.Theneuronalmechanismsofthisresponseincludeactivationofthecarotidchemoreceptors,re-leaseofnoradrenalineandnitricoxide intherostralventromedialpreopticarea,andglutamatergicactivationinthelateralpreopticarea(LPO). Here, Iexaminedthehypothesis thatglutamate-sensitiveneuronsintheLPOactivateGABAergictransmissionthatmediateshypoxia-inducedhypothermia intherostralventromedialmedulla(RVMM)inurethane-chloralose-anesthetized,neuromuscularlyblocked,artificiallyventilatedrats. UnilateralmicroinjectionofGABA(15nmol/50nl)intheRVMMelicitedapromptincreaseintailskintem-peratureanddecreasesinTc,oxygenconsumptionrate,andheartrate.TheGABA-sensitivesitewaslocalizedintheRVMM,includingtherostralraphepallidusandadjacentparapyramidalarea.PretreatmentwiththeGABAAreceptorblockerbicucullinemethiodide(10pmol/100nl),butnotvehiclesaline,bilaterallymicroinjected intotheRVMMgreatlyattenuatedthehypothermicresponsesevokedbyhypoxic(10%O2and90%N2for5min)stimulationorbybilateralmicroinjectionsofglutamate(5nmol/100nl)intotheLPO.Theresultssuggestthathypoxia-inducedhypothermiawasmediated,atleastinpart,byacti-vationofGABAAreceptorsintheRVMM.NoCOI.

Poster PresentationsPathophysiology

3P-105Topographical study of activated microglia and c-Fos immunoreactive neurons following acute restraint stressSugama, Shuei(Department of Physiology, Nippon Medical School)

Recentstudieshaveshownthatexposuresofanimalstostress,eitheracuteorchronic,inducerobustmicroglialactivationinthebrain.Thestress-inducedmicroglialactivationhasbeenwelldocumentedinre-gionswhicharesusceptibletoclinicalneurodegenerativediseases,suchasParkinson’sdisease,Alzheimer’sdisease,anddepression.Inthepresentstudy,weinvestigatedthespatialdistributionofc-Fosandactivatedmicrogliainvariousbrainregionsbyemployingsham-oper-ated,adrenalectomized,andadrenalectomizedandoralcorticosteronetreatedrats,following2hperiodofrestraintstress.Thepresentstudyshowedthat:1)acute2hrestraintstresssignificantlyincreasedc-Fosimmunoreactiveneuronnumberinvariousregions,suchasthethal-amus,hypothalamus,periaqueductalgray(PAG),substantianigra(SN),but,not inthehippocampus.Theacutestress inducedmorec-Fosimmunoreactiveneuronsinthesebrainregionsinadrenalectomizedratsthaninsham-operatedrats;2)themicrogliaactivationoccurredinaccordancewithc-Fosimmunoreactiveneuronsmostofthesebrainregionsexcept forthehippocampus.Themicroglialactivationwassignificantlyenhancedbytheadrenalectomy;3)oraladministrationofcorticosteroneconsistentlysuppressedbothc-Fos immunoreactiveneuronsandmicroglialactivationintheseregions.Thus,thepresentstudydemonstratesthatneuron-microgliamayhavecloseinteractionseachotherduringthetimeofacutestressandthatglucocorticoidsmayprovideinhibitorysignalstobothneuronalandmicroglialactiv-ity.NoCOI.

3P-106Neuroprotective effect of water-soluble vitamin E de-rivatives as radical scavengers/antioxidantsSuezumi, Koki1; Yamada, Kuniya1; Tokumaru, Osamu2; Ogata, Kazue2; Kitano, Takaaki3; Yokoi, Isao2(1Medical student, Oita University Faculty of Medicine, Yufu, Japan, 2Department of Neurophysiology, Oita University Faculty of Medicine, Yufu, Japan, 3Medical Education Center, Oita University Faculty of Medicine, Yufu, Japan)

Weevaluatedneuroprotectiveeffectofnewlydevelopedwater-solublevitaminEderivatives,ETSGS(glutathione+vitaminE)andEPCK1(vitaminC+vitaminE)againstischemia-reperfusioninjury(IRI)byphosphorousNMRspectroscopy(31P-NMR)andbyelectronspinreso-nance(ESR)spectrometry.Brainsliceswere incubated inaNMRsampletubewithstandardartificialcerebrospinalfluidwithETSGSorEPCK1(0,10,100µM)at27.5°C.BrainsliceswereexposedtoIRIbyhaltingtheperfusionfor1hr,followedbythereperfusionfor3hrs.High-energyphosphatesinbrainslices,phosphocreatine(PCr)andATP,andintracellularpHwereseriallymeasured.Directfreeradicalscavengingactivitywasevalu-atedbyESRusingspintrappingmethod.BothETSGSandEPCK1demonstratedneuroprotectiveeffectagainstIRIat100µM,butnotat10µM;PCrhadsignificantlybetterrecoverythancontrol(p<0.05).InESRstudy,thosederivativessignificantlyscavengedmultiplefreeradicalsexamined(HO·,O2·-,ascorbatefreeradicals,t-BuOO·-,NO,DPPHradicals)withEC50’sfrom10-4to10-2M.EC50foreachradical(scavengingprofile)variedbetweenETSGSandEPCK1.ItisconcludedthatvitaminEderivativesmightbeneuroprotectiveagainstIRIthroughtheirantioxidativeactivity,whichisatleastpar-tiallyattributabletotheirdirectscavengingactivityagainstmultiplefreeradicals.NoCOI.


3P-107A new therapeutic approach for glioblastoma using tumor selective cell-penetrating peptideHiga, Moritoshi; Matsushita, Masayuki(Dept. of Mol. Cell. Physiol. Grad. Sch. Med. Univ. Ryukyus. Okinawa, Japan)

Cell-penetratingpeptides(CPPs)hasbeenexpectedasanewbiomed-icaltoolfromtheperspectiveofitshighefficiencyandminimalinva-siveness.However,sincelowspecificitytotargetcells,clinicalappli-cationoftheCPPsforcancerremainschallenging.Inthisstudy,weattempted identificationofnovelcancer-specificCPPsfortargetingglioblastomamultiforme(GBM),whichisoftenrefractoryandresistancetotreatment.Here,weconductedscreeningofCPPswhichhavingaffinitytohumanGBMcelllineU87MG,frommRNAdisplayrandompeptideslibrary.Basedonamino-acidsequenceofthecandidateCPPsobtainedfromthescreening,wesynthesizedfluorescentpeptidesandexaminedtransductionefficiencyforvariouscelllinesderivedfromhistologicallydifferenttypes.Inaddition,somemodificationsofami-no-acidsoftheCPPresultedinenhancementofcell-penetratingactiv-ity.Furthermore,GBM-selectiveCPPfusionp16Ink4afunctionalpep-tideinducedcellularapoptosis,whichdemonstratethatthisCPPcoulddeliverp16peptideintoU87MGcells,andconsequentlyhadananti-tumoreffectagainstGBM.TheseresultsindicatethatthenovelCPPidentifiedinthisstudypermeatewithhighlyaffinityintoGBMcellsandsuggest itspotential for imagingandtherapeuticbyselectivedeliveryofanycargomoleculestotumortissue.FurtherstudiesarerequiredtoevaluatethetissuespecificityandthedistributionoftheCPPsinGBMmodelmouse.NoCOI.

3P-108Tumor-associated macrophages in experimental glio-mas in the rat brainGotoh, Katsuhiro; Kusakawa, Akari; Horiuchi, Mika; Sugimoto, Kana; Takahashi, Hisaaki; Yano, Hajime; Tanaka, Junya (Dept. of Molecular and Cellular Physiology, Graduate School of Medicine, Ehime Univ., Ehime, Japan)

Weanalyzedthefunctionsoftumor-associatedmacrophages(TAMs)inexperimentalgliomasintheratbrain.ThegliomaswereinducedbytransplantingC6gliomacellsintotheneonatalratbrainswithin24hafterbirth.Immunohistochemicalstainingonthecryostatsectionsofthebrainwithgliomatumorrevealedhighlydenseaccumulationofmacrophagesthatweredescendantsofbonemarrowcellsbutnotofresidentmicroglialcellsasrevealedbybone-marrowtransplantationexperiments.TREM2,aproteinresponsibleforrecognitionofapoptot-iccells,wereexpressedbyTAMslocatedintheperipheryofthetumormass.TAMsinandaroundnecrotictumormassstronglyexpressedCD68,amarkerforphagocytes.Progranulin,acellgrowthfactorwasabundantlyanddiffuselyexpressedbyalmostallTAMs.IsolatedTAMsexpressedproinflammatoryfactorssuchas inetreleukin-1betaandchemokinesCCL2,CCL3,andCCL4atquitehighlevels,whereastheexpressionofthefactorsweremuchsuppressedinthetumormass,evenwhentheexpressionlevelswerenormalizedwiththelevelofIba1(amarkerformacrophages).Theseresultsindicatethatproin-flammatorynatureofTAMsinthetumormasswerehighlysuppressed,resultingintheprotectionoftumorcellsfromthedestructiveinfiltra-tionofinflammatorycells.NoCOI.

3P-109Oct-3/4 promotes tumor angiogenesis in glioblastoma through VEGF productionTakahashi, Hisaaki; Kawabe, Yuya; Iwata, Shinji; Hosokawa, Yuki; Sugimoto, Kana; Yano, Hajime; Tanaka, Junya(Molecular and Cellular Physiology, Ehime Univ Graduate School of Medicine, Ehime, Japan)

AccumulatingevidenceshowsthattheexpressionlevelofOct-3/4,aself-renewalregulatorinstemcells,ispositivelycorrelatedwiththeprogressionofvarioussolidtumors.However,littleisknownregardingtheinfluenceofOct-3/4inthetumorangiogenesisofglioblastomas.Inthepresentstudy,wesubcutaneouslytransplantedOct-3/4-over-expressinghumangliomaU251cells(U251/EGFP-Oct)intotherightthighsofnudemicetoevaluatetherolesofOct-3/4intheglioblastomaangiogenesis.Bothtumorsizeandthenumberoflargevesselsgrow-inginthetumorweremarkedlyincreased.Inaninvitromodelofangiogenesis,conditionedmediafromU251/EGFP-Octcellssignificant-lyacceleratedcapillary-liketubeformationcomparedwithconditionedmediafromU251/EGFPcellsthatweredevoidofOct-3/4overexpres-sion.IncomparisonwithU251/EGFPcells,U251/EGFP-Octcellshadmarkedlyelevatedexpressionofvascularendothelialgrowthfactor-mR-NAunderthecontrolofHIF1alpha.InU251/EGFP-Octcells,enhancedproteinexpressionandnucleartranslocationofHIF1αwereobservedinnormoxicconditions.OurresultsdemonstratethatOct-3/4-express-inggliomacellshavetheabilitytoadapttolow-oxygenenvironmentswithintumormassesbypromotingtumorangiogenesis,andthusOct-3/4maybeapromisingtargetfortreatmentofmalignantgliomas.NoCOI.

3P-110Contributions of TGFbeta1 on insidious glioma cell invasions in the experimental glioma and exploration of the source of the TGFbeta1Yaguchi, Haruna; Shiota, Kohei; Shimoda, Takefumi; Yano, Hajime; Sugimoto, Kana; Takahashi, Hisaaki; Tanaka, Junya(Dept. of Molecular and Cellular Physiology, Graduate School of Medicine, Ehime Univ., Ehime, Japan)

Insidious invasionsofgliomacells fromprimarytumorsitetowardsurroundingnormalbrainparenchymacauseuntreatablepathologicalconditionsattherecurrenceaftersurgicalresection.Althoughsup-pressionofthisinvasionhaslongbeendesiredasapromisingthera-peutictarget,ithasyettobedeveloped.Wehaveestablishedthein-sidious invasionmodel inKSNnudemicebyxenograftingC6ratgliomacellsintothebrain,andobservedtheinvasionaccompaniedbyCD105/Endoglin (+)butCD309/VEGFR2(-)bloodvessels, implyingdominantcontributionofTGFbeta1ratherthanVEGFin insidiousgliomainvasions.Moreover,theinvasionwaspreventedbyinhibitionofSodiumion/protonexchanger1(NHE1)usinganNHE1inhibitor.NowweareonthewaytodeterminewhetherC6cellsthemselvesarethesourceofTGFbeta1inthegliomabrainaccompanyinginsidiousinvasions.Ontheonehand,NHE1hasbeenreportedtoactsontheconversionof latent formofTGFbeta1secretedanddepositedonextracellularmatrix(ECM)towardactiveform,viaregulationofECMconstruction. InhibitionofNHE1byan inhibitordidnotaffectthemRNAlevelsofTGFbeta1.WearealsoonthewaytodeterminetheconsequenceofNHE1 inTGFbeta1conversion in insidiousgliomainvasions,andwouldliketodiscussaboutthepossibilityofNHE1astherapeutictargetfortheinvasions.NoCOI.


3P-111Exploration of the mechanism of TGFbeta1 produc-tion from glioma associated macrophage-like cells in experimental gliomaNomura, Noriko; Yaguchi, Haruna; Shiota, Kohei; Shimoda, Takefumi; Yano, Hajime; Sugimoto, Kana; Takahashi, Hisaaki; Tanaka, Junya(Dept. of Molecular and Cellular Physiology, Graduate School of Medicine, Ehime Univ., Ehime, Japan)

Insidious invasionsofgliomacells fromprimarytumorsitetowardsurroundingnormalbrainparenchymacauseuntreatablepathologicalconditionsattherecurrenceaftersurgicalresection.Primarygliomamasscontainssignificantnumberofmacrophage-likecellsaswellasgliomacells.Themacrophage-likecellsaredifferent fromresidentmicrogliainmanyaspects,forexamplethehigherexpressionofsodi-umion/protonexchanger1 (NHE1),whiletheirroles ingliomacellinvasionsareyettobeelucidated.WehaveestablishedtheinsidiousinvasionmodelinKSNnudemicebyxenograftingC6ratgliomacellsintothebrain,andobservedthe invasionaccompaniedbyCD105/Endoglin(+)butCD309/VEGFR2(-)bloodvessels,implyingdominantcontributionofTGFbeta1ratherthanVEGFininsidiousgliomainva-sions.Moreover,theinvasionwaspreventedbyinhibitionofNHE1usinganNHE1inhibitor.Nowweareonthewaytodeterminewheth-ergliomaassociatedmacrophage-likecellsarethesourceofTGFbeta1inthegliomabrainaccompanyinginsidiousinvasions.Ontheonehand,NHE1hasbeenreportedtoactsontheconversionoflatentformofTGFbeta1secretedanddepositedonextracellularmatrix(ECM)towardactive form,viaregulationofECMconstruction.Wewould liketodiscussastotheconsequenceofNHE1inTGFbeta1conversion ininsidiousgliomainvasions,andthepossibilityofNHE1astherapeutictargetfortheinvasions.NoCOI.

3P-112Effects of interactions of microglia/macrophages and C6 glioma cells in co-culture on the proinflammatory functions of microgliaHoriuchi, Mika; Gotoh, Katsuhiro; Kawasaki, Shun; Takahashi, Hisaaki; Yano, Hajime; Tanaka, Junya(Dept. of Molecular and Cellular Physiology, Graduate School of Medicine, Ehime Univ., Ehime, Japan)

Microglia/macrophagesmassivelyaccumulateinandaroundatumormassofanexperimentalratgliomamodel,inwhichC6gliomacellsaretransplantedintotheneonatalratbrainparenchyma.Manystud-ieshaveshownthattumor-associatedmacrophages(TAMs)enhancethetumorgrowthandinvasion.Althoughalargenumberofactivatedmicrogliawerelocatedastheyweresurroundingthetumormass,ithasnotbeenwellelucidatedwhatkindsofeffectsthemicrogliaelicitonthegliomas.Inthisstudy,wecomparedthechangesinnatureofmicroglia/TAMswhencoculturedwithC6gliomacells.Quantitativereal-timeRT-PCRexperimentsrevealedthatcoculturedmacrophagesdownregulateexpressionofmRNAencodingproinflammatorycyto-kinessuchasinterleukin-1beta,tumornecrosisfactoralpha,andinter-feron-alpha,whereasmicroglialcellsinthecoculturedidnotshowanysignificantfunctionalchanges.ImmunohistochemicalstainingrevealedthatTAMsbutnotmicrogliacellsincocultureoftenexpressedphago-cytemarkersCD68andTREM2.Furthermore,thereisnoapparentinflammatoryreactionsaroundthetumormass,inspiteofthemarkedaccumulationofactivatemicroglialcells.TheseresultssuggestthatmicroglialcellsandTMAsplaydistinctrolesinthegrowthofglioma.NoCOI.

3P-113Oct-3/4 promotes chemoresistance of glioblastoma cells through the expression of ABC transportersHosokawa, Yuki; Takahashi, Hisaaki; Sugimoto, Kana; Yano, Hajime; Tanaka, Junya(Molecular and Cellular Physiology, Ehime Univ Graduate School of Medicine, Ehime, Japan)

Glioblastomaisthemostmalignanttypeofprimarybraintumorthathasbeenshowntocontainasmallpopulationofcancerstemcells(CSCs).CSCssharemanyofthepropertiesofnormalstemcells,suchasself-renewal,differentiationintomultilineagecells,anddrugresis-tance.Althoughchemotherapykillsmostofcellsintumorstochemo-therapeuticagents,CSCsmaybeleftbehind,whichthencauselocaltumorrecurrence.RecentstudieshavedemonstratedincreasedOct-3/4expression,aself-renewalregulator instemcells, inglioblastomas.However, little isknownregardingthe influenceofOct-3/4 inthechemoresistancepropertyofglioblastoma.Inthisstudy,weestablishedOct-3/4-overexpressingglioblastomacells(U251/Oct-3/4cells)toassessthechemoresistanceproperty.TodeterminethechemosensitivityinU251/Oct-3/4cells,weexposedthesecellstovariousconcentrationsofchemotherapeuticreagentsincludingtemozolomide,carboplatin,andetoposide(VP16)for48hr,andperformedcellviabilityassaydeter-minedbyLDHrelease.Comparedwithcontrolcells,U251/Oct-3/4cellsexhibitedgreaterresistancetotheseagents.ThisabilitywascanceledinthesecellsbysuppressingtheexpressionofOct-3/4usingtet-offsystem.Furthermore,U251/Oct-3/4cellsexpresshigherlevelsofmultidrugresistance-relatedgene,ABCG2.TheseresultssuggestedthatfuturetreatmentsshouldtargetOct-3/4positiveCSCsintumorstoimprovethesurvivalofbraintumorpatients.NoCOI.

3P-114Response of glial cells to IL18 produced in the isch-emic rat brainsMise, Ayano; Sugimoto, Kana; Nishioka, Ryutaro; Takahashi, Hisaaki; Yano, Hajime; Tanaka, Junya(Dept. of Molecular and Cellular Physiology, Graduate School of Medicine, Ehime Univ., Ehime, Japan)

Wehavefoundthat interleukin-18(IL-18)andtransforminggrowthfactor-beta1(TGFb)arethemostabundantlyproducedcytokinesincorelesionsoftheischemicratbrainswhosemiddlecerebralarterytransientlyoccludedfor90min.It isplausiblethattheseabundantcytokines infiltrate intotheadjacentperi-infarcttissueandactonglialcells.IthasbeenshownthatTGFbinducesexpressionofNG2chondroitinsulfateproteoglycan(NG2)byprimaryculturedmicroglialcellsalsotostimulatetheirmigrationactivities.Infact,NG2+residentmicroglialcellsarelocatedintheperi-infarcttissueandtheyappearedengaged ineliminationofdegeneratedneurons. Inthisstudy,weparticularlyaddressedtheactionsofIL-18onglialcellsintheperi-in-farcttissue.Toexaminetheresponseofglialcellsinvitro,weusedmixedglialculturesstartedfromtheneonatalrat forebrains.Theculturecontainedastrocytes,microglialcellsandNG2glialcells (oroligodendrocyteprogenitorcells)butnotneurons.Whenmixedglialcultureswere incubatedwithIL-18,mRNAexpressionthatencodetypeIinterferons,nestin,NG2andhepatocytegrowthfactor(HGF)wasincreasedinadose-dependentmanner.TheseresultssuggestapossibilitythatIL-18maybeatleastpartlyinvolvedinactivationofglialcellsand/orglialscarformationintheischemicbrains.NoCOI.


3P-115Treadmill exercise as rehabilitation for stroke model; its effects through increased serum corticosterone levelNishioka, Ryutaro1; Sugimoto, Kana1; Mise, Ayano1; Kojyo, Katsura; Mohammed E, Choudhury; Takahashi, Hisaaki; Yano, Hajime; Tanaka, Junya(Dept. of Molecular and Cellular Physiology, Graduate School of Medicine, Ehime Univ., Ehime, Japan)

Althoughrehabilitationmaybethemosteffectivetherapyforstroke,mechanismsunderlyingthecurativeeffectsarenotfullyelucidated.Weaddressedtheshort-termeffectsof lighttreadmillexerciseonischemicbrainedemaandneurologicaldysfunction.Wistarratsweresubjectedtotransient(90min)rightmiddlecerebralarteryocclusion(MCAO)thatproducedlargestrokelesion.Theareaofthelesionwasmeasuredwithmagneticresonanceimaging(MRI)onthenextdayor1day-postreperfusion(1dpr),andonlyratswithsubstantiallylargeischemic lesionweregrouped intoexerciseandnon-exerciseones.Treadmillwashorizontallysetanditsspeedwasat4–6m/secandtheratsranonlyfor10min/dayat2,3,and4dpr.Onthe5dpr,thebrainlesionswereagainexaminedwithMRI.Theseverityofbrainedemawasevaluatedbydividingthevolumeoftherighthemispherebythatoftheleftone.Consequently,thelightexercisewassignificantlyre-ducedbrainedemaandamelioratedthemotor functionthatwasevaluatedonemonthafterMCAO.Theamelioratingeffectoftheex-ercisewasabolishedwhenanti-glucocorticoidagentmifepristoneoranti-mineralocorticoidagentspironolactone.Orallyadministeredlowdoseofcorticosteronesuppressedthebrainedemainthenon-exercisegrouprats.Theseresultsshowthattheforcedlightexercisecontrolledbrainedemabyenhancingsecretionofadrenalcorticosterone.NoCOI.

3P-116The light treadmill exercise after the cerebral infarc-tion controls cellular edema: the involvement of aqua-porin 4 and sodium/hydrogen exchanger 1Kojyo, Katsura; Nishioka, Ryutaro; Sugimoto, Kana; Aono, Hitomi; Mohammed E, Choudhury; Takahashi, Hisaaki; Yano, Hajime; Tanaka, Junya(Dept. of Molecular and Cellular Physiology, Graduate School of Medicine, Ehime Univ., Ehime, Japan)

Wehaverecentlyfoundthatlighttreadmillexerciseamelioratesbrainedemausingaratstrokemodel,inwhichtherightmiddlecerebralarteryofamaleWistarratwastransientlyoccludedfor90min.Theameliorationofbrainedemamayberelatedtotheincreasedlevelofbloodcorticosterone.WeinvestigatedexpressionofmRNAencodingaquaporin4(AQP4)andsodium/hydrogenexchanger1(NHE1)inthethreepartsofcerebralcortexthatwerecontralateral,peri-infarctandischemiccoretissues.AQP4-mRNAwasmuchincreasedintheperi-in-farcttissueandNHE1-mRNAintheperiandcoretissues.LighttreadmillexercisesuppressedthesemRNAexpressions.Forcedtread-millexercisehasbeenshowntoincreasebloodcorticosteronelevel.Invitrostudysuingratprimarymixedglialcultureshowedthatcorti-costeroneat10nMbutnot100nMsuppressedtheexpressionofAQP4-andNHE1-mRNA.Anti-glucocorticoidoranti-mineralocorticoidagentspartlypreventedthecorticosterone-inducedenhancedexpressionofthemRNAexpression.TheseresultssuggestthattreadmillexercisesuppresstheinfluxofsodiumionandthesubsequentwaterintothecytoplasmofglialcellsbysuppressingexpressionofAQP4andNHE1,leadingtoameliorationofedemaofthestrokebrain.Wearecurrent-lyconducting invitroexperimentstodeterminewhetherNHE1 isactuallyinvolvedincellularedemausingC6gliomacells.NoCOI.

3P-117Role of glutamate transporters in functional compen-sation at the intact hemisphere contralateral to a strokeTakatsuru, Yusuke1; Kaneko, Ryosuke2; Obi, Kisho1; Shimokawa, Noriaki1; Nabekura, Junichi3; Koibuchi, Noriyuki1(1Dept. Integrative Physiol., Gunma Univ. Grad. Sch. Med., 2Inst. Exp. Animal Res., Gunma Univ. Grad. Sch. Med., 3Div. Homeostatic Develop., NIPS)

Afterischemicbrainstroke,thecorrespondingareacontralateraltothelesionmaypartlycompensatesforthelossoffunction.However,theunderlyingprocessesinthecontralateralhemisphereafterstrokehavenotyetbeenfullyelucidated.Recentstudieshaveshownthatastrocytesmayplaycriticalrolesinsynapticreorganizationandfunc-tionalcompensationafterastroke.Thus,weaimtoclarifythecontri-butionofastrocytesusingamousestrokemodel.InvivoCa2+imagingshowedasignificantlylargenumberofastrocytesinthecontralateralSSCrespondingtoipsilaterallimbstimulationatthefirstweekafterinfarction.Simultaneously,extracellularglutamine level increased,indicatingtheinvolvementofastrocytesintheconversionofglutamatetoglutamine.Suchprocessmaybeimportantforfunctionalrecovery.Thishypothesiswassupportedfurtherbytheobservationthatappli-cationofTFB-TBOA,aglialglutamatetransporterblocker,disturbedthefunctionalrecovery.Thesefindingsindicatetheinvolvementofastrocytesinfunctionalremodeling/recoveryintheareacontralateraltothelesion.Ourstudyhasprovidednewinsightsintothemechanismsunderlyingsynapticremodelingaftercerebral infarction,especially,criticalroleoftheglutamatetransportersonfunctionalrecoveryafterstroke.Thistime,wepresenttherecentresults foractivatingtheastrocyteandglutamatetransportersusingseveraldrugs.NoCOI.

3P-118Pre-conditioning exercise upregulated superoxide dismutase activity and prevented sensori-motor dys-function after brain ischemia in ratsNoguchi, Taiji1; Tamakoshi, Keigo1; Takamatsu, Yasuyuki1; Toda, Takuya1; Waseda, Yuya1; Kato, Hiroaki1; Akatsuka, Shinya2; Toyokuni, Shinya2; Ishida, Kazuto1(1Department of physical therapy, Nagoya University Graduate School of Medicine, Aichi, Japan, 2Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Aichi, Japan)

Weinvestigatedpre-conditioningexercisecanenhanceantioxidantactivityandamelioratemotordysfunctionfollowingtransientmiddlecerebralarteryocclusion(MCAO).MaleWistarrats(5weeksold)wereassignedto4groups:Sham(n=6),Ex+Sham(n=6),MCAO(n=8),andEx+MCAO(n=12).Ex+ShamandEx+MCAOwereforcedtorunonamotorizedtreadmillataspeedof15m/minfor30mineverydayfor3weeks.MCAOandEx+MCAOweredonebya90minleft-MCAOusinganintraluminalfilamentafter3weeks.24hoursafterthesurgery,animalswereassessedforneurologicaldeficits(ND),laddertest,limbplacingtest(LP)andbeamwalkingtest(BW).Then,superoxidedismutase(SOD)activityofallgroups(Ex+Sham:n=6,Sham:n=6,MCAO:n=7,Ex+MCAO:n=7)wasquantifiedtoevaluateantioxidantenzymeactivity.Leftsomato-sensorycortexwasremovedandassayedSODactivitiesusingSODassaykit-WST(Do-jindo,Kumamoto,Japan).ND,laddertest,andLPwereimprovedby3weeksexercise,butBWwasnotsignificantlyimproved.SODactiv-ityofEx+MCAOwashigherthanMCAO.Thesedatasuggestpre-conditioning exercise attenuatesmotor dysfunction followedMCAO.TheseresultsmightbecloselyassociatedwithreductionofoxidativebraindamageviatheprotectiveeffectofSODactivity.NoCOI.


3P-119Role of K -Cl cotransporter(KCC2) for neural reorga-nization after cerebrovascular disease Nakamura, Kayo1; Wake, Hiroaki2; Nabekura, Junichi1(1National institute for physiological sciences, Division of Homeostatic Development, Okazaki, Japan, 2National institute for basic biology)

KCC2hasbeenknowntoplayan importantrole inneuralcircuitformationthroughtheregulationofintracellularchlorideconcentration.Moreover,down-regulationofKCC2hasbeenobservedinimmatureand injuredneurons,whichresults inanexcitatoryresponse fromGABAtransmission(Moorhouse,A.J.andNabekura,J;ISBN,2011).MyresearchisfocusedonunderstandinghowchangesinKCC2expressionareassociatedwiththerecoveryphaseafterstroke.ThisisespeciallyimportantsincethephysiologicalsignificanceofKCC2expressionlevelsonbehaviourisnotknownyet.ToelucidatethepossibleroleofKCC2,wegeneratedTet-offtransgenicmicewhoseKCC2geneex-pression isunderthecontrolofthecalciumcalmodulindependentproteinkinase2andtetracyclineinduciblesystem.Whencomparingthesetransgenicmicetocontrolmiceinamodelofcerebralischemiawefounddifferencesincerebralinfarctsizeintheacutephaseandmotorparalysisintherecoveryperiod.InthissessionIwilldiscusstheroleofKCC2reductionaftercerebralinfarction.Inthefuture,IwouldliketostudythepossibleroleofKCC2inneuronalplasticity.NoCOI.

3P-120Suppression of draining lymph node metastasis of oral squamous cell carcinoma by inhibition of the fac-tors secreted from primary tumor site at pre-metastat-ic phaseYano, Hajime1; Kaminota, Teppei1,2; Nomura, Noriko1; Kobyashi, Yusuke1; Kirino, Yui1; Sugimoto, Kana1; Takahashi, Hisaaki1; Tanaka, Junya1(1Dept. of Molecular and Cellular Physiology, Graduate School of Medicine, Ehime Univ., Ehime, Japan, 2Dept. of Otolaryngology, Graduate School of Midicine, Ehime Univ.)

Progressionoforalsquamouscellcarcinomalargelydependsonthemetastasisontodraininglymphnode.Sincethemetastasistowarddraining lymphnodeprecedessystemicmetastasis inmanycases,suppressionofthismetastasisisexpectedasapromisingstrategyforpreventionofthiscancerprogression.Lymphnodemetastasishaslongbeenunderstoodasaresultoffilterfunctioninlymphnodesforlymphfluid,andthemoleculareventsconcernswiththismetastasisattract-edlessattentionthanpre-metastaticeventsinotherorganssuchaslung.Wehavefoundpre-metastatictissuereorganizationindraininglymphnodesassumedaspre-metastaticnicheformation,byestablish-ingmetastasismodelbyxenograftinghighlymetastatichumansqua-mouscellcarcinomacelllineSASL1m,towardKSNnudemicetongue.Moreover,weobservedsecretionofthefactorsincludingTGFbeta1frompre-metastaticprimarytumorsiteinducesCD31-positivebloodvessel-likestructureindraininglymphnode.Nowweareonthewaytoassesstheeffectsof inhibitionofsecretedfactors fromSASL1mprimarytumorontolymphnodemetastasis,andwouldliketodiscussastothepossibilityofanti-metastatictherapyNoCOI.

3P-121In vitro effects of nicorandil on lower esophageal sphincter tone in ratsShimbo, Tmonori; Fujisawa, Susumu; Ohno, Yoshitake; Sato, Kaito; Yokota, Koji; Ohba, Takayoshi; Ono, Kyoichi(Department of Cell Physiology, Akita University Graduate School of Medicine, Akita, Japan)

Theloweresophagealsphincter(LES)isaspecializedregionoftheesophagealcircularsmoothmusclethatallowspassagesofaswallowedbolustothestomach.Functionaldisorderofanesophagussuchasachalasiadisplaysadiminishedperistalsisinloweresophagus.IthasbeenreportedthatnitricoxideplaysamajorroleinLESrelaxation.However,thedetailedmechanisminvolvedintheregulationofLESactivityisstillelusive.NicorandilpossessesdualpropertiesofanitrateandK+ATPchannelagonist.WehypothesizedthatnicorandilreducedtheLEScontractionbyNOand/orK+channeldependentmechanism(s).TheaimofthisstudywastoinvestigatetheeffectsofnicorandilonLEStoneintheratisolatedLESpreparationcontractedwithcarbacholorhighconcentrationofexternalK+(50mM).LEStissuesofratswereplacedinastandardorganbathandactivitieswererecordedusingthe softwareChart Pro v 4.0.After contractionwith carbachol,nicorandilwasaddeddirectlytothetissuebathincumulativelyin-creasingconcentrations,resultinginasignificantrelaxationoftheLESinaconcentration-dependentmanner.Ontheotherhand,nicorandilfailedtocausearelaxationwhentheLEStissueswerecontractedwithhighK+solution.RT-PCRrevealedthatKir6.1,Kir6.2,SUR1andSUR2Bsubunit,whichcomposeK+ATPchannel,wereexpressedinratloweresophagus.ThesefindingssuggestthatnicorandilhasasuppressoreffectonLEScontractionsassociatedwithK+ATPchannelfunction.NoCOI.

3P-122Oxidative stress in rats with monocrotaline-induced pulmonary hypertensionMizukami, Tomoe1,4; Shimouchi, Akito1; Taniguchi, Kentaro4; Jinno, Naoya1; Sawano, Makoto2; Kondo, Takaharu3; Seiyama, Akitoshi4; Shirai, Mikiyasu1(1Department of Cardiac Physiology, NCVC, Suita, Osaka, Japan, 2Department of Emergency Medicine and Critical Care, Saitama Medical Center, Kawagoe, Saitama, Japan, 3College of Life and Health Sciences, Chubu University, Kasugai, Aichi, HJapan, 4Human Health Sciences, Graduate School of Medicine, Kyoto University, Kyoto City, Kyoto, Japan)

Monocrotaline (MCT)causespulmonaryhypertension (PH)and/oracutehepaticinjuryinadose-dependentmanner.ForPHmodel,nu-merous investigatorshavestudiedmainlyonthecardiopulmonarychangesinratswithasinglestandarddoseofMCT(60mg/kg).WerecentlyreportedthatMCT-PHinratsaccompaniedmarkedCOproductionintheliver,suggestingMCT-inducedhepaticinjurywereassociatedwithROS.However,pathophysiologicalrolesofoxidativestressintheMCT-PHratshavenotbeenestablished.ThepresentstudyexaminedalterationsofoxidativestressinMCTrats.BasedonthestandardmethodsforPHstudies,MCT-PHandcontrolrats(maleSPF-SD)weremade.Threeweeks later,animalsunderanestheticsandmechanicalventilationweresubjectedtothemeasurementsofplasmaBAPandd-ROM,andofurine8-OHdG/creatinineratio.InMCTrats,urine8-OHdG/creatinineratioswereincreasedandplasmaBAP/d-ROMratios(latentanti-oxidantpotential)decreased.TheseresultsindicatedthatoxidativestresswereinvolvedinratswithMCT-inducedPH.Wewillalsopresentthe localizationofoxidativestressbytheimmuno-histochemicalstudiesanddiscusstheassociationwiththeCOproductionintheliver.NoCOI.


3P-123Anti-inflammatory effects of dexamethasone on mi-croglia/macrophages: inhibition on JAK/STAT path-wayEnomoto, Masato1; Wada, Aiko1; Kawasaki, Shun1; Kikuchi, Satoshi2; Sugimoto, Kana1; Takahashi, Hisaaki1; Yano, Hajime1; Tanaka, Junya1(1Dept. of Molecular and Cellular Physiology, Graduate School of Medicine, Ehime Univ., Ehime, Japan, 2Dept. of Emergency Medicine, Graduate School of Midicine, Ehime Univ.)

Activatedmicrogliamaybeonepotentialcauseofneuronaldeathinvariousneurologicaldisorders.This isat leastpartlyduetotheirproductionofpro-inflammatorymediators.Glucocorticoidshavebeenshowntosuppresssuchharmfulreactionsofmicroglialcellsonneuronsveryeffectively.Althoughtheanti-inflammatoryeffectsofglucocorti-coidshavebeenbelievedtoexertthroughtheirbindingtoglucocor-ticoidreceptor(GR),someliteraturesshowGR-independenteffectsofglucocorticoids.Wehaverecentlyfoundthatasyntheticglucocorticoiddexamethasone(Dex)suppressesphosphorylationofsignaltransducerandactivatoroftranscription1(STAT1)inratprimarymicrogliaandperitonealmacrophagesthatwere incubatedwithratrecombinantinterferongammafor15or30min.ItmaybepresumablethatDexelicitsthe inhibitoryeffectonJAK/STATpathwaythroughawayindependentofGR.Wehavepreparedmicroglialcells/macrophagesthataredevoidofGRinordertoinvestigateGR-independenteffectsofDexusingsiRNA.OurpreliminaryresultsshowthatDexcansup-pressphosphorylationofSTATs intheabsenceofGR inthesiR-NA-treatedmicroglialcells.DexcompletelysuppressedLPS-inducedNOproductionbymacrophages,andthesuppressionwasstillpartlyobservedeveninthesiRNA-treatedcells.ThepresentstudymightrevealtheeffectsofglucocorticoidthatareindependentofGR.NoCOI.

3P-124Activated microglia in the substantia nigra pars retic-ulata of rats with 6-OHDA-induced ParkinsonismAono, Hitomi; Mohammed E, Choudhury; Higaki, Hiromi; Sugimoto, Kana; Takahashi, Hisaaki; Yano, Hajime; Tanaka, Junya (Dept. of Molecular and Cellular Physiology, Graduate School of Medicine, Ehime Univ., Ehime, Japan)

Injectionof6-hydroxydopamine(6-OHDA)intothesubstantianigracausesParkinson’sdiseasesymptomssuchasbradykinesiaandrigid-ity.6-OHDAinjectionalsocausedactivationofglialcellsaswellasdopaminergicneuronaldegeneration inthesubstantianigraparscompacta(SNpc).Inthisstudy,wehaveconductedimmunohistochem-icalanalysesonglialcellreactionswithspecialemphasisonmicrogli-alcells.MicroglialcellactivationwasobservednotonlyintheSNpcbutalsotheSNparsreticulata(SNpr),wherenoneuronaldeathwasobserved.Microglialcells intheSNprwerecharacterizedwiththeexpressionofCD68,amarkerforphagocytes,suggestingthatthecellsmaybeengagedinphagocytosis.Immunoblottingstudyusinganti-syn-aptophysinantibodyshowedthedecreaseofsynapses intheSN.Furthermore,quantitativereal-timeRT-PCRdemonstratedthede-creaseinthelevelofmRNAsencodingglutamatereceptors,NR2D(onetyp2ofNMDAreceptors),mGluR1andmGluR4.Takentheob-servationthatmicroglialcellssometimeseliminatesynapsesthroughphagocytosis.CD68+activatedmicroglialcells intheSNprmaybeinvolved insuppressionofglutamate-dependentneurotransmission,whilesuppressingtheover-activationofGABArgicneuronsandpre-ventingthemanifestationoftheneurologicsymptoms.NoCOI.

3P-125An outdated hypnotic bromvalerylurea ameliorates murine model of dermatitisSekioka, Akihiro; Mohammed E, Choudhury; Islam, Afsana; Sugimoto, Kana; Takahashi, Hisaaki; Yano, Hajime; Tanaka, Junya (Dept. of Molecular and Cellular Physiology, Graduate School of Medicine, Ehime Univ., Ehime, Japan)

Bromvalerylurea(BU)isanoutdatedhypnotic/sedativewithamolec-ularweight223.Recently,wehavefoundthatBUsuppressednitricoxide(NO)productionbyLPS-stimulatedmicroglia/macrophages.TheinhibitoryeffectmaybemediatedthroughinhibitionofphosphorylationofSTAT1.Inthisstudy,BUwasusedtotreatmurinemodelsofatopicdermatitisandcontactdermatitis;theformerwasinducedwithanointmentconatinigdeadmitebodiesandthelatterwith2,4,6-trini-tro-1-chlorobenzene(TNCB).BUwasdissolvedinvaselineat1%w/wandspreadoverthebackskin(atopicmodel)ortheearskin(TNCBmodel).BUointmentamelioratederosionandbleedingintheatopicmodelanddecreasedthethicknessoftheearsoftheTNCBmodelwithin5hafterthefirsttreatment.ImmunoblottingstudyhasrevealedthatBUointmentsuppressedthephosphorylationofJAK1andSTATs1,5and6intheskinsamplesofTNCBmodelsthatwasprepared1hafterthespreadoftheointment.Furthermore,BUointmentincreasedSOCSexpressionintheskin.ThesedatashowthatBUwithasmallmolecularsizerapidlyinfiltratesintotheskinandamelioratesderma-titisbysuppressingphosphorylationofSTATs.NoCOI.

3P-126An outdated hypnotic bromvalerylurea as a novel agent for sepsis: the inhibitory action on JAK/STAT pathwaySakurai, Yuko1; Kikuchi, Satoshi1,2; Nishihara, Tasuku1; Kawasaki, Shun1; Takahashi, Hisaaki1; Yano, Hajime1; Aibiki, Mayuki2; Tanaka, Junya1(1Dept. of Molecular and Cellular Physiology, Graduate School of Medicine, Ehime Univ., Ehime, Japan, 2Dept. of Emergency Medicine, Graduate School of Midicine, Ehime Univ.)

Bromvalerylurea(BU)isanoutdatedhypnotic/sedativeandcurrentlyisrarelyusedinclinics.RecentlywefoundthatBUsuppressednitricoxide(NO)productionbyLPS-stimulatedmicroglia.Inthisstudy,BUwasusedtotreataratmodelofsepsisinducedbycecum-ligationandpuncture(CLP).Peritonitisprogressedrapidlywithmarkedswellingoftheileum24hafterCLP.Measurementofseruminterleukin(IL)-6proteinlevelsinsepticratsbyELISAwasusedasanindexofsever-ityofsepsis,andwaselevated20fold.Theincreaseofserumcreatininelevelsanddeteriorationofarterialbloodgasdatainsepticratssug-gestedtheyhaddevelopedmultipleorganfailureasaconsequenceofsepsis.Themortalityrateofsepsisratswas80%1weekafterCLP.Progressofthesefatalsymptomswasmarkedlyinhibitedbysubcu-taneousinjectionofBUtwice/day,resultinginthedecreasemortalityby40%at1week.BUsuppressedthephosphorylationofSTAT1whenperitonealmacrophages (PMs)were incubatedwith interfer-on-gamma(IFNg).WhenPMswereknocked-downoftheirJAK1orSTAT1expressionbytheuseofrespectivesiRNA,theeffectsofBUwereabolished.ThesedataindicatethatBUpreventedtheprogressofsystemicinflammationthroughtheinhibitionofIFNg-inducedJAK1/STAT1activationthatmayleadtothesystemicinflammation.NoCOI.


3P-127Celastrol inhibits interleukin-17A-stimulated rheuma-toid fibroblast-like synoviocyte migration and invasionLiu, Yan-Qing1,2; Guo, Shi-Yu2; Li, Guo-Qing1,2; Sunagawa, Masataka2; Hisamitsu, Tadashi1,2(1Sch. Clin. Med. Univ. Yangzhou, Yangzhou, China, 2Dept. Physiol. Sch. Med. Univ. Showa, Tokyo, Japan)

Interleukin-17A (IL-17A)-inducedmigrationand invasionoffibro-blast-likesynoviocytes(FLSs)iscriticalforthepathogenesisofrheu-matoidarthritis(RA).Morethan30%ofRApatientsareresistanttoavailabletherapies,despitetheintroductionofnovelbiologicagents.Therefore,itisnecessarytodevelopnewanti-arthriticagents.Recentstudieshavedemonstratedthatcelastrolhasanti-arthriticactivityinanadjuvant-inducedarthritis (AIA)model.However,theeffectandmolecularmechanismsofcelastrolonthemigrationandinvasionofRA-FLSsarenotyetunderstood.ResultsshowedthattreatmentofRA-FLSswithcelastrolsuppressedtheIL-17A-inducedmigrationandinvasionabilitiesofthecells. Inaddition,celastrol inhibitedIL-17A-inducedmatrixmetalloproteinase(MMP)-9mRNAandproteinexpres-sion,andtheproteolyticactivityofMMP-9inRA-FLSs.Furthermore,ourresultsrevealedthatcelastrolinhibitedthetranscriptionalactivi-tyofMMP-9bysuppressionofthebindingactivityofnuclearfactor-κB(NF-κB)intheMMP-9promoter,andinhibitedIκBαphosphorylationandnucleartranslocationofNF-κB.Inconclusion,celastrolcaninhib-itIL-17A-inducedmigrationandinvasionbysuppressingNF-κB-me-diatedMMP-9expressioninRA-FLSs.TheseresultsprovideastrongrationaleforfurthertestingandvalidationofcelastrolasanadjunctwithconventionaldrugsforthetreatmentofRAinhumans.NoCOI.

3P-128Roles of a transcription factor interferon regulatory factor 8 (IRF8) in proinflammatory reactions of macro-phages and microgliaKawamoto, Chisato; Yano, Hajime; Kawasaki, Shun; Takahashi, Hisaaki; Tanaka, Junya(Dept. of Molecular and Cellular Physiology, Graduate School of Medicine, Ehime Univ., Ehime, Japan)

Interferonregulatoryfactor8(IRF8)isbelongingtoIRFfamilyproteinsthatmediatesignalsoriginatingformtypeIinterferon.IRF8hasbeenimplicatedinthedifferentiationprocessofmyeloidlineagecellsintomonocytesandmacrophages.However,therolesofIRF8inthefunc-tionsofmacrophagesandrelatedcellshavenotwelldocumented,comparedtootherIRFproteins.Recently,ithasbenshownthatIRF8playsacriticalroleinthetransmissionofpaininthespinalcordbycontrollingmicroglialfunctions.Inthisstudy,weareaddressingtherolesofIRF8inproinflammatoryreactionsofmacrophagesandmi-croglialcells.Macrophageswere isolatedfromthe lungs (alveolarmacrophages;AMs)andtheperitonea(peritonealmacrophages;PMs)ofWistarrats.Microglialcellswereobtainedfromratprimarymixedglialcultures.TotalRNAwaspreparedfromAMs,PMsandmicrogliaandreversetranscribedtocDNA.mRNAencodingIRFswerequan-titativelyanalyzedusingquantitativereal-timeRT-PCR.AlthoughthethreetypesofcellsexpressedIRF8-mRNAsignificantly,thelevelwaslessthanthoseof IRF1,3and7mRNA.WhenIRF8-mRNAwasknockeddownbytheuseofsiRNAandIRF8proteinwasactuallyreduced,LPS-inducedNOproductionwasreducedinPMsandmicrog-lia.Bycontrast,AMsincreasedNOproduction.TheseresultssuggestthatIRF8playdistinctrolesindifferentcelltypes.WearecurrentlyinvestigatinghowIRF8isinvolvedintheproinflammatoryreactions.NoCOI.

3P-129Iba-1 positive macrophage is activated in the anterior pituitary in the chronic inflammatory pain model ratYagasaki, Yuki; Katayama, Yoko; Nagata, Tomonari; Kinoshita, Yoko; Kawakami, Yoriko(Dept Physiol, Sch. Med, Tokyo Women's Medical Univ, Tokyo, Japan)

Non-hormonalcells,suchasamacrophageandafolliculostellatecell,existintheanteriorpituitarylobe(AP),however,thefunctionofthesecellsremainslargelyunclear.Inthisstudy,weexaminednon-hormon-alcellresponsestochronicpainusingtwochronicpainmodels:CFAinducedchronicinflammatorypainmodelandachronicneuropathicpainmodel (Seltzermodel).WedeterminedtheNon-hormonalcellsproportionandmorphologyusingantiIba-1antibody(markerofpanmacrophages)andantiS-100antibody(markeroffolliclostellatecell).WerevealedthatIba-1positivemacrophageoccupied3%ofproportionofthetotalAPcellsintheadultintactmalerat.Inthechronicinflam-matorycondition,Iba-1positivecellproportionwasincreasedandthecellsmorphologicallychanged:moreroundedsomawithenhancedlamellipodia.Thesechangeswerenotobservedinthechronicneuro-pathicpainmodelrat.WealsodeterminedtheS100positivefolliculo-stellatecellaswell,butcellproportionandmorphologywerenodif-ferenceamongthatofacontrolgroupandtwochronicpainmodels.Furthermore,werevealedthatIL-1βpositivecellwasincreasedintheAPinthechronicinflammatorypainrat,andmostofIL-1βimmuno-reactivecell(89%)mergedIba-1immunoreactivecell.TheseresultsindicatethatthechronicinflammatorypainhadactivatedIba-1positivemacrophageintheanteriorpituitary,whichsynthesizedIL-1βmodu-latingpainsensation.NoCOI.

3P-130Oct-3/4 induces CpG demethylation in MGMT pro-moter to acquire temozolomide resistance in glioblas-toma cellsFunahashi, Yu; Takahashi, Hisaaki; Sugimoto, kana; Yano, Hajime; Tanaka, Junya(Molecular and Cellular Physiology, Ehime Univ Grad Sch of Med, Ehime, Japan)

Temozolomideisthemosteffectivecytotoxicalkylatingagentsusedformalignantgliomas.AcellularDNA-repairenzyme,MGMT,revers-esalkylationattheO6positionofguanine, therebytheexpressionlevelofMGMTiscloselyrelatedtothesensitivityofbraintumorstoTemozolomide.MGMTexpression iscontrolledbyamethylation/demethylationoftheCpGislandsinthepromoterregionofMGMTgene.Oct-3/4,aself-renewalregulatorinstemcells,hasbeenknowntoexpressonvariouskindsofsolidtumorsincludingglioblastoma,andhasbeeninvolveintumorprogression,malignancyinglioblastomas.Therefore,inthepresentstudy,weinvestigatedwhetherOct-3/4in-volvesinthesensitivityoftemozolomidethroughtheexpressionofMGMT.Comparedwithcontrolcells,Oct-3/4overexpressionresultedindecreasedsusceptibilitytotemozolomide,assessedbyLDHassay.InOct-3/4-expressingcells,expressionofMGMTmRNAwasupreg-ulatedbyqPCRanalysis.Themethylationstatusof27CpGsiteswithintheMGMTpromoterwasanalyzedbygenomicsequencingofbisulfite-modifiedDNA.Oct-3/4-expressingcellsshowedenhanceddemethylationofCpG islands.TheseresultssuggestthatOct-3/4promotestoacquiretemozolomide-resistantphenotypeofglioblastomacellsbyupregulationofMGMTexpressionthroughtheepigeneticchangeofMGMTpromoterregion.WealsoinvestigatetheeffectofSox-2,anessentialgeneforstemness,againsttheepigeneticregulationofMGMTgene.NoCOI.


3P-131Role of plasminogen activator inhibitor-1 in the devel-opment of insulin resistance and osteoporosis in obese female miceTamura, Yukinori1; Kawao, Naoyuki1; Yano, Masato1; Okada, Kiyotaka1; Matsuo, Osamu2; Kaji, Hiroshi1(1Department of Physiology and Regenerative Medicine, Kinki University Faculty of Medicine, Osakasayama, Japan, 2Kinki University Faculty of Medicine, Osakasayama, Japan)

Wepreviouslydemonstratedthatplasminogenactivator inhibitor-1(PAI-1),aninhibitoroffibrinolysis,isinvolvedintype1diabeticbonelossinfemalemice.PAI-1iswellknownasanadipogenicfactorinducedbyobesity.WethereforeexaminedtheeffectsofPAI-1deficiencyonbone,glucoseandlipidmetabolisminhighfatandhighsucrosediet(HF/HSD)-inducedobese femalemice.Femalewild-type (WT)andPAI-1-deficient(KO)micewerefedwithHF/HSDornormaldietfor20weeksfrom10weeksofage.HF/HSDincreasedthelevelsofplas-maPAI-1inWTmice.PAI-1deficiencyimprovedglucoseintolerance,insulinresistanceandhyperlipidemiainducedbyobesity.Bonemin-eraldensity(BMD)attrabecularboneaswellasthelevelsofosteo-genicgenes intibiaweredecreasedbyHF/HSDinWTmice,andthosechangesbyHF/HSDwerenotaffectedbyPAI-1deficiency.HF/HSDincreasedthelevelsofplasmatumornecrosisfactor-alpha(TNF-α)bothinWTandPAI-1KOmice,andthelevelsofplasmaTNF-αwerenegativelycorrelatedwithtrabecularBMDintibiaoffemalemice.Inconclusion,wedemonstratedthatPAI-1deficiencydoesnotaffectthetrabecularbonelossinducedbyobesitydespitetheameliorationofinsulinresistanceandhyperlipidemiainfemalemice.Thus,thechang-esofBMDandbonemetabolismbyobesitymightbepartlythroughTNF-αandindependentofPAI-1,glucoseandlipidmetabolism.NoCOI.

3P-132Voluntary Exercise Suppresses Development of Dia-betes Mellitus and Improves Exercise Performance in OLETF RatsSakata, Susumu1; Hanaoka, Tomoko1; Takada, Yoshihiro1; Okuda, Syunji1; Yasui, Toshihide1; Washio, Hiroe1; Takeshita, Daisuke1; Iwami, Keiko1; Imagita, Hidetaka1; Minematsu, Akira1; Waki, Hidefumi2; Nakatani, Akira3(1Kio Univ. Grad. Sch. Health Sci, Nara, Japan, 2Dept. Physiol., Wakayama Med. Univ. Sch. Med., Wakayama, Japan, 3Lab. Exer. Physiol., Nara Univ. Edu., Nara, Japan)

Theaimofthisstudywastoexaminewhethervoluntarywheel-running(WR)suppressdevelopmentofdiabetesmellitus (DM)and improveexerciseperformance(EP)inatype2DMmodelratOLETF.Five-week-oldOLETFratswerehousedeither incagesequippedwithwheels(OLETF-WR)orinstandardcages(OLETF-SED)formorethan1year.Eleven-month-oldratsunderwentanoralglucosetolerancetest(OGTT)andanexaminationofHbA1c.Fourteen-month-oldratsun-derwent10-dayvoluntaryWR,treadmillperformance,hangingandgrip-strengthtests.TherewasanegativecorrelationbetweentotalWRdistanceandbodyweightinOLETF-WR.InOLETF-SED,bloodglucose(BG)increasedaftertheageof33weeks,whileBGinOLETF-WRandLETOremainedunchangedthroughouttheexperimentalperiod.InOGTT,BGofOLETF-WRandLETOwithnormalHbA1clevels,returnedtonormallevel2hoursafterglucoseadministration,whereasBGofOLETF-SEDwithhigherHbA1clevels,remainedhigh.LETOandOLETF-WRshowedthehighestandmoderateEP intreadmillperformance,hangingandgrip-strengthtests,respectively,whileOLETF-SEDdiabeticratsthelowestEP.Thus,long-termvol-untaryWRcouldsuppressDMdevelopmentand improveEP inOLETFrats.NoCOI.

3P-133Secondary Structure and Thermal Stability of FcαRI β Chain Polymorphism (L172I, L174V, E228G) Terada, Tomoyoshi; Takahashi, Teppei; Arikawa, Hajime; Era, Seiichi(Department of physiology and biophysics, Gifu University Graduate School of Medicine, Gifu, Japan)

TheβchainofthehighaffinityIgEFcreceptor(FcεRI)actsduringanallergicreactionasasignalamplifierinmastcells.Geneticpoly-morphismsthatresultinthreeaminoacidchangesinFcεRIβchainhavebeenidentifiedascandidatesthatassociatewithallergicreaction.WehaveinvestigatedthestructureofmouseFcεRIβchainwildtype(aa143-235)andpolymorphisms(L172I,L174VandE228G).Thefar-UVCDspectraoftheβ-wildtype(WT)andpolymorphismsareindicativeofanα-helicalproteinandpolymorphismsdonothaveany lossorcollapseofα-helicalcontent.Weinvestigatedthetransitioncurveforthermaldenaturationofβ-WTandβ-polymorphisms,andcalculatedtheGibbsfreeenergy(ΔG).ΔGvaluesofβ-L172Iandβ-L174Varealmostthesameasthatofβ-WT,however,ΔGvalueofβ-E228Gislowerthatthatofβ-WT.Thesedatasuggestthatβ-E228GaffectsthethermalstabilityofFcεRIβchain.β-E228GmayplaysomesortofrolesinallergicreactionsviaFcεRIβchaininmastcells.NoCOI.

Poster PresentationsOral Physiology (3)


3P-134Gustatory neuron profiles in rat reticular thalamic nu-cleusKato, Risako; Koshikawa, Noriaki; Kobayashi, Masayuki(Department of Pharmacology, Nihon University School of Dentistry, Tokyo, Japan)

DepartmentofPharmacology,NihonUniversitySchoolofDentistryTheparvicellularportionoftheventroposteromedialnucleusofthalamus(VPMpc)relaysgustatoryinformationfromtheparabrachialnucleustothegustatoryinsularcortex(GC).Inaddition,theVPMpcprojectstothereticularthalamicnucleus(RTN),whichalsoreceivesexcitatoryinputsfromlayerVIneuronsintheGC.TheprincipalneuronsintheRTNareGABAergic,andtheyprojecttotheVPMpc,andtherefore,theVPMpc,RTN,andGCconstitutea feedbackcircuittoprocessgustatory information.However, theneuralresponsesoftheRTNneuronstogustatorystimulationhavenotbeenwellunderstood.Thepresentstudyaimedtoexamine(1)whichregionoftheRTNconnectstotheGC,and(2)theneuralprofilesofRTNneuronsinresponsetogustatorystimuli.Ananatomicalexperimentusingaretrogradetrac-er,FluoroGold,showedthattherostroventralpartoftheRTN(RTN-rv)receivedprojectionfromtheGC.ApartofneuronsintheRTNrvrespondedtogustatorystimulationtotheoralcavity:~15%and~8%ofneuronsincreasedanddecreasedtheirfiringfrequency,respective-ly.Wecouldnotfindtherelationshipbetweenthespontaneousfiringfrequencyandthechangeoffrequencybygustatorystimuli.Theseresultssuggestthatneurons intherostroventralpartof theRTNneuronsreceivegustatoryinformation,andlikelytomodulateneuralactivitiesintheVPMpc.NoCOI.

3P-135Changes of intensity and palatability by mixing taste solutionsKatagawa, Yoshihisa1; Yasuo, Toshiaki2; Suwabe, Takeshi2; Gen, Keika1; Sako, Noritaka2(1Dept. Dent. for the Disability and Oral Health, Asahi Univ. Sch. Dent. Mizuho, Japan, 2Dept. Oral Physiol., Asahi Univ. Sch. Dent. Mizuho, Japan)

Therearesmallstudiestoevaluateintensityandpalatabilityforthemixedtastesolutionsinhuman.Inthisstudy,weinvestigatedhowhumansubjectsevaluatedthembyusingtheLabeledMagnitudeScale(Greenetal.,1993)andtheLabeledHedonicScale(Limetal.,2009).Atotalof30malesevaluatedintensityandpalatabilityforsometastesolutions.Followingsolutionswereusedastastestimuli;0.3–10mMsaccharinNa(Sacc),0.03–1.0MNaCl(Na),0.1–0.3mMQHCl(Q),themixedtastesolutionsof5mMSaccandoneof0.03–1.0MNa,andthemixedtastesolutionsof5mMSaccandoneof0.1–0.3mMQ.Asresults;therewassignificantmaineffectforsaltorbitterintensitybetweenpureNaorQ,andmixedsolutionswithSacc.WhenSaccwasmixedwithNaorQ,palatabilitiesformixedsolutionswerehigherthanthoseforpuresolutions.Theseresultssuggestthatmixingofdifferenttypeoftastestimuluschanges intensityandpalatability fororiginal tastestimulus.NoCOI.

Poster PresentationsPhysical Fitness, Sports Medicine

3P-136High concentration CO2-water bath may reduce the muscle hardness and soreness after resistance exer-ciseYamamoto, Noriyuki1; Hashimoto, Masaaki2(1Dept Health Sci, Jpn Red Cross Hokkaido Coll Nurs, Kitami, Jpn, 2Lab Physiol, Dept Phys Ther, Fac Med Sci, Teikyo Univ Sci, Tokyo, Jpn)

ThesedativeeffectonsympatheticnervefunctionofCO2-waterbathmayimplyapossibilityofthefacilitationofmusclefatiguerecovery.Weinvestigatedwhethertheimmersionofextremitiesincludingag-onistmusclesintoartificiallymadeCO2-water(CO2>1000ppm)influ-encesrecoveryofmusclehardnessandsorenessinfatigueafterresis-tance exercise in twelvemale subjects (Ss).On the first day ofexperiment,Ssperformed100timescalfraiseexerciseandimmersedlowerlegsintotap-waterorCO2-water(35°C,for10min)aftertheexercise.Hardnessofthegastrocnemiuswasdeterminedbyindenta-tionmethodatpre-exercise,immediatelyafterexercise,andafter10minrecovery.Themusclesorenesswasassessedusingavisualana-loguescale.Thelegbathwasperformedevery24hfor5consecutivedaysaftertheexerciseday,andthemuscleparametersweremeasuredbeforeandafterthelegbatheachday.Asamesubjectwastestedwithanotherbath-waterafteraseriesofmeasurement.Maximummusclehardnesslastedfor3daysafterexerciseirrespectiveofthekindofbath-water,thendecreasedgraduallytobaseline.InCO2-waterimmersioncomparedwithtap-water immersion,musclehardnessdecreasedquicker.Musclesorenessintap-waterpeakedatthesecondday,andthenreducedgradually.InCO2-watercase,musclesorenessdisappearedatthethirdday.Thepresentresultssuggestthatbathingintoartificialhigh-concentrationCO2-watermaycontributetoarecov-eryfromthemusclefatigue.NoCOI.


3P-137LDL-C / HDL-C ratio may be a factor of cardiac sud-den death during exerciseFukada, Kihachiro1; Kushi, Hidehiko1; Takashina, Terue1; Onuma, Naoko1; Oki, Kazuma2(1Graduate School of Literature and Social Sciences, Nihon University, Tokyo, Japan., 2Institute of Humanities and Social Sciences, Nihon University, Tokyo, Japan)

[Introduction] Increasing inthePlasminogenactivator inhibitor -1(PAI-1) levelmaybea factorofcoronaryarterydisease.Coronaryarterydiseaseisoneofthefactorsofcardiacsuddendeathduringexercise.TheaimofthisstudywastoevaluatewhetheradifferenceintheLDL-C/HDL-CratioaffectsthePAI-1levelduringacutestren-uousexercise.[SubjectsandMethods]Thirteenhealthytrainedmenaged19to23yearsparticipatedinthisstudy:7ofthesewerecategorizedintheLDL-C/HDL-Cratio<2.0group(L/H<2.0group),and6intheLDL-C/HDL-Cratio>2.0group(L/H>2.0group).VenousbloodsampleswerecollectedfromthesubjectsbeforeandaftertheyperformedtheCooper12-mintest(runningasfaraspossiblewithin12min).LDL-Cconcentrations (mg/dL),HDL-Cconcentrations (mg/dL),PAI-1level(ng/mL)weremeasuredbyusingbloodsamples.[Results]LDL-C/HDL-CratiowassignificantlyhigherinL/H>2.0group(2.3±0.07)thanintheL/H<2.0group(1.2±0.07;P<0.05).PAI-1levelbefore(30.4±1.9ng/mL)andafter(25.8±3.4ng/mL)exercisedidnotchangesignificantlyintheL/H<2.0group,whereasitincreasedsignificantlyintheL/H>2.0group(41.6±10.0ng/mLto61.8±11.7ng/mL;P<0.05).[Conclusions]PAI-1levelincreasedafterstrenuousexerciseinhighLDL-C/HDL-Cratiogroup.Consequently,thepresentstudysuggest-edthatLDL-C/HDL-Cratiomaybeafactorofcardiacsuddendeathduringexercise.NoCOI.

3P-138Exercise training improves peripheral vascular func-tion in type I diabetic mice as measured by X-ray mi-croangiographySonobe, Takashi; Tsuchimochi, Hirotsugu; Shirai, Mikiyasu(Dept of Cardiac Physiol, Natl Cerebral & Cardiovasc Ctr Res Inst, Osaka, Japan)

WeinvestigatedwhetherexercisetrainingimproveshindlimbbloodflowandendothelialfunctioninstreptozocininducedtypeIdiabeticmice.Miceweredividedintothesedentaryandexercisetraininggroups2weekspoststreptozocininjection(200mg/kg).Theexercisetrainingwasperformedwithtreadmilllevelrunningfor60min/day,5days/week,for4weeks.Thereafter,1)invivohindlimbvascularimagingusingX-raymicroangiography,or2)femoralbloodflowmeasurementusingtransonicflowprobewereperformed.Themicewereanesthetizedwithurethane-α-chloralose,thentracheostomizedandcannulatedintocarotidarteryandjugularvein forbloodpressuremonitoringandacetylcholine(ACh)administration,respectively.Anadditionalcatheterwasinsertedintothefemoralarteryforinjectionofiodinatedcontrastagent,ortheflowprobewasplacedaroundthefemoralartery.Themicehindlimbarteries (innerdiameter:50to200µm)wereclearlyvisualizedbyX-rayimaging,furthermoredynamicchangesinvesseldiameterinresponsetoAChwereobserved.TheexercisetrainingimprovedtheACh-inducedvasodilationindiabeticmice,andtheeffectwasmostlyfoundinthesmallerarteries(<100µm).However,ACh-in-ducedincreasesinfemoralbloodflowwerenotsignificantlydifferentbetweendiabeticandexercisegroups.Thesedatasuggestedexercisetraininghaspotential toamelioratetheendothelialvasodilatordys-functionoftheperipheralskeletalmusclearteriesindiabetes.NoCOI.

3P-139Effectiveness of autonomic control of the circulation is enhanced with increased plasma volume after in-tense exerciseOkazaki, Kazunobu1,2; Takeda, Ryosuke2; Suzuki, Akina2; Imai, Daiki1,2; Naghavi, Nooshin2; Yamashina, Yoshihiro2; Yokoyama, Hisayo1,2; Miyagawa, Toshiaki1,2(1Research Center for Urban Health and Sports, Osaka City Univ, Osaka, Japan, 2Dept of Environmental Physiology for Exercise, Osaka City Univ Grad Sch of Med, Osaka, Japan)

Weassessedwhether increasedplasmavolume (PV)enhancestheeffectivenessofautonomiccontrolofthecirculationafterintenseex-ercise.METHODS:Sevenhealthymen(~23yrs)underwenttwotrialsthoseofwhichdifferinthetimingofpost-exerciseintakeofmacronu-trients.Inbothtrials,subjectskeptseatedrestforbaseline(BL),thenperformeda72-minintenseexercise(8setsof4minat80%VO2max+5minat20%VO2max)andkeptseatedrestduring0-5thand22–23rdhourofrecovery.Theytookproteinandcarbohydrate(3.2kcaland0.18gprotein/kg)justafterexercise(0H)or2hourslater(2H).PercentchangeinPVfromBL(δ%PV)wasdeterminedandbeat-to-beatbloodpressure,R-Rinterval,heartrate,andstrokevolume(SV)weremea-suredduring5-minspontaneousandfixedrate(0.2Hz)respirationatBLandeveryhourduringrecoveryphases.Dynamicarterial-cardiacbaroreflexsensitivity(BRS)wascalculatedbysequenceanalysis.RE-SULTS:δ%PVin0Hwashigherthan2Hat5thand23rdhourofre-covery(P<0.05).TherewasnodifferenceinBRSbetweentrials,whileSVandeffectivearterial-cardiacbaroreflexgainestimatedasBRSxSVwerehigherin0Hthan2Hat5thand23rdhourofrecovery(P<0.05).CONCLUSIONS: IncreasedPVenhancestheeffectivenessofautonomiccontrolofthecirculationwithanincreasedSVafterintenseexercise.NoCOI.

3P-140Effects of the gum chewing on cardiac autonomic bal-ances and anxiety levels during the origami competi-tion in the young people. Sakakibara , Yoshikazu1; Yoshida, Yuka1; Masuda, Atsuko2; Tanaka, Michiko3(1Department of psychological informatics, Kanazawa Institute of Technology, 2Medical Education Center, Faculty of Health Science, Ryotokuji Univ., 3Fukuoka prefectural Nursing Univ.)

Weaimedtoexploreeffectsofthegumchewingoncardiacautonom-icbalanceswithusingHRVduringtheorigamicompetition(origamiC)in13maleand5femalestudents.Subjectswerepairedtobalancetoeachotherinorigamiskills.EachpairrepeatedtheorigamiContwodifferentdays.Oneofeachpairwasthefirstgum-chewerandtheotherwasthesecond.Numbersofthefirstandthesecondgum-chew-erswerebalancedbetweenboth.Subjectswereexplainedabouttheexperimentalflow.Duringthe10minrestingperiod,apairsatonchairsdirectedinparallelwitheachotherandsawtheirownsceneryphotoontheroomwall.Thegumchewerchewedduringthelatterhalfofthistimeandthrewoutitattheendofthisperiod.Then,theycor-rectedthesittingdirectiontofaceeachotherandplayedorigamiCfor5min.AquestionnaireoftheSTAI(A-state)wasansweredaftertheexplanation,andaftertheexperiment,too.Gumchewingdidnotimprovetheperformanceoftheorigamiandwinnerswerethesamein7of9pairs.TheparasympatheticactivityassessedbyHFwassignificantlyreducedinwinnerswithgumchewing(p<0.05,n=9),butnot inwinnerswithoutgum(p=0.23,n=8).ThegumchewinghassignificantlyreducedanxietyscoresoftheA-state,inbothloserandwinner.Presentresultsmaymeanthatgumchewingcouldleadthemindtoconcentrateonthecompetition,particularlyintheadvanta-geouscase.NoCOI.


3P-141Combined effects of hyperthermia and mental fatigue on endurance exercise capacity in the heat Otani, Hidenori1; Kaya , Mitsuharu2; Tamaki, Akira3; Tsujita, Junzo4 (1Faculty of Health Care Sciences, Himeji Dokkyo University, Himeji, Japan, 2General Education Center, Hyogo University of Health Sciences, Kobe, Japan, 3Department of Rehabilitation Science, Graduate School of Health Sicence, Hyogo Uiversity of Health Sciences, Kobe, Japan, 4Department of Health and Sport Science, Hyogo College of Medicine, Nishinomiya, Japan)

Theaimofthepresentstudywastoexaminetheeffectsofhyperther-miaandmentalfatigueonenduranceexercisecapacityintheheat.Eightmalevolunteerscompletedfourcyclingtrialsat80%maximumoxygenuptakeuntilvolitionalexhaustioninaclimaticchamber(30°C,50%RH).Volunteerscycledafter:90minseatedrest(CON),90minofademandingcognitivetaskto inducemental fatigue (MF),30minimmersion in40°Cwaterto inducehyperthermiafollowing60minseatedrest(HT),or30minimmersionin40°Cwaterwithademandingcognitivetaskfollowing60minofademandingcognitivetasktoinducebothmentalfatigueandhyperthermia(MF+HT).Rectaltemperatureatthestartofcyclingwas36.7±0.5(±SD),36.8±0.2,38.1±0.4,and38.0±0.1°CinCON,MF,HT,andMF+HT,respectively.ExercisetimetoexhaustionwassignificantlylessinMF+HTthaninCONandMF(CON18±7min;MF17±7min;MF+HT9±3min,P<0.05).Atthepointofvolitionalexhaustion,rectaltemperature,meanskintempera-ture,heartrateandcutaneousvascularconductancewerenotdifferentbetweentrials. Inconclusion,acombinationofhyperthermiaandmental fatigueelicitssignificantreductions inenduranceexercisecapacityintheheat.NoCOI.

3P-142Ubiquitin ligase Nedd4 expression and atrophy in un-loaded rat plantaris muscleYamauchi, Hideki; Takemori, Shigeru(Div. Physical Fitness, Dept. Molecular Physiol., The Jikei Univ. Sch. of Med., Tokyo, Japan)

Purpose:Itiswelldocumentedthattwomusclespecificubiquitinli-gases,atrogen-1/MAFbxandMuRF1,areinvolvedprominentlyintheskeletalmuscleatrophy.Recentstudieshavesuggestedthatotherubiquitinligase,Nedd4(neuralprecursorcellexpresseddevelopmen-tallydown-regulatedprotein4)playsamoreencompassingrole indenervation-inducedmuscleatrophy.Therefore,weinvestigatedtheinvolvementofNedd4inunloading-inducedmuscleatrophywithspe-cificfocussetonagedependency.Methods:F344femalerats(4-,10-,and20-month)weredividedintothreegroups;cagedcontrolgroup,hindlimb-unloadedgroup,andhindlimb-unloadedand intermittentlyreloadedgroupineachage.Ratsofreloadedgroupwereexercisedonresistanceexercisedevicewithaloadof30%ofbodymassfor3weeks(30min/day,6days/week).Plantarismuscleswereanalyzed.Result:Musclemassandmaximumforceincagedcontrolgroupincreasedfrom4-monthto10-monthanddecreasedfrom10-monthto20-month.Thehindlimb-unloadingdecreasedmusclemassandmaximumforceatanyage,butmostprominentlyin20-monthrats.SignificantincreaseintheexpressionofNedd4wasfoundonlyin20-monthrats.Intermit-tentreloadingduringhindlimb-unloadingperiodinhibitedmuscleat-rophy independentofage.However, theeffectofreloadingontheNedd4expressionwasnotsignificantlyobservedeven in20-monthrats.Conclusion:TheubiquitinligaseNedd4mayparticipateinseveremuscleatrophyatleastattheoldage,althoughitmaynotintimatelylinkedwithatrophyprocess.NoCOI.

3P-143Abdominal obesity augments the reduction in cough peak flow by supination in middle-aged and elderly womenYamashina, Yoshihiro1; Tabira, Kazuyuki2; Yokoyama, Hisayo1,3; Okazaki, Kazunobu1,3; Suzuki, Akina1,3; Takeda, Ryosuke1,3; Nooshin, Naghavi1,3; Miyagawa, Toshiaki1,3(1Department of Environmental Physiology for Exercise, Osaka City University Graduate School of Medicine,Osaka,Japan, 2Department of health science, Kio University, 3Reserch Center for Urban Health and Sports, Osaka City University)

Theaimofthepresentstudywastoexaminewhetherabdominalobesityandthedifferentposture,sittingorsupination,affectedonthecoughcapacity inmiddle-agedandelderlywomen.Method:Twen-ty-fourmiddle-agedandelderlywomenconsistedof10with (obesegroup)and14without(non-obesegroup)abdominalobesitywerein-cludedinthisstudy.Inallsubjects,weevaluatedthechangesinvitalcapacity(VC)andcoughpeakflow(CPF)intwodifferentpositions,sittingandsupination.Results:CPFaswellasVCweresignificantlylower insupinationthan insitting inbothgroups (280.3±67.0vs.313.1±70.2L/mininobesegroup,298.1±82.1vs.316.8±77.4L/mininnon-obesegroup,p<0.05).AlthoughnodifferencewasfoundinVCorCPFbetweenthegroupsateachposition,thechangesinbothVCandCPFbyposturalchangeweregreaterinobesegroupthaninnon-obesegroup (5.2±4.4%vs.3.7±5.3% inVC,10.7±3.9%vs.7.0±5.6% inCPF,p<0.05).Inobesegroup,thechangesinCPFwaspositivelycorrelatedwithwaistcircumference(r=0.63,p<0.05).Conclusion:Ourresultssug-gestedthat,inthesubjectswithabdominalobesity,coughcapacitywasaffectedmoremarkedlybytakingsupination,becausetheirdia-phragmresistancewasfurtherincreasedinsupinationduetostuffedabdominalcavitywithexcessivefatcontents.NoCOI.

3P-144Post-transcriptional suppression of lipopolysaccha-ride-stimulated inflammatory responses by macro-phages in middle-aged miceShirato, Ken; Imaizumi, Kazuhiko(Laboratory of Physiological Sciences, Faculty of Human Sciences, Waseda University, Tokorozawa, Japan)

The intensitiesofmacrophage inflammatoryresponsestobacterialcomponentsgraduallydecreasewithage.Giventhatareducedrateofproteinsynthesisisacommonage-relatedbiochemicalchange,whichispartiallymediatedbyincreasedphosphorylationofeukaryoticiniti-ationfactor-2α(eIF-2α),weinvestigatedthemechanismresponsibleforthedeteriorationofmacrophage inflammatoryresponses, focusingspecificallyontheage-relatedbiochemicalchanges inmiddle-agedmice.Peritonealmacrophagesisolatedfrom2-month-old(young)and12-month-old(middle-aged)maleBALB/cmicewerestimulatedwithlipopolysaccharide(LPS).AlthoughLPS-stimulatedsecretionoftumornecrosisfactor-α(TNF-α)bythemacrophagesfrommiddle-agedmicewassignificantlylowerthanthatfromyoungmice,LPScausedmarkedincreasesinlevelsofTNF-αmRNAinmacrophagesfrommiddle-agedaswellasyoungmice.Moreover,LPSevokedsimilarlevelsofphos-phorylationofc-JunN-terminalkinase (JNK)andnuclear factor-κB(NF-κB)inyoungandmiddle-agedmice.Incontrast,thebasallevelofphosphorylatedeIF-2αinmacrophages frommiddle-agedmicewashigherthanthatinmacrophagesfromyoungmice.Salubrinal,anin-hibitorofthephosphataseactivitythatdephosphorylateseIF-2α,sup-pressedtheLPS-stimulatedinflammatoryresponsesinamurinemac-rophage cell line RAW264.7. These results suggest thatpost-transcriptionalsuppressionofmacrophageinflammatoryrespons-esduringmiddleagerequiresphosphorylationofeIF-2α.NoCOI.


3P-145Evaluation of functional reach in aged people by the correlation with the reach and height of young adult peopleNakamoto, Naoko; Tamura, Takumi; Harami, Maiko; Tsubota, Yuji (Dep. Physiol. Osaka Kawasaki Rehabil. Univ. Osaka, Japan)

Thefunctionalreachtestisusedtomeasurebalanceabilitiesintheclinicalfield.Thefunctionalreach(FR)wascorrelatedwiththeheightinyoungadultpeople(Morishitaetal.,89thAnnualMeetingofPSJ,2011).Inthisstudy,weevaluatedFRinagedpeoplebytheregressionlinewiththeheightinyoungadultpeople.FRdistancewasmeasuredin58healthyagedparticipants(58.7±19.1yearsold).RateofFRwascalculatedbythecomparisonwithestimatedFRbythefurtherre-gressionanalysisbetweenFRandheightinyoungadultpeople.TherateofFRwassignificantlyreducedwithage(r=-0.31,p<0.02).ButrawFRdistancesshowedweakcorrelationwithage(r=-0.23,p=0.08).Inthefourparticipantswithafallshowedsmallerratiosuchas39±17%.ThereforetherateofFRestimatedbyourregression linemaybeusefultoevaluatebalanceabilitiesinagedpeople.NoCOI.

Poster PresentationsCell Physiology, Molecular Physiology

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3P-146The naftopidil analogue HUHS1015 induces apopto-sis in gastric cancer cellsKaku, Yoshiko; Tsuchiya, Ayako; Kanno, Takeshi; Nishizaki, Tomoyuki(Division of Bioinformation, Department of Physiology, Hyogo College of Medicine, Nishinomiya, Japan)

Thepresentstudy investigatedtheantitumoreffectof thenewlysynthesizednaftopidilanalogueHUHS1015(1-[2-(2-methoxyphenylami-no)ethylamino]-3-(naphthalene-1-yloxy)propan-2-ol)onhumangastriccancercells.HUHS1015reducedcellviability inatreatmenttime(24-48h)-andconcentration(1-100µM)-dependentmanner,theviabil-ityreachingalmost0%ofcontrolat100µMboth inMKN-28andMKN-45humangastriccancercells. Cisplatin,ananticancerdrugwidelyused,alsoreducedviabilityofMKN-28andMKN-45cellsinaconcentration(1–100µM)-dependentmanner,buttoa lesserextentthanthatforHUHS1015.HUHS1015significantlyincreasedthenum-berofTUNEL-positivecellsascomparedwiththatforcisplatin.More-over,HUHS1015apparentlyincreasedthepopulationofPI-positiveandannexinV-positivecells,whichcorrespondstolateapoptosis/secondarynecrosis.Takentogether,theseresultsindicatethatHUHS1015ex-hibitsamorebeneficialantitumoreffectonhumangastriccancersthancisplatin.NoCOI.

3P-147Regulatory mechanisms of the G1 to S phase cell cy-cle progression via changes in the intracellular con-centration of Cl- in MKN28 human gastric cancer cellsMiyazaki, Hiroaki1,2; Marunaka, Yoshinori1,2(1Department of Molecular Cell Physiology, Kyoto Prefectural University of Medicine, Kyoto, Japan, 2Japan Institute for Food Education and Health, Heian Jogakuin (St.Agnes') University, Kyoto, Japan)

Wepreviouslyclarifiedthatthereductionof intracellularchlorideconcentration ([Cl-]i) inhibitsthecellproliferationofgastriccancerMKN28cellsbydiminishingthetransitionratefromG1toScellcyclephase.IfthereareoscillatorychangesintheactivityofCl-transport-ersand/orCl-channelsduringthecellcycleprogression,oscillatorychangesofthe[Cl-]iwouldalsooccur.However,mechanismsinvolvedinthemodulationofcellcycleprogressionbyCl-transportersand/orCl-channelsarestillpoorlyunderstood.Toclarifytheunderlyingmechanisms,wemeasuredthemRNAandproteinexpressionofCl-transporters(NKCC1andKCC1)andCl-channels(CLC-2andCLC-3).Wealsoanalyzedthe[Cl-]iofthecellsineachcellcyclestage(G1,SandG2/M)releasedfromsynchronizationbyusingadoublethymidineblockmethod.The[Cl-]iwasreducedintheMphasefollowedbytheelevationof [Cl-]i intheG1andSphase.TheproteinexpressionofNKCC1waslowintheMandearlyG1phases.However,proteinex-pressionsofKCC1,CLC-2andCLC-3wereunchangedduringcellcycleprogression.Wealsotriedtoclarifyplasmamembraneexpres-sionsofNKCC1,KCC1,CLC-2,andCLC-3.Theseresultsstronglysuggestthatcellcycle-dependentexpressionofNKCCleadingtoos-cillatorychangesof[Cl-]iplaysimportantrolesintheG1toSphasecellcycleprogressioninMKN28cells.NoCOI.


3P-148Intracellular localization of fluorescent glucose deriv-atives taken up into mammalian tumor cellsTakigawa, Shota1,2; Ono, Kouki1; Ngatomo, Katsuhiro1; Sasaki, Ayako1; Yoshida, Takashi2; Yamada, Katsuya1(1Hirosaki University Graduate school of medicine Department of Physiology, 2Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University)

D-glucoseisafundamentalenergyaswellascarbonsourceforcells.However, itsdynamismincellularuptake isyettobethoroughlyunderstoodatthemicroscopiclevelinmanyorganisms.FluorescentderivativesofD-glucosesuchas2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose(2-NBDG)hasbeeneffectivelyusedasatooltoinvestigatesuchuptakeprocessesespeciallyinmammals,althoughtheirintracellularfateisstillunclear.Inthepresentstudy,weexam-inedintracellularlocalizationof2-NBDGinmammaliantumorcellsbyusingaconfocalmicroscopyincombinationwithvariousmarkersfororganelles.Thesedatamaybeusefulasabasis forunderstandingphosphorylation,degradation,andextinctionofthefluorescentprobe.NoCOI.

3P-149Regulation of Helix Repeat protein function by S100 proteins -Regulatory mechanism of tumor cell growth by beta-catenin-Yamaguchi, Fuminori; Hirata, Yuko; Hossain, Akram; Kamitori, Kazuyo; Dong, Youyi; Noguchi, Chisato; Katagi, Ayako; Tokuda, Masaaki(Department of Cell Physiology, Faculty of Medicine, Kagawa University, Kagawa, Japan)

S100proteinsaresmallcalciumbindingproteinswith“Helix-loop-He-lix”structure.Inhuman,morethan25differentS100proteinshavebeenreportedandtheyshoweddifferenttargetsandphysiologicalfunctions.HelixRepeatproteinscontainrepeatedhelixstructuresandincludeHEAT,Armadillo,TPR(Tetratricopeptiderepeat),andAnkyrinrepeatfamilies.WehavepreviouslyreportedthatS100proteinsreg-ulatetheactivityofproteinphosphatase5thatcontainsTPRrepeat.Here,wereportthatS100proteinsregulatethefunctionofbeta-cat-eninwhichcontainstheArmadillorepeat. Inthenormalcells, theamountofbeta-cateniniskeptlowbythedestructioncomplexinthecytoplasm.However,thegeneticmutationsdysregulatethefunctionofdestructioncomplexand increased levelofbeta-cateninproteinenhancesthetranscriptionofcellgrowthrelatedgenesresultedintheunlimitedtumorcellgrowth.WefoundthatS100A2andS100A6boundtobeta-cateninandinhibitedtheinteractionbetweenbeta-cateninandBCL9.S100A6decreasedthetranscriptionalactivityofbeta-catenin-TCFcomplexandinhibitedthetumorcellgrowth.OurfindingssuggestthatS100proteinscouldregulatethebeta-cateninfunctionbyinhibit-ingthetranscriptionalactivityofbeta-catenin-TCFcomplexthroughBCL9.NoCOI.

3P-150Regulation analysis of thioredoxin interacting protein (TXNIP) for the development of new cancer therapiesKamitori, Kazuyo1; Yamaguchi, Fuminori1; Dong, Youyi1; Hossain, Akram1; Hirata, Yuko1; Katagi, Ayako1; Noguchi, Chisato1; Hisaoka, Masahiro2; Tokuda, Masaaki1 (1Department of Cell Physiology, Faculty of Medicine, Kagawa University, Kagawa, Japan, 2Faculty of Medicine, Yamaguchi University, Yamaguchi, Japan)

Thioredoxin interactingprotein (TXNIP) isananti-tumorproteindown-regulated incancercells.MolecularanalysisconcerningtheregulationofTXNIPamountcouldleadtodevelopnovelcancerther-apies.WehavereportedthatamonosaccharideD-allosesignificantlyup-regulatestheTXNIPexpression,resultingintheinhibitionofcellcycleprogressioninvariouscancercelllines.HereweelucidatedthemechanismsregulatingTXNIPamount inhepatocellularcarcinomacelllineHuH-7.WeanalyzedsignalingmechanismsofTXNIPup-regulationcausedbyD-allose.D-allosetransientlyactivatedp44/p42MAPKpathway.Inhibitionofp44/p42MAPKphosphorylationbyPD98059reducedexpressionofTXNIP.D-allosealsoactivatedp38MAPK,andinhibitionofp38MAPKbyLY2228820reducedtheexpressionlevelofTXNIP.These results suggest that both p44/p42 MAPK pathway andp38MAPKpathwayparticipateintheTXNIPup-regulationcausedbyD-allose.WealsoexaminedmechanismsofTXNIPdecreasecausedbyserumstimulation.Upontheadditionoffetalcalfserum,TXNIPrapidlyde-creases.Aproteasomeinhibitor lactacystin inhibitedthisdecrease.ThisresultsuggeststhatTXNIPisdegradedthroughtheubiquitin-pro-teasomepathwayupontheserumstimulation.FurthermolecularanalysisandinvivoadministrationofD-allosewouldleadustohavebetterunderstandingofthenewcancertherapiesusingD-allose.NoCOI.

3P-151PPARα modulation of Ca2+-regulated exocytosis in guinea pig antral mucous cells: activation of NOS1 via PI3K/Akt pathway.Mantoku, Daiki1; Tanaka, Saori1; Matsumura, Hitoshi1; Nakahari, Takashi2(1Laboratory of Pharmacotherapy, Osaka University of Pharmaceutical Sciences, Takatsuki, Japan, 2Department of Physiology, Osaka Medical College, Takatsuki, Japan)

Inantralmucouscells,theCa2+-regulatedexocytosisisactivatedbyacetylcholine(1µM)consistedofaninitialtransientincrease(initialphase)followedbyasecondslowerdecline(latephase).OurpreviousreportdemonstratedthatanautocrinemechanismviaPPARαstimu-latesNOS1andenhancestheinitialphaseofCa2+-regulatedexocytosismediatedviaNO/cGMPaccumulation. Inhibitionof thisautocrinemechanismbyinhibitorsofPKG,NOS1orPPARαabolishedtheen-hancementofinitialphaseandinducedatransientincreaseinthelatephase.However,atpresent,wedonotknowhowPPARαactivatesNOS1 inantralmucouscells.Ontheotherhand,PPARsactivatesphosphatidylinositol3kinase(PI3K)/Aktpathwayinvascularendo-thelialcells.WeexaminedtheeffectsofaninhibitorofPI3K(50nMWortmannin)andaninhibitorofAkt(100nMAkt2/2kinaseinhibitor)ontheCa2+-regulatedexocytosisinantralmucouscells.BothinhibitorsabolishedtheenhancementoftheinitialphasestimulatedbyaPPARαagonist(50nMGW7647)buttransientlyincreasedthelatephase.TheseobservationssuggestthatPPARαstimulatesNOS1mediatedviaPI3K/Aktpathwayleadingtoanenhancementofmucinreleaseinantralmucouscells.NoCOI.


3P-152Ca2+-regulated exocytosis enhanced by AA/PPARα autocrine mechanism in guinea pig antral mucous cellsTanaka, Saori1; Shimamo, Chikao1; Matsumura, Hitoshi1; Nakahari, Takashi2(1Laboratory of Pharmacotherapy, Osaka University of Pharmaceutical Sciences, Takatsuki, Japan, 2Department of Physiology, Osaka Medical College, Takatsuki, Japan)

Inantralmucouscells,acetylcholine(ACh,1µM)activatesCa2+-regu-latedexocytosis,consistingofaninitialpeakthatdeclinerapidly(initialphase)followedbyasecondslowerdecline(latephase)lastingduringAChstimulation.WehavereportedthattheinitialphaseofCa2+-reg-ulatedexocytosisismaintainedbyanautocrinemechanismviaAA/PPARαinantralmucouscells.However,itstillremainsuncertainhowPPARαenhancesCa2+-regulatedexocytosisinantralmucouscells.TheaimofthisstudyistoclarifythemechanismfollowingthePPARαactivationinantralmucouscells.GW7647(aPPARαagonist)enhancedthefrequencyoftheinitialphasestimulatedbyACh.GW6471(aPPARαblocker)abolishedtheenhancementofthe initialphase inducedbyGW7647,butproducedadelayedandtransientincreaseinthefrequen-cyofthelatephase.Moreover,aPKGinhibitororaNOS1inhibitorabolishedtheenhancementoftheinitialphaseandinducedadelayedandtransientincreaseinthefrequencyofthelatephase,similarlytoGW6471.Inantralmucosae,AChorGW7647increasedNOproductionandcGMPcontents.AnalysisofWesternblottingandimmunohisto-chemistrydemonstratedthatNOS1existsinantralmucouscells.Thus,theAA/PPARαautocrinemechanismstimulatesNOproductionviaNOS1andcGMPaccumulation,whichenhancestheinitialphaseofCa2+-regulatedexocytosisinantralmucouscells.NoCOI.

3P-153Involvement of MARCKS phosphorylation by PKCδ in exocytotic amylase secretion in rat pancreatic acinar cellsSatoh, Keitaro1; Narita, Takanori2; Sugiya, Hiroshi2; Seo, Yoshiteru1(1Dept. Regul. Physiol., Dokkyo Med. Univ. Sch. Med., Mibu, Japan, 2Lab. Vet. Biochem., Nihon Univ. Coll. Bioresource Sci., Fujisawa, Japan)

Inpancreaticacinarcells,stimulationofcholecystokinin(CCK)induc-esexocytoticamylasesecretion.Intheamylasesecretion,activationofproteinkinaseC(PKC)isthoughttobeanessentialstep.However,regulationofamylasesecretionviatheactivationofPKCisunclear.Myristoylatedalanine-richCkinasesubstrate(MARCKS)isknownasamajorcellularsubstrateforPKC.MARCKShasbeenimplicatedinvariouscellularfunctionssuchasmotility,phagocytosis,mitogenesis,andmembranetrafficking.TheMARCKSphosphorylationbyPKCδhasbeenreportedinhumanpancreaticcarcinoidtumorcells.Here,we investigatedthe involvementofMARCKSphosphorylationbyPKCδinratpancreaticamylasesecretion.MARCKS,phosphorylat-ed-MARCKSandPKCδintheacinarcellsweredetectedbyWesternblotting.TranslocationofMARCKSintheaciniwasobservedbyim-munohistochemistry.Amylaseactivity inthemediumandthecelllysatewasmeasuredbyBernfeld’smethod.MARCKSproteinwasdetectedinratpancreaticacinarcells.CCK(100pM)inducedMARCKSphosphorylation.CCKhadnoeffectonthetotalamountofMARCKS.CCKinducedPKCδactivation.APKCδinhibitor,rottlerin,inhibitedtheCCK-inducedMARCKSphosphorylationandamylasesecretion.MARCKS-relatedpeptideastheMARCKSinhibitor inhibitedtheCCK-inducedamylasesecretion.Thesefindingssuggest that theMARCKSphosphorylationbyPKCδis involved inthepancreaticamylasesecretion.NoCOI.

3P-154CAFFEINE INHIBITS FLUID SECRETION IN INTER-LOBULAR DUCTS ISOLATED FROM GUINEA-PIG PANCREASMochimaru, Yuka; Yamamoto, Akiko; Nakakuki, Miyuki; Yamaguchi, Makoto; Kondo, Shiho; Taniguchi, Itsuka; Ishiguro, Hiroshi(Laboratory of Human Nutrition, Nagoya University Graduate School of Medicine)

HighconcentrationsofethanolandbileacidsinducesustainedelevationofintracellularCa2+concentration([Ca2+]i)inpancreaticacinarandductcells,whichisthoughttoberelatedtothepathogenesisofacutepan-creatitis.Previousstudiesreportedthatcaffeineinhibitedethanol-orbileacid-induced [Ca2+]ielevation inpancreaticacinarcells. Inthepresentstudyweexaminedtheeffectsofcaffeineonfluidsecretionand[Ca2+]iinductcells.Interlobularductswereisolatedbymicrodis-section.During3hourculturebothendsoftheductssealedsponta-neouslythus isolatingthe luminalspacefromthebath.ThesealedductsweresuperfusedwiththestandardHCO3

--bufferedsolutionandtherateoffluidsecretionwascalculatedfromtheincrementintheluminalvolumeandexpressedassecretoryrateperunitareaofepi-thelium(nlmin-1mm-2).[Ca2+]iwasmeasuredbymicrofluorometryinductsloadedwithfura2.Caffeine(2mM)reversiblyinhibitedsecretin(1nM)-stimulatedfluidsecretionfrom1.27±0.04to0.59±0.06,respec-tively.Caffeine(2mM)reversiblyinhibitedacetylcholine(10µM)-stim-ulatedfluidsecretionfrom0.71±0.07to0.28±0.07(p<0.05),respec-tively.Caffeine(2mM)reducedthelevelof[Ca2+]iinsecretin-stimulatedducts.Caffeine(2mM)reversiblyinhibitedacetylcholine-inducedele-vationof[Ca2+]i.Insummary,caffeineinhibitedsecretin-andacetylcho-line-stimulatedfluidsecretioninpancreaticductcells,whichwasasso-ciatedwithdecreaseof[Ca2+]i.NoCOI.

3P-155Elastic fiber formation of ductus arteriosus in Brown-Norway rat has a unique character trait Akaike, Toru1; Hsieh, Yi-Ting2; Liu, Norika Mengchia2; Ohmori, Eriko2; Yokota, Tomohiro2; Kajimura, Ichige1; Minamisawa, Susumu1(1Dept. of Cell Physiol., The Jikei Univ. Sch. of Med., Tokyo, Japan, 2Dept. of Life Sci. and Med. Biosci., Waseda Univ., Tokyo, Japan)

Patentductusarteriosus(PDA)isacommoncongenitalcardiovasculardiseaseinnewborn.Itspathogenesisisstronglyconcernedinstruc-turalremodelingdisordersofDAconstitution like internalelasticlaminae(IEL),extracellularmatrix,andsmoothmusclecells.Brown-Nor-way(BN)inbredrathaswithahighincidenceofPDAcomparedtootherlaboratoryratstrains.BNratdevelopsseveralelastin-relatedarterialimpairmentssuchasrupturesoftheIELinabdominalaortaandaorticelastindeficit inadult.ThereforeBNrathassystemicelastin-relatedarterial impairmentsthatcancausePDA.However,molecularmechanismsintheDAofBNrathavenotbeensufficientlycleared.Wethenaimedtoidentifythemechanisms.First,wefoundthattheDAinfourneonatesofsixBNneonatesstillremainedopenalthoughthatinallF344neonatesclosedwithinonehourafterbirth.Next immunostainingrevealedthat intimalcushion formationwaspoor,andmigrationofsmoothmusclecellsfromthemediaintosub-endothelialspacewasobscureintheDAoftheBNneonates.TheIELwasthickerandtheelasticlamellaeinthemediawerescarcerthanthoseoftheDAintheF344neonates,suggestingthattheirregularelasticfiberformationwasacommonfeatureoftheDAintheBNratstrain. Inconclusion, the irregularityofelasticfiber formation isauniquevascularremodelingthatmaycontributetothecauseofPDAinBNratstrain.NoCOI.


3P-156Vidarabine, a selective cardiac adenylyl cyclase in-hibitor, prevents ventricular arrhythmias in Calseques-trin 2 knockout miceSuita, Kenji1; Suita, Kenji1; Fujita, Takayuki1; Jin, Hui-Lin1; Cai, Wenqian1; Okumura, Satoshi2; Ishikawa, Yoshihiro1(1Cardiovas. Res. Inst., Yokohama City Univ., Yokohama, Japan, 2Dept. Physiol., Tsurumi Univ. Sch. of Dent. Med, Yokohama, Japan)

Thebeta-adrenergicreceptor/adenylylcyclase(AC)signalingiscrit-icalforcatecholamine-inducedphysiologicalresponsesofcardiomyo-cytes.Previously,wefoundthatVidarabine,ananti-herpesvirusagent,isoneoftheselectiveinhibitorsofcardiacACsubtype.Cardiacisoformofcalsequestrin,calsequestrin2 (Casq2),playsan importantrole inCa2+handling.Casq2gene-knockout(Casq2KO)mousehasbeenknownasamodelofcatecholaminergicventriculararrhythmias.First,weassessedtheantiarrhythmiceffectofVidarabine inCasq2KOmice.Intraperitoneal(ip)injectionofisoproterenol(ISO,1.5mg/kg)inducedprematureventricularcontractions(PVCs)inCasq2KOmice.Vidara-binereducedthenumberofPVCoccurredduring20minutesafterISOchallenge (56%reductionat60mg/kg ip,P<0.01). Inaddition,norepinephrine-inducedphosphorylationofRyanodinereceptor2(RyR2),whichisrelatedtotheleakyRyR2,wasbluntedbyVidarabineinventriclesofwildtype(WT)mice(18%reduction,P<0.05).Consis-tently, inventricularmyocytes isolatedfromWTmice,VidarabineattenuatedISO-inducedCa2+leakandspontaneousCa2+releasefromSR,whichhasbeenconsideredasanpotentialarrhythmogenictrigger(27%reduction,P<0.05and58%reduction,P<0.01).OurresultssuggestthatVidarabinesuppressthedevelopmentofcatecholamine-inducedventriculararrhythmiasviapreventionofabnormalCa2+releasefromSR.NoCOI.

3P-157Mitochondria-endoplasmic/sarcoplasmic reticulum Ca crosstalk and cellular functionTakeuchi, Ayako1; Kim, Bongju2; Matsuoka, Satoshi1(1Integr. Physiol. Fac. Med. Sci. Univ. Fukui, 2Inst. Genome Res. Univ. Tokushima)

MitochondrialCaplaysimportantrolesinregulatingmitochondrialaswellascellularfunctionssuchasenergymetabolismandapoptosis.ThedynamicchangeofcytoplasmicCaalsodependsonmitochondri-alfunction,whichinturnregulatescellularfunction.Werecentlyre-portedthatNCLX(slc24a6) functionsasamitochondrialNa-Caex-changerinBlymphocytesandcardiomyocytes.Inbothtypesofcells,NCLXprovidesCafrommitochondriatoendoplasmic/sarcoplasmicreticulum(ER/SR), therebymodulatingthecellularCaresponsetoantigenreceptorstimulationinBlymphocytesandtheautomaticityincardiaccelllineHL-1(Kimetal.,J Physiol,2012;Takeuchietal.,Sci Rep,2013).WeproposedthatCaextrudedfrommitochondriaviaNCLXaccumulatesinanarrowmitochondria-ER/SRsubspace,fromwhichER/SRCapump(SERCA)efficientlytakesupCa.Inthepresentstudy,wefurtherexaminedthishypothesisbyperform-ingsimulationanalysesusingcomprehensivecellmodelsofBlympho-cytesandcardiacpacemakersinoatrial(SA)nodecells,bynewlyin-corporatingmitochondria-ER/SRsubspaceCadynamics.Itwasshownthatthelargerthefractionofmitochondriafacingmitochondria-ER/SRsubspace,thelargertheER/SRCacontentbecomes.Contributionofmitochondria-ER/SRsubspaceonthecellular functionswillbediscussedindetail.NoCOI.

3P-158Involvement of K+ channel activity in the cytotoxicity of TNF-α in cultured human proximal tubule cellsNakamura, Kazuyoshi; Komagiri, You; Kubokawa, Manabu (Department of Physiology, Iwate Medical University School of Medicine, Yahaba, Japan)

TNF-αisknowntocausecellinjuryinvariousorgans,includingthekidney.SincetherearesomereportssuggestingthatchangesinK+channelactivityplayedrolesintherenaltubularcellinjuryinducedbyendotoxemiaorischemia,itispossiblethatthecytotoxiceffectsofTNF-αwouldpartlybemediatedbyitsactiononK+channelsinrenaltubularepithelia.However,littleinformationisavailable,regardingtherelationshipbetweentheeffectsofTNF-αonK+channelactivityandcellviability.Inthisstudy,weinvestigatedtheeffectsofTNF-αonK+channelactivityandcellviabilityinculturedhumanproximaltubulecells(RPTECs),usingthepatch-clamptechniqueandfluorescentim-aging.Incell-attachedpatches,TNF-α(20ng/ml)acutelystimulatedtheactivityofaninwardlyrectifyingK+channel,whichwasabolishedbyasolubleTNFreceptoranalog,etanercept(10µg/ml).Furthermore,aMAPKinhibitor,U0126(20µM),blockedthestimulatoryeffectofTNF-α.ThecytotoxicityofTNF-αwasexaminedbyusingtwofluo-rescentdyes,calceinandpropidiumiodide(PI).WhenthecellsweretreatedwithTNF-αfor24h,PI-staineddeadcellsincreased,whichwaspartlyblockedbytheconcomitantpresenceofU0126oraK+channelinhibitor,tertiapin(1µM).TheseresultssuggestedthatTNF-αstimulatedtheactivityofaninwardlyrectifyingK+channelinRPTECsthroughitsbidingtothespecificreceptorandactivationofMAPK,andthatthestimulationofK+channelwouldpartlybeinvolvedinthecytotoxicityofthiscytokine.NoCOI.

3P-159Enhancement of ciliary beating by Carbocystein (CCys) in distal airway ciliary cells of mice.Ikeuchi, Yukiko1; Tanaka, Saori1; Matsumura, Hitoshi1; Nakahari, Takashi2(1Laboratory of Pharmacotherapy, Osaka University of Pharmaceutical Sciences, Takatsuki, Japan, 2Department of Physiology, Osaka Medical College, Takatsuki, Japan)

TheeffectsofCCys(amucolytic)onthebendangle(CBA)andthefrequency(CBA)ofciliarybeatingwereexaminedinairwayciliarycellsisolatedfrommouselungs.OurpreviousstudyrevealedthatanincreaseinintracellularpH(pHi)increasesCBFandCBA,andade-crease in intracellularCl-concentration ([Cl-]i) increasesCBA.CCys(100µM)increasedCBAby30%,whileitincreasedCBFby5%inthepresenceofHCO3

-.ThepresentstudydemonstratedthatCCysdecreases[Cl-]iviaactivationofCFTRresultinginanincreaseinCBA.Ontheotherhand,weinhibitedtheNa+/HCO3

-cotransport(NBC),theCl-/HCO3

-exchange(AE)orbothusinganNa+-freesolution,aCl--freesolutionorDIDS.TheresultssuggestthatCCysactivatesNBCandAEleadingtoanincreaseinHCO3

-(anincreaseinpHi).ThismaycauseasmallincreaseinCBF(5%).Inconclusion,CCysincreasesCBAby30%viaadecreasein[Cl-]iandCBFby5%viaasmallincreaseinpHi.NoCOI.


3P-160Modulation of ciliary beat frequency by PDE1 during procaterol stimulation in distal airway ciliary cells of mice.Kogiso, Haruka1; Tanaka, Saori1; Shimamoto, Chikao1; Nakahari, Takashi2(1Laboratory of Pharmacotherapy, Osaka University of Pharmaceutical Sciences, Takatsuki, Japan, 2Department of Physiology, Osaka Medical College, Takatsuki, Japan)

Weexaminedtheeffectsofprocaterol(Proc,anβ-agonist)onciliarybendangle(CBA)andciliarybeatfrequency(CBF)inairwayciliarycells.ProcinitiallystimulatesanincreaseinCBAandthenCBFviacAMPaccumulation.Thus,uponstimulatingciliarycellswithProc,aCBFincreasewasdelayed.However,IBMXincreasedbothCBAandCBFinasimilartimecourse.Ontheotherhand,adecreasein[Ca2+]istimulatedan increaseCBF,whereasan increase in [Ca2+]ididnot.Moreover,intheexperimentsofW-7(acalmodulininhibitor),there-coveriesofciliarybeatingafterremovingW-7weremarkedlydelayedinPKI-treatedcells,comparedwithnon-treatedcells.Theseobserva-tionssuggestthattheconcentrationofcAMPinthemicrodomainregulatingCBF(outerdyneinarm,ODA)iscontrolledbyaCa2+-de-pendentphosphodiesterase(PDE1).AselectivePDE1inhibitor(8MmIB-MX)increasedCBF,notCBA,andthenafurtherstimulationwithProcsimilarly increasedbothCBAandCBF.An immunohistochemicalexaminationusingelectronmicroscopydemonstratedthatPDE1existsinthemicrodomainneartheODAsinthecilia.Inconclusion,PDE1modulatesCBFbyregulatingODAsinairwayciliarycells.NoCOI.

3P-1612-photon FLIM imaging of SNARE assembly in endo-crine and neuron cells.Takahashi, Noriko; Sawada, Wakako; Watanabe, Satoshi; Ohno, Mitsuyo; Kasai, Haruo(Sturctural Physiology, CDBIM, Graduate School of Medicine,The University of Tokyo, Tokyo, Japan)

WehaveinvestigatedtheSNAREconfigurationsintheplasmamem-branesofbetacellsinthepancreaticisletsandpresynapticterminalsofcorticalneurons,usingtwo-photonfluorescence lifetime imaging(FLIM)ofForsterresonanceenergytransfer(FRET).WeconstructedtheFRETprobesofSNAREs,VAMP2andsyntaxin1andSNAP25,bylabelingwithTurquoise(donor)orVenus(acceptor).Inpancreaticbetacells,mostSNAREswereunassembledintheplasmamembranes,exceptforasmallfraction(6%)ofSNAP25formingabinarycomplexwithsyntaxin.Incontrast,asignificantfractionofSNAP25andsyn-taxinformedtheternarytrans-SNAREcomplexesintheboutons,andwasreversiblyconvertedintocis-formuponstimulation.Furthermore,theactivezonehadapropensitytoassembletrans-SNAREcomplexeventhougheitherSNAP25orsyntaxinwereinactivatedbybotulinumtoxins,andthatphorbol-esterfacilitatedformationofthetrans-SNAREcomplexes.WealsofoundtheoligomerizationofSNAP25atsynapticterminals inthedomain-swappedconfiguration.Thus, theSNAREconfigurationsarediverseandregulatedtoallowtheultrafastexocy-tosisintheactivezone,whileslowexocytosisintheisletbetacells.NoCOI.

3P-162Measurement the history of the neural activity with AIME-MRIKikuta, Satomi1; Yanagawa, Yuchio2; Homma, Noriyasu1; Osanai, Makoto1(1Tohoku Univ. Grad. Sch. Med., Sendai, Japan, 2Gunma Univ. Grad. Sch. Med., Maebashi, Japan)

Theactivation-inducedmanganese-enhancedMRI(AIME-MRI)isoneofthecandidatemethodsformeasuringtheneuronalactivityinvivo.Thismethodhasthepotentialtomeasurethehistoryoftheneuronalactivity,sincetheMn2+canentertheactiveneuronthroughvoltage-de-pendentCa2+channels.However,ithasnotbeenclearthattherelationbetweentheneuronalactivityandtheaccumulationofMn2+inthecell.Therefore,weevaluatetheabilityofAIME-MRIformeasurementoftheneuronalactivity.Atfirst,weconfirmedthatthelongitudinalrelaxationtimeofH+(T1)measuredbymeansofMRIapparatuswasrelatedtothe [Mn2+].The inverseofT1 (1/T1)wasproportionalto[Mn2+],thus[Mn2+]canbemeasuredbyMRIquantitatively.Next,weinvestigatedtherelationbetweentheneuronalactivityandtheaccu-mulationofMn2+inthestriatalGABAergicneurons.TheamountoftheCa2+influxwascorrelatedtotheneuronalactivityevokedbyte-tanicstimulationswithvariousfrequencies.Inthesamesliceprepa-ration,theamountsofthefluorescencequenchinducedbythetetan-icstimulationsundertheconditionof50µMMnCl2administrationwererecorded.Asaresult,theamountofthequenchwasproportion-altotheamountoftheCa2+influx.Theseresultssuggestedthatacell,whichhashigheractivity,accumulatedlargeramountofMn2+intheneuron.Thus,ourresultssupportedthatAIME-MRIcanmeasurethehistoryoftheneuronalactivitiesinthewholebrainnoninvasivelyinvivo.Inadditionwehaveattemptedthismethodforneuraldiseasemousemodel.NoCOI.

3P-163Effects of thyroid hormone on microglial functionNoda, Mami1; Mori, Yuki1; Kalashnikova, Anastasia2; Churilov, Leonid P2(Laboratory of Pathophysiology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan, 2Department of Pathophysiology, School of Medicine, St. Petersburg State Medical University)

L-tri-iodothyronine (3,3’,5-triiodothyronine;T3) isanactive formofthyroidhormone(TH)essentialfordevelopmentandfunctionofthecentralnervoussystem(CNS).Clearanceofdebrisandapoptoticcellsbymigratoryresponseandphagocytosisofmicrogliaisacriticalstepforrestorationofdamagedneuralnetwork.HerewereporteffectsofT3onmicroglial functionstudiedwithtime-lapsevideomicroscopy,48-wellmicrochemotaxisBoydenchamberandimmunocytochemistrystainingusingprimaryculturedmurinemicroglia.ExposuretoT3increasedmicroglialmotility,chemotaxisandmembraneruffling.TheT3-inducedmicroglialmigrationwasinhibitedbyantagonistsofphos-phoinositide3-kinase(PI3K),mitogen-activatedprotein(MAP)/extra-cellularsignal-regulatedkinase (ERK),Na+/K+-ATPase,Gi/o-protein,andbyinhibitorsofGABAAandGABABreceptors.InhibitionofTHtransportersandreceptors (TRs)suppressedT3-inducedmicroglialmigrationandmorphologicalremodelling.Moreover,controlandT3-in-ducedmigrationofmicrogliaisolatedfromwild-typemicewasgreat-erthanthatofTRα-knock-outmice.In vivostabwoundmodelshowedthatattractionofmicrogliatothesiteoflesionwasalsoaffectedbyT3.TheseresultsdemonstratethatT3regulatesmultiplefunctionalresponsesofmicroglia.NoCOI.


3P-164High Extracellular Ca2+ Enhances the Adipocyte Dif-ferentiation of Bone Marrow Stromal Cells Through Suppression of ERK ActivityMiyamoto, Yuki1; Hashimoto, Ryota1; Katoh, Youichi2; Okada, Takao1(1Dept. of Physiology, Juntendo Univ. Med., 2Dept. of Cardiology, Juntendo Univ. Med. Urayasu Hosp.)

Mesenchymalstemcellsfoundinbonemarrowstromalcells(BMSCs)arethecommonprogenitorsforbothadipocyteandosteoblast.Wehaverecentlyreportedthattreatmentwithinsulinanddexamethasone,high [Ca2+]oenhancedadipocytebutnotosteoblastaccumulation inBMSCs,suggestingthatincreasesin[Ca2+]ocausedbyboneresorptionmightaccelerateadipocyteaccumulationinaginganddiabeticpatients.Inthisstudy,weusedprimarymouseBMSCsto investigate themechanismbywhichhigh[Ca2+]oenhanceadipocyteaccumulation.10.8mMof[Ca2+]opartiallysuppressedthephosphorylationofERKacti-vatedbyPMA(phospho-ERKpositivecells;50%vs.65%comparingto1.8mMof[Ca2+]ogroup).WenextdeterminedwhetherinhibitionofERKbyinhibitorsforMEK(U0126andPD0325901)altertheexpressionlevelsofadipocytemarkers.TreatmentwithinsulinanddexamethasoneforBMSCssignificantlyincreasedthemRNAlevelsofC/EBPα(1.8times)andPPARγ(1.3times),whichareimportantadipogenictran-scriptionfactors.5µMofU0126enhancedthemRNAexpressionofC/EBPα(2.5times)andPPARγ(2.3times).Similareffectswereobservedin20nMofPD0325901-treatedgroup.ThesedatasuggestthathighextracellularCa2+enhancestheadipocytedifferentiationofBMSCsthroughsuppressionofERKactivity.Increased[Ca2+]o-suppressionofERKsignalingmaybeanewtargetfortherapyforanemiaandfrac-turescausedbyacceleratedmarrowadipocyteaccumulationinaginganddiabeticpatients.NoCOI.

3P-165High Extracellular Ca2+ Enhances the Adipocyte ac-cumulation of Bone Marrow Stromal Cells Through a decrease in cAMPHashimoto, Ryota1; Katoh, Youichi2; Iesaki, Takafumi1; Okada, Takao1(1Dept. of Physiology, Juntendo Univ. Med., 2Dept. of Cardiology, Juntendo Univ. Med. Urayasu Hosp.)

[Background]Mesenchymalstemcellsfoundinbonemarrowstromalcells (BMSCs)arethecommonprogenitors forbothadipocyteandosteoblast.Wehaverecentlysuggestedthatincreasesin[Ca2+]ocausedbyboneresorptionmightaccelerateadipocyteaccumulationinath-erogeniccondition.Inthisstudy,weinvestigatedthemechanismbywhichhigh [Ca2+]oenhanceadipocyteaccumulation. [Methods and Results]WeusedprimarymouseBMSCsandevaluatedthelevelofadipocyteaccumulationbymeasuringOilRedOstaining.Highcon-centration(10.8mM)of[Ca2+]oenhancedtheaccumulationofadipocytesinBMSCsbyabouttwotimesunderthetreatmentofbothinsulinanddexamethasone(atherogeniccondition),whereashigh[Ca2+]oalonedidnot induceadipocyteaccumulation.UsingtheELISAmethod,weshowedthathigh [Ca2+]odecreasedtheconcentrationofcAMPbyabout20%inBMSCs. Increasingthe intracellularconcentrationofcAMP(1µMforskolinand1µMdb-cAMP)hamperedtheenhancementinadipocyteaccumulationbyabout40–50%underhigh [Ca2+]o inBMSCs.Ontheotherhand,thishamperedeffectwasnotobservedinBMSCsculturedinbasalconcentration(1.8mM)of[Ca2+]o.Usingre-al-timeRT-PCR,weshowedforskolinanddb-cAMPsuppressedtheexpressionofimportantadipogenictranscriptionfactors(PPARγandC/EBPα)underhigh[Ca2+]oinBMSCs.[Conclusions]Thesedatasuggestthat increases in [Ca2+]omightaccelerateadipocyteaccumulationthroughadecreaseincAMPinatherogeniccondition.NoCOI.

3P-166RANKL-induced osteoclast differentiation on RAW264.7 cells is suppressed by the direct effect of IL-17AInoue, Hiroshi; Uchihashi, Kenji; Nishikawa, Yasuo(Department of physiology, Osaka Dental University, Osaka, Japan)

Boneiscontinuouslyremodeledbyboneformationandresorption,andthecooperativebonemetabolismistightlyregulatedtomaintainho-meostasis.But,boneresorptionmechanismhasnotbeenfullyeluci-dated.IL-17AisaproinflammatorycytokinethatismainlysecretedbyactivatedTcells.IL-17Aplaysanimportantroleinmanyautoim-muneandinflammatorydiseases.IL-17Asensitizesosteoclastprecur-sorstothekeyosteoclastfactorRANKLbyincreasingRANKexpres-siononosteoclastprecursors.However,thedirecteffectsofIL-17Aonthedifferentiationofosteoclastprecursorsintoosteoclastshavenotbeenclarified.Therefore,westudiedtheroleofIL-17Ainthedirectdifferentiationandactivationofosteoclastprecursors.WetestedtheeffectofIL-17AontheproliferationandthedifferentiationofRAW264.7cellsandalsoontheexpressionofIL-17receptors.ExpressionofIL-17RCwasconfirmedonRAW264.7cellsbyFACSanalysis.IL-17Adidnot affect theproliferation, but suppressed thedifferentiation ofRAW264.7cellsinthepresenceofsolubleRANKLintoosteoclastinadose-dependentmanner.Phosphorylationofp38MAPkinasewasfurtherenhancedinthepresenceofRANKLcomparedwithcontrol,whichwasreducedbyIL-17Ainadose-dependentmanner.TheseresultssuggestthatthedirecteffectofIL-17AsuppressedRANKL-in-ducedosteoclastdifferentiation.Furthermore,itissuggestedthatin-hibitionofp38MAPkinasephosphorylationbyIL-17Amaybeoneofthefactorsthatsuppressthedifferentiationofosteoclastprecursorsintoosteoclasts.NoCOI.

3P-167Effects of 365 nm LED light on cell growth of cultured RAW264.7 cellsIkehara, Toshitaka1; Aihara, Mutsumi2; Akutagawa, Masatake3; Tsuchiya, Kohichiro2; Tkahashi, Akira2; Kinouchi, Yohsuke3(1Dept Welfare, Fac Health Welfare, Tokushima Bunri University, 2Inst Health Biosci, Univ Tokushima Grad Sch, Tokushima, Japan, 3Inst Socio Tchno Sci, Univ Tokushima Grad Sch, Tokushima, Japan)

WestudiedeffectsofultravioletA(UV-A)irradiationusinglight-emit-tingdiodeoncellgrowthofRAW264.7cells.Cellswereplatedon96wellplatesatadensityof105–106cells/ml.After24hr,thesecellswereirradiatedforvariedtime(0–5min)andmaintainedagainfor0–72hrat37°C.Cellsimagesweretakenbyamicroscope,andthecellnumberwascalculatedfromtheseimages.Irradiationfor2minsignificantlysuppressedcellgrowth,andadditionofN-acetylcysteine(NAC)re-coveredfromthecellgrowthinhibitionandremovedintra-andextra-cellularROS(reactiveoxygenspecies)inducedbytheirradiation.This2min-irradiationdidnotsostronglydecreasedglutathioneandgluta-thionereductaseactivity,anddidnotstumulatetheLDHreleaseintomediumandlipidperoxydation.Finally,todetectROSinducedintheculturemediumbyUV-Alight iradiation for2min,wemeasuredelectronparamagneticresonance(EPR)signalsinthepresenceofspintrappingagents (TPCandDMPO)byEPRspectrometer.NaN3de-creasedthespinpeaksformedbyTPCandhistidinedecreasethepeaksbyDMPO.Thesemeasurementsindicatethatsingletoxygen(1O2)isinitiallyinduced,and1O2reactsfirstwithDMPO,andthere-sultingDMPO-1O2 intermediate is immediatelydecomposedtogivehydroxylradical.TheseresultssuggestthattheseROSinduced incytoplasmorculturedmediuminhibitthecellgrowthofRAWcells.NoCOI.


3P-168Analysis of cold stress induced cell damage in HeLa cellMikai, Masataka; Araki, Miya; Yamada, Misato; Kobayashi, Daisuke; Hazama, Akihiro(Cellular Integrative Physiology, Graduate School of Medicine, Fukushima Medical University, Fukushima, Japan)

Acellwasdamagedbyexposuretoextracellular factorssuchasphysicalandchemical factors.Wefocusedonthecoldstressasanextracellularfactortocelldamage,andevaluatedcellviabilitiesofcoldstressexposedHeLacell.ControlHeLacellswereincubatedat37°C,5%CO2inDMEMwith10%FBSforovernight.HeLacellswereplacedunder4°C(coldtreatment)for1and3days.Bothcellswerestainedwithcalcein-AM(Cal)andpropidiumiodide(PI)andthecellviabilitywasanalyzedbyflowcytometry.Theviability(Calpositive/PInega-tive)of3-daycoldtreatmentcellwas10%,ontheotherhand,thatof1-daycellwas85%,whichwasthesameastheviabilityofcontrolcells.Flowcytometricviabilityof1-daycoldtreatmentdidnotdecrease,however,thecellproliferationwassuppressedbycoldtreatmentandthecellgrowthdidnotrecoveredafterthetemperaturesettingat37°Cagain.Weobservedtime-dependentcellswellingafter24hcoldtreatment.TheswollencellswerenotstainedwithacridineorangeandJC-1,whichwaslysosomeandmitochondriaindicator,respective-ly.Theswollencellalsodidnotstainedwithanti-lysosomalassociatedmembraneprotein-1.Lactosedehydrogenaseactivityof1-daycoldtreatmentextracellularmediumwaslow.Thereforethecellmembranewasnotdamagedduring1-daycoldtreatment.Theseresultsindicat-edthatcoldstresscelldamageoccurredonintracellularorganelle.NoCOI.

3P-169Mechanically induced ATP release in 3D culture of normal and cancerous mammary cell lines and effects of TGF-β1Takahashi, Yuko1,2,3; Furuya, Kishio2,4; Nakahari, Takashi3; Sokabe, Masahiro2,4(Dept Breast surgery, Osaka Medical College, 2Dept Physiol Nagoya Univ, Grad sch Med, 3Dept Physiol, Osaka Medical College, 4Mechanobiol lab, Nagoya Univ, Grad sch Med)

Cancercellsareaffectedbyvariousfactorsfromtheirmicroenviron-mentincludingmechano-signaling,suchasextracellularmatrixrigid-ity,aswellaschemo-signaling,suchasgrowthfactors.ATP,aubiq-uitouscell-cellsignalingmolecule,isusuallyreleasedbymechanicalstimulation,anditsreceptorsareexpressedevenincancercellsandaffectcellconditions.TGF-β1issecretedincancermicroenvironment,andrecentlyappearedtoinducecellmigrationandactinremodelingviaATPreleaseandactivationofitsreceptorsinhumanlungcancercells.Fromthesefacts,wepostulatedthatmechano-andchemo-sig-nalingincancermicroenvironmentinteractviaATPsignaling.Here,weusedhumanmammaryepithelialcell line (HuMEC)andbreastcancercellline(MDA-MB231)in3Dor2Dcollagen-gelculture,andmeasuredATPreleasebystretchandhypo-osmoticstimulationsusingtherealtimeATPbioluminescenceimagingsystem.ResultsshowedbothmechanicalstimuliinducedtransientandsignificantATPreleasefromseveralcellswithapeakconcentrationofnearly10µM.Itspreadtothesurroundingsandseemedtokeephigherconcentrationinthemicroenvironment.WithTGF-β1treatmentforseveraldays,cellshapeschangedremarkablytoelongatedfibrousmorphologyandATPre-sponse increasedboth insizeandfrequency.Cellmorphologyalsoseemedtochangeaftermechanicalstimuli.Webelievethesefindingssupportourhypothesis.NoCOI.

3P-170Analysis of the binding mode of AIF to the membraneYamashita, Tetsuo; Hashimoto, Takeshi; Igarashi, Junsuke; Kosaka, Hiroaki(Faculty of Medicine, Kagawa Universit, Kagawa, Japan)

Mitochondrialflavoprotein,apoptosis-inducingfactor(AIF),isknownasoneofthekeycaspase-independentdeatheffector.Inresponsetoseveralapoptoticstimuli,AIFisreleasedfrommitochondriaandtrans-locatetothenuclei.Instaticconditions,AIFisattachedtothemito-chondrialmembrane;uponstimulation,itmayneedtoundergopoly-peptidecleavage inorder toacquiresolubilityandpro-apoptoticproperties.Sofar, twoconflictingmechanismshavebeenproposedregardingtothemechanismswherebyAIFisboundtothemembranecomponents.TheoneisthatAIFisanintegralmembraneprotein,whereitsN-terminalregion(mouse,residues67–85)penetratesintotheinnermembrane.TheotheristhatAIFisaperipheralmembraneprotein.ToelucidateexactbindingmodeofAIFtothemembrane,wedevelopedrecombinantE. colistrainswhichoverexpresstwodistinctformsmouseAIF,mitochondria-typeAIF(delta1–53)orcleaved-typeAIF(delta1–102).Wefoundthatmorethan80%ofmitochondria-typeAIFwasassociatedtothemembranefractionswhentheionicstrength(I)ofpreparationbufferwaswithinphysiologicalrange(I=150mM).However,mostenzyme(95%)wasrecoveredwithinthesolublefrac-tionsathigherI(300mM).Itisnoteworthythatcleaved-typeAIFispartlyassociatedtothemembrane(about50%)atlowI,andwasalsodissociatedbyincreasesinI.TheseresultssuggestthattheN-terminalregion(residues67–85)ofAIFisnotpenetratedintothemembraneandisindirectlyinvolvedinthemembrane-binding,andthatbindingofAIFtothemembranearelargelydependentonionicbond.NoCOI.

3P-171UV-induced apoptosis is inhibited in LPS-activated BV-2Kaneko, Yoko S; Nakashima, Akira; Nagasaki, Hiroshi; Kodani, Yu; Ota, Akira(Department of Physiology, Fujita Health University School of Medicine, Toyoake, Japan)

Wepreviouslyreportedtheoptimaldoseoflipopolysaccharide(LPS)markedlyextendsthelifespanofmouseprimary-culturedmicrogliabysuppressingapoptoticandautophagiccelldeath.Inthepresentstudy,weinvestigatedtheeffectsofLPSonapoptosisbyultraviolet(UV)irradiationinmicroglialcellline,BV-2.Apoptosiswasobservedwithin1handmorethanhalfofBV-2wereinanapoptoticstateat3hafterUVirradiation.Procaspase-3wascleavedtobeactive-forminUV-irradiatedcells.Bycontrast,inBV-2treatedwithLPSfor24h,caspase-3wasnotcleavedbyUVirradiation.TreatmentwithLPSeffectivelyprotectedBV-2againstapoptosisinducedbyUVirradiation.LPStreatmentofBV-2increasedthep21Waf1/Cip1cyclin-dependentkinaseinhibitorprotein levelat6handgrowtharrestandDNAdamage(GADD)45αproteinlevelat24h.Becausep21andGADD45αarrestthecellcycleinG1orG2/Mstage,respectively,andregulateapopto-sisonstress, theproteinexpressionsofp21andGADD45αweresuppressedbysmallinterferingRNA.Whileneithercaspase-3cleavagenorapoptosiswasinducedineitherp21orGADD45αknockdownBV-2,caspase-3wasactivatedtoinduceapoptosisinbothp21andGAD-D45αdoubleknockdowncells.Thesedatasuggestthatp21andGAD-D45αcooperatetoregulateapoptosiscausedbyUVirradiation.Becausemicrogliaactivatedexcessivelymayplaycriticalrolesintheexacer-bationofneurodegeneration,theregulationofapoptosisinmicrogliacouldbeanewandpromisingstrategytoinhibitthedeteriorationofneurodegenerativedisease.NoCOI.


3P-172High oxygen condition facilitates the differentiation of human iPS cells into pancreatic progenitors and insu-lin-producing cellsHakim, Farzana; Kaitsuka, Taku; Jamiruddin, Mohd. Raeed; Wei, Fan-Yan; Tomizawa, Kazuhito(Department of Molecular Physiology, Kumamoto University, Kumamoto, Japan)

Pluripotentstemcellshavepotentialapplications inregenerativemedicinefordiabetes.Differentiationofstemcellsintoinsulin-produc-ingcellshasbeenachievedusingvariousprotocols.However,boththeefficiencyofthemethodandpotencyofdifferentiatedcellsareinsuf-ficient.Oxygentension,thepartialpressureofoxygen,hasbeenshowntoregulatetheembryonicdevelopmentofseveralorgans,includingpancreaticβ-cells. Inthisstudy,wetriedtoestablishaneffectivemethodforthedifferentiationofinducedpluripotentstemcells(iPSCs)intoinsulin-producingcellsbyculturingunderhighoxygen(O2)con-ditions.TreatmentwithahighO2conditionintheearlystageofdif-ferentiationincreasedinsulin-positivecellsattheterminusofdifferen-tiation.WefoundthatahighO2conditionrepressedNotch-dependentgeneHes1expressionandincreasedNgn3expressionatthestageofpancreaticprogenitors.Thiseffectwascausedbyinhibitionofhypox-ia-induciblefactor-1α(HIF-1α)proteinlevel.Moreover,ahighO2con-ditionactivatedWntsignaling.Optimalstage-specifictreatmentwithahighO2conditionresultedinasignificantincreaseininsulinproduc-tioninbothmouseembryonicstemcells(mESCs)andhumaniPSCs(hiPSCs),andyieldedpopulationscontainingupto50%C-peptide-pos-itivecellsinhiPSCs.TheseresultssuggestthatculturinginahighO2conditionataspecificstage isuseful fortheefficientgenerationofinsulin-producingcells.NoCOI.

3P-173Analysis of biphasic Ca2+ uptake by the endoplasmic reticulum during Ca2+ oscillations in mouse eggsKikuchi, Takashi; Shirakawa, Hideki(Department of Engineering Science, The University of Electro-Communications, Tokyo, Japan)

Inmammalianeggs,repetitiveincreasesincytosolicCa2+concentration([Ca2+]cyt),orCa2+oscillations,areinducedbyspermatozoa,andtriggeraseriesofeventsleadingtoeggactivation.EachCa2+transientintheoscillationsisduetoCa2+releasefromtheendoplasmicreticulum(ER)throughinositol1,4,5-trisphosphatereceptor/Ca2+channels.Forcom-prehensiveunderstandingofthemechanismofCa2+oscillations,there-fore,theinformationaboutCa2+concentrationintheERlumen([Ca2+]ER)isessential.Inthepresentstudy,wemeasuredthechangesin[Ca2+]ERduringCa2+oscillationsinmouseeggsinducedbyspermortheex-pressionofphospholipaseCζ,usingageneticallycodedCa2+probe,D1ER.Bysimultaneousmonitoringof[Ca2+]ERand[Ca2+]cytwithD1ERandfura-2,itwasrevealedthat,aftertherapiddecreasebyCa2+releaseateachCa2+transient,[Ca2+]ERincreasedwithabiphasictimecourse,consistingofanimmediateincreaseasfastasthedecreasein[Ca2+]cyt,followedbyaslowandgradualincreaseuntilthenextCa2+transientoccurred.Whereastherate intheslowphaseofrecoverywasnotinhibitedbythapsigargin, itwasdependentonextracellularCa2+concentration,indicatingthatitislimitedbytherateofCa2+influx,notbythatofCa2+uptakebyCa2+pumpsontheERmembrane.TheseresultsareconsistentwiththeideathattheintervalofCa2+oscillationsatfertilizationisdeterminedbythesupplyingrateofCa2+fromoutsidetheeggtorefillthedepletedCa2+stores.NoCOI.

3P-174Numerical simulation of fast and slow Ca2+ oscilla-tions in fertilized mouse eggsShirakawa, Hideki; Tan, Moutou(Department of Engineering Science, The University of Electro-Communications, Tokyo, Japan)

Manytypesofcellrespondtoexternalstimuliwithrepetitiveincreas-esinthecytoplasmicCa2+concentration([Ca2+]cyt),orCa2+oscillations.Inmouseeggs,changesin[Ca2+]cytinducedbythefusionwithspermshowcomplexpattern:slowoscillationsattheintervalof10–30min,eachofwhichconsistsoffastoscillationsattheintervalofseveraltensofseconds.Inthepresentstudy,weexaminedthefactorsthatdeter-minethetemporalcharacteristicsofCa2+oscillationsinmouseeggs,bynumericalsimulationbasedonthemodelbyDeYoungandKeiser(D-Kmodel).WiththeoriginalD-Kmodel,Ca2+oscillationsweregen-eratedbyincreasingtherateofIP3production,assumingthetranslo-cationofPLCξfromsperm.Although,bychangingparameter(s)rep-resenting thekinetics of inhibition of IP3 receptorsbyCa2+, thefrequencyoftheoscillationswasabletobeadjustedsimilartothatofeitherfastorslowoscillations,theoscillationscontainingbothfastandslowcomponentscouldbereproducedonlybyaddingthetermforCa2+influxfromoutsidetheeggtoD-Kmodel.SimulatedCa2+oscilla-tionswithsuchmodelagreedwellwithmeasuredresultsnotonlyforthechangesin[Ca2+]cytand[Ca2+]intheintracellularstores,butalsoforthedependenceofslowfrequencyontheextracellular[Ca2+].To-getherwiththefactthatthedependenceofPLCξactivityon[Ca2+]cythadessentiallynoeffectonthesimulationresults,itwassuggestedthatslowCa2+oscillationsinfertilizedeggsaredrivenbyCa2+influx,notbycharacteristicenzymeactivityofPLCξ.NoCOI.

3P-175Important role of cell membrane microdomain for Ca2+-sensitization of vascular smooth muscle contrac-tionKajiya, Katsuko1,2; Nojimoto, Kazutaka1; Kishi, Hiroko1; Takada, Yuichi1; Zhang, Ying1; Kobayashi, Sei1(1Yamaguchi University Graduate School of Medicine, Ube, Japan, 2Faculty of Agriculture, Kagoshima University, Kagoshima, Japan)

Therearetwotypesofvascularsmoothmuscle(VSM)contractions;1)Ca2+-dependentcontractionofVSMregulatesphysiologicalvasculartoneandmaintainsbloodpressure,2)Rho-kinase-mediatedCa2+-sensi-tizationofVSMcontractioncontributestovasospasm.Wepreviouslyidentifiedsphingosylphosphorylcholine(SPC)asanupstreammediatoroftheCa2+-sensitization.ThedegreesofSPC-inducedCa2+-sensitizationcorrelatedwellwithserumandVSMtissuecholesterol(Chol)levels.Furthermore,depletionofVSMCholdestroyedChol-enrichedmem-branemicrodomainssuchascaveolaeandlipidrafts,andabolishedtheSPC-inducedCa2+-sensitization.However,mechanismsbywhichSPCtransducestheCa2+-sensitizingsignalsexclusivelythroughmem-branemicrodomainsareunknown.Thus,weinvestigatedthepossiblerolesofcellmembranemicrodomainintheSPC-inducedCa2+-sensiti-zationofVSMcontraction.First,wemeasuredtheinteractionofSPCwithhumanVSMcellsusingthesurfaceplasmonresonance,provid-ingthefirstdirectevidenceforChol-dependenthighaffinityofD-eryth-ro-SPC(d-SPC)fortheVSMcellsascomparedwithL-threo-SPC(l-SPC)andothersphingolipids.Secondly,weexaminedtheinteractionofSPCwithmodelmembranesforastronglinkagebetweenCholandtheaffinityofSPCforthemembrane.TheseresultssupportthefirstdirectevidencethatVSMcellshaveveryhighaffinityford-SPC,butnotl-SPC,indicatinghighlystructuralspecificityofSPC.NoCOI.


3P-176Mechanisms of regulation of skeletal muscle prolifer-ation and differentiation Yamazawa, Toshiko1; Ohno, Tetsuo1; Ohkido, Makiko2; Yamamoto , Hiromasa1; Yamaguchi, Maki1; Takemori, Shigeru1 (1Dept Mol. Physiol., Jikei Univ. Sch. Med., Tokyo, Japan, 2Dept Mol. Biol., Jikei Univ. Sch. Med., Tokyo, Japan)

Thepolyaminesputrescine,spermidineandsperminearelowmolec-ularweightorganicpolycations,wellknownasmediatorsinvolvedincellhomeostasis.Theproposedrolesofpolyaminesarethefunctioningofionchannels,nucleicacidpackaging,signaltransduction,autophagy,DNAandproteinsynthesis,cellproliferation,anddifferentiation,aswellasregulationofgeneexpression.Althoughregulationofpolyam-inelevelsisassociatedwithmusclehypertrophyinskeletalmuscle,yettheunderlyingmechanismsofpolyaminearenotwelldefined.Here,westudiedhowpolyaminesmayaffecttheproliferationand/ordifferentiationofmurinemyoblastprogenitorC2C12cellline.Toeval-uatetheroleofpolyaminesintheproliferationprocess,wecountedthenumbersofmyoblastsevery24hours,butthepolyaminedidnothaveinfluenceformyoblastsproliferation.Ontheotherhand,duringinductionofmyogenicdifferentiation,uponpolyaminetreatmentofC2C12cellsthenumberofmyotubessignificantlyincreased.Morpho-logically,polyamine-treatedC2C12cellsexhibitelongatedcellbodyandbecomemulti-nucleatedmyotubes.Ultrastructuralanalysisundertransmissionelectronmicroscoperevealedthatpolyamine-treatedmulti-nucleatedmyotubesexhibitedanabundantpresenceofmyofil-aments.Therefore,ourstudydemonstratesthatpolyaminesmayplayanimportantroleinregulatingmyogenicdifferentiationratherthanmyoblastsproliferation.NoCOI.

3P-177Lowered extracellular pH is involved in the pathogen-esis of skeletal muscle insulin resistanceHayata, Hiroki1; Miyazaki, Hiroaki1,2; Niisato, Naomi1,2; Yokoyama, Noriko1; Marunaka, Yoshinori1,2(1Department of Molecular Cell Physiology, Kyoto Prefectural University of Medicine, Kyoto, Japan, 2Japan Institute for Food Education and Health, Heian Jogakuin (St. Agnes') University, Kyoto, Japan)

Insulinresistanceintheskeletalmuscleismanifestedbydiminishedinsulin-stimulatedglucoseuptakeandisacorefactorinthepathogen-esisoftype2diabetesmellitus,butthemechanismcausinginsulinresistanceisstillunknown.OurrecentstudyhasshownthatpHofinterstitialfluidwasloweredinearlydevelopmentalstageofinsulinresistanceinOLETFrats,knownasamodeloftype2diabetesmelli-tus.Therefore,inthepresentstudy,weconfirmedeffectsoftheex-tracellularpHontheinsulinsignalingpathwayinaratskeletalmus-cle-derivedcellline,L6cell.ThephosphorylationleveloftheinsulinreceptorwassignificantlydiminishedinlowpHmedia.Thephosphor-ylationlevelofAkt,whichisadownstreamtargetoftheinsulinsig-nalingpathway,wasalsodeclined in lowpHmedia.Moreover, thebindingaffinityof insulintothe insulinreceptorwasreducedbyloweringextracellularpH,whiletheexpressionofinsulinreceptorsontheplasmamembranewasnotaffectedbytheextracellularpH.Final-ly,insulin-induced2-deoxyglucoseuptakeinL6cellswasdiminishedinlowpHmedia.Ourpresentstudysuggeststhatloweredextracel-lularpHconditionsmayproducethepathogenesisoftheinsulinresis-tanceinskeletalmusclecells.NoCOI.

3P-178Disassembly of actin stress fibers is crucial for the growth arrest induced by cell compression.Asahi, Manaho1; Hayakawa, Kimihide2; Sokabe, Masahiro1,2(1Dept. of Physiol., Nagoya Univ. Grad. School of Medicine. Nagoya, Japan, 2Mechano-biology Lab. Nagoya Univ. Grad. School of Medicine. Nagoya, Japan)

Adherentcellsrequirearigidsubstrateontowhichtheycanattachforcellgrowthandsurvival.Itisgenerallythoughtthatmechanicalforce iscrucial forthisanchoragedependentcellgrowth,butthemolecularmechanismsunderlyingtheforce-dependentcellgrowthremainstobesolved.Thepresentstudyexaminedtherolesofactincytoskeletonandtyrosinephosphorylationoffocaladhesionproteinsintheretardedcellgrowthinducedbycellcompression.HaCaThumankeratinocytesor3Y1ratfibroblastswereculturedonabi-axiallystretchedPDMSchamber,andtensioninthecellswasreducedbyreleasingthechambertotheoriginalsize.Thiseffectivecellcompres-siondecreasedthenumberofEdU-positiveS-phasecells,whichwasretardedbyinhibitingactinstressfiberdisassemblywiththeactindepolymerization inhibitor jasplakinolide.Bycontrast,cellgrowthundercompressionwasarrestedwhentyrosinedephosphorylationoffocaladhesionproteinswas inhibitedbythetyrosinephosphataseinhibitorphenylarsineoxide(PAO).Similarly,cellgrowthwasarrest-edwhenactincytoskeletonwasdisruptedbytheactin-polymerizationinhibitorlatrunculinA,regardlessofthepresenceofPAO.Thesere-sultssuggestthatdisassemblyofactinstressfibersiscrucialforthegrowtharrestinducedbycellcompression,buttyrosinedephosphor-ylationoffocaladhesionproteinsisnotessentialforthisprocess.NoCOI.

3P-179Role of Steroidogenic acute regulatory protein-related lipid transfer domain containing 10 (STARD10) in the regulation of PPARα activityIto, Masanori; Seki, Yoshinari; Adachi-Akahane, Satomi(Department of Physiology, School of Medicine, Faculty of Medicine, Toho University, Tokyo, Japan)

Steroidogenicacuteregulatoryprotein (StAR)-related lipidtransfer(START)domaincontaining10(STARD10)isamemberoftheSTARTdomaincontaininglipidtransferproteinfamily.STARD10ishighlyexpressedintheliver,gallbladder,andintestine.WehavereportedthatSTARD10isinvolevedintheregulationoftheconjugation,secre-tion,andabsorptionofbileacidsinthestudyusingStard10knockout(Stard10-/-)mice.GeneexpressionassaysuggestedthatSTARD10regulatesthegenesthatareregulatedbythetranscription factorPPARα(peroxisomeproliferator-activatedreceptoralpha).TheaimofthisstudywastoclarifytheroleofSTARD10 intheregulationofPPARαactivity. Inthisstudy,weanalysedtheeffectofSTARD10overexpressionandknockdownonthePPARαactivitybyluciferasereporterassayandqRT-PCRofPPARαtargetgenesusingmousehepatomacelllineHepa1-6andhumancoloncancercelllineCaco-2.OverexpressionofStard10geneenhancedthePPARαactivity inHepa1-6cells.Ontheotherhand,theknockdownofStard10geneusingsiRNAloweredthePPARαactivity.OverexpressionofSTARD10enhancedthegeneexpressionofPPARαtargetgenesinCaco-2cells.Theseresults indicatethatSTARD10 is involved inregulatingthegeneexpressionbymodulatingPPARαactivity.NoCOI.


3P-180Decrease of pyruvate kinase activity by cesium caused cesium dependent cell growth inhibitionKobayashi, Daisuke; Kakinouchi, Kei; Hazama, Akihiro(Cellular Integrative Physiology, Graduate School of Medicine, Fukushima Medical University, Fukushima, Japan)

DistributionofCsinthewholebodiesaftertheintakeofCscellshadbeenalreadyinvestigated.However,westilldonotknowthetransportpathwayofCsintothecellsandtheeffectofCsonthecellmetabolisms.WeexaminedtheproliferationofHeLacellsculturedinDMEMwith10%FBSat37°C,5%CO2andsupplementedwith10mMofdifferenttypesofalkalimetals.Amongthem,onlyCsinhibitedtheproliferationofthecells.TheproliferationdecreaseisdependentonCsconcentra-tion.Twodifferentmethodsofliveanddeadassaywereperformed(i.e.,LDHassayandflowcytometricassay).TheHeLacellmembranewasnotdamagedby10mMCs.ToconfirmextracellularCsuptakeintothecell,intracellularcationswereanalyzedbycapillaryelectro-phoresis.The intracellularcationcontentwasanalyzedasmagne-sium-basedintracellularcationratio.TheintracellularCswasdetectedinCs-treatedcellsbutnotincontrolcell.PyruvatekinaseactivityfromcrudeextractofHeLacellshoweddose-dependentinhibitionbyCs.TheintracellularpHwasmeasuredbyBCECF,thepH-sensitivedyeindicator.Alkalizationof intracellularpHwasoccurred inthecellsculturedwithCsCl,butnotinthecellsculturedwiththeotheralkalimetals.Generally,intracellularpHoftumorcellswaslowerthannor-malcells.OurresultssuggetedthatCsaddedtoextracellularmediauptakeintothecells,andinhibitionofcellproliferationpossiblyduetodecreaseofglycolysisand intracellularalkalization inducedbyCsadministration.NoCOI.

3P-181Comparative studies on the post-translational modifi-cation of recombinant and plasma-derived human se-rum albumin: thiol oxidation, glycation and carbonyla-tion.Takahashi, Teppei; Terada, Tomoyoshi; Minami, Takeshi; Arikawa, Hajime; Matsuyama, Yukie; Era, Seiichi(Department of Physiology and Biophysics, Gifu University Graduate School of Medicine, Gifu, Japan)

Commerciallyavailablehumanserumalbumin(HSA)productshavebeenwidelyusedinbothlaboratoryandclinicalfields.Weexaminedthepost-translationalmodifications(thioloxidation,glycationandcar-bonylation)ofrecombinantalbumin(rHSA),comparedwiththoseofplasma-derivedalbumin(pHSA).ProductswereobtainedfromSigmaCo.USA(productnos.A9731forrHSA;A1653andA3782forpHSA).TherHSAisexpressed inrice.ForpHSA,theA1653 isan initialproductobtainedfromlarge-scalepooledhumanseraandtheA3782isafinalproductwhichisfattyacidfreeandglobulinfree.Thioloxi-dationwasanalyzedbyanHPLCmethod.GlycationandcarbonylationwereexaminedbyusingeachanalyticalELISAmethod.Valuesforthiolcontent(%)ofallproductswerelessthan40%,andthesevalueswerequitelowcomparedwiththoseofhealthysubjectspreviouslyreported.Dimer fractionwasobserved inallproducts.Values forglycation(%)andcarbonylation(nmol/mgprotein)ofallproductswerehigherthanthoseofhealthysubjects.TheseresultssuggestthattheheterogeneityofaminoacidmodificationofcommercialpHSAproductsappearstooccurduringmanufacturingprocessofHSAfromlarge-scalepooledplasma,andkindsofproductspeciesmaybealsoimportantforrHSAproducts.Elucidationof theexactrelationshipbetweenthepost-translationalmodificationandfunctionsofHSArequiresfurtherstudy.NoCOI.

3P-182Systemic screening of compounds targeting aberrant protein synthesis caused by dysregulation of 2-meth-ylthio modification in tRNATajiri, Tkaaki; Wei, Fan-Yan; Kaizuka, Taku; Tomizawa, Kazuhito (Kumamoto University Department of Molecular Physiology, Kumamoto, Japan)

GeneticvariationsinCdk5regulatorysubunitassociatedprotein1-like1 (CDKAL1)havebeenassociatedwiththedevelopmentoftype2diabetes(T2D).WehavepreviouslyfoundthatCdkal1catalyzestheformationof2-methylthiomodificationspecificallyatA37ofcytosolictRNALys(UUU).The2-methylthiomodificationiscriticalforaccurateandefficientdecodingoflysinecodon.Inbothmouseandhuman,thedeficiencyof2-methylthiomodificationcausedaberranttranslationofproinsulin,andresultedinimpairmentofinsulinsecretion.Giventhecriticalcontributionof2-methylthiomodificationinproteinsynthesisandT2Ddevelopment, targetingaberrantproteinsynthesiswouldthereforebeabeneficialstrategyfortreatmentofT2Dpatientscar-ryingriskCDKAL1variations.However,noneofcurrentanti-diabeticdrugsisdevelopedtodirectlytargetproteinsynthesis.Inthepresentstudy,wedevelopedauniqueluciferase-basedsystemtoscreencom-poundsthatwouldpotentiallyreduceaberrantproteinsynthesiscausedbytheabsenceof2-methylthiomodification.Utilizingthissystem,wehavescreenedasyntheticcompoundlibraryandidentifiedanumberofeffectivecompounds.Thepositivecompoundswerefurtherexaminedfortheeffectininsulinsecretioninisolatedpancreaticisletsandinvivo.Astheresults,wehaveidentifiedseveralcompoundsthatarebeneficialforbothproteinsynthesisandinsulinsecretion.NoCOI.

Poster PresentationsBlood


3P-183A recombinant habutobin fragment has the inhibitory effect on collagen-induced platelet aggregationNakamura, Mariko1; Sunagawa, Masanori1; Kosugi, Tadayoshi2 (1Department of Molecular and Cellular Physiology, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan, 2Meio University, Graduate School of Nursing)

Fourfragmentsofrecombinanthabutobin (r-habutobin) (F1,F2,F3andF4)wereproducedbythetruncationofhabutobincDNAinordertoidentifythefunctionaldomainresponsibleforitsanti-plateletaction.Toexaminewhetherther-habutobinfragmentspreventplateletag-gregation,F2andF3r-habutobinweretestedfortheireffectsontheaggregationofwashedrabbitplatelets.Uponcollagenstimulationofwashedplatelets,weassessedtheeffectsofther-habutobinfragmentsontheconformationalchangeofglycoprotein (GP)IIb/IIIaandtheexpressionofP-selectinusingflowcytometry(FCM)withPAC-1(whichonlybindstoactivatedplatelets)andP-selectinantibodies.ThepercentinhibitionofaggregationbyF3r-habutobinwas22.6±5.99%(n=7),andthatbyF2r-habutobinwas14.75±13.93%(n=4).F3r-habutobinalsoprolongedthelagtimeofcollagen-inducedplateletaggregationandslightlyinhibitedcollagen-inducedATPreleasefromthewashedplatelets.TheseresultssuggestthatthereductioninATPreleaseandtheactivationofGPIIb/IIIaandP-selectinonwashedplateletsmayinhibitcytoskeletalrearrangementinthepresenceofF3r-habutobin.SinceanumberofprolinesarepresentinF3r-habutobin,proline-re-lateduniquestructuremightplayimportantroleintheinhibitionofbothP-selectinexpressionandtheactivationofGPIIb/IIIa.NoCOI.

3P-184Treatment with fibrinogen γ-chain peptide-coated, ADP-encapsulated liposomes as an infusible hemo-static agentHagisawa, Kohsuke; Ohta, Hiroyuki; Kemuriyama, Takehito; Tashiro, Akimasa; Hiruma, Megumi; Nishida, Yasuhiro(Physiology, National Defense Med Coll, Japan)

Background:Wedevelopedfibrinogenγ-chain (HHLGGAKQAGDV,H12)-coated,adenosine-diphosphate (ADP)-encapsulated liposomes[H12-(ADP)-liposomes]thataccumulateatbleedingsitesviainteractionwithactivatedplateletsviaGPIIb/IIIaandaugmentplateletaggrega-tionby releasingADP.We evaluated thehemostatic efficacy ofH12-(ADP)-liposomesinthesettingofactiveliverbleedingindilution-althrombocytopenicrabbitsfollowingmassivetransfusion.Methods:Acutethrombocytopenia(platelets<50×103/µL)wasinducedinrabbitsbyrepeatedbloodwithdrawalandisovolemictransfusionofautologouswashedredbloodcells.Liverhemorrhagewasinitiatedbypenetratingliverinjuryinthem.Subsequently,theyreceivedastrictiontreatmentagainsttheliverhemorrhageforfiveminutesandwereintravenouslyadministeredH12-(ADP)-liposomeswithplatelet-poorplasma (PPP),platelet-richplasma (PRP),PPPaloneorH12-(PBS)-liposomes/PPPduringtheastriction.Results:AdministrationofH12-(ADP)-liposomesrescued60%oftherabbitsfromtheliverhemorrhageaswellasPRPadministrationdid50%ofthem.Incontrast,rabbitsreceivingPPPorH12-(PBS)-liposome/PPPachieved10%or17%survivalinthefirst24hrs,respectively.H12-(ADP)-liposomeswereobservedatthebleedingsiteintheliverwiththrombusformation,suggestinganinductionofthrombi.Conclusions:H12-(ADP)-liposomesmaybeasafeandeffectivetherapeutictoolduringdamagecontrolsurgeryforacutethrombocy-topenictraumapatientswithmassivebleeding.NoCOI.

3P-185Effect of Ginsenosides from Panax Ginseng on pro-tection mechanisms against oxidative stress in blood preservationOkada, Nanae1; Kono, Yusuke1; Izumi, Ryo1; Kono, Hiroki1; Suzuki, Yoji1; Ohkubo, Nobutaka1; Samukawa, Keiichi2; Aoto, Mamoru1; Mitsuda, Noriaki1(1The Department of Circulatory Physiology, Graduate School of Medicine. Ehime University. Toon, Japan, 2The Department of Functional Histology, Graduate School of Medicine. Ehime University. Toon, Japan)

Westudiedtoevaluateprotectiveeffectsagainstoxidativestressonstoredbloodat4°CwithginsenosidesextractedfromPanaxginseng.Deformabilityoferythrocytes,whichwerestoredwithcitrate-phos-phate-dextrose(CPD)inoneweek,decreased.However,erythrocytedeformabilityofstoredCPDbloodwithginsenoside-Rg2orRh1lesserdecreasedthanwithoutthem.Theyinhibitedtheoxidation-induceddecreaseofthiol-groupofmembraneproteins.Thesetwoginsenosidesmadelactateproductiontodecrease,butglucoseconsumptionnottodecrease.ErythrocytesproducedCO2byoxidativephaseinPentosePhospatePathway(PPP).Inoneweek,pCO2ofCPDbloodinclosedtube increased,pCO2ofCPDbloodwithginsenoside-Rh1 increasedmorethanwithoutit.SoitissuggestedtheginsenosidesinducedPPPintheerythrocyte,thenreducedglutathioneregenerationincreasedagainstoxidativestress.NoCOI.

3P-186Effect of lignans on protective mechanisms of erythro-cyte against oxidative stressSato, Masatoshi1; Okada, Nanae1; Suzuki, Yoji1; Ohkubo, Nobutaka1; Aoto, Mamoru1; Yamauchi, Satoshi2; Mitsuda, Noriaki1(1The Department of Circulatory Physiology, Graduate School of Medicine. Ehime University. Toon, Japan, 2The Department of Bioresources, Faculity of Agriculture, Ehime University. Matsuyama, Japan)

Wearestudyingtoevaluatetheeffectsofiron-inducedoxidativestressandprotectiveeffectsoflignan,whichisakindofpolyphenolfromflaxseeds,againstoxidativedamageonredoxstatusoferythrocytemembrane.Heparinizedvenousbloodwasobtainedfromhealthydo-nors,andimmediatelycentrifuged.Afteracarefulremovalofplasmaandbuffycoat,erythrocyteswerepurifiedbythreecyclesofresus-pensionandwashingwithisotonicHEPES-bufferedsaline(HBS)andresuspendedatadjustablehematocritinHBS.Theerythrocytesweretreatedwith0–2mMFeSO4/0–10mMascorbicacidcontainingalignan(Secoisolariciresinol(SECO)orMatairesinol(Mat),Lariciresinol(Lar))and incubatedat37°Cfor1hour.Usingthiolgroupofmembraneproteinsoferythrocyteasanindex,wehavescreenedlignans.(-)-SECOinhibitedtheoxidation-induceddecreaseofthiol-groupoferythrocytemembrane.Iron-ascorbatetreatmentimpairederythrocytesuspensionsviscosity.(-)-SECOpartiallypreventedincrementofviscosity.Eryth-rocytesuspensionswereincubatedat37°Cin2-3hourstoevaluateaeffectoflignansonglyco-metabolism.SECOwereincreasedGlucoseconsumptionatleast,howeverlactateproductiondidnotincrease.Tofurtheranalyzethereducingmechanismoferythrocyte,wearenowtryingtoevaluateoxidativephaseofPentosePhosphatePathway.NoCOI.


3P-187Dehydroepiandrosterone protected against oxidative stress on rheological function of erythrocytesIzumi, Ryo; Murakami, Yoshimasa; Takechi, Kana; Suzuki, Yoji; Ohkubo, Nobutaka; Aoto, Mamoru; Mitsuda, Noriaki(The Department of Circulatory Physiology, Graduate School of Medicine. Ehime University. Toon, Japan)

Dehydroepiandrosterone(DHEA)hasapreventiveeffectovernervessystem,underwhichoxidativestressispathologicallyimportant.Wearestudyingtoevaluatetheeffectsofiron-inducedoxidativestressandprotectiveeffectsofDHEAagainstoxidativedamageonredoxstatusoferythrocytemembrane.Heparinizedvenousbloodwasob-tained fromhealthydonors,and immediatelycentrifuged.Afteracarefulremovalofplasmaandbuffycoat,erythrocyteswerepurifiedbythreecyclesofresuspensionandwashingwithisotonicHEPES-buff-eredsaline(HBS)andresuspendedatadjustablehematocritinHBS.Theerythrocytesweretreatedwith0-1mMFeSO4/0–5mMAscorbicacidcontainingaDHEAandincubatedat37°Cfor1hour.Iron-ascor-batetreatment impairederythrocytesuspensionsviscosity.DHEApartiallypreventedincrementofviscosity.DHEAshowedtheinhibi-toryeffectfromoxidation-induceddecreaseofmembranethiolgrouponerythrocytemembraneproteins.DHEAareefficaciousinprotectingerythrocytesagainstoxidativestressasreducersorradicalscavengers.NoCOI.

3P-188The exposure of phosphatidylserine on the surface of mouse erythroblastKono, Ryoma; Onji, Hiroshi; Suzuki, Yoji; Ohkubo, Nobutaka; Mitsuda, Noriaki; Aoto, Mamoru(Depertment of Circulatory Physiology, Ehime University Graduate School of Medicine)

Inallanimalcells,phospholipidsareasymmetricallydistributedbe-tweentheouterand inner leafletsof theplasmamembrane.Thisasymmetricdistributionisdisruptedduringapoptosis,andexposedphosphatidylserine(PtdSer)ondyingcellsservesasan“eatme”signaltofacilitatephagocytosis.Itisdisruptedinotherbiologicalsystems,too.Forexample,whenbloodplateletsareactivated, theyexposePtdSertotriggertheclottingsystem.ItisthoughtthattheexposureofPtdSercontributestothecell-cellinteraction.ThePtdSerexposureisbelievedtobemediatedbytheactivationofCa2+-dependentphos-pholipidscramblasesthattransportphospholipidsbidirectionallyortheinactivationofATP-dependentflippasesthatproducetheasym-metricdistributionofphospholipid,butitsmolecularmechanismisstillunknown.WefoundthatPtdSerwasexposedtothesurfaceoferyth-roblastswhichpreparedfromspleensofphlebotomizedmice.BecausetheconcentrationofATPwasmaintainedhighlyandtheconcentrationofCa2+wasdecreasedinthem,theexposureofPtdSertothesurfaceoferythroblastswasmediatedbyCa2+-dependentphospholipidscram-blases.NoCOI.

3P-189The both α2-antiplasmin and plasminogen activator inhibitor type-1 gene deficient mice induces high IgE productionOkada, Kiyotaka1; Ueshima, Shigeru1,2; Yano, Masato1; Tamura, Noriyuki1; Kawao, Naoyuki1; Sakamoto, Akemi3; Hatano, Masahiko3; Arima, Masafumi3; Nagai, Nobuo4; Tokuhisa, Takeshi3; Kaji, Hiroshi1; Matsuo, Osamu1(1Department of Physiology and Regenerative Medicine, Kinki University Faculty of Medicine, Osaka, Japan, 2Department of Food Science and Nutrition, Kinki University Faculty of Agriculture, Nara, Japan, 3Department of Clinical Pathological Biochemistry, Chiba University Graduate School of Medicine, Chiba, Japan, 4Department of Animal Bioscience, Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Japan)

Thepathophysiologicalchangesinducedbyalackofbothα2-antiplas-min(α2-AP)andplasminogenactivatorinhibitortype-1(PAI-1)genewereinvestigatedusingdoubleknockout(KO)mice.PlasmaIgElevelsintheα2-AP/PAI-1-doubleKOmiceincreasedwithageandexceeded1,000ng/mLafter6monthsofage.TheplasmacellsthatproducedIgEweredetectedinperivascularassembledlymphocytes.Intheα2-AP/PAI-1-doubleKOmice,perivascularlymphocyteinfiltrationwasobservedinthelung,liver,andkidneysandperibronchiallymphocyteinfiltrationwaspresentinthelung.Whenthebonemarrowcellsfromα2-AP/PAI-1-doubleKOmiceweretransplanted into10-GyXxayirradiatedwild-type(WT)mice,thephenotypesoftherecipientsweresimilartothoseofα2-AP/PAI-1-doubleKOmice.Thesimultaneousexpressionofboththeα2-APandPAI-1genescontributetothemain-tenanceofimmunologicalfunctionsthatarerelatedtoIgE.Moreover,itissuggestedthatbothα2-APandPAI-1areinvolvedintherecruit-mentoflymphocytesintheperipheraltissues.NoCOI.

3P-190Essential role of Macrophage MHC receptor (MMR) in allograft rejection: generation and analysis of MMR2 knockout miceYamaji, Junko Tashiro1; Maeda, Shogo1; Ikawa, Masahito2; Okabe, Masaru2; Daikoku, Eriko1; Saito, Masahisa1; Shiraiwa, Yuka1; Yoshida, Ryotaro1; Kubota, Takahiro1(1Dept. Physiol., Osaka Med. Coll., Takatsuki, Japan, 2Res. Inst. for Microbial Diseases, Osaka, Japan)

Wepreviouslyreportedmousemacrophage(Mφ;C57BL/6;H-2DbKb)-me-diatedanddhaplotype-specificlysisofallografts(H-2DdKd)inthere-jectionsiteandisolatedtwonovelreceptorsonMφormonocytes(MO)forH-2DdandH-2Kd(mouseMHCs),MMR1and2.Inthepresentstudy,wegeneratedMMR2knockout(MMR2-/-)C57BL/6micetoexaminethebiologicalconsequencesofMMR1and2inallograftrejection.TheMMR2-/-miceshowednormalbodygrowthandfertilityandhadnoobviousabnormalities intermsofcellnumber inorcompositionoftheirlymphoidtissuesorinTlymphocyteresponsestoalloantigenornon-alloantigen.TheylackedMMR2mRNAorproteinexpressionintheirMOandfailedtorejectKd-transgenicskingrafts.Surprisingly,theyalsolackedMMR1mRNAandproteinexpressionintheirMOandfailedtorejectDd-orDdKd-transgenicskingrafts.Meanwhile,theydidrejecttheskingraftsfromC3H[third-party],B10D.2[allogeneicMHCclassII]orBALB.B[allogeneicminorhistocompatibilityantigen]mice.However,H-2Dd-,H-2Kd-orH-2DdKd-EL-4cells (lymphomaofC57BL/6origin)intradermallyorintraperitoneallyinjectedintoMMR2-/-micewererejectedbywerelysedbyCD8+TCRαβTcellpopulationinatransgene-numberdependentway.TheseresultsindicatedthatMMRsonMO/MφandTCRsoncytotoxicTcellpopulationinmicewereessential forrecognitionandrejectionofallograftedskinandlymphoma.NoCOI.


Poster PresentationsRespiration

3P-191Respiratory and locomotor activities in the phrenic and abdominal nerves in the neonatal rat in vitroIizuka, Makito1; Ruangkittisakul, Araya2; Ballanyi, Klaus2(1Cent. Med. Sci., Ibaraki Pref. Univ. Health Sci., Ibaraki, Japan, 2Dept. Physiol., Univ. Alberta, Edmonton, AB, Canada)

InspiratoryactivephrenicmotoneuronsarelocatedintheC4-6spinalsegmentsamongothertypesofmotoneurons innervatingforelimbmuscles.Abdominalmuscleshavevariousrolessuchasrespirationandlocomotion.Inthein vitrobrainstem-spinalcordpreparationfromneonatalrat,phrenicnerveandabdominalmusclesshowinspiratoryandexpiratoryactivity,respectively.Hereweexaminedintheisolat-edpons-spinalcordpreparations fromneonatalratwhetherfictivelocomotion,inducedby5–10µMNMDAplus10–20µM5-HT,occurredintheC4ventralroot(VR),phrenicandabdominalmusclenerves(il-ioinguinalnerve,IIG).Thehindlimbflexormuscleactivitywasmoni-toredfromL2VR.IncontrolsolutionphrenicandC4VRshowedinspi-ratoryactivityconsistently,whileIIGshowedexpiratoryactivityonlyatthebeginningoftheexperiment.Duringfictivelocomotion,bothC4VRandIIGshowedlocomotoractivity.EspeciallyIIGshowedtheflexoractivity.Ontheotherhand,phrenicnervedidnotshowloco-motoractivityin4preparationsandweaklocomotoractivityinone.Weconcludethatinneonatalrats,boththecentralpatterngeneratorforlocomotionandthemedullaryrespiratorycenterprovidesynapticinputtothefunctionallypropermotoneuronsexistingatthesamesegmental levels.Theresultsalsoshowedabdominalmotoneuronshavereceivedinputsfrombothcenters,andthispreparationcouldbeusefultoexplorehowtwocentersinteractwithatthelevelofmoto-neuronsandspinalinterneurons.NoCOI.

3P-192Hypoxic modulation on respiratory neuron and TRPA1 channel during perinatal period Mari, Shiga1; Kobayashi, Kimiko2; Shimomura, Hideki1,3; Tanizawa, Takakuni3; Noguchi, Koichi2; Arata, Akiko1(1Dept. of Physiology,Hyogo College of Medicine,Nishinomiya, Japan, 2Dept of ,Anatomy and Neiroscience, Hyogo Col of Med,Nishinomiya, Japan, 3Dept. of Pediatrics,Hyogo College of Medicine,Nishinomiya, Japan)

TRPA1channelplayedaroleofdetectionofhypoxiawithitsconfor-mationalchangeintheperipheralchemoreceptor.However,thecentraleffectsofhypoxiaandTRPA1channeloncentralchemoreceptionduringperinatalstagehadnotbeendeeplyunderstood.WeexaminedtheeffectsofhypoxiaandTRPA1channelonrespiratoryneuronsusingisolatedbrainstemspinalcordpreparations.Respiratoryrhythmwasfacilitatedinhypoxiafromembryonicday18(E18)topostnatalday1(P1)rats,butrespiratoryrhythmdecreasedinP2–4.TheeffectsofTRPA1channelagonistsfacilitatedtherespiratoryrhythminE18-P1rats,butdepresseditinP2-4rats;thatwerethesameashypoxia.Weinvestigatedextracellularrecordingsofrespiratoryneurons intherostralventrolateralmedulla (RVLM). Inhypoxia, thefrequencyofPre-Inspiratory(Pre-I)andtonicneuronsshoweddepressioninP2–P4;thoseofPre-IandtonicneuronsshowedfacilitationinE18-P0.ThefrequencyofexpiratoryandtonicneuronswasfacilitatedinE18-P0,andwasdepressed inP2–P4bytheapplicationofTRPA1agonist.TheseresultssuggestedthathypoxiaandTRPA1agonistshowedsameeffectsonC4rhythm,buttheeffectersofneuronsweredifferentbetweenhypoxiaandTRPA1agonist.TonicneuronsmightplayacrucialroleofthemodulationofbothhypoxiaandTRPA1channelontherespiratorynetwork.NoCOI.

3P-193Post-apneic respiratory responses in patients with ob-structive sleep apneaKanamaru, Mitsuko1; Yokoe, Takuya2; Watanabe, Yoshio2; Sagara, Hironori2; Izumizaki, Masahiko1(1Department of Physiology, Showa University, School of Medicine, 2Department of Internal Medicine, Division of Allergy and Respiratory Medicine, Showa University, School of Medicine, Tokyo, Japan)

Apnea/hypopnea(AH)stimulateschemicalrespiratorydriveinpatientswithobstructivesleepapnea(OSA).Generally,chemicalrespiratorycontrolisstrongerduringwakefulnessthansleep.Wepostulatedthatthisisalsotrueofchemicalrespiratorydriveonresumptionofrespi-rationafteranepisodeofAH.Weuseddiagnosticpolysomnographydatatoanalyzerespiratoryrate,asanindexofstrengthofrespiratorydrive,infourOSApatientswithAHindexesofapproximately30.Wedefinedperiodswhenstage2NREMsleepwasmaintainedbefore,throughandafterepisodesofAHasresumptionofrespirationduringsleep(RESPs)andthosewhenstage2NREMsleepbeforeandthroughAHshiftedtowakefulnessthroughandafterepisodesofAHasre-sumptionofrespirationduringwakefulness(RESPw).Wemeasuredrespiratoryrate,calculatedfromrespiratoryduration,andpercutane-ousoxygensaturationbeforeandafterAHforthefirsteventofRESPsorRESPwafterfallingasleep.InthefourstudypatientswithOSA,therespiratoryratewasincreasedandpercutaneousoxygensaturationdecreasedforthefirstepisodeofRESPs.However, therespiratoryratewasalmostconstantandindependentofpercutaneousoxygensaturationforthefirstsuchepisodeofRESPw.Thus,inOSApatients,respirationiscontrolledbyseverityofhypoxiaduringsleepandisindependentofhypoxicchemoreceptiverespiratorycontrolduringwakefulness.NoCOI.


3P-194Neuronal mechanisms of shortening of respiratory neuron burst by eugenolKotani, Sayumi; Onimaru, Hiroshi(Department of Physiology, Showa University School of Medicine, Tokyo, Japan)

Eugenoliscontainedinseveralplantsincludingcloveandisusedasananalgesicdrug. Inperipheralandcentralnervoussystem, thiscompoundmodulatesneuronalactivitythroughactiononvoltage-gat-ed ionicchannelsand/orontransientreceptorpotentialchannels.However,itisunknownwhetheritexertsanyeffectsontherespira-torycenterneuronsinthemedulla.Weexaminedeffectsofeugenolorcarvacrolonrespiratoryrhythmgenerationinthebrainstem-spinalcordpreparationfromnewbornrat(P0–P3).ThepreparationsweresuperfusedbymodifiedKrebssolutionat25–26oC,andinspiratoryC4ventralrootactivitywasmonitored.Membranepotentialsofrespira-toryneuronswererecorded intheparafacialregionoftherostralmedulla.Bathapplicationofeugenolorcarvacrol(0.5–1mM)decreasedrespiratoryrhythmaccompaniedwithstronginhibitionofburstactiv-ityofpre-inspiratoryneurons.Afterwashedout,respiratoryrhythmgraduallyrecoveredbutthedurationofinspiratoryburstwasextreme-lyshortenedandthiscontinuedformorethan1hrafterwashedout.TheshortingofrespiratoryneuronburstwaspartiallyblockedbyGABAAantagonistbicuculline,glycineantagoniststrychnineandGABABantagonistphaclofen.Spiketrainofactionpotentialsinrespi-ratoryneuronsinducedbydepolarizingcurrentpulsewasdepressedbyapplicationofeugenolorcarvacrol,withinductionofonlytheinitialspike.Theseresultssuggestthatchangesinbothofmembraneexcit-abilityandsynapticconnectionsareinvolvedintheshorteningofre-spiratoryneuronburstbyeugenolorcarvacrol.NoCOI.

3P-195Cellular mechanisms of capsaicin actions on the re-spiratory neurons in brainstem-spinal cord prepara-tions from the newborn ratTani, Mariho; Lin, Shih-Tien; Onimaru, Hiroshi(Department of Physiology, Showa University School of medicine, Tokyo, Japan)

Capsaicinisknownasanagonistforheat-sensitivetransientreceptorpotentialvanilloid1 (TRPV1)channel.Weexaminedtheeffectsofcapsaicinonrespiratoryrhythmgeneration inthebrainstem-spinalcordpreparationsfrom0–3dayoldWistarrats.Preparationsweresuperfusedatarateof3.0ml/minwiththefollowingartificialcerebro-spinalfluid (inmM):124NaCl,5.0KCl,1.24KH2PO4,2.4CaCl2,1.3MgCl2,26NaHCO3and30glucose,equilibratedwith95%O2and5%CO2,pH7.4,at26–27°C.Inspiratoryactivitywasmonitoredfromthefourthcervicalventralroot(C4).BathapplicationofcapsaicininducedbiphasicresponsesintheC4rate;initialdecreaseandsubsequentin-crease,withdose-dependentmanner(1–10µM).Effectsofcapsaicinweredesensitizedinresponsetotherepeatedapplication.Thisdesen-sitizationwasobservedevenaftertreatmentwithcalmodulinantag-onist,W-7(50µM)orinthelowCa2+/highMg2+solutionwith0.5mMEGTA.Pre-inspiratoryneuronsweredepolarizedin2minaftercapsaicinapplication.Effectsof10µMcapsaicinweresignificantlyattenuatedaftertreatmentwith1µMthapsigarginthatspecificallyinhibits theendoplasmicreticulumCa2+-ATPaseand inducesthereleaseofintracellularstoredCa2+fromtheendoplasmicreticulum.OurfindingssuggestthatactivationofTRPV1channelsinthemedul-lahadthevariousinfluencesontherespiratorycenterandthattargetsofcapsaicinareintracellularCa2+storesitesofendoplasmicreticuluminadditiontothecellmembrane.NoCOI.

3P-196Effects of riluzole on respiratory rhythm generation in the brainstem-spinal cord preparation from newborn ratLin, Shih-Tien; Onimaru, Hiroshi(Department of Physiology, Showa University School of Medicine, Hatanodai, Shinagawa-ku, Tokyo, Japan)

Thecontributionsofioniccurrentsplayakeyroleinelucidatinghowtherespiratoryrhythmisgeneratedinthebrainstem.Riluzoleisoneofthetherapeuticagentsforamyotrophiclateralsclerosis(ALS)andisalsoknownasapersistentsodiumchannelblocker.Inthepresentstudy,weexaminedeffectsofriluzoleonpre-inspiratory(Pre-I)neuronsintherostralmedullaaswellasonthe4thcervicalventralroot(C4)-inspiratoryactivitiesintheinvitrobrainstem-spinalcordprepa-rationsfromnewbornrats.Preparationswereisolatedfrompostnatalday0(P0)-P3Wistarratsandweresuperfusedatarateof3.0ml/minwiththefollowingartificialcerebrospinalfluid(inmM):124NaCl,5.0KCl,1.24KH2PO4,2.4CaCl2,1.3MgCl2,26NaHCO3and30glucose,equilibratedwith95%O2and5%CO2,pH7.4,at26–27°C.TherateofC4inspiratoryburstwasinhibitedinadose-dependentmanner(1–200µM)after15minapplicationofriluzole.Inaddition,riluzolecausedastrongreductioninthedrivepotentialofPre-Ineuronsbutnotofin-spiratoryneurons.Theinhibitoryeffectsofriluzolewerenotreversibleespecially inhigherdoses.Afterwashedout,C4 inspiratoryburstgraduallychangedintoepisodicpatterninwhichoneburstconsistedof2–4shortseparatebursts.Ourfindings indicatedthattheburstgenerationofPre-Ineuronsismoresensitivetoriluzolethaninspira-toryburstgeneration,andthussuggestedanimportantroleofper-sistentsodiumchannelsintheburstgenerationofPre-Ineurons.NoCOI.

Poster PresentationsOthers


3P-197Development of a new cancer-therapeutic method us-ing a nano-magnetic particle for malignant pleural me-sotheliomaFeng, Xianfeng1; Umemura , Masanari1; Sato, Itaru1; Ito, Kayoko1; Miyajima, Akiyoshi1; Ohtake, Makoto1; Makino, Ayako1; Eguchi, Haruki2; Ishikawa, Yoshihiro1(1Department of Cardiovascular Research Institute, Yokohama City Univ. of Med.,Yokohama, Japan, 2 IHI corporation,Yokohama, Japan)

Background:Malignantpleuralmesothelioma(MPM)isoneoftheworstpoor-prognosistumorsofserosalsurface,suchasthepleuraandtheperitoneum.Howevertheincidenceofthistumorisincreasingintheworldasresultofwidespreadexposuretoasbestos.BecauseoflackoftheestablishedregimenforMPM,wehavedevelopedanewhyper-thermiatherapyusinganano-magneticparticle,EI236,whichweidentified.EI236exhibitsnotonlyanti-cancereffectbutalsoferromag-netism,whichcouldgenerateheatpowerinanalternatingcurrentmagneticfield(AMF).MethodandResults:WehaveexperiencedwithuseofEI236fortreatingMPMcells.EI236promotedreactiveoxygenspecies(ROS)ofMPMcellsinadose-dependentmanner.WeperformedtheelectrophoresisofsupercoiledplasmidDNAinthepresenceofvariousconcentrationsofEI236orcisplatin.TheresultshowedthatEI236 inducedDNAnicking,whichwassimilartothatofcisplatin.EI236exhibitedpotentanti-cancereffectonseveralMPMcellsinadose-dependentmannerbyMTTassay.Theanti-cancereffectofEI236wasgreaterthanthatofcisplatin.EI236promotedapoptosisofvariousMPMcells,andfurtherbyexposedtoAMF.Conclusion:OurstudyshowEI236actssimultaneouslyasanti-cancerdrugandhyperthermiceffectinMPMcells,suggestingthatEI236canassistusindevelopinganewtreatmentmethodforMPMinthefuture.NoCOI.

3P-198Brain activation during recalling sensory by EEG mea-surementsNakamura, Koki1; Tsureishi, Akihiro1; Kobayashi, Takakazu2(Graduate School of Engineering and Science, Shibaura Institute of Technology and Science, Tokyo, Japan, 2Department of Electronic Engineering, Shibaura Institute of Technology, Tokyo, Japan)

Aftermaintainingcontinuoussensorystimulus,itmightbeabletofeellikeactualstimulusevenifthereisnostimulus.Atthistime,wethoughtthatpersonsrecallthestimulusunconsciously.ThepurposeofthisstudyistoinvestigatewhethertherearefeaturepointsonEEGandtocontributetotheEEGstudiessuchasBMIinthefuture.Tosignalthelightstimulus,thesubjectsrecalltheelectricalstimulus.Wealreadyfoundthatthere isa linearitybetweenthevisualevokedpotential(VEP)andsomatosensoryevokedpotential(SEP)frompreviousstud-ies.Byusingthisprinciple,itispossibletoobtainonlytheEEGofanassociationtime.Asaresultoftheestimation,likeanevent-relatedpotential(ERP)wasobservedatabout300msafterperformingarecallontheEEG.Furthermore,weestimatedthesignalsourcebysLORE-TA,andfoundthatinsularcortexisstronglyactivated.InthestudiesoffMRI,itwasfoundthatinsularcortexhasanimportantroleintheexperienceofemotionsuchasdiscomfort,emotions,fearandexperienceofpain.Especially,therearregionoftheinsularcortexisstronglyrelatedtoauditory,somatosensory,andskeletalmusclemovement.Basedonthephysiologicalfacts,inthisstudy,wefoundthatthefeaturepointssuchastheERPappearstoEEGatrecallofsenses.Itissug-gestedthatthisisprobablyrelatedtoapartofthebrainthatreactswhenstimulatedactually.NoCOI.

Poster PresentationsStudy Methodology

3P-199Development and application examples of selective injection system into hippocampus CA1 via monitored theta oscillationWakazono, Yoshihiko; Tsutajima, Jyoji; Takamiya, Kogo(Department of Neuroscience, University of Miyazaki, Miyazaki, Japan)

Methodsofcellbiologyandelectrophysiologyusingdissociatedprima-ryculturedneuronsallowinvitrostudyofmolecularfunctions;how-ever,analysisofintactneuronalcircuitryisoftenpreferable.Toinves-tigateexogenousgenes,viralvectorsaremostcommonly injectedusingapipettethatisinsertedfromthetopofthecortex.Althoughtherearefewreportsthatdescribethesuccessrateof injectionindetail, it issometimesdifficult to locatethepipettetipaccuratelywithintheCA1pyramidalcelllayerbecausethelayerisonly0.1mmthick.Inthepresentstudy,wehavedevelopedasystemtoinjectviralvectorsaccuratelyintothemousehippocampalCA1pyramidalcelllayerusingastereotaxicinjectionsystemwithsimultaneouselectro-physiologicalmonitoringofthetaoscillation.Thepipettetipwasposi-tionedreliablybasedonintegratedvaluesofthethetaoscillationinthehippocampalCA1pyramidalcelllayer.Usingthissystem,trans-fectionofexogenousgenes,GFP,intohippocampusCA1byinjectingGFP-expressing lentivirusvector indicatedthatGFPsignalswererestrictedintheregion.Moreover,lentivirusinfectionofGluA1cDNA,whichisanAMPAreceptorsubunit,intoGluA1KOmice,whicharereportedtobeunabletoinduceLTP,revealedtorescueLTPexpres-sionintheKOmice.Thisapproachallowsaccurateinjectionofsolutionsandprovidesanefficientmethodofgenetransferusingviralvectorsintothehippocampus,whichcanbeausefultoolforstudiesinvolvingthemolecularmechanismsofneuronalfunctions.NoCOI.


3P-200Visualizing dendritic spine morphologies along single dendrites of hippocampal neuron by a high-resolution confocal microscopy with a novel clearing reagentOsanai, Hisayuki1; Aoyagi, Yuka1; Kawakami, Ryosuke2; Nemoto, Tomomi2(1Graduate School of Information Science and Technology, Hokkaido University, Hokkaido, Japan, 2Research Institute for Electronic Science, Hokkaido University, Sapporo, Japan)

Morphologicalchangesofdendriticspinesarethoughttobeimplicat-edinlearningandmemoryprocesses.However,itisquitedifficulttoexaminedetailedmorphologiesofallthespinesthroughoutawholesingleneuronbytheconventionalopticalmicroscopy,sincethespatialresolutionorthetransparencyis insufficientduetothediffractionlimitorduetothe lightscattering inthefixedbrain,respectively.Previously,wefoundthat2,2’-tiodiethanol(TDE)renderedfixedmousehippocampalslicesopticallytransparentafterbriefimmersionintoaTDEsolution.Thisallowedthatthedendritesandthespinesbecameclearlyvisibleatthedeeperregionintheslices.Here,wedemonstrat-edthatthecombinationofaTDEsolutionandahighnumerical-aper-tureoil-immersionobjective lensprovidedhigh-resolutionconfocalimagesofthespinesthroughoutthesingledendrites.Bymeasuringthelengthsofthemajorandminoraxesofthespinehead,themor-phologiessuggested largervariations intheshapeofthedendriticspineheadintheapicaldendritethanthoseinthebasaldendrite.Thisresultsuggeststhatourtechniquecouldprovideanewinsightintosingleneuronalfunctions.NoCOI.

3P-201Modification of a novel continuous flow-through cell separation method for the isolation of mouse dendritic cellsShiono, Hiroyuki1; Matsui, Takuya1; Niwata, Satoru2; Ito, Yoichiro3 (1The Department of Physiology, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan, 2Technical Research Laboratory, Kurabo Industries, Ltd., Neyagawa, Osaka, Japan, 3Laboratory of Bioseparation Technology, Biochemistry and Biophysics Center, NIH, Bethesda, MD, USA)

Wehavedevelopedanovelcontinuousflow-throughcellseparationmethodfortheseparationofhumanbloodcells.Thisapparatuscon-tinuouslyseparatescellsintofivefractionsaccordingtotheirdensitiesbyflow-throughdensitygradientcentrifugation.Mousedendriticcells(DCs)wereisolatedbyFACSpositiveselectionafterMACSnegativeselectionoflow-densitycellsrecoveredfromthedigestedspleenbybatchdensitygradientcentrifugation.DCsarepresentinthedigestedspleenatabout2%withlargenumberoflymphocytes.Atthesepara-tionofthecellsuspensionunderhypertonicosmolalityof350mOsm/l,bothdensitiesofDCsandlymphocyteswereshiftedhigher,whiletheirdensitydifferencewasnotexpanded.AttheseparationthroughasetofOptiPrepwiththedensitiesof1.066,1.071,1.078,1.085and1.095g/ml,thecellsuspensionadjustedto1.066g/mlindensitywaspumpedintothelayerwiththesamedensity.Then,DCswereretainedandflowedoutfromthesamelayeratahighconcentrationofabout80%,whileallothertypesofcellsmigratedintotheneighboringlayers.SincetheconcentrationofDCsbythepresentmethodwasneartothatbyMACSnegativeselection,thisalternativemethodwouldbetime-sav-ingandcost-effectiveforthecellisolationbyFACSandmightmakethecellisolationbyMACSpossible.NoCOI.

3P-202A new method, flow analysis, to quantify the activity of axonal transport of cultured neuronsKatakura, Takashi; Isonaka, Risa; Kawakami, Tadashi(Department of Physiology, Kitasato University School of Medicine, Sagamihara, Japan)

TakashiKatakura,RisaIsonaka,TadashiKawakamiDepartmentofPhysiology,KitasatoUniversitySchoolofMedicine,Sagamihara,Japan-Wehaverecentlydevelopedanewmethodtoevaluateaxonaltrans-port.Weusedapublicdomain,Java-basedprogram,ImageJwithKBIplugin.Enhancedvideoimageswerecapturedandrotatedsomede-greesinordertoholdthemainorientationofthemovingorganellesholizontally,andthensavedasTiff-formatvideoclips.128×128pixelareaiscropped,inwhichaxonaltransportisdisplayedappropriately.UsingKBIplugin,weperformedFlowanalysis.Theactivityofaxonaltransportisquantifiedasthesumofparticles(organelles)whichmovesmorethan3pixelspersecond.Velocityvectorsarecalculated(thevelocityandthedegreeofanangleofthemovingparticlewithinafixedareaatagivenresolutionpixels,suchas8,16,32pixels).Wecaneasilydistinguishandsumupthenumberofanterogradetransportingparticlesandretrogradetransportingparticlesbythesignofanangleofvelocityvector,wheretheformerhasaplussignandthelatterhasaminussignrespectively.With8pixor16pixresolutionvalue,theresultsfromFlowanalysisarecoincidedwiththeresultsobtainedfromourtraditionalquantificationmethod.32pixvaluesometimesdismissedmovingparticlesandthusthecalculatedvaluesarenotconsistentwithourpreviousresults.NoCOI.

3P-203Evaluation of reporter rats which conditionally ex-press red fluorescent protein (tdTomato) under Cre-loxP systemIgarashi, Hiroyuki1; Koizumi, Kyo4; Kaneko, Ryosuke2; Yanagawa, Yuchio2; Muramatsu, Shin-Ichi3; Ishizuka, Toru4; Yawo, Hiromu1,4(Tohoku Univ. Grad. Sch of Med, Sendai, Japan, 2Gunma Univ. Grad. Sch of Med, Gunma, Japan, 3Jichi Med Univ, Tochigi, Japan, 4Tohoku Univ. Grad. Sch of Life Sci, Japan)

TheCre/loxPrecombinationsystemisoneoftheconditionalchromo-somalmimicstoinvestigatesystemicfunctionoftargetedgenes,andhasbeenadoptedtoexamineafunctionofspecificgene.Forinvivoexperiments,mousemodeliscurrentlypopularandadvancedgeneticmanipulationhasprogressedlessfarintherat.However,ratofferspotentialadvantagesof largerbodysizeandprogressedabilitytoaccomplishmorecomplexbehavioraltask.Inthisstudy,weevaluatedthreefounderratswhichhaveredfluorescentprotein(tdTomato)geneinthedownstreamofloxP-flankedSTOPcassette.Oneofthemphe-notypicallyexpressedtdTomatowiththefollowingadministrationofCrerecombinase.Firstlywe injectedAAV-Cre intostriatumofF1pupstotesttheconditionalexpressionoftdTomato.EachCre-immu-no-positivecellssparselylocatedintheinjectionpartandmergedwithtdTomato.Secondly, thefibroblasts,whichwereprimarilyculturedfromthetail,alsoexpressedcytosolictdTomatoupontransfectionwithCrerecombinasegene.ItissuggestedthatthereporterratlinewhichconditionallyexpressestdTomatoissuccessfullyestablished.Itwouldfacilitatetheneurophysiologicalstudiesandtheconnectomicsofiden-tifiedneuronsbyexpressingCreunderacertainpromoter.AllanimalprocedureswereconductedinaccordancewiththeguidingprinciplesofPhysiologicalSocietyofJapanandNIH.NoCOI.


3P-204The development of novel electroporation for cell physiological researchNumano, Rika1,2; Kurita, Hirofumi1; Matsuo, Minako1; Mizuno, Akira1(1Environmental and Life Sci, TOYOHASHI Univ. of Tech., Aichi, Japan, 2EIIRIS, TOYOHASHI Univ of Tech, Aich, Japan)

Wehaveinvestigatedanovelgenetransfectioncombiningelectropo-rationtechniquesanddropletsciencewithhightransfectionefficiencyandcellviability.AnaqueousdropletcontainingmammaliancellsandforeignplasmidDNAperformsabouncingmotionbetweenapairofelectrodesbyCoulombforceindielectricoilwithapplicationofaDCelectricfield.Whenadropletmovesbetweenapairofelectrodesfor2–5minbyapplicationofa1–3kVDCelectricfield,thelocalintenseelectricfieldfacilitatesgenetransfectionduringtheperiodicbouncingmotionofthedroplet.Thismethodhasseveraladvantagescomparedwithprevioustransfectiontechniques,includingsimultaneoustrans-fectionofvarioustypesofDNAintoevenasfewas1000cells,trans-fectionintodifferentiatedneuralcells,andtheestablishmentofstablecell lines.Nothermal loadisappliedduetotheverysmallelectriccurrentinaDCelectricfield,whilethedropletmakescontactwiththeelectrodethatcouldinduceapulsedelectricfield.Thisisbecausethedropletelectroporationelectrodesfordisposable96-and24-wellplateshavebeenimprovedforconcurrentperformance.Itispossiblethatthiselectroporationtechniqueprovidesanovelmeansforcellphysiologicalresearchbyhigh-throughputscreening.NoCOI.

3P-205Quasi-real time recording of biomagnetic fields using a magnetically linear MI gradiosensorNakayama, Shinsuke1; Kondo, Masao1; Atsuta, Satoshi3; Uchiyama, Tsuyoshi2(Nagoya Univ. Grad. Sch. Med., Nagoya, Japan, 2Nagoya Univ. Grad. Sch. Eng., Nagoya, Japan, 3Fujidenolo Corp., Komaki, Japan)

Therearenumeroustissuesandorganswhichemployelectricsignals,distributedoverthebody.For instance,neuronscode informationusingspikeactivity,whilemusclescontractwithariseofintracellularCa2+triggeredbyactionpotentials.Amagneticsensoreasilyappli-cabletolivingsystemswillbeaconvenienttooltoevaluatephysiolog-icalfunctionsnon-invasively.Hereweshowbiomagneticfieldmea-surements using a magnetically linear magnetoimpedance (MI)gradiosensor.Thismagneticsensorheadismadeofasingleamorphouswireandacoupleoffinecoilsmountedinthebothendofthiswire.Asaresult,applicationsofanexcitationpulseproduceverysimilarinductionpotentialsinthepaireddetectorcoils,therebysubtractionofthepairofinductionpotentialsenablestodetectchangesinmag-neticfields lessthan100pT.UsingthisnewMIgradiosensor,wesuccessfullymeasuredoscillatingmagneticfieldspresumablyunder-lyingpropagationofpacemakerelectriccurrentingutmusculaturesisolatedfromguinea-pigs.Whenthedirectionofmuscle layerwasreversedagainstthesensorhead,thedirectionofmagneticfieldswasalsoreversed.Wesimulatedtheoscillatingmagneticfieldsassumingintercellularelectriccurrentandextracellularreturncurrent.Fur-thermore,wemeasuredoscillatingcardiacmagneticfieldsaccompaniedbyECG.Magneticfieldscorrespondingtoprematureventricularcontractionswereoccasionallyrecorded.COIproperlydeclared.

3P-206Physiological data recording and analyzing system to improve the score on the small bore rifle shooting tar-get during the training and matchShimada, Youichi(College of Informatics and Human Communication, Kanazawa Institute of Technology, Ishikawa, Japan)

Toinvestigatetheinfluenceofthephysiologicalparameterssuchaspulserateandrespirationrhythmtothescoreonthetargetofthesmallboreriflepracticeandthematch,physiologicaldatarecordingandanalyzingsystemhadbeendevelopedwiththemicroprocessors.Thissystemincludestheweakpulselaserbeamfloodlighttoirradiatesynchronouslywithtriggering,scoreindicatingsystemequippedtheopticalsensorsonthetargetandmicroprocessor.RespirationrhythmisrecordedthroughthethermalICsensorlocatednearthenostril.Thepulserateisdetectedwiththemicrophoneattachedontherightwristandamplified.ThebarreldisturbanceisdetectedthroughthethreedimensionalaccelerationsICsensor.Thesedataarestoredinthememorycardonthemicroprocessorboardandprocessedonthepersonalcomputerformoredetailstatisticalanalysisforrelationshipbetweenthescoreandthephysiologicalparameters.NoCOI.

Poster PresentationsEducation


3P-207The yearly changes of scores of written examinations of physiology and the lowering of the levels of aca-demic performance of medical students in JapanMaeda, Masanobu1; Hano, Takuzo2(1Department of Physiology, Wakayama Medical University School of Medicine, Wakayama, Japan, 2Center for Medical Education and Development, Wakayama Medical University, Wakayama, Japan)

Theaimofthisstudyistoinvestigatetheyearlychangesofscoresofwrittenexaminationsofphysiologyandtofindevidenceofthelower-ingofthelevelsofacademicperformanceofmedicalstudentsinJapanaftertheincreaseofthenumberofmedicalstudentsfromthepointofviewofscoresofwrittenexaminationsofphysiology.Usingscoresofexaminationsofphysiologyfrom2002to2012,themean,SD,mean+SD,mean-SD,thenumberofthestudents,thenumberoftheun-successfulstudents,andtherateoftheunsuccessfulstudentswereexamined.Thescoresofexaminationsofphysiologyofmedicalstudentsdroppedsignificantlyafterthe increaseof thenumberofmedicalstudents.OneofthecausesoftheloweringofthelevelsofacademicperformanceofmedicalstudentsinJapanistheincreaseofthenum-berofmedicalstudents.NoCOI.

Author Index


AUTHOR INDEX

ＡＵＴＨＯＲＩＮＤＥＸ（Boldface indicates presenting author．）

A

AbeChikara 1P-038,1P-064,2P-088,2P-089,2P-165,2S30H1-4,3O4G-3

AbeHiroshi 1P-142AbeManabu 1P-145AbeOsamu 3P-068AdachiTakeshi 1P-073Adachi-AkahaneSatomi

3P-179AgarieHiromi 3P-085AgataNobuhide 1P-178AhmedMd.SalehYoussef

2O3G-1AibikiMayuki 3P-126AidaTomomi 1S7G-2AiharaMutsumi 3P-167AijimaReona 2P-208AkagawaKimio 1P-172,

2P-059AkagiTakumi 1P-162AkaikeToru 2P-040,

2P-041,3P-155AkaoTeppei 1P-192AkasakaKeisuke 2P-121AkatsukaShinya 3P-118AkedaYukihiiro 3S49G-5AkihoHirotada 2S31J-4AkimotoNozomi 3P-019AkitaHisanao 2P-110,

3P-026,3P-075AkitaTenpei 2S33K-4AkiyamaTsuyoshi 1P-041,

1S17F-3,2P-049,2P-050AkiyoshiJotaro 3P-073AkizawaFumika 3P-057AkramHossain 2P-128AkutagawaMasatake

3P-167AllenMAndrew 3S52K-1AlexanderABachnanov

1P-153AmanoAkira 1P-062,

2P-031,2SL09F,3P-002AmanoIzuki 2P-143,

2P-173AmitaniHaruka 2S35B-5AmitaniMarie 2S35B-5AnderssonKarl-Erik

1P-215AndoHiroshi 3P-034AndoHiroshi 3P-029,

3P-061AndoTsugunobu 3P-023AnthonyEPickering

3P-020AnzaiNaohiko 1S10H2-2,

2P-204AokiHiroki 1S19H1-4AokiRiko 3O7J-3AomineMasahiro 3P-102AonoHitomi 3P-116,

3P-124AonoShuichi 3P-031

AonumaHitoshi 2P-117AonumaKei 1P-055AotoMamoru 3P-185,

3P-186,3P-187,3P-188AouShuji 2S23B-1,

3P-086AoyagiHidekazu 2P-127AoyagiKunihiko 2P-137,

2S31J-1AoyagiToshifumi 1P-139AoyagiYuka 3P-200AoyamaMineyoshi 1P-169ArakawaMayu 2P-121,

2P-169ArakawaTetuso 2S29H1-3ArakiMiya 3P-168ArakiNaomi 1S3E-3ArataAkiko 2S36C-5,

3P-192ArikawaHajime 3P-133,

3P-181ArimaHiroki 1P-034ArimaMasafumi 3P-189AsahiManaho 3P-178AsaiKiyofumi 1P-169AsakawaAkihiro 2S35B-5AsakuraKeiichi 2S27F-5AsanoKazuhito 2P-185,

2P-186,3P-040AsanumaNaokazu 3P-029,

3P-061AsayamaEmi 1P-120AshcroftFrances 2S43K-2AshiharaTakashi 2P-026,

3S46D1-3AsmaraHadhimulya

2S34K-1AtsumiToshiyuki 2P-189AtsumiYusuke 3P-055AtsutaSatoshi 3P-205AwakaShun-ya 3O7J-2AzamiTakuya 2S41H1-4

B

BabaAsuka 3O7J-1BabaHironori 3P-083BabaHiroshi 3P-021BaiJiayu 1P-021,

1P-023,1P-024BaiXiaopeng 2S31J-4BaimoukhametovaVDinara

2S38E-3BainsSJaideep 2S38E-3BallanyiKlaus 3P-191BannaiHiroko 2S38E-1BannaiMakoto 2P-123BarlowA.Linda 1S2D1-1BatuKeceli 1P-004BellierJean-Pierre 1P-023,

1P-173BereiterDavid 1P-124BillyTChen 1P-137BlessingWWilliam3S52K-3BoldbaatarDamdindorj

2P-148BonciAntonello 1P-137BongjuKim 2S27F-3BoudakaAmmar 2P-018BrodskyMFrances2P-205BrzoskaTomasz 2S41H1-3

C

CaiWenqian 1S17F-4,3P-156

CamusMStephane2P-205ChaChaeYoung 2S27F-5ChalupeckyVladmir

2S39F-3ChenT.Billy 1P-137ChenXin2P-175ChibaAtsushi 3P-082ChikahisaSachiko 1P-143,

2P-176,3P-088ChimotoSohei 2P-072ChisatoKatagi 2P-128ChooMyeongJeong

3O6F-6ChurilovPLeonid 3P-163CohenBLawrence 3SL15H1CorfasGabriel 2S23B-3CrottiLia 3S46D1-1CuiHong 3S49G-2CuiXiaoke 2S39F-3

D

DaikokuEriko 1P-214,2P-076,2P-168,3P-190

DanjoTeruko 1S18G-3DasPartha 1P-132DewaTakehisa 1P-029DezakiKatsuya 2P-148DingWei-Guang 1P-021,

1P-023,1P-024,1P-053,1P-173

DoiHirokazu 3P-076DongYouyi 2P-197,

3P-149,3P-150DonishiTomohiro 2P-075,

3P-030,3P-074,3P-077DoyaKenji 2S39F-1DruckerJ.Daniel 1S2D1-4DuCheng-Kun 1P-041,

2P-049,2P-050DubelStefan 1P-053

E

EbiharaShizufumi 1P-142EbinaKoudai 2P-097EdamuraKazuya 3O5J-1EgashiraYoshihiro 1S5F-4EguchiHaruki 3P-197EguchiKohei 2P-130EifukuSatoshi 1P-133EkaSunarwidiPrasedia

2P-152ElmquistKJoel 2S35B-2EmaMasatsugu 2S41H1-4EndoKana 1P-036,

1P-047,1P-077,3O4G-1,3O4G-2

EndoNaoto 3P-022EndoYamaokaMasako

2P-032,2P-130EnokiRyosuke 3S58H1-2EnomotoKo-ichi 2P-058EnomotoMasato 3P-123EraSeiichi 1P-217,

3P-133,3P-181EshimaHiroaki 1P-190EshimaNobuoki 3P-035EslerMurray 2S30H1-3EspositoGianluca 1P-154EtoKei 3O6F-8EtoTamon 1P-114EtoriKeishi 1P-145

F

FanGuoping 2S35B-2FengRui 2P-020,

2P-210,2S34K-1,2S43K-3FengXianfeng 3P-197FisherA.N.Jonathan

2S24C-5FlucherEBernhard1P-187FontoniniAlfredo 1P-137FoskettJKevin 3S48F-4FrankPark 2P-047FuchuuHidetaka 2O3G-1FujiharaChizuko 2P-130FujiharaHiroaki 1P-106FujiiKosuke 3P-073FujiiNobuharu 3S47E-4FujiiNobumi 3P-029,

3P-061FujiiTakuto 1P-032,

1S13K-3,2P-014,3O7J-2FujiiTeruyuki 1P-176FujiiYutaka 1P-063,

2P-043FujikiNobuhiro 1P-106,

3S55D1-3FujimotoTetsuya 3P-086FujinokiMasakatsu2P-118,

2P-119FujiokaYoichiro 2P-161FujisawaSusumu 1P-073,

3P-121FujitaMasako 2P-079,

2P-094FujitaReiko 1P-057FujitaSayaka 1P-141FujitaTakayuki 1S17F-4,

3P-156FujitaTakuya 2P-133FujitaTsugumi 1P-116,

1P-117,2P-012,2P-013,3S54C-1

FujitaniYoshio 1S15D2-3Fujita-YoshigakiJunko

1P-202,1P-206FujiwaraHiroshi 2P-189FujiwaraKen 2S23B-4


AUTHOR INDEX

FujiwaraTomonori1P-172,2P-059

FujiwaraYuichiro 1P-011,1P-012,3S48F-2

FujiwaraYurika 2P-175FujiyamaRie 3P-051FukadaKihachiro 3P-137FukadaToshiyuki 1S15D2-1FukamiHideyuki 3P-039FukataMasaki 2P-010FukubaYoshiyuki 1P-152,

2P-032,2P-130FukudaAtsuo 1S8H1-2,

2S33K-4FukudaNorio 1P-066,

1P-067,1P-175,1P-176,2S26E-2

FukudaSatoshi 1P-098,1P-100,1P-103,1P-15,2P-103

FukunagaKoji 2S23B-1FukuokaYoshiyuki2P-032FukuokayaWataru2S23B-3FukushiIsato 3O7J-7FukushimaHidefumi

3O5J-3FukushimaMasaya2P-191FukushimaTeruyuki

1P-089FukutaNaomi 2P-007,

2P-015,3P-024FukuyamaNaoto 3O4G-4,

3O7J-6FunahashiMakoto 1P-079FunahashiYu 3P-130FunatoYosuke 1S16E-4FurueHidemasa 2P-194,

3P-008,3P-018,3P-019,3P-020,3P-023,3P-036

FurukawaYasuo 1P-084FurukawaYuko 1P-016FurutaTakahiro 3S51J-4FurutaTumugu 3P-029FurutaniKazuharu2P-030FuruyaKishio 3P-169FuzesiTamas 2S38E-3

G

GaoQinghua 2P-210GenKeika 3P-135GodaNobuhito 1P-074,

2P-041GomiNorihiro 2P-185,

2P-186,2P-187GotoChikara 2S29H1-2GotoKatsumasa 1P-183GotoKazunori 2S32J-2GotoKei 2P-085GotoNaomi 1P-203GotohKatsuhiro 3P-108,

3P-112GouraudSSabine 1P-040,

1P-043,1P-049,1P-132GuoFeng 2P-020,

2P-210GuoShi-Yu 3P-127GUPTARUPALI 2P-001

H

HabataToshiya 2P-110HagaShouhei 1P-114HagiharaMKenta 3P-014HaginomoriShin-ichi

2P-076HagisawaKohsuke 1P-124,

2P-084,3P-184HagiwaraNobuhisa1S1A-4HaijimaAsahi 2P-143,

2P-173HakimFarzana 3P-172HakozakiAtsushi 3P-018HamaNoriyuki 3P-013HamadaHironobu 1P-077,

3O4G-2HamadaYuka 2P-138,

3P-090HamaguchiShigeto3S49G-5HamanoueMakoto 1P-168HanDong-Yun 2S34K-1HanJin 　　 2S27F-2HanafusaManami 1P-086HanaokaTomoko 3P-132HanashimaAkira 1P-184HanaueMayu 2P-120HanoTakuzo 3P-207HaoLiying 2P-020,

2P-021,2P-210,2S43K-3HaraS. 3P-056HaraToshiko 3P-094HaradaChizuru 3P-035HaradaKeita 2P-147HaradaShuitsu 1S2D1-1HaraguchiAtsushi 3P-099HaraguchiRyo 2P-026,

3S46D1-3HaraguchiShogo 1P-213HaramiMaiko 3P-145HaranoNozomu 1P-209HarataMisato 1P-057HarimaMiki 1P-079HarperJMatthew 2S35B-2HarrelToshioDaniel

3S46D1-3HaruyamaNaoto 1P-139,

1P-140HaseHideharu 2P-004HasegawaJun 2S39F-4HasegawaKayoko 3P-015HasegawaKeiko 2P-076HashiguchiMitsuko1P-171HashiguchiToshio 1P-171HashimotoHirofumi2P-141,

2P-164HashimotoHisashi 2S31J-2HashimotoIzumi 1P-132HashimotoKen 1P-069HashimotoMasaaki2P-166,

3P-136HashimotoMichio 3P-094,

3P-097HashimotoRyota 3P-164,

3P-165HashimotoSadamitsu

1P-199HashimotoTakeshi 2P-042,

2P-150,3P-170HashimotodaniYuki3O6F-5

HashitaniHikaru 1P-042,1P-188,1P-193,2P-033,2P-037

HashizumeMiki 1P-136HasunumaItaru 1P-213HataTadayoshi 2P-027HataYukiko 1P-055HatakeKatsuhiko 3S45C-4HatanakaKohei 2S39F-3HatanakaYusuke 3S53B-1HatanoMasahiko 3P-189HatanoMasahiko 3S49G-3HattaAzusa 1P-126HattoriMotoyuki 2S43K-4HattoriSatoko 1P-009HattoriTomoe 2P-161HayakawaKimihide3P-178HayashiHidetoshi 1S22K-2HayashiHisaki 1S17F-1,

2P-046,2P-047HayashiKeitarou 2P-204HayashiKenshi 3S46D1-1HayashiKoei 1P-178HayashiMikio 2P-200HayashiNaoyuki 1P-048,

2P-032,2P-138,3P-090HayashiRyotaro 1P-120HayashiTakagiAkiko

1P-090HayashiTakuya 2P-107HayashiTokumasa 1P-194HayashiYasunori 3P-053,

3P-067HayashiYukio 3P-018HayashidaMichikata

2P-053HayashidaYuki 1S09H1-1,

1S09H1-3,2S39F-4HayataHiroki 3P-177HazamaAkihiro 1P-208,

2P-045,2P-151,2P-152,3P-168,3P-180

HeGuilin　　 2P-020HeoHye-Jin 2S27F-2HibinoHiroshi 1O2J-4,

2S24C-1HidaAkiko 1S21J-4HidaHideki 1P-169

2P-101,2P-102,2P-106,2P-125,2P-196,3P-062,3P-080

HidakaSoh 1O2J-3HidakaYuko 1S17F-4HidemaShizu 1P-120HidetoshiNiwa 1P-037HigaMoritoshi 3P-107HigakiHiromi 3P-124HigakiSayuri 3P-072HigashijimaShin-ichi

2S40G-3HigoNoriyuki 2P-104,

2P-107HiguchiSatomi 3P-039HiguchiTaiga 2P-014HikidaTakatoshi 1S18G-3,

1S18G-4HIkidaMasaki 2S27F-3HimenoYukiko 2P-031HimiNaoyuki 1P-163HinoKokoro 2P-129

HinoYoshiko 1P-018HiraRiichiro 2P-115HirabaKatsunari 1P-210HirabayashiToshiyuki

2S28G-1HiraharaTakao 2S39F-3HiraiHirokazu 1S5F-1,

1S5F-2,2P-183HiraiYasuharu 1P-119HiranoKatsuya 1P-070HiranoMayumi 1P-070HiranoMitsuru 2P-022HiranoTomoo 2S38E-4HiraoMizuho 3P-101HiraokaYuichi 1P-120HirasawaHiroyuki 3S49G-3HirashimaMasanori2S41H1-4HirataYoshihiro 1P-134HirataYuko 2P-128,

2P-197,3P-149,3P-150HirokawaErisa 1P-066HironoChikara 2P-199,

2S36C-3HironoMoritoshi 2P-062HiroseKenzo 1S14D1-2HiroseMasamichi 1P-057HiroseSatoshi 3P-068HiroseShinichi 3O6F-2HiroseTomoya 3S49G-5HirotaAkihiko 3P-013HirumaHiromi 2P-160HirumaMegumi 3P-184HisadaToshiaki 2S39F-3HisajimaNozomi 1P-055HisamitsuNaoko 2P-191HisamitsuTadashi 1P-195,

1P-215,2P-185,2P-186,2P-187,2P-188,2P-189,2P-190,2P-191,3P-033,3P-040,3P-127

HisamitsuTakashi 1P-002,2S43K-1

HisanoSetsuji 3P-079HisanoSuguru 1P-170HisaokaMasahiro 3P-150HisatomeIchiro 2P-028,

2P-099HishidaRyuichi 1P-110,

1P-128,2P-073,3P-021,3P-022

HishikawaYoshio 3LS6D1HitomiSuzuro 1P-149,

1P-205,1P-209HoireMinoru 3S46D1-1HokiAyaka 3O7J-8HommaChihiro 3P-054HommaIkuo 3SL11CHommaKazuhiro 3O7J-6HommaNoriyasu 1P-078,

3P-162HondaAkira 2P-029,

2P-154HondaKenji 3P-019HondaKuniya 3P-004,

3P-009HongoKenichi 1P-051HongoMaiko 1P-073HonjoHaruo 1S1A-3HonmaAya 1P-151HonmaChihiro 1P-162


AUTHOR INDEX

HonmaKen-ichi 1P-134,1P-151,1S21J-3,2P-174,3S58H1-2

HonmaMasashi 2S39F-2HonmaSato 1P-134,

1P-151,1S21J-3,2P-174,3S58H1-2

HonmaSatoru 3S51J-4HonmaYusuke 3P-083HoriMasatoshi 2S31J-2HoriTetsuya 1S14D1-1HoriYuuichi 1P-089,

2P-060HorieSawa 1P-203,

3P-039HorieTakeo 2S40G-1HoriguchiKazuhide2S31J-2HorikawaJunsei 3P-081HorikawaKazuki 3S58H1-1HorikawaYuusuke 2S27F-5HorikoshiYuko 2P-045HorioKayo 1S8H1-2HoriuchiJouji 2P-098,

2P-100HoriuchiMika 3P-108,

3P-112HoriuchiTakatoshi 2P-098HosogaiMasae 2P-091HosogiShigekuni 2P-162HosoiNobutake 1S5F-2HosokawaYuki 3P-109,

3P-113HosokawaYutaka 3P-081HosokiYukari 3P-002HosonoTakayoshi 2P-121,

2P-169,2P-170HososhimaShoko 3P-003HossainAkram 2P-197,

3P-149,3P-150HossainMdShamim

2O3G-1HotchkissSRichard3S49G-3HottaHarumi 3P-056,

3P-010HottaNorio 3P-012HoutmanMarien 1O1H2-4HozumiKatsuto 3S49G-4HsiehYi-Ting 3P-155HuHuiyuan 2P-020,

2P-210HuYaopeng 2S25D1-2,

3S57G-1HudspethA.J. 2S24C-5HungHsichin 2P-137,

2S31J-1HyodoMasamitsu 3P-046

I

IchiharaKosuke 2P-095IchikawaHiroyuki 2P-079IchikawaJun 1S4E-1,

2P-005IchikawaYasuhiro 3S55D1-1IchimaruToru 2P-122,

2P-123IchinoheT 1P-198IchinoseMitsuyuki 2P-177IchiseNobutoshi 1P-068IdeRyoji 2P-080

IdesakoMitsuhiro 1P-036,1P-047,1P-077,3O4G-1,3O4G-2

IeharaTakahiro 1P-032IesakiTakafumi 2P-048,

3P-165IfukuMasataka 2O3G-1IgarashiHiroyuki 3P-203IgarashiJunsuke 2P-042,

2P-150,3P-170IgarashiMasakazu 2P-110IgataSachiyo 1P-030IgiChihiro 2P-121,

2P-169IHARAEikichi 2S31J-4IidaTetsuo 2P-197IinoMasamitsu 2P-023,

2P-024IinoSatoshi 2S31J-2IizukaMakito 3P-191IkawaMasahito 3P-190IkebeMitsuo 1S12K-3IkedaAzusa 3P-036IkedaMasaaki 1S21J-1IkedaMayumi 2P-096IkedaYuko 3P-101IkeharaToshitaka 3P-167IkemotoHideshi 2P-187,

2P-191IkemuraTsukasa 1P-048,

2P-138IketaniMitsue 1S17F-3IkeuchiYukiko 3P-159IkeyaYoshimori 3O4G-4IkomaYoko 2P-163ImadaHideki 1P-127ImagitaHidetaka 3P-132ImahashiRisa 1S7G-2ImaiDaiki 3P-092,

3P-139ImaiKenji 2P-129,

2P-134,2P-135ImaiYuko 2P-004ImaizumiKazuhiko1P-180,

3P-144Imaizumi-OhashiYoshie

1P-071ImanagaIssei 1S1A-1Imanaka-YoshidaKyoko

１S19H1-3ImbeHiroki 2P-075,

3P-030ImotoKeiji 1P-166,

2P-063,2P-194,3P-008,3P-018,3P-019,3P-020,3P-023,3P-036,3S46D1-1

ImuraYoshio 1P-016InadaShin 3S46D1-3InagakiAkihiro 2P-200,

2P-202,2P-203InagakiTadakatsu 1P-050,

2P-035,2P-043,2P-049,2P-050

Inagaki-OharaKyoko2S31J-3

InanobeAtsushi 1P-025InaseMasahiko 2P-105,

3P-059,3P-082,3P-084InenagaKiyotoshi 1P-149,

1P-205,1P-209

InokuchiSadaki 3S49G-4InoueAkio 3P-069InoueHana 2P-009,

2P-198InoueHidekazu 3P-008InoueHiroshi 1S6G-3InoueHiroshi 3P-166InoueMai 1P-162InoueMasashi 1P-153InoueMasumi 2P-147InoueMayuko 1S2D1-4InoueRentarou 2P-203InoueRitsuko 1P-092InoueRyuji 1S4E-1,

2P-004,2P-005,2P-137,2P-154,2S25D1-2,2S31J-1,3O6F-2

InoueShigeaki 3S49G-4InoueTakafumi 1P-108,

2P-036InoueTomio 3P-098InoueWataru 2S38E-3InuiAkio 2S35B-5InuiTadashi 3P-007,

3P-047InukaiYoko 1S17F-1,

2P-047,2P-087,3P-100InutsukaAyumu 3P-054,

3P-071InuzukaHiroyuki 3O5J-3IraPastan 1S18G-3IribeGentaro 1P-052,

3S57G-3IsaTadashi 2O3G-4,

3P-016IshiakwaS. 3P-033IshibashiHitoshi 2P-064,

3O6F-7,3P-026,3P-075IshidaTakuya 3P-077IshidaAkimasa 2P-101,

2P-102,2P-196,3P-062IshidaAndrew 1S09H1-1IshidaHiroaki 3P-084IshidaKazuto 3P-118IshidaMinori 1P-192IshidaYukisato 1S12K-4IshigamiTomoaki 1S3E-3IshiguroAkio 2P-117IshiguroGo 1P-113IshiguroHiroshi 2P-136,

2P-201,3P-154IshiguroMasanori 1P-088IshiharaJun 2P-100IshiharaKeiko 1P-030,

1P-194,1S16E-1IshiiHisayoshi 1P-196,

1P-204IshiiKei 1P-036,

1P-047,1P-077,2S29H1-1,3O4G-1,3O4G-2

IshiiMTakashiro 3P-050IshiiNaokata 3S59K-2IshiiNobuhito 1P-005IshiiRitsuko 2P-194IshiiShuya 2S26E-2IshiiToshiyuki 1S8H1-3IshiiYoshiki 1P-179IshijimaAkihiko 2S26E-1IshikaneHiroshi 1S09H1-2IshikawaAkinori 3P-063

IshikawaJunko 3P-063IshikawaNaohiro 1S09H1-3IshikawaShintaro 2P-185,

2P-188,2P-189,2P-190,3P-040

IshikawaTatsuya 3O6F-7IshikawaYoshihiro 3F1F-1,

1S17F-4,2P-036,2P-038,3P-156,3P-197,3S55D1-1

IshikuboHarumi 1S7G-2IshikuraToru 2P-141IshiwataRyo 2P-038IshiwataShin’ichi 1P-066,

1P-067,1P-175,1P-176IshizakiYasuki 3O6F-3IshizukaKen’Ichi 1P-039,

1P-212IshizukaToru 2P-019,

3P-003,3P-203IsidaAkimasa 3P-080IsikawaJunko 3P-060IslamAfsana 3P-125IslamShafiqulMd 2S31J-2IsodaMasaki 2S28G-3IsomuraMinoru 1S4E-3IsonakaRisa 2P-069,

3P-202IsooNoriko 1P-098,

1P-099,1P-100,2P-055IsowakiMutsumi 1P-098,

1P-099,1P-100ItagakiYuya 2P-152ItamiNoritomo 1P-217ItoAkira 1P-139ItoChihiro 2P-184ItoH SP-1ItoHiroaki 3P-046ItoKayoko 3P-197ItoMasanori 3P-179ItoSadayoshi 1P-217ItoShin-Ichi 3P-013ItoYasuhiro 2P-180ItoYoichiro 3P-20１ItohHideki 2S26E-2ItohHideki 3S46D1-1ItohMasayuki 1P-030,

1P-031,1P-059,1P-194,1S16E-1

ItohYamato 2P-096ItoiKeiichi 1P-133ItoigawaMasataka 2P-184ItsukiKyohei 2P-004IwamiKeiko 3P-132IwamotoMasayuki 1P-019,

1P-028,1P-029IwamotoTakahiro 1P-058,

1S16E-3IwamuraTakashi 2S39F-3IwanagaKoichi 2S31J-2IwasakiHirohide 3S51J-5IwasakiShin-ichi 1P-212,

2P-127IwasakiTakami 3P-029IwasakiToshiharu 2P-146IwasakiYusaku 2S23B-2IwasawaTakahiro 1P-167IwaseSatoshi 1S17F-1,

2P-047,2P-086,2P-087,2P-167,3P-100

IwataKoichi 1P-125,


AUTHOR INDEX

3P-004,3P-009,3P-028IwataShinji 3P-109IwataShusuke 1S2D1-4,

1S3E-1IwataniJun 3P-077IwatsukiKen 1S2D1-3IzumiRyo 3P-185,

3P-187IzumizakiMasahiko3P-193

J

JamiruddinMohd.Raeed3P-172

JeongBeomLee 3P-095,3P-096

JiaShuSheng 2P-174JiangChang-Yu 1P-116,

1P-117,2P-012,2P-013,3S54C-1

JIAOQibin 2P-040JimuraKoji 3P-068JinHui-Lin 1S17F-4,

3P-156JinYu 3P-069JinnoNaoya 1P-131,

2P-136,3P-122JinnoYuka 1P-020JohnPO’Doherty 3P-066JunJaeYeoul 2S32J-3JungCha-Gyun 2P-101,

2P-102,2P-106,2P-125,2P-196,3P-062,3P-080

JutabhaPromsuk 1S10H2-2,2P-204

JyotakiMasafumi 1S2D1-2,1S3E-1

K

KabaHideto 3P-037,3P-038,3P-041,3P-045,3P-046

KabayamaShigeru 1P-217KadookaYoshimasa2S39F-3KadorikuFumiya 2P-178,

2P-179KadowakiTakako 2P-130KadowakiTakashi 2P-140KaiharaKeiko 1P-052,

1P-052KaitsukaTaku 1P-160,

2P-126,2P-144,3P-001,3P-172,3P-182

KajiHiroshi 3P-131,3P-189

KajimuraIchige 2P-040,2P-041,3P-155

KajiyaFumihiko 3SL12D1KajiyaHiroshi 2P-207,

3O5J-3KajiyaKatsuko 3P-175KakegawaWataru 1S14D1-4KakigiRyo 1P-186,

2P-048KakigiRyusuke 3P-052KakinokiYasuaki 1P-149KakinouchiKei 3P-180KakinumaYoshihiko

1S17F-3KakitaHiroki 1P-169

KakiuchiNobuko 2P-189KakizoeYutaka 1S3E-4KakuYoshiko 3P-146KalashnikovaAnastasia

3P-163KamataniDaiki 1P-110KambeTaiho 1S15D2-5KamedaHiroshi 1P-099,

1P-103,2P-055,2P-103KamedaSeiji 1S09H1-3,

2S39F-4KameiYasuhiro 2S26E-4KameyamaAsako 2S43K-3,

2P-021KameyamaMasaki 2S34K-1,

2S43K-3,2P-020,2P-021KamijoYoshi-ichiro3S59K-4KamikuboYuji 1S22K-1KaminotaTeppei 3P-120KamitoriKazuyo 2P-128,

2P-197,3P-149,3P-150KamiyaHaruyuki 1P-087,

1S14D1-3KamiyaKaichiro 1S1A-3KamiyaKentaro 2S29H1-4KamiyamaTsutomu

2P-103KamoshidaAtsushi3P-054KanamaruMitsuko 3P-193KandelBabuMunal1P-007kanedamakoto 1P-122,

1S8H1-2,1S8H1-3KanekoKentaro 1P-089KanekoRyosuke 3P-117,

3P-203KanekoSYoko 2P-139,

3P-171,2S33K-3KanekoShuji 2S37D1-2KanekoTakeshi 3S51J-4KanemaruKazunori

2P-023,2P-024KaneokeYoshiki 3P-077,

2P-075,3P-030,3P-074KangQin 1P-116KangawaKenji ２SL05BKanikowskaDominika

3P-100KanmuraYuichi 3P-043,

3S52K-2KannoTakeshi 2P-155,

2P-193,3P-069,3P-146KanoMasanobu 1S5F-1,

3O6F-4,3O6F-5,3O6F-6KanoTakeshi 2P-117KanoYutaka 1P-190KansakuKenji 3P-085KarakiShin-Ichiro 2P-132,

2P-133KarinaHajar 1P-181KariyaTaro 2S39F-3KasaiHaruo 1P-090,

2P-057,2P-061,3P-161KASAIMasatoshi 3P-016KashiharaToshihide

1P-187KashimaHideaki 1P-152,

2P-032,2P-130,3P-090KashioMakiko 2P-006,

2P-011,2P-208KashiwadaniHideki3P-043,

3S52K-2KashiwayanagiMakoto

3P-042KatafuchiTetsuro 2P-096KatafuchiToshihiko2O3G-1,

2S23B-1KatagawaYoshihisa3P-135KatagiAyako 3P-149,

3P-150KatagiChisato 2P-128KatagiriAyano 1P-125KatagiriChiaki 2P-008KatagiriHideki 3S53B-2,

3S53B-1KatahiraHaruto 2P-191KatakuraA 1P-197KatakuraMasanori 3P-094,

3P-097KatakuraTakashi 2P-069,

3P-202KatanosakaKimiaki3P-005KatanosakaYuki 3P-005KataokaNaoya 1S18G-1KataokaTsuyoshi 1P-077,

3O4G-2KatayamaYoko 1P-104,

3P-129KatoEiko 1P-089KatoFusao 1P-080,

1S5F-5,2S36C-1,3P-032,3P-064,3S53B-4

KatoGo 2P-085KatoHiroaki 3P-118KatoRikako 2O3G-4KatoRisako 3P-134KatoSatoru 1P-158,

1P-159,1P-170KatoShin 1P-169KatohAkiko 2P-164KatohYouichi 3P-164,

3P-165KatsudaShin-ichiro2P-045Katsumata-KatoOsamu

1P-202,1P-206KatsurabayasiShutaro

2EL2A-1KawabeYuya 3P-109KawadaTeruo 2P-017KawadaToru 1P-041,

2S30H1-1,2S30H1-3KawaguchiA 1O1H2-2,

1P-198KawaguchiMakoto 2P-125KawaguchiNorihiko

3P-058KawaguchiShin-ya 2S27F-4KawaguchiYasuo 2P-113KawaharaIsao 2P-085KawaharaKatsumasa

1S10H2-1KawaharaYukio 1S11J-2KawaiKensuke 3S53B-3,

3S53B-3KawaiMakoto 1P-051KawaiMinako 3P-013KawaiTakafumi 1P-016KawakamiKeisuke 1P-178KawakamiKoichi 2P-134,

2P-135KawakamiNozomi 1P-156

KawakamiRyosuke3P-200KawakamiTadashi 2P-069,

3P-202KawakamiYoriko 3P-129KawamotoChisato 3P-128KawamuraNaoki 1P-133KawanabeAkira 1P-015,

3S48F-2KawanoFuminori 3S47E-2KawanoMasako 3P-067KawanoYoshihisa 3P-073KawaoNaoyuki 3P-131,

3P-189KawasakiHiroshi 3S44B-３KawasakiMakoto 2P-141KawasakiShun 3P-112,

3P-123,3P-126,3P-128KawasetsuTakumi2S39F-4KawashimaKoichiro

2P-124KawashimaYoshiyuki

2S24C-2KawataRyo 2P-076KawataShiyori 2P-178,

2P-179KawataniMasahito 3P-018KawawakiJunko 1P-018KayaMitsuharu 3P-141KazamaItsuro 2P-016,

3O7J-1KazuyoshiHirota 1P-037KeceliBatu 3S48F-1KeceliSumru 3P-052KemuriyamaTakehito

1P-124,2P-084,3P-184,3S49G-1

KidaHiroyuki 3P-006,3P-065

KidoMizuho 2P-208KijimaTakeshi 1P-195KikuchiSatoshi 3P-123,

3P-126KikuchiTakashi 3P-173KikutaSatomi 3P-162KikuyamaSakae 1P-213KimBongju 3P-157KimHae 3S57G-4KimHyoungKyu 2S27F-2KimJuhyon 2P-149KimKiWoo 2S35B-2KimNari　　 2S27F-2KimSung 3S57G-4KimotoKatsuya 1P-055KimuraAkihisa 2P-075,

3P-030KimuraEiji 2S26E-4KimuraJunko 1P-058,

SP-2KimuraKumi 1S6G-3KimuraMasako 1P-185,

2P-211,1S12K-4KimuraShingo 1P-057KimuraSumiko 1P-184KimuraTadashi 2P-121KimuraTomohiko 1P-177KimuraYukiko 2S40G-3KinoshitaKoshi 1P-055KinoshitaYoko 3P-129KinouchiYohsuke 3P-167KirinoYui 3P-120


AUTHOR INDEX

KishiHiroko 1P-177,3P-175

KitaSatomi 1P-058,1S16E-3

KitadaMasaki 1P-195KitagataYuta 1P-038,

2P-088,2P-089KitagawaMichinori2P-199KitagawaYuichiro 1P-157KitaguchiTetsuya 2P-158,

2P-159KitakojiHiroshi 2P-134,

2P-135KitamaToshihiro 2P-111,

2P-114KitamoriKazuya 1P-161KitamuraAkihiko 1P-153KitamuraAtsuko 2P-191KitamuraKenichiro1S3E-4KitamuraMitsuo 2P-178,

2P-179KitamuraTadahiro 1S11J-3KitamuraTaiko 3P-017KitanoTakaaki 3P-106KitaokaKazuyoshi 2P-178,

2P-179KitazawaHiromasa3P-017KitazawaMasahiro 1P-027KitazawaShigeru 3P-034KitoYoshihiko 2S32J-1KittakaHiroki 3P-024KiyonakaShigeki 1S16E-2KiyonariHiroshi 1P-009KoShigeru 2P-201KobaSatoshi 2P-099KobashiMotoi 2P-079,

2P-094KobayashiDaisuke 2P-151,

2P-152,3P-168,3P-180KobayashiKatsunori

1S5F-3KobayashiKazuto 1P-133KobayashiKimiko 3P-192KobayashiKiyoka 1P-156,

1P-157KobayashiMasayoshi

3P-044KobayashiMasayuki

1P-211,3P-049,3P-134KobayashiSei 1P-177,

3P-175,1S12K-4KobayashiSoushi 3O5J-4KobayashiSuguru 1P-088,

1P-115KobayashiTakakazu

1P-129,1P-181,3P-198KobayashiTakeshi 1P-068KobayashiYasushi 1S1A-4Kobirumaki-ShimozawaFuyu

1P-066,1P-067,2P-040KoblingerKathrin 2S38E-3KobyashiYusuke 3P-120KodamaItsuo 1S1A-3KodamaS 1O1H2-2KodaniYu 2P-139,

2S33K-3,3P-171KofujiTakefumi 1P-172,

2P-059KogaKeisuke 3P-008KogaShunsaku 2P-032

KoganezawaTadachika3O7J-8

KogisoHaruka 3P-160KohnoDaisuke 2S35B-2KohnoTatsuro 3P-021KohsakaAkira 1P-040,

1P-043,1P-049,1P-132KohsakaHiroshi 2S40G-2KoibuchiNoriyuki 1P-155,

2P-143,2P-146,2P-173,3P-117

KoikeChieko 3P-002KoiwaiMegumi 2P-152KoizumiHidehiko 3S54C-4KoizumiKyo 3P-203KoizumiSchuichi 3O6F-8KojimaHaruki 1P-078KojimaNaoki 1P-150KojimaToshio 3P-072KojyoKatsura 3P-115,

3P-116KomagataJunya 2P-111,

2P-114,2P-116KomagataSeiji 3P-021KomagiriYou 3P-158KomatsuMasatoshi1P-187KomatsuTakuto 1P-055KomatsuYukio 2S38E-2KomatsuzakiYoshimasa

1P-114KomoriYukako 3S49G-4KomukaiKimiaki 1P-051KomuraYutaka 2S28G-4KondoAyaka 2P-032KondoMasao 3P-205KondoShiho 3P-154KondoShinji 3P-072KondoTakaharu 2P-136,

3P-122KonishiMasato 2P-009,

2P-198KonishiRyoji 2P-042KonishiSeiki 3P-068Konishishiro 1P-086KonnoKohei 1P-133KonnoKohtaro 1P-010KonoHiroki 3P-185KonoRyoma 3P-188KonoYusuke

3P-185KoppensteinerPeter

3S53B-1KoriyamaYoshiki 1P-158,

1P-159,1P-170KosaiKen-ichiro 1LS2EKosakaHiroaki 2P-042,

2P-150,3P-170Koshiba-TakeuchiKazuko

2S41H1-4KoshikawaNoriaki 1P-211,

3P-134KosugiTadayoshi 3P-183KotaniSayumi 3P-194KoyaTomoyuki 1P-183KoyamaMiharu 2P-067KoyamaYoshimasa1P-133,

1P-139,1P-140KozaiDaisuke 1S16E-2KozakiYasuko 1P-161KubaHiroshi 1P-111,

1P-112,1P-113KuboAsako 3P-012KuboKazuhiko 3P-086KuboReika 1P-084KuboTakuya 2P-126KuboToshikazu 1P-147KuboYoshihiro 1P-003,

1P-004,1P-010,1P-026,1P-027,3F1F-3,3S48F-1,1P-022

KubokawaManabu1P-057,2P-156,3P-158

KubokiRyosuke 3P-057KubotaMichinori 3P-081KubotaNaoto 2P-140KubotaTakahiro 1P-214,

2P-076,2P-168,3P-190KubotaYamato 3P-083KubotaYasuo 2P-042KubotaYoshiyuki 3S51J-1KudoFumiyo 1P-152KudoKazutoshi 1P-148KudoRisa 3S45C-4KudohMasaharu 2P-074KumagaiMegumi 1S21J-1KumagayaShun 2P-036KumamotoEiichi 1P-116,

1P-117,2P-012,2P-013,3S54C-1

KumanoYuri 2P-175KumarHarish 3O5J-2KumasakaReon 2S29H1-3KumeShin-ichiro 1P-022KunimatsuAkira 3P-068KuniyaMayu 3O7J-7KuniyasuHiroki 2P-085KunoMiyuki 1P-018KuorseHitoshi 1S17F-2KurachiYoshihisa 1O2J-4,

1P-025,2P-026,2P-030,2P-065

KuraharaHaiLin 2P-137,2S25D1-2,2S31J-1,3S57G-1

KurahashiTakashi 3S48F-3KuraishiYasushi 3P-023KuramitsuSachiko 1P-209KuraokaKoji 3P-059KurataEiko 2S23B-3KurataKiyoshi 3F1F-2KurataYasutaka 2P-028,

2P-044KurebayashiNagomi

2P-023,2P-024,2P-029KurganovErkin 2P-002KuriharaKinji 1P-201KuriharaSatoshi 1P-051,

1P-075KuritaHirofumi 3P-204KuriuToshihiko 1P-086KurodaOKumi 1P-154KurodaShigeru 3S58H1-2KurokawaTatsuki 1P-013,

1P-014,3S48F-2KurokiChihiro 3P-073KurokiK 2P-056KurosawaMieko 1P-076,

2P-181,3S59K-1KuroseMasayuki 1P-126KusakabeGTakehiro

2S40G-1

KusakabeMoriaki 2S31J-2KusakariYoichiro 1P-051,

1P-074,1P-075,2P-040KusakawaAkari 3P-108KusanagiMasahiko2P-045KushiHidehiko 3P-137Kusumoto-YoshidaIkue

1P-137KusuzakiKatsuyuki2P-162KuwabaraYoshihiro

1P-059KuwaharaAtsukazu

2P-132,2P-133KuwaharaKoichiro1P-059KuwaharaYuko 2P-086,

2P-087KuwakiTomoyuki 2P-163,

3P-043,3S52K-2,3S54C-2

L

LamMichelleHoi-Shung1P-188

LeeSyann 2S35B-2LeemChaeHun 2S27F-2LesmanaRonny 2P-146,

2P-173LiAndrew 1S20H2-3LiGuangshuai 1P-018LiGuo-Qing 3P-127LiSui 2P-128LiangNan 1P-036,

1P-047,1P-077,3O4G-1,3O4G-2

LinShih-Tien 3P-195,3P-196

LiuHaixin 1P-137LiuMengchiaNorika

3P-155LiuShuang 2P-210LiuShunyuan 2P-021,

2S43K-3LiuYan-Qing 3P-127LoriAnnBirder 1P-215LuFeng 1P-163LuHaiyan 3S51J-3LUPing 2P-040LuXiaohan 1S20H2-2LuoHuan 3P-001,

3P-015LuoQing-Tian 1P-116

M

MaFangFang 1P-056MaHanlu　　 3P-078MabuchiKaori 1P-146,

3S45C-3MadhyasthaRadha 3O5J-2MaedaHitoshi 1P-100,

1P-103MaedaKazutaka 3P-084MaedaMasanobu 1P-040,

1P-043,1P-049,1P-132,3P-207

MaedaSachiko 1P-068MaedaShogo 3P-190MaedaTakuya 2P-177MaejimaYuko 2P-140,

2S35B-1,2S43K-2MaekawaFumihiko2P-140,


AUTHOR INDEX

2S23B-4MaekawaMasao 1P-089,

2P-060MaenoTakashi 2P-058MaenoYoshimi 3O7J-7MagillJ.Peter 2P-109MajimaHideyuki 3EL3A-1MakinoAyako 3P-197MakinodanManabu2S23B-3MakitaNaomasa 3EL3A-3,

1S1A-2,3S46D1-2,3S46D1-3

MakiyamaTakeru 3S46D1-1,3S46D1-4

MalletNicolas 2P-109MamunAlAbdullah

1S17F-1,2P-047MangoniEMatteo 1P-053ManitaSatoshi 3P-053,

3P-054,3P-071ManiwaKeiichi 3P-022MantokuDaiki 3P-151MargolskeeF.Robert

1S2D1-4MariaLTheodorides

1P-153MarinaNephtali 3S54C-3MarkWSherwood 2S38E-1MaruiShuri 1P-144,

2P-092,3P-093MarunakaYoshinori

1S13K-4,1S3E-2,1S20H2-4,2P-162,2P-206,3P-147,3P-177,3S48F-4

MaruyamaHitoshi 1P-076MaruyamaIkuro 2LS4D1MaruyamaMasugi 3O5J-2MaruyamaSatoshi 2P-171MaruyamaTadashi1P-071MaruyamaTakashi2P-141,

2P-164MaruyamaTokumi1P-163,

2P-042MaruyamaYoshio 2P-016,

3O7J-1MasamizuYoshito 2P-113,

2P-115MasuYujiro 2P-116MasubuchiSatoru 1S21J-3,

2P-184MasudaAtsuko 2P-172,

3P-140MasudaKimiko 1P-056MasudaShinosuke 1P-155MasudaTadashi 2P-196MasukiShizue 3S59K-4MasumotoT 2P-056MasuyamaShigeru 2P-172MatobaMotohiro 1P-209MatsubaraChie 3P-053,

3P-054,3P-070,3P-071MatsudaHiroko 1P-083,

2P-200MatsudaMayumi 1P-144,

2P-092,3P-093MatsudaMitsuyoshi

3P-042MatsudaShohei 1P-139MatsudaTeru 1P-191MatsufujiHiroshi 2LS3B

MatsuiHideki 1P-156,1P-157,2P-056

MatsuiTakuya 2P-184,3P-201

MatsukawaKanji 1P-036,1P-047,1P-077,2S29H1-1,3O4G-1,3O4G-2

MatsukiYuka 1P-019Matsuki-FukushimaMiwako

1P-199,1P-202,1P-206MatsumotoAkihiro1P-130,

1S8H1-4MatsumotoMasayuki

2S28G-2MatsumotoNaoki 2P-093,

2P-095MatsumotoNarihisa3P-057MatsumotoSatoshi 2S31J-2MatsumotoShigeji 2P-080MatsumotoTakaaki2P-167MatsumotoTakashi3P-054,

3P-071MatsumuraHitoshi 3P-151,

3P-152,3P-159MatsunagaShigeki 1P-019MatsunagaTomoko1P-210MatsuoKou 1P-205MatsuoMinako 3P-204MatsuoOsamu 3P-131,

3P-189MatsuoRyuji 1P-207,

2P-079,2P-094MatsuoSatoshi 2P-091MatsuokaHidetada2P-147MatsuokaSatoshi 2S27F-3,

2S34K-4,3P-157MatsuokaTomomi 3P-102MatsushitaAkitomo1P-116,

1P-117,2P-012,2P-013,3S54C-1

MatsushitaMasayuki2P-008,3P-107,3S55D1-2

MatsutaniShinji 1S8H1-1MatsuuraHiroshi 1P-021,

1P-023,1P-024,1P-053,1P-060,1P-061,1P-173,1S17F-5

MatsuuraTakanori2P-141,2P-164

MatsuyamaKiyoji 2P-112MatsuyamaYukie 1P-217,

3P-181MatsuzakaYoshiya2P-117MatsuzakiKentaro 3P-094,

3P-097MatsuzakiMasanori2P-113,

2P-115,3S58H1-5MatuuraTetsuyu 2P-177MawatariKazuhiro1P-170MayanagiTaira 2P-156MazahirHasan 3S58H1-2McAllenM.Robin 3S52K-4McKinleyJ.Michael3S52K-4MemidaHiraku 2P-025,

2S27F-5MengDIan 1P-126MenuetClement 3S52K-1MesircaPietro 1P-053MiakeKiyotaka 2O3G-1MichikawaMakoto 2P-125

MichiueHiroyuki 1P-156,1P-157,2P-056

MidorikawaMitsuharu2P-054

MidorikawaRyosuke1P-007

MiedaMichihiro 3S58H1-2MiharaHiroshi 2P-018,

2P-208,3S57G-2MikaiMasataka 3P-168MikiHiroaki 1S16E-4MikiKenju 1P-044,

1P-045MikiTakafumi 3O6F-1MikiTakashi 1S6G-1MikoshibaKatsuhiko

2S38E-1MikuniTakayasu 1S5F-1MimuraMasaru 2P-066MinYoungKi 3P-095,

3P-096MinamiMasabumi 1S18G-2MinamiTakeshi 3P-181MinamiYoichi 1P-147MinamisawaSusumu

1P-066,1P-074,1P-075,1P-176,1S19H1-5,2P-036,2P-041,2P-093,2P-095,3P-155

MinegishiShintaro 1S3E-3MinematsuAkira 3P-132MinobeEtsuko 2P-020,

2P-021,2S34K-1,2S43K-3MinokoshiYasuhiko1S6G-4,

2P-145,2S31J-3,2S35B-3MisawaHidemi 2O3G-2,

2P-124MiseAyano 3P-114,

3P-115MishimaTatsuya 1P-172,

2P-059MishinaMasayoshi 1P-098,

1P-099,1P-100MisonouHiroaki 2P-062MisumiSachiyo 2P-101,

2P-102,2P-196,3P-062,3P-080

MitohYoshihiro 2P-079,2P-094

MitsudaNoriaki 3P-185,3P-186,3P-187,3P-188

MitsuhashiRyosuke1P-180MitsuiRetsu 2P-033,

2P-037MitsushimaDai 1P-082,

3P-006,3P-063,3P-065,3P-060

MitsuyamaS SP-1MiuraAkira 2P-130MiuraHirohito 1S2D1-1MiuraKouhei 2P-032MiuraMasami 1P-092MiuraYutaka 2P-125MiwaNaofumi 2P-120MiwaYoshihiro 3S58H1-3MiyachiEi-ichi 1P-127,

1S8H1-2MiyagawaToshiaki3P-092,

3P-139,3P-143MiyagawaTsuyoshi3F1F-4,

1P-009MiyajiAkane 1P-048,

2P-138MiyajimaAkiyoshi 3P-197MiyakawaNaohisa 2S42J-4MiyakawaTsuyoshi1P-068MiyakeMasao 1P-208MiyamotoAki 2S28G-4MiyamotoAkiko 3O6F-8MiyamotoOsamu 1P-163MiyamotoToshikazu

3S56E-1MiyamotoYuki 3P-164MiyanariKenji 1P-177MiyanoKanako 1P-209MiyashitaYasushi 3P-068MiyataKohei 1P-148MiyataMariko 1P-104,

1P-105MiyawakiNana 1P-013,

1P-016MiyawakiShouichi 1P-207MiyazakiHiroaki 2P-162,

3P-147,3P-177MiyazakiMariko 1P-217MiyazakiMitsunori3S47E-3MiyazakiShinji 1P-120MiyazakiTadaaki 2P-161MiyazakiTaisuke 3O6F-6MiyazakiTakefumi1P-094MiyazakiToshihiro 3P-051MiyazawaYuta 3P-032,

3P-064MiyazonoSadaharu3P-042MiyazuMotoi 1P-005,

1P-192MiyoshiKazuchika 1S10H2-4MiyoshiTomomitsu1P-123MizoguchiNaoko 3P-049MizokamiTakuya 1S8H1-1MizuhikiTakashi 3P-057MizukamiHiroaki 2P-103MizukamiTomoe 1P-131,

2P-136,3P-122MizukamiYukako 1P-161MizumuraKazue 1P-182,

1P-191,3P-005,3P-012MizunoAkira 3P-204MizunoTomohito 1P-179MizutaKotaro 3P-067MizutamiTakahiro 3P-001MizutaniSatoshi 2P-094MochidaSumiko 2P-051,

2P-052,2P-053MochimaruYuka 3P-154MochizukiAyako 3P-098MochizukiH 1O1H2-2MohammedEChoudhury

3P-115,3P-116,3P-124,3P-125

MohammedMazher3S52K-3MohriSatoshi 1P-069MomiyamaToshihiko

1P-093Momose-SatoYoko 1P-102,

1P-118MoriFutoshi 2P-105MoriHidezo 3O4G-4MoriMichinori 2P-052MoriRintaro 2P-100


AUTHOR INDEX

MoriTomohiro 1P-178MoriXMasayuki 2P-004,

2P-022,2S25D1-2MoriYasuo 2P-003,

2P-004,2P-006,2P-022,2S25D1-3,2S25D1-4,3O6F-1,3O6F-2

MoriYuki 3P-163MoriguchiShigeki 2S23B-1MorimotoKeiko 1P-146,

2P-175,3S45C-3,3S45C-4MorimotoSachio 1P-041MorimotoSatoshi 1P-051MorimotoYuji 1P-091MorishimaMasaki 1P-050,

1P-056MoritaAtsumi 3S59K-4MoritaHironobu 1P-038,

1P-064,2P-088,2P-089,2P-165,2S30H1-4,3O4G-3

MoritaShin-ya 1S13K-2MoritaTakumi 1P-210MoritoYusuke 3P-034MoriuraYoshie 1P-018MoserTobias 2S24C-4MukaiHideo 1P-136MurabeNaoyuki 1P-099,

1P-101,1P-103,2P-055,2P-103

MurakamiManabu 1P-037MurakamiMasataka

1P-071,1P-199,1P-200MurakamiShingo 2P-065MurakamiTomonari

3P-014MurakamiYoshimasa

3P-187MurakoshiHideji 3O6F-8MurakoshiTakayuki

1P-136MuramatsuKen 2P-116MuramatsuShin-Ichi

3P-203MuramotoKazuyo 1P-201,

3P-049MuraokaYoshihiro 3O7J-7MuraseShiori 1P-182MurataAkira 2P-105,

3P-084MurataK. 3P-033MurataMiyahiko 1P-216MurataTakahisa 2S31J-2MurataTakuya 2P-122,

2P-123MurataYoshihiro 3P-038,

3P-041MurataYoshimichi 2P-016,

3O7J-1MurataYumi 2P-104,

2P-107MurayamaMasanori

1P-162,3P-053,3P-054,3P-055,3P-070,3P-071

MurayamaNamie 1S09H1-4MurayamaTakashi1P-184,

2P-009,2P-023,2P-024,2P-029

MushiakeHajime 1P-078,3P-058

MutaKazumasa 2S31J-4

N

NabekuraJunichi 2P-064,2P-085,3O6F-7,3O6F-8,3P-117,3P-119

NabikaToru 1S4E-3NagaiHisashi 3S45C-4NagaiNobuo 3P-189NagaiRyozo 2S39F-3NagaiYuko 1P-038,

2P-088,2P-089NagamineTakashi 1P-088,

2P-112NaganoMasatoshi 1P-097NagaoKenji 2P-123NagaoSoichi 2SL08ENagaoYozoh 1S10H2-4NagaokaShogo 1P-165NagaokaYuya 2P-098NagasakiHiroshi 2P-139,

2S33K-3,3P-171NagaseMasashi 3S53B-4NagashimaKei 1P-144,

2P-092,3P-091,3P-093,3S45C-2

NagataRina 2P-027NagataTomonari 3P-129NagatomoKatsuhiro

1P-006NagayaYoshiaki 1P-169NagayoshiYu 1P-160NaghaviNooshin 3P-092,

3P-139NaitoHisashi 1P-186NaitoTomoyuki 1P-123NaitoYasuhiro 3O7J-3NaitouKiyotada 2P-081,

2P-082,3P-025NakadaTomoaki 1P-213NakadaTsutomu 1P-187NakagawaMasashi 2S40G-1NakagawaOsamu 2P-046NakagawaTakayuki

2S37D1-2NakagawaMachiko2S39F-3NakaharaNaoya 1P-185,

1S12K-4NakahariTakashi 3P-151,

3P-152,3P-159,3P-160,3P-169

NakahataYoshihisa2P-064NakaiAkira 1P-183NakaiGaku 1P-075,

2P-040NakaiJunichi 2P-115,

2S40G-1,3P-053,3P-067NakaiShota 1P-062NakajimaKatsumi 2P-105,

3P-084NakajimaKazuki 2P-149NakajimaKazuyuki1P-168NakajimaSora 2P-131NakajimaTadashi 3S46D1-1NakajimaYuichi 3O5J-2NakajoKeisuke 2P-093NakajoKoichi 1P-022,

1P-026,1P-027NakakukiMiyuki 3P-154NakamaruYuji 1P-151NakamoriHiroyuki 2P-081,

2P-082,3P-025NakamotoMiki 2P-121,

2P-169NakamotoNaoko 3P-145NakamuraC.Kouichi

2P-109NakamuraHiroko 1P-211NakamuraHitomi 2P-121NakamuraKayo 3P-119NakamuraKazuhiko

2S31J-4NakamuraKazuhiro2P-183NakamuraKazuhiro1S18G-1

2S36C-2,3O7J-5NakamuraKazuyoshi

2P-156,3P-158NakamuraKei-ichiro

1P-194,3S51J-2NakamuraKoki 3P-198NakamuraMariko 1P-205NakamuraMariko 3P-183NakamuraShiro 3P-098NakamuraTakami 3P-061NakamuraTatsuro 2S31J-2NakamuraTomoyuki

１S19H1-2NakamuraWataru 1S11J-5,

1S21J-3NakamuraYoshiko 3O7J-5NakamuraYosuke 2P-091NakamuraYuki 3O6F-2Nakamura-MaruyamaEmi

1P-163Nakamura-NishitaniYTomoe

2P-209,2S34K-3,2S43K-1NakanishiMichio 2S29H1-3NakanishiNibuo 1P-201NakanishiShigetada1S18G-3NakanishiTakako 1P-195Nakanishi-MatsuiMayumi

1P-203NakanoRei 3O5J-1NakanoTamami 3P-034NakaoAkito 3O6F-1NakaoKazuki 1P-009NakaoShu 2P-209,

2S34K-3NakaoTomomi 1P-132NakasekoIzumiHiroko

2S42J-3NakashimaAkira 2P-139,

2S33K-3,3P-171NakashimaKeisuke1P-205NakashimaNoriyuki

3P-050NakashimaToshihiro

1P-085NakataniAkira 3P-132NakataniIkuko 3S51J-4NakataniTakeshi 2S29H1-3NakayaMichio 1S17F-2NakayamaAyumi 1S2D1-1NakayamaKiyomi 3P-098NakayamaMasaaki1P-217NakayamaShinsuke3P-205,

2S32J-4NakayamaTomohiro

3O5J-1NakazatoMasamitsu

2S23B-3

NakazawaKazuo 2P-026,3S46D1-3

NakazawaRyoichi 1P-217NambaToshiharu 3P-037,

3P-038NanboAsuka 2P-161NaoyaMatsumoto 3S49G-5Naritakazuhiko 1P-163NaritaKazumi 1P-164,

2P-122,2P-123NaritaShinya 1P-078NaritaTakanori 1P-199,

3O5J-1,3P-153NaruseKeiji 1P-052,

1S19H1-1,2S25D1-1,3P-005

NarushimaMadoka1P-105NasuTeruaki 3P-027NekiDaisuke 1O2J-1NemotoTomomi 3P-200NgatomoKatsuhiro3P-148NigoRyosuke 3P-102NiisatoNaimo 1S20H2-4NiisatoNaomi 1S3E-2,

2P-162,2P-206,3P-177NiizekiKyuichi 1P-046,

2P-097NikiMayu 1S3E-1NikkuniAkihiko 2S28G-4NiliusBernd 2P-014NinFumiaki 1O2J-4,

2S24C-5NinomiyaYuzo 1S2D1-2,

1S2D1-4,1S3E-1NishiEri 3P-035NishiYohei 1S10H2-4NishidaKeigo 1S15D2-4NishidaMotohiro 2S37D1-1NishidaYasuhiro 1P-091,

1P-124,2P-084,2P-171,3P-184,3S49G-1

NishidaYoko 2P-074NishideKohki 1P-055NishieTomomi 2S41H1-4NishiharaTasuku 3P-126NishiiKiyomasa 1S1A-4NishijoHisao 1P-191,

2P-106NishijoTakuma 1P-093NishikawaYasuo 3P-086,

3P-166NishikawaYuri 3P-035NishikiTei-ichi 1P-156,

1P-157,2P-056NishimaruHiroshi 2S40G-4NishimoriAtsuko 3P-102NishimoriKatsuhikio

1P-120NishimotoRei 2P-011NishimotoTakaaki 3P-069NishimuraHironobu2P-180NishimuraKunihiro1P-139,

1P-140NishimuraMasataka

2P-071NishimuraNaoki 1S17F-1,

2P-047,2P-086,2P-087,2P-167,3P-100

NishimuraRumiko 2P-167NishimuraYukako 2P-121,


AUTHOR INDEX

2P-169,2P-170NishimuraYuri 1P-146,

3S45C-3NishinoEri 1P-119NishinoHitoo 2P-106NishinoYuka 1P-060,

1P-061NishioMiki 2P-027NishiokaHaruna 2S35B-4NishiokaRyutaro 3P-114,

3P-115,3P-116NishiyamaA 1O1H2-2,

1P-197NishiyamaFumiaki1S09H1-4NishizakiTomoyuki2P-155,

2P-193,3P-069,3P-146NishizawaSono 1P-183NisimaruNaoko 2S36C-4NiwaFumihiro 2S38E-1NiwaMasatoshi 2P-083,

2P-108NiwataSatoru 3P-20１NodaKazuko 2P-110,

3P-026,3P-075NodaMami 2EL1A-2,

3P-019,3P-163NoguchiChisato 3P-149,

3P-150NoguchiJun 2P-061NoguchiKoichi 3P-192NoguchiTaiji 3P-118NoguchiTeruo 2S29H1-3NoguchiTomohiro 3P-042NoharaKeiko 2S23B-4NojimotoKazutaka 3P-175NomaAkinori 2P-031,

2S27F-1,2S27F-5,2SL09FNomiyaMasanori 1P-215NomuraHiroko 2P-027,

2P-180NomuraNoriko 3P-111,

3P-120NorihisaYamamoto3S49G-5NoseAkinao 2S40G-2NoseHiroshi 3S59K-3NoseKazutoshi 2P-136NozakiKanako 1P-084NozuchiNozomi 1P-060,

1P-061Numaga-TomitaTakuro

1S4E-3NumanoRika 3P-204NumataTomohiro 1S16E-2,

2S25D1-3,3O6F-1,3O6F-2NyamdavaaEnkhjargal

2O3G-3

O

ObataKoji SP-1,1P-038,1P-064,2P-165

ObiKisho 3P-117OdaMasataka 1P-126OdaYoichi 1O1H2-3,

1O2J-1OdagawaMaya 1P-162,

3P-054,3P-055,3P-070,3P-071

OdagiriSaori 3S53B-1OezkucurNurdan 1P-013

OgaiKazuhiro 1P-158,1P-159,1P-170

OgasawaraChie 2P-129OgataGenki 1O2J-4,

1S09H1-1OgataKazue 2P-192,

3P-106OgataMasanori 2P-110,

3P-026,3P-075OgataToru 1P-168OgawaGo 2P-074OgawaYoshihiro 1S6G-2OgawaYui 2P-190OgonIzaya 1P-068OguchiKatsuji 2P-023OguraAkihiko 1P-165OguraK 1O1H2-2OharaHiroki 1S4E-4OharaYuki 1P-121OhashiHiroki 2P-098OhbaTakayoshi 1P-037,

1P-073,3P-121OhbaYusuke 2P-161,

3S58H1-4OhgiKimiko 2P-207Oh-horaMasatsugu1S4E-2OhiraYoshinobu 1P-183,

3SL13EOhishiKazue 1P-071OhkiKenichi 3P-014OhkiTakashi 1P-175OhkidoMakiko 3P-176OhkitaMasashi 2P-015OhkuboFuki 2P-115OhkuboJunichi 2P-141,

2P-164OhkuboNobutaka 3P-185,

3P-186,3P-187,3P-188OhkumaMahito 1S8H1-2OhkuraMasamichi 2P-115,

2S40G-1,3P-053,3P-067OhkuriTadahiro 1S3E-1OhkusaTomoko 1S1A-3OhmoriEriko 3P-155OhmoriHarunori 1P-111,

1P-119,2P-077,2SL06C,3P-050

OhmoriIori 1P-156,1P-157,2P-056

OhmuraAyano 2P-098OhnishiHideo 2P-141OhnoHiroshi 1S8H1-1OhnoMitsuyo 2P-057,

3P-161OhnoNobuhiko 3S51J-3OhnoSeiko 3S46D1-1OhnoTakae 1P-098,

1P-099,1P-100OhnoTetsuo 2P-211,

3P-176OhnoYoshitake 3P-121Ohno-ShosakuTakako

2P-182,3O6F-5OhnukiYoshiki 1P-174OhsakiYasuyoshi 2P-208OhshitaKensuke 1S16E-1OhsimaShinsuke 3P-083OhtaHiroyuki 3P-184,

1P-091,1P-124,2P-084OhtaKeisuke 3S51J-2

OhtaToshio 2P-015OhtakeMakoto 3P-197OhtomoKuni 3P-068OhtsuTeiji 3P-101OhtsuboSena 1P-116,

1P-117,2P-012,2P-013,3S54C-1

OhwadaTomohiko 1S16E-2OiHanako 1S09H1-1OikawaShino 1S17F-3OikawaShota 2P-027OikiShigetoshi 1P-019,

1P-028,1P-029,2SL07D1OishiTakao 3P-072OisoYutaka 2P-139OkabeKoji 2P-008,

2P-207,3O5J-3OkabeMasaru 3P-190OkabeNaohiko 1P-163OkabeShigeo 3S51J-5OkabeTakashi 1S21J-1OkadaJun-ichi 2S39F-3OkadaKiyotaka 3P-131,

3P-189OkadaMayumi 2P-191OkadaNanae 3P-185,

3P-186OkadaTakao 1P-186,

2P-048,3P-164,3P-165OkadaTakashi 2P-115OkadaYasumasa 3O7J-7OkadaYasunobu 1P-032,

2P-153,2S33K-1OkadaYukio 3P-051OkamotoFujio 2P-008,

2P-207,3O5J-3OkamotoShiki 2P-145,

2S31J-3,2S35B-3OkamotoTadao 1P-142OkamotoYasuo 2S41H1-2,

3S49G-2OkamotoYosuke 2P-039OkamotoYuji 2P-054OkamuraJun-ya 3S50H1-3OkamuraYasushi 1P-011,

1P-012,1P-013,1P-014,1P-015,1P-017,1P-020,1P-034,2P-004,2S25D1-2,3S48F-2

OkazakiKayo １S17F-3OkazakiKazunobu 3P-092,

3P-139,3P-143,3S59K-4OkazakiYuka 1S09H1-3OkiKazuma 3P-137OkochiYoshifumi 1P-016,

1P-017OkuShinichiro 2P-010OkuboNaoki 1P-147OkudaHiroko 1P-012OkudaSyunji 3P-132OkuiToshiyuki 3P-097OkumuraSatoshi 1P-174,

3P-156OkunoHirotsugu 2S39F-4OkutaniFumino 3P-038,

3P-045,3P-046Omatsu-KanbeMariko

1P-060,1P-061,1S17F-5OmiKaori 3P-027OmoriEriko 2P-040,

2P-041OmoriKoichi 1P-208OmotoSayo 3S45C-4OmuraSayuri 3O5J-2OnakaTatsushi 2P-142ONDAAkiko 2P-040OnimaruHiroshi 3P-194,

3P-195,3P-196,3S54C-5OnjiHiroshi 3P-188OnoDaisuke 3S58H1-2OnoKatsushige 1O1H2-4,

1P-050,1P-056OnoKentaro 1P-149,

1P-205,1P-209OnoKouki 3P-148OnoKyoichi 1P-037,

1P-073,2P-039,3P-121OnoTaketoshi 1P-191OnoYusuke 3S56E-2OnoderaMiki 1P-046OnoeHirotaka 2P-107OnogiChikao 1P-112OnumaNaoko 3P-137OoharaTakahiro 2S29H1-3OokiMakoto 1S2D1-1OomuraYutaka 2S23B-1OonamiHiroki 1P-140OotsuMao 1P-121OotsukaAyano 1P-120OotsukaYouichirou2P-163,

3S52K-3OouraShunsuke 1P-087OrieKoga 2S27F-3OsadaKazumi 3P-042OsagawaSatoshi 1P-050OsakaToshimasa 3P-104OsakoYoji 3P-091OsamuTasaki 3S49G-5OsanaiHisayuki 3P-200OsanaiMakoto 1P-008,

1P-078,3P-162OshioKen-ichi 3P-082OshitaKensuke 1P-059OtaAkira 2P-139,

2S33K-3,3P-171OtaKeisuke 3P-055,

3P-071OtaNaomi 2P-077OtaniHidenori 3P-141OtsuAyaka 2S41H1-4OtsukaAiri 1P-143,

3P-088OtsukaWakako 2P-126OtsukiLucia 2P-151OuraKanna 1P-143OwadaYuji 3P-006OyaManami 2P-158,

2P-159OyamaKotaro 1P-066,

1P-067,1P-175,2S26E-2OyamadaHideto 2P-023OzakiMakoto 1P-144OzawaKeiya 2P-103

P

PartidaGloria 1S09H1-1PascaleGuicheney 3S46D1-1PearsonJames 2P-043PengjamYutthana 3O5J-2


AUTHOR INDEX

PiaoHulin 2S25D1-1PicheM. 3P-056PittmanJQuentin 2S38E-3PrasedyaSunarwidhiEka

1P-208

Q

QinLin 　　 3P-078

R

RansohoffMRichard3S51J-3

RedgravePeter 2O3G-4ReichenbachTobias2S24C-5RitaSukmaRauza 2P-148RossantJanet 2S41H1-4RuanXiong,Zhong 1S20H2-3RuangkittisakulAraya

3P-191

S

SadakiyoKaori 1P-076SadamotoHisayo 1P-086SaekiYasutake 1P-174SagaraHironori 3P-193SaharaYoshinori 1P-203,

3P-039SaikiChikako 2P-080SairenjiTaku 1P-155SaitoAiko 3P-031SaitoHiroyuki 2P-186,

2P-185,2P-187SaitoKazuto 3LS7ESaitoMasahisa 1P-214,

2P-076,2P-168,3P-190SaitoMinoru 1P-114SaitoShigeru 2P-001,

2P-002,2P-015SaitoT.Claire 2P-007,

2P-015,3P-024SaitoTakehiko 1S09H1-4SaitoToshiyuki 3P-087SaitoYuki 1P-145SaitohTadashi 1P-046,

2P-097SaitowFumihito 1P-096,

1P-097,3S44B-1SajiMakoto 2P-110SakabaTakeshi 2P-054SakagamiMasamichi

3P-066SakaguchiHirofumi2S24C-3SakaguchiShota 3S58H1-3SakaiAtsushi 3S44B-1SakaiHideki 1P-032,

1S13K-3,2P-014,3O7J-2SakaiHiromu 1P-018SakaiKazuyoshi 1P-127SakaiShigekazu 1P-191SakaiTetsuro 1P-065SakakibaraKazumasa

2P-106SakakibaraNorikazu

1P-163,2P-042SakakibaraYoshikazu

2P-172,3P-140SakamotoAkemi 3P-189SakamotoKazuhiro2P-117,

3P-058

SakamotoKazuho 2P-205SakataSouhei 1P-013,

3S48F-2SakataSusumu 3P-132SakaueYuko 1P-173SakimaMiho 1S17F-1,

2P-047SakimotoYuya 1P-082SakimuraKenji 1P-016,

1P-145,3O6F-4,3O6F-6SakoNoritaka 3P-135SakumaKazuya 2S35B-1SakumaTetsushi 1S7G-2SakuragiSokichi 2P-131SakuraiHiroki 3P-031SakuraiKaoru 1P-217SakuraiMasaki 1P-098,

1P-099,1P-100,1P-101,1P-103,2P-055,2P-103

SakuraiTakashi 2P-023,2P-024

SakuraiTakeshi 1P-138,1P-145,1P-150

SakuraiYuko 3P-126SamiosNikolaosVinicius

1P-108SamukawaKeiichi 3P-185SanadaMasumi 2P-059SanadaTadashi 2S39F-4SanematsuKeisuke1S3E-1SanoHideto 2S41H1-3SanoIHitomi 3O7J-3SasaiNobuaki 1P-179SasaiYoshiki 2P-139SasajimaHitoshi 3P-042SasakiAyako 3P-148SasakiEiji 3P-018SasakiHiroyuki 3P-099,

3P-101SasakiMakoto 3P-039SasakiSei-Ichi 2P-083,

2P-108SasakiTakehiko 2P-161SasakiTakeshi 2P-112SasakiTsutomu 1S11J-3SasamotoKouhei 2P-206,

1S20H2-4SaskiJunko 2P-161SataYusuke 2S30H1-3SatakeShin’Ichiro 2P-063SatoAkira 3P-072SatoEiki 2P-117SatoFumitaka 2P-100SatoHiromichi 1P-123SatoItaru 3P-197SatoJun 3P-031SatoKaito 3P-121SatoKaoru 1P-121SatoKatsushige 1P-102,

1P-118,3S50H1-1SatoMaki 2P-047,

2P-087,3P-100SatoMasaaki 2S26E-1SatoMasaaki 3P-053,

3P-067SatoMasahiro 1S10H2-4SatoMasaki 1O1H2-2,

1P-197,1P-198,1P-199SatoMasaru 1S5F-5SatoMasatoshi 3P-186

SatoMotohiko 1S17F-1,2P-047,2P-087,2P-167,3P-100

SatoNobuo 1P-144,2P-092,3P-093

SatoOsamu 1S12K-2SatoRei 2P-037SatoSo 1P-208SatoTadasu 2P-079SatoTakao 2P-189SatoTakayuki 1S17F-3SatoTakehito 3S49G-4SatoToshiya 1P-196,

1P-204SatoYoko 3S50H1-1SatoYoshiaki 1P-091SatoYu 2P-072SatohHiromasa 1P-096SatohKeitaro 3P-153SatohYoshihide 1P-039,

1P-212,2P-127Sato-NumataKaori 2P-153,

2S33K-1SatouChie 2S40G-3SawadaNorifumi 1P-215SawadaWakako 2P-057,

3P-161SawaiHajime 1P-123SawanoMakoto 3P-122SchlaichMarkus 2S30H1-3SchwartzJPeter 3S46D1-1ScottK.Jennifer 1S2D1-1SeiHiroyoshi 1P-143,

2P-176,3P-088SeiyamaAkitoshi 1P-131,

3P-122SekiAkiko 1S1A-2,

1S1A-4SekiJunji 1P-131SekiMasafumi 3S49G-5SekiYoshinari 3P-179SekiguchiMasayuki1P-081,

3S53B-1SekineYukiko 3P-067SekinoYuko 1P-121,

2S42J-5SekiokaAkihiro 3P-125SeoEriko 1P-071SeoYoshiteru 1P-071,

2P-119,3P-153SeraMayuko 1P-159SeradaNoriyuki 1P-195SetogawaTsuyoshi3P-057ShaoDong-Xue 2P-020SharottAndrew 2P-109ShiXiuyu 3O6F-2ShibaYoshiki 2P-199,

2S36C-3ShibamotoToshishige

2P-028,2P-044,2P-078ShibasakiKoji 3O6F-3ShibataHideshi 2P-181ShibataKeisuke 3O6F-8ShibataMinoru 3P-022ShibataShigehiro 1P-073ShibataShigenobu 3P-099,

3P-101ShibataYosaburo 1S1A-4ShibukawaYoshiyuki

1P-197,1P-198,1P-199

ShibukiKatsuei 1P-110,1P-128,2P-073,3P-021,3P-022,3P-083,3S50H1-4

ShibuyaMasabumi 2S41H1-4ShidaraMunetaka 3P-057ShidoOsamu 3P-094,

3P-097ShigaMari 3P-192ShigemuraNoriatsu1S2D1-2,

1S2D1-4,1S3E-1ShiinaTakahiko 2P-081,

2P-082,3P-025ShimaTakeshi 3P-025ShimadaYouichi 3P-206Shimada-ShimizuNaoko

2S43K-1ShimamoChikao 3P-152,

3P-160ShimazakiTakashi 1O1H2-3ShimboTmonori 3P-121ShimegiSatoshi 1P-109,

3P-011ShimizuHiroshi 1P-072ShimizuNoriyuki 1P-143,

2P-176,3P-088ShimizuShuji 1P-041,

2S30H1-1,2S30H1-3ShimizuShunichi 2S25D1-3,

2S25D1-4ShimizuTakahiro 1P-032,

1S13K-3,2P-014,3O7J-2ShimazuTakeshi 3S49G-5ShimizuYasutake 2P-081,

2P-082,3P-025ShimizuYuko 2P-101,

2P-102,3P-062ShimizuYuuki 2P-047,

2P-086,2P-087,3P-100ShimodaTakefumi 3P-110,

3P-111ShimodouzonoMegumi

1LS1D1ShimojuRie 1P-076,

2P-181,3S59K-1ShimokawaNoriaki1P-155,

2P-146,2P-173,3P-117ShimomuraHideki 3P-192ShimomuraKenju 2S35B-1,

2S43K-2ShimonoKen 3O6F-1ShimouchiAkito 1P-131,

2P-136,3P-122ShimozawaTogo 1P-066ShimuraDaisuke 1P-074,

1P-075,2P-040ShimuraTsuyoshi 2S35B-4,

3P-007,3P-089ShinYoungOh 3P-095ShinbaraHisashi 2P-129ShinboTomonori 1P-073ShinodaMasamichi 3P-009,

3P-028ShinodaYoshikazu 1O2J-2,

3SL10BShinoharaKazuyuki3P-076ShinosakiKazuhiro 3P-077ShinozakiRina 1P-136ShintaniA.Seine 1P-067,

1P-175,1P-176,2S26E-2ShionoHiroyuki 2P-184,


AUTHOR INDEX

3P-20１ShiotaKohei 3P-110,

3P-111ShioyaTakao 2P-029,

2P-157ShiraiMikiyasu 1P-041,

1P-045,1P-050,1P-063,1P-131,2P-035,2P-043,2P-049,2P-050,3P-122,3P-138,2P-136

SHIRAISHIRyosuke2P-040

ShiraiwaYuka 1P-214,2P-076,2P-168,3P-190

ShirakawaHideki 3P-173,3P-174

ShirakawaHisashi 2S37D1-2ShirakawaTetsuo 3P-028ShirakiMotoyuki 2P-106ShiraoTomoaki 1P-121ShiratoKen 1P-180,

3P-144ShiromotoTakashi 1P-163ShiuchiTetsuya 1P-143,

2P-176,3P-088ShojiKazuyo 2P-042ShozibHabibulBari2S32J-4ShutoYachiko 2P-192ShutohFumihiro 3P-079SimizuYuko 3P-080SobueKenji 2P-156SohmaYoshiro 1S10H2-3,

2P-201SokabeMasahiro 3P-169,

3P-178SomaShogo 1P-109,

3P-011SomeyaNami 1P-152,

2P-130SoneHideyuki 2S35B-2SongWen-Jie 2P-070,

2P-071,3P-001,3P-015,3S50H1-2

SonobeTakashi 2P-035,2P-043,3P-138

SonomuraTakahiro3S51J-4SoyaM 1O1H2-2SoyaShingo 1P-138,

1P-150Stary-WeinzingerAnna

1O1H2-4StewardMartin 2P-201StoraceDoug 3SL15H1StradleighTyler 1S09H1-1StriessnigJoerg 1P-053SuematsuNaofumi 1P-109,

1P-123,3P-011SuezumiKoki 3P-106SugaHidetaka 2P-139,

2S33K-3SugaHiroki 2P-186,

2P-187,2P-191SugaMayu 1P-207SugaSechiko 1P-006SugamaShuei 3P-105SuganamiTakayoshi

1S6G-2SuganoEriko 1S09H1-4SugawaraYuto 2P-182SugayaYuki 3O6F-4

SugenoyaJunichi 2P-087,3P-100

SugetaShingo 1P-079SugiHaruo 1P-181SugimachiMasaru 2S30H1-1,

2S30H1-3SugimotoKana 3P-108,

3P-109,3P-110,3P-111,3P-113,3P-114,3P-115,3P-116,3P-120,3P-123,3P-124,3P-125,3P-130

SugimotoKoji 3P-079SugimotoMariko 3P-032SugimotoShunji 3P-081SugimuraKaitoYae1P-080SugitaMakoto 2P-199,

2S36C-3SugitaRiko 2P-081SugitaniKayo 1P-158,

1P-159,1P-170SugiuchiYuriko 1O2J-2SugiuraMidori 2S42J-5SugiuraSeiryo 2S39F-3SugiuraTakao 1P-183SugiyaHiroshi 3O5J-1,

3P-153SugiyamaToshiro 2P-018,

3S57G-2SugiyamaYuka 3S58H1-3SuiLi 2P-197SuitaKenji 1S17F-4,

3P-156,3P-156SukeguchiChie 1P-044SuminoAyumi 1P-029SumiyaEiji 2P-129SumiyoshiMiho 2P-137,

2S31J-1SunDeri 2P-210SunLingling 2P-044SunWuping 2P-017SunXuefei 2P-020,

2P-210SunYu 2P-020SunagawaMasanori3P-183SunagawaMasataka

1P-215,2P-187,2P-189,2P-191,3P-033,3P-127

SungUhna 3SL15H1SuwabeTakeshi 3P-135SuyamaShigetomo 2P-140SuzukiAkina 3P-092,

3P-139,3P-143SuzukiAzumi 3P-028SuzukiEtsuko 1S14D1-3SuzukiHaruhiko 2S32J-4SuzukiHideki 1P-180SuzukiHidenori 1P-096,

1P-097,3S44B-1SuzukiHiroko 1S17F-1,

2P-047SuzukiHiroshi 2S39F-2SuzukiJunji 2P-023,

2P-024SuzukiMadoka 2S26E-2,

2S26E-3SuzukiMayuko 3P-085SuzukiNaoya 2P-068SuzukiTakayuki 1P-162,

3P-054,3P-070,3P-071SuzukiYoji 3P-185,

3P-186,3P-187,3P-188SuzukiYoshiro 2P-007,

2P-017SuzukiYuko 2S41H1-3SuzukiYutaka 2P-111

T

TabataKitako 1S09H1-4TabiraKazuyuki 3P-143TachibanaMasao 1P-130,

1S8H1-4TachiyashikiKaoru1P-180TagawaMasami 3EL4A-1TagawaYoshiaki 3P-014TaguchiAkiko 2S23B-3TaguchiIsamu 2P-100TaguchiMika 1S12K-4TaguchiToru 1P-178,

1P-191,3O7J-4TaharaYu 3P-099TaiShinobu 2P-198TajiriTkaaki 3P-182TakadaYoshihiro 3P-132TakadaYoshinori 2P-022TakadaYuichi 3P-175TakagakiRyodai 1P-177TakagiNoriaki 2P-181TakagishiMiwa 1P-040,

1P-043,1P-049TakagoHideki 2S24C-4TakahashiHirokazu3S53B-3TakahashiHisaaki 3P-108,

3P-109,3P-110,3P-111,3P-112,3P-113,3P-114,3P-115,3P-116,3P-120,3P-123,3P-124,3P-125,3P-126,3P-128,3P-130

TakahashiIchiro 2P-055TakahashiKazumi 1P-133TakahashiKen 2S25D1-1TakahashiKuniyuki3P-083TakahashiMaki 1S09H1-4TakahashiMayu 1O2J-2TakahashiNobuaki 1S16E-2,

2P-003TakahashiNobuyuki

2P-017TakahashiNoriko 2P-057,

2P-061,3P-161TakahashiSugata 3P-083TakahashiTeppei 3P-133,

3P-181TakahashiTomoyuki

1S14D1-1TakahashiTomoyuki

2P-100TakahashiWaka 3S49G-3TakahashiYasuhito1P-217TakahashiYukari 1P-080,

3P-032,3P-064TakahashiYuko 3P-169TakaiAkira 1P-005,

1P-192,1S12K-2TakaiShingo 1S2D1-2,

1S2D1-4,1S3E-1TakakiAkiko 3P-085TakakiMiyako 1P-064,

2P-085,SP-1TakakuwaNorihiro2O3G-4

TakamataAkira 1P-146,2P-175,3S45C-3,3S45C-4

TakamatsuKen 1P-168,2P-120

TakamatsuYasuyuki3P-118

TakamiyaKogo 3P-199TakamoriShigeo 1S5F-4TakamotoKouich 1P-191TakanariHiroki 1O1H2-4,

1P-056TakanashiYurie 1P-155TakanoHiromichi 1P-042,

2P-034,2P-037TakanoMakoto 1P-030,

1P-031,1P-059,1P-194,1S16E-1

TakanoYukio 3P-019TakaoTomoka 2P-208TakaokaYutaka 3S56E-3TakaseMiki 1S5F-4TakashimaMasashi2P-189TakashinaTerue 3P-137TakasuEtsuko 2S40G-2TakataMaki 2P-042TakataNorio 2P-066TakatsuSatomi 3P-005TakatsuruYusuke 2P-143,

2P-173,3P-117TakayamaYasunori2P-195TakayanagiRyoichi2S31J-4TakayanagiYuki 2P-142Takazawakenji 2P-045TakechiKana 3P-187TakedaAtsushi 1S15D2-2TakedaKotaro 3O7J-7TakedaRyosuke 3P-092,

3P-139,3P-143TakedaShinichi 2S37D1-4TakedaYukari 2S27F-1,

2S27F-1,3P-002TakeiLeonGen 2P-119TakemasaTohru 3S47E-1TakemoriShigeru 1P-185,

2P-023,2P-211,3P-142,3P-176,1S12K-4,3S56E-4

TakemotoMakoto 2P-070TakemotoMariko 2P-191TakemotoYumi 2P-090TakeoJiro 1P-081TakeshimaHiroshi 2S34K-2TakeshimaNobuo 2EL2A-2TakeshitaDaisuke 3P-132,

SP-1TaketoMegumi 1P-083TakeuchiAyako 2S34K-4,

3P-157,2S27F-3TakeuchiHiroko 3S48F-3TakeuchiJun 2S41H1-4,

2S41H1-5TakeuchiKazuhiko1P-058TakeuchiKazuhiko3P-044TakeuchiKouzou 1S09H1-3TakeuchiYuichi 1P-104TakewaYoshiaki 1P-063TakeyaKosuke 1P-192,

1S12K-2TakeyaMitsue 2EL1A-1,

1P-194TakigawaShota 3P-148


AUTHOR INDEX

TakumiToru 1S21J-5,3S44B-４

TakuwaNoriko 2S41H1-2,3S49G-2

TakuwaYoh 2P-042,2S41H1-2,3S49G-2

TamagawaTakaaki3P-009TamaiMakoto 1S09H1-4TamakiAkira 3P-141TamakiMisako 2P-188,

2P-190TamakoshiKeigo 3P-118TamaluFuminobu 1P-107TamariKengo 3P-044TamaruTeruya 1S21J-2TamiyaJunko 2P-080TamuraAtsushi 1P-008TamuraNoriyuki 3P-189TamuraRisa 1P-091TamuraTakashi 3P-069TamuraTakumi 3P-145TamuraTetsuya 1P-169TamuraYukinori 3P-131TanMoutou 3P-174TanakaAoi 2P-175TanakaEtsuro 3O4G-4,

3O7J-6TanakaF.Kenji 3O6F-3,

1S7G-3TanakaHYasuyo 2P-113TanakaHiroki 1S09H1-3TanakaJunko 3S58H1-3TanakaJunya 3P-108,


TanakaKenji 2P-066TanakaKohichi 1S7G-2,

1S7G-4TanakaKunihiko 2P-096,

2P-165TanakaM SP-1TanakaMasaki 2S28G-5TanakaMichiko 3P-140TanakaMutsumi 3S52K-4TanakaRYasuhiro2P-113TanakaSaori 3P-151,

3P-152,3P-159,3P-160TanakaSayoko 1S22K-3TanakaShingo 3P-066TanakaYoshimasa 2S31J-4TanakaYuichi 1P-114Tandai-HirumaMegumi

1P-124,2P-084,3S49G-1TaniMariho 3P-195TanidaMamoru 2P-028,

2P-044,2P-078TanifujiShota 2P-051,

2P-052,2P-053TaniguchiHiroki 3P-060TaniguchiHiroshi 2P-129,

2P-134,2P-135TaniguchiItsuka 3P-154TaniguchiKentaro 1P-131,

3P-122,2P-136TaniguchiMutsuo 3P-037,

3P-038TaniguchiS SP-1

TaniguchiSazu 2P-129,2P-134,2P-135

TanimuraAsami 3O6F-6TanizawaTakakuni3P-192TanjiJun 1SL03FTaoShengChen 2S34K-2TarunoAkiyuki 3S48F-4TasakiOsamu 3S49G-5TashiroAkimasa 2P-084,

3P-184TashiroMichiko 2P-198TashiroShogo 3P-043,

3S52K-2TateokaTakashi 2P-133TateyamaMichihiro1P-003TatsumiEisuke 1P-063TawaratsumidaY. 3P-033TazakiMasakazu 1O1H2-2,

1P-197,1P-198TazumiShoko 3S45C-4TazunokiShun 2P-072TehBoonLoongDaniel

2P-019TeradaMasaki 3P-074,

3P-077TeradaMiki 2P-131TeradaTomoyoshi 1P-217,

3P-133,3P-181TerawakiHiroyuki 1P-217TeruiNaohito 3P-027TeruiTakako 1P-066TestuyaKushikata 1P-037TheodoreMNelson

1P-153TkahashiAkira 3P-167TodaChitoku 1S6G-4TodaKazuo 3P-051TodaMayumi 3P-031TodaSatoshi 3P-075TodaTakuya 3P-118TodorokiKikue 3O4G-4,

3O7J-6TohmiManavu 1P-128TohseNoritsugu 1P-068TokiTamami 3O7J-3TokudaMasaaki 2P-128,

2P-197,3P-149,3P-150TokuhisaTakeshi 3P-189TokumaruOsamu 2P-192,

3P-035,3P-048,3P-106TokunagaRyota 2P-181TombazTuce 3SL15H1TomidaMihoko 3P-029,

3P-061TominaHiroshi 1S09H1-4TominagaMakoto 2P-001,

2P-002,2P-006,2P-007,2P-010,2P-011,2P-015,2P-017,2P-018,2P-195,2P-208,3P-024,2S26E-5,2S37D1-5,3S57G-2

Tominaga-YoshinoKeiko1P-165

TomitaAkiko 1P-020TomitaItsuka 2P-095TomitaMasaru 3O7J-3TomizawaKazuhito1P-160,

2P-126,2P-144,3P-001,3P-172,3P-182

TomomuraAkito 1P-201

TomonariHiroshi 1S2D1-1TomonoKazunori 3S49G-5TongJia 3P-045TooyamaIkuo 1P-173ToriyaMasako 2S23B-4TorresAJorge 2P-205ToshimaHiroko 2P-093,

2P-095ToyaSyutaro 2P-143ToyodaFumiyo 1P-213ToyodaFutoshi 1P-023,

1P-053ToyokuniShinya 3P-118TrappDBruce 3S51J-3TrevorPowell 2S27F-5TrillerAntoine 2S38E-1TshiroAkimasa 1P-124TsuboiTakashi 2P-158,

2P-159TsubosakaMiku 3P-101TsubotaYuji 3P-145TsuchimochiHirotsugu

1P-045,1P-050,2P-035,2P-043,3P-138

TsuchiyaAyako 2P-155,3P-146

TsuchiyaKohichiro3P-167TsuchiyaSaki 3P-035TsuchiyaTeizo 1P-179TsudaMakoto 3P-008TsudaMasayuki 1S17F-3TsudaMasumi 2P-161TsudaYasumasa 3P-006,

3P-065TsujiTadataka 3P-047TsujiYukiomi 3S46D1-2TsujimtoHisaya 1P-180TsujinoNatsuko 1P-145TsujitaJunzo 3P-141TsukadaKazuhiro 3O7J-2TsukaharaReiko 2P-086TsukamotoIkuko 1P-163,

2P-042,2P-197TsukamotoSeiichi 1P-067,

1P-176TsukanoHiroaki 1P-128,

2P-073,3P-021,3P-022,3P-083

TsukitaSohei 3S53B-2TsumotoKunichika2P-026,

2P-030TsumuraM 1O1H2-2,

1P-197TsumurayaTomoyuki

2P-008TsunematsuTomomi

1S11J-4TsureishiAkihiro 3P-198TsurudaKeiko 2P-090TsushimaEikichi 1P-055TsutajimaJyoji 3P-199TsutsuiHidekazu 1P-020,

1P-034TsutsuiHiroshi 2P-097TsutsuiKazuyoshi 1P-213TsuzukiChizuru 1P-025TurnerMichaelJames

2S30H1-1

U

UchidaKunitoshi 2P-017,3P-024,2S26E-5

UchidaSae 2O3G-2,2P-124,3P-056

UchidaYuki 3P-091UchigashimaMotokazu

1S5F-1UchihashiKenji 3P-166UchimuraMotoaki 3P-034UchimuraNaohisa 3EL3A-2UchiyamaAsako 2S42J-2UchiyamaHiroyuki1S8H1-1UchiyamaTsuyoshi3P-205UedaHirotaka 1P-207UedaRika 1P-085UedaYoshitomo 2P-101,

2P-102,2P-196,3P-080UedaYuhki 2P-194UeharaAkira 2P-029UenoKaori 2P-199UenoKazumi 3O4G-2UenoMunehisa 1S21J-1UesakaNaofumi 1S5F-1,

3O6F-6UeshimaShigeru 3P-189UesugiKen 1P-075,

2P-040UetaYoichi 3EL1A-3,

2P-141,2P-153,2P-164,2S33K-2

UetsukaSatoru 1O2J-4UeyamaTakashi 3S45C-1UezonoYasuhito 1P-209UjiharaIzumi 1P-149UjiharaYoshihiro 1P-069UmedaKazuyoshi 1P-167UmekiSayaka 3P-027UmemuraMasanari1S3E-3,

3P-197UmetsuRena 1P-161UrakawaSusumu 1P-191,

2P-106UrakuboTomoyoshi

1P-165UranoTetsumei 2S41H1-3UrashimaTakashi 1P-075UsamiKenichi 3S53B-3UsamiTakako 1S7G-2UshidaTakahiro 3P-031UshijimaKazuo 1P-059UtaDaisuke 2P-167,

2P-194,2P-195,3P-019,3P-023

UtsugiChizuru 3P-042

V

vanderHeydenMarcel1O1H2-4

W

WadaAiko 3P-123WadaKeiji 1P-081,

3S53B-1WadaMakoto 3P-085WadaSatoshi 3P-093WakabayashiHidetaka

3S59K-4


AUTHOR INDEX

WakabayashiShigeo1P-002,2P-209,2S34K-3,2S43K-1,3O5J-4

WakabayashiYuji 2P-149WakamoriMinoru 3O6F-1WakanaNoriaki 3O7J-6WakasugiKeisuke 1P-159WakazonoYoshihiko

1P-007,3P-199WakeHiroaki 2P-113,

3P-119WakiHidefumi 1P-040,

1P-043,1P-049,1P-132,3P-132

WamsteekerCusulinIJaclyn2S38E-3

WangBing 2P-208WangChi 2P-070WangFei 1S20H2-2WangGang 3S50H1-3WangJing 2S25D1-1WangMofei 2P-044WangYan 1P-056WasedaYuya 3P-118WashioHiroe 3P-132WashioTakumi 2S39F-3WatabeMAyako 1P-080,

1S5F-5,3P-064,3S53B-4WatanabeDaishi 2P-189WatanabeEizo 3S49G-3WatanabeHiroshi 1P-058WatanabeHiroyuki1P-037WatanabeKazuto 1P-135WatanabeKenji 1P-110WatanabeMakino 2P-048WatanabeMasahiko1P-010,

1S5F-1,3O6F-6WatanabeMasahiro2S39F-3WatanabeMasaki 2P-007WatanabeMasaru 1S12K-1WatanabeNobuhiro3P-056,

3P-010WatanabeOsamu 2LS5EWatanabeSachiko 2P-032WatanabeSaori 2O3G-2,

2P-124WatanabeSatoshi 1P-090,

2P-057,2P-061,3P-161WatanabeSayaka 2P-144WatanabeShiori 2P-111WatanabeShu-Ichi 1P-107WatanabeTae 1P-077,

3O4G-2WatanabeTakaki 1O1H2-3WatanabeTatsunori

3P-021WatanabeTatsuo 2P-099WatanabeYasuhide1P-058WatanabeYoshio 3P-193WatayaTakafumi 2P-139WeiFan-Yan 1P-160,

2P-126,2P-144,3P-001,3P-172,3P-182

WeiFei 1P-199,1P-200

WeiJia-zhang 1P-058WhelanJPatrik 2S38E-3WhiteMorris 2S23B-3WiersmaJelle 1P-126Wolf-JohnstonAmanda

1P-215WookKimTae 3P-096

X

XieYu 1P-021,1P-023,1P-024

XuJianjun 2P-020,2P-021,2S34K-1,2S43K-3

XuMing 2P-066XuZhi-Hao 1P-117,

2P-012,2P-013,3S54C-1

Y

YadaTakeshi 3P-048YadaToshihiko 2P-140,

2P-148,2S23B-2,2S23B-4,2S35B-1,2S43K-2

YagasakiYuki 3P-129YagiKohei 3P-058YagiTakakazu 1P-207YagiTakeshi 1P-110,

1P-128YagiTetsuya 1S09H1-3,

2S39F-4YagishitaSho 1P-090YagitaKazuhiro 1P-147,

1S11J-1,1S21J-3YaguchiHaruna 3P-110,

3P-111YaguchiYuichi 1P-008YamadaDaisuke 1P-081,

3S53B-1YamadaJinzo 3P-017YamadaKatsuya 1P-006,

3P-148YamadaKazuhiro 3P-073YamadaKazuyuki 1P-162,

3P-054YamadaKuniya 3P-106YamadaMisato 3P-168YamadaMitsuhiko 1P-187YamadaNaohiro 1P-008YamadaRei 1P-111,

1P-112,1P-113YamadaTetsuya 3S53B-1,

3S53B-2YamadaYoichi 1P-068YamagataYoko 1P-166YamagishiMasakazu

3S46D1-1YamagishiTatsuya3P-083YamaguchiAoi 1P-038YamaguchiErina 2S35B-4,

3P-089YamaguchiFuminori

2P-128,2P-197,3P-149,3P-150

YamaguchiJunya 2P-064YamaguchiKaori 2P-003YamaguchiKazuhiko

1P-095YamaguchiKazuma2P-022YamaguchiKen’ichi1P-167YamaguchiKiichiro1P-209YamaguchiMaki 2P-211,

3P-176YamaguchiMakoto2P-136,

2P-201,3P-154YamaguchiRie 2P-181

YamaguchiTakashi1S18G-3YamaguchiYuji 1P-048YamajiJunko 1P-214,

2P-076,2P-168YamajiTashiroJunko

3P-190YamamotoAkiko 2P-201,

3P-154YamamotoDaisuke1P-029YamamotoHiromasa

3P-176YamamotoHiromi 2S30H1-2YamamotoHiroyuki1P-179YamamotoIzumi 1P-010YamamotoKuniyo 2S36C-3YamamotoMegumi2P-121,

2P-169YamamotoNorihisa3S49G-5YamamotoNoriyuki2P-166,

3P-136YamamotoSachiho 2P-175YamamotoTakashi1S7G-1,

1S7G-2YamamotoTakashi1SL02D1,

3P-047YamamotoTatsuya2P-107YamamotoTomomi1P-009,

1P-010YamamotoYo 3S51J-4YamamotoYoshimichi

2P-034YamamotoYoshio 1P-006YamamotoYui 3P-006YamamotoYuki 2P-044YamamuraAya 1P-161,

2S37D1-3YamamuraHisao 2S37D1-3YamamuraKensuke

1P-126YamanaHisako 1S3E-3YamanakaAkihiro 3EL4A-2,

3P-054,3P-071YamanakaYuki 1P-179YamaokaHiroyo 1P-054YamaokaHiroyo 2S27F-5YamaokaSeiya 1S8H1-1YamasakiMasao 2P-027,

2P-180YamasakiMaya 3O6F-6YamashinaYoshihiro

3P-092,3P-139,3P-143YamashitaHaruyoshi

3P-022YamashitaHayato 1S10H2-3YamashitaHiroshi 2S39F-3YamashitaKanna 1P-058YamashitaKenya 3P-079YamashitaMasayuki

1P-035YamashitaTetsuo 2P-042,

2P-150,3P-170YamatoTakako 3P-102YamauchiHideki 3P-142YamauchiMasateru2P-106YamauchiSatoshi 3P-186YamazakiDaiju 2S34K-2YamazakiDaisuke 1S16E-4YamazakiFumio 3P-103YamazakiHiroyuki 1P-121YamazakiMaya 3O6F-4

YamazakiTakashi 2S39F-3YamazawaToshiko2P-023,

3P-176YanagawaYuchio 1P-166,

1S5F-4,3P-162,3P-203,3S44B-1,3P-070

Yanagi(Ishihara)Keiko1P-059

YanagidaMitsuhiro1SL04AYanagidaYurie 1P-129YanagisawaMasashi

3S52K-3YanamakaAkihiro 1S11J-4YanaseMasanobu 2S29H1-3YangHunMo 3P-095,

3P-096YangTianxin 1S20H2-2YanoHajime 3P-108,


YanoMasato 3P-131,3P-189

YasoshimaYasunobu2S35B-4,3P-089

YasuharaOsamu 1P-173YasuiMasato 1S10H2-3YasuiToshihide 3P-132YasukochiMidori 2P-029YasumatsuKeiko 1S2D1-4,

1S3E-1YasuoToshiaki 3P-135YasuokaYukiko 1S10H2-1YawoHiromu 2P-019,

3P-003,3P-203YinChengzhu 1P-122YokawaT. 3P-056YokoeTakuya 3P-193YokoiIsao 2P-192,

3P-035,3P-048,3P-106YokoiMari 2P-172YokoiMotoo 3P-080YokotaKoji 3P-121YokotaShigefumi 2P-145YokotaTomohiro 3P-155YokoyamaHisayo 3P-092,

3P-139,3P-143YokoyamaMegumi1P-202,

1P-206YokoyamaNoriko 3P-177,

1S13K-4,1S20H2-4YokoyamaUtako 1S19H1-5,

2P-036,2P-038,2S41H1-1,3S55D1-1

YokoyamaYoshihiro3P-062,3P-080

YonedaKazunori 2S39F-3YoneharaYoshiyuki3P-009YooHae 3S57G-4YoshidaAtsumasa 3P-040YoshidaKeitaro 2P-066YoshidaKen-ichi 3S45C-4YoshidaNorio 2P-186,

2P-188YoshidaRyotaro 3P-190YoshidaRyusuke 1S2D1-2,

1S2D1-4,1S3E-1YoshidaSachine 1P-154


AUTHOR INDEX

YoshidaTakamasa 1O2J-4YoshidaTakashi 3P-014YoshidaTakashi 3P-148YoshidaTakayuki 3S44B-2YoshidaYuka 3P-140YoshidaYuri 2P-188YoshiharaToshinori1P-186YoshikawaTomoko2P-174YoshikawaY SP-1YoshimotoJunichiro2S39F-1YoshimotoMisa 1P-044,

1P-045,2P-035,2P-043YoshimuraMichihiro

1P-051YoshimuraMitsuhiro

2P-141,2P-164YoshinoMasami 1P-033YoshiokaDaisuke 1P-109YoshiokaKazuaki 2S41H1-2,

3S49G-2YoshiokaNoboru 1P-103,

2P-055,2P-103YoshiokaToshitada1P-183YoshitakeKohei 1P-128YosidaSachiko 1S8H1-2YoumJaeBoum 2S27F-2YouyiDong 2P-128YuYing-Chun 1S10H2-3YuanX.-J.Jason 2S37D1-3YuriKazunari 3P-091YuzakiMichisuke 1S14D1-4

Z

ZamponiW.Gerald 3SL14FZhanDong-Yun 1P-041,

2P-049,2P-050ZhangGX SP-1ZhangJingqi 2P-208ZhangYing 1P-177,

3P-175ZhaoJuanjuan 3S49G-2ZhaoMeimi 2P-020,

2P-210ZhaoYan　　 2P-210ZhongRenjia 3P-078ZhouYiming 2P-002,

2P-010,2P-017ZhuWan-Jun 1P-217

plenary lecture memorial lectures...tawara memorial lecture (march 18, 9:00–10:00, room c) 3sl11c...

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