2

TABLE OF CONTENST

Committees 3

MessagefromthePresidents 5

Sponsors 6

ProgramSummary 7

Conferences 8

Symposia 16

OralPresentationI 61

OralPresentationII 67

SOFARCHIMembersIncorporation 73

PostersI 77

PostersII 200

Map 323

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COMMITTEES

Sociedad Chilena de FarmacologíaPresident RamónSotomayor,UniversidadValparaísoVice-President EdgarPastene,UniversidaddeConcepciónPastPresident RafaelBurgos,UniversidadAustraldeChileTreasurer VivianaNoriega,UniversidadAndrésBelloSecretary GeorginaRenard,UniversidaddeValparaísoDirectors PabloJara,UniversidaddeSantiagodeChile RodrigoCastillo,UniversidaddeChile JorgeFuentealba,UniversidaddeConcepción

Sociedad Chilena de Ciencias FisiológicasPresident MauricioBoric,PontificiaUniversidadCatólicadeChileVice-President BredfordKerr,CentrodeEstudiosCientíficos-CECPastPresident JuanReyes,PontificiaUniversidadCatólicadeValparaíso.Treasurer MauricioRetamal,UniversidaddelDesarrolloSecretary FelipeSimón,UniversidadNacionalAndrésBelloDirectors MarceloGonzález,UniversidaddeConcepción. AlexisGonzález,PontificiaUniversidadCatólicadeValparaíso, XavierFigueroa,PontificiaUniversidadCatólicadeChile JulioAlcayaga,UniversidaddeChile

Sociedad Chilena de Neurociencia

President JuanBacigalupo,UniversidaddeChileVice-President AdriánPalacios,UniversidaddeValparaísoPastPresident AlanNeely,UniversidaddeValparaísoTreasurer PatricioRojas,UniversidaddeSantiagodeChileSecretary MagdalenaSanhueza,UniversidaddeChileDirector PaulDélano,UniversidaddeChileDirector MiguelReyes,UniversidaddeSantiagodeChile

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Organizing and Scientific Committee

JuanBacigalupo UniversidaddeChileMauricioBoric PontificiaUniversidadCatólicadeChileVivianaNoriega UniversidadAndrésBelloAdriánPalacios UniversidaddeValparaísoGeorginaRenard UniversidaddeValparaísoMauricioRetamal UniversidaddelDesarrolloJuanReyes PontificiaUniversidadCatólicadeValparaísoPatricioRojas UniversidaddeSantiagodeChileMagdalenaSanhueza UniversidaddeChileRamónSotomayor UniversidaddeValparaíso

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Dearcolleaguesandstudents,

We like to welcome you to the first joint conference of the Chilean Societies for Pharmacology, Physiological Sciences andNeuroscienceheldinthisbeautifulregionofourcountry.Inputtingtogetherthismeetingwehavetakenadvantageofourwidelycommoninterests,withacertainemphasisindrugaddiction.Wehavechosenthisplacenotonlyforitsmysticbeauty,butalsotoreinforceourcommitmentasscientiststotransmitourendeavortoregions.Aspartofthispledge,severalreachoutactivitieswillbeconductedwithyounghighschoolstudentsonthemainissueofaddiction.Thisproblemwillalsobediscussedfromvariousperspectivesinaroundtablewiththeparticipationofrelevantnon-scientificcommunitymembers.

Wehavemadeeveryefforttoputtogetheranambitiousprogramcoveringavarietyoftopics,withthehopethateachoneofyouwillfindinterestingaspectsandwillgobackhomewiththefeelingofhavinglearnednewthings,aswellashavinghadagreattime.Themeetingcomprisestheparticipationofveryprominentinvitedforeignandlocalspeakers,whowillpresentustheircurrentworkinconferencesandsymposia.WewanttounderscoretheYoungNeuroscientistSymposiumsponsoredbytheChileanSocietyforNeuroscience, inwhichthespeakersareadvancedgraduatestudentsandpostdoctoralfellows.Wealsohaveincludedtwosessionsofshortoralpresentations,andanspecialincorporationsessionwithparticipantsapplyingtobecomemembersoftheChileanSocietyforPharmacology.Therearealsotwoposterpresentations,whichholdmostoftheworkstobepresentedatthisJointMeetingandwheretheparticipationofstudentsisparticularlyrelevant.

Weareverysatisfiedbythenumberofpeople thatareattendingthemeeting.Specialattentionwaspaid to thestudents,asourSocietiesareprovidinganimportantnumberoffellowshipstoallowthemtotakepartofthismeeting,addingtofellowshipsgenerouslysponsoredbytheComisiónNacionaldeCienciayTecnología,CONICYT.Gladly,theseeffortsresultedinthatnearlyhalftheattendeesarestudents.

Themeetingrepresentsamajorfinancialendeavorforoursocieties,whichwouldnotbepossiblewithouttheparticipationofseveral commercial companies sponsoring it.We like to thankArquimed,Galénica, Lab-Tec, Loncotec, Sigma,GrupoBiosandValquimandweencourageyoutovisitthestandswheretheyshowtheirproducts,andtoattendacoupleoftechnicaltalksthefirstdayoftheMeeting.

Weanticipatecountingwithyourpresence inasmanyactivitiesasyoucan,hoping thismeetingwillofferanopportunity forintenseandfruitfulinteractionsbetweenestablishedandyoungscientistsandstudents.YouarecordiallyinvitedtopartakeinthegettogetherreceptiononTuesdayevening,andtohavefunintheclosingDinnerandDancingonFriday.UseyourleisuretimeonThursdayafternoontovisitthemanyinterestinganduniqueplacesofthisregionofCoquimbo,ElquiValleyandnearbycityofLaSerena.

Haveagoodtime!

RamónSotomayorSociedadde

FarmacologíadeChile

MauricioBoricSociedadChilenadeCienciasFisiológicas

JuanBacigalupoSociedadChilenade

Neurociencia

Message from the Presidents

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SPONSORS

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PROGRAM SUMMARY

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CONFERENCES

9

STIMULATING NEURONS WITH LIGHT AND GOLD

Bezanilla, Francisco1.,Treger,Jeremy2.,Carvalho-de-Souza,Joao2.,Dang,Bobo3.,Kent,Stephen2.,Pepperberg,David4.,1BiochemistryandMolecularBiology,professor,UniversityofChicago.2BiochemistryandMolecularBiologyUniversityofChicago.3ChemistryUniversityofChicago.4OphtalmologyandVisualSciencesUniversityofIllinoisatChicago.

Stimulationofneuronswithlightisausefultoolinunderstandingthecircuitryofthenervoussystem.Forexample,ithaslongbeen known that pulses of infrared light (IR ofwavelength ~ 2000 nm) are effective in exciting neurons and other excitabletissues.RecentlywehavefoundthatthemodeofactionofIRpulsesisviaarapidincreaseintemperature.Thefasttemperatureincreasechangesthemembranecapacitance(C)producingacurrentproportionaltodC/dt,whichdepolarizesthemembraneandinitiatesanactionpotential.Thistechniquehastwoimportantdrawbacks.First,itlackslocalizationofthedeliveredlightenergy,consequentlyishardtoaimatcertaintypeofneuronsandsecondIRof2000nmdoesnotpenetratebeyond0.1mmoftissue.Wehavesoughttoovercometheseproblemsusingvisiblelightbutstilltakingadvantageofthesamemechanismtoexcitethecells.Hereweshowthatgoldnanoparticles(20nmindiameter)canbeconjugatedtohigh-avidityligandsforavarietyofcellulartargets.Onceboundtoaneuron,theseparticlestransducemillisecondpulsesoflightintoheat,viasurfaceplasmonresonance,whichchangesmembranecapacitance,depolarizingthecellandelicitingactionpotentials.Thetemperatureincreasedoesnotexceed2CbutitchangesfastenoughtoinduceacurrentproportionaltodC/dt.Comparedtonon-functionalizednanoparticles,ligand-conjugatednanoparticleshighlyresistconvectivewashoutandenablephotothermalstimulationwithlowerdeliveredenergyandresultingtemperatureincrease.Ligandstargetingthreedifferentmembraneproteinsweretested;allshowedsimilaractivityandwashoutresistance.Thissuggeststhatmanytypesofligandscanbeboundtonanoparticles,preservingligandandnanoparticlefunction,andthatmanydifferentcellphenotypescanbetargetedbyappropriatechoiceofligand.Combiningopticaldetectionofmembranepotentialandtargetedgoldnanoparticleswecouldstimulateandrecordfrombrainslicesinanall-opticalexperiment.Ourfindingshaveapplicationsasanalternativetooptogenetics(withoutgenetics)andpotentiallyfortherapiesinvolvingneuronalphotostimulation.SupportedbyNIHgrantsEY023430,GM030376,andEY001792.

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PHARMACOLOGY: TOOLS TO UNDERSTAND SPERM PHYSIOLOGY

Trevino, Claudia1.,Torres,Paulina1.,Sánchez-Carranza,Oscar1.,Darszon,Alberto1.,López-González,Ignacio1.,1GenéticadelDesarrolloyFisiologíaMolecularUniversidadNacionalAutónomadeMéxico. (SponsoredbyCONACyT128566,PAPIIT IN204914AndTheAlexanderVonHumboldtFoundation)

Spermatozoaareuniquelyequippedtoreach,recognizeandfusewiththeegg.Toperformthesetasks,spermatozoamustbepreparedto face constantly changing surroundings, and toovercome several physical barriers. Since theyarebasically transcriptionallyandtranslationallysilent,spermcellsrelyprofoundlyondiversesignalingmechanismstoswimtowardstheegg,andtoadjusttochallengingenvironmentalconditions.Decipheringthesesignalingpathwaysisofutmostimportancetounderstandtheprocessoffertilizationandcontributetosolveinfertilityproblemsordesignnovelcontraceptivemethods.Popularmethodologiessuchasgenesilencingormutagenesiscannotbeappliedinspermcells.Thereforetodeterminethemolecularentitiesthatparticipateinspermfunctions,mostofthestudiesrelyontheuseofdrugs.Wehavespecialinterestintwospermspecificionchannelswhoseabsenceproducessterility.OneofthemisaK+channelnamedSlo3,inmousespermSlo3(mSlo3)isresponsiblefortheplasmamembranehyperpolarizationassociatedtocapacitation(CAH),aprerequisiteforfertilization.Recently,usingflowcytometry,wereportedthathumanspermatozoaalsoundergoCAHandourpharmacologicalstudiesinhumanspermsuggestedtheparticipationofatleasttwoSlofamilyK+channels.Meanwhile,controversyexistsastowhetherSlo3,Slo1,orboth,areexpressedinhumansperm.SincethespecificpharmacologicalprofileofheterologouslyexpressedhumanSlo3(hSlo3)K+channelshadnotyetbeenexplored,wewereunabletoestablishthepreciseidentityoftheinvolvedK+channel(s).Inthepresentstudy,wefirstdeterminedthepharmacologicalprofileofheterologouslyexpressedhumanhSlo3inChineseHamsterOvarycells,andthencompareditwiththehumanCAHpharmacologicalprofile.WefoundthatheterologouslyexpressedhSlo3possessapharmacologicalprofilethatdiffersfromthatofmSlo3,consistentwithspecies-specificdifferencesobservedamongotherspermionchannels.WhileboththecorrelationanalysisofhSlo3currentsandtheCAHpharmacologicalprofileconfirmedtheparticipationofhSlo3,theyalsosuggestthatadditionalK+channelsmaybeinvolvedinCAH,inparticularSlo1channels.

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STRESS AND THE BRAIN: FROM ADAPTATION TO DISEASE

De Kloet, Ron1., 1Endocrinology and Metabolic Disease, Medicine, Leiden University Medical Center. (Sponsored by RoyalNetherlandsAcademyOfArtsAndSciences)

Cortisolandcorticosterone(CORT),secretedfromtheadrenalsasendproductofthehypothalamus-pituitary-adrenal(HPA)axis,haveaprofoundactiononstructure,plasticityandfunctionofspecificbraincircuitsunderlyingstressadaptation.Thisactionexertedbythestresshormonesismediatedbytworeceptorsystems:glucocorticoidreceptors(GR)andmineralocorticoidreceptors(MR)thatmodulateslowgenetranscriptionaswellasfastnon-genomicresponses.MRhasaprincipallocalizationinlimbiccircuitryincludinghippocampusandamygdala;GRisexpressed,unevenly,ineverybraincell,particularlyifinvolvedwithregulationofthestressresponse.MRandGRoperateincomplementarymannerduringthreedifferentphasesofthestressresponse.First,theonsetofthestressreactionwhenMRmediatesCORTactiononappraisalofnovelinformationandemotionalreactivity.Second,the termination characterized by CORTpromotingbehavioural adaptation andmemory storage. Third, the basal phasewhenultradianandcircadianoscillationsofCORTpermitrecoveryandgrowth,whilemaintainingthebrain’sresponsivitytostress.UponimbalanceofMR:GR-regulatedlimbic-corticalsignalingpathwaystheinitiationand/ormanagementoftheneuroendocrinestressresponseiscompromised.AtacertainthresholdthismayleadtoaconditionofHPAaxisdysregulationandimpairedbehaviouraladaptation,whichcanenhancesusceptibilitytostress-relatedpsychopathology.Suchimbalancecanbeinheritedthroughgeneticvariationofthereceptors,orisimposedbystressfulexperiences,notablyinearlylife,thatmoderategeneexpressionbyepigeneticprocesses: DNAmethylation and/or histone acetylation. In chronic stressmodels of human psychopathologywe observed inhippocampalneuronsa largegenomicreorganizationthat isrevealeduponacutechallengewithCORTand ischaracterizedbyactivationofintracellularsignalingpathwaysinvolvedinchromatineorganizationandepigeneticprocesses.Inbehaviouralrealmsuch stressful conditions shift resources fromamygdala–hippocampus/prefrontal cortex toamygdala- striatalpathways, thatunderliemoresimplehabitbehaviourforcopingpurposes.ThisswitchcanbepreventedbymanipulatingtheMR,supportingtheevidencethatthisCORTreceptorisaprominenttargettopromotestressresilience.DeKloetER(2014)FromReceptorBalancetoRationalGlucocorticoidTherapy.Endocrinology 155:2754-2769.

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NEW SIGNALING MECHANISMS IN THE ACTIONS OF AMPHETAMINE

TORRES, G1.,1Pharmacology,AssociateProfessor,UniversityofFlorida.

The dopamine transporter (DAT) regulates extracellular dopamine (DA) levels and signaling via uptake and efflux. Addictivepsychostimulants such as amphetamine increase extracellular DA levels in motivational and reward brain areas by targetingDAT.Examiningthebasicmechanism(s)thataffectDATeffluxiscriticalforunderstandingbothfundamentalaspectsofDAregulationand forclinical intervention inDA-relatedbraindisordersassociatedwith the therapeuticuseandabuseofpsychostimulants.Severalstudieshaverevealedaplethoraofprotein-proteininteractionsinfluencingDATdistributionandactivity;suggestingthatthefine-tuningofDAhomeostasisoccursviaanelaborateinterplayofmultiplemechanisms.WerecentlyreportedthatβγsubunitsofGproteinsbinddirectlytotheC-terminus(residues582-620)ofDATtodown-regulateuptakeactivity.HerewereportthatthenovelDAT/GβγinteractionalsopromotesDAeffluxthroughDAT.Specifically,activationofGβγsubunitsusingmSIRKincreasedDAeffluxthroughDATusingbothheterologouscellsandprimaryneuronsinculture.Thiseffectwasblockedinthepresenceofgallein,aGβγinhibitor.Likewise,aTAT-peptidecontainingtheGβγinteractingdomainofDATblockedtheabilityofmSIRKtoinduceDAefflux,suggestingtheeffectofGβγoneffluxisaresultofadirectinteractionwiththetransporter.Basedonthesedata,wehypothesizedthatGβγmayalsobeinvolvedintheactionsofamphetamine.Insimilareffluxexperiments,amphetamineinducedadose-dependentincreaseinDAeffluxinbothheterologouscellsandprimaryneuronsinculture.Moreimportantly,amphetamine-inducedeffluxwasbluntedinthepresenceofeithergalleinortheTAT-peptidecontainingtheDATinteractingdomain.Furthermore,inhibitionofGβγwithgalleinalsoattenuatedtheamphetamine-inducedlocomotoractivityin vivo.Collectively,ourdatasuggestthatthedirectinteractionofGβγsubunitwithDAThasanimportantroleinbothphysiologicalandamphetamine-induceDAefflux.

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GAP JUNCTION CHANNELS AND HEMICHANNELS: FROM FUNDAMENTAL SCIENCE TO THEIR INVOLVEMENT IN INFLAMMATORY RESPONSES OF CHRONIC HUMAN DISEASES

Sáez,JuanC1,.1DepartamentodeFisiología,PontificiaUniversidadCatólicadeChile,Santiago-ChileandInstitutoMilenio,CentroInterdisciplinariodeNeurocienciasdeValparaíso,Valparaíso,Chile.

Most cells of the animal kingdom express gap junction channels (GJCs), which formmembrane specializations found at cellinterfacescalledgapjunctions.GJCsallowtransferofionsandsmallmoleculesbetweencontactingcells.Inmammals,theyareformedbyproteinscalledconnexinsandtheirphysiologicalfunctionistocoordinatemetabolicandelectricalresponsesamongmembersofcellcommunities; theyalsoplayarelevant,but lessstudiedfunctional roleascell-celladhesionstructures.Morerecently,anewformofmembranechannels,termedhemichannels(HCs;halfofGJchannel)havebeenfoundinthecellsurfaceofmostcellswheretheytransientlycommunicatetheintraandextracellularcompartments;also,theycanbeformedbypannexins(unrelatedtoconnexins). Nowdays,HCsarerecognizedascellmembranepathwaysforreleasingautocrineandparacrinecellsignalsaswellasfortransferringmetabolites.GapjunctionchannelsandHCspresentdistinctunitarycurrentevents,permeabilityfeaturesandregulatoryproperties.Inseveralinheritedhumandiseaseslossofgapjunctionalcommunicationand/orgainofHCfunctionhavebeenshowntobedirectlyrelatedtothepathologicalcondition.InhibitionofHCspreventscelldegenerationmostlikelyduetothereductionofCa2+influxthatpromotesinflammasomeactivation.Accordingly,selectiveHCblockersareeffectivein reducingcellulardysfunctionanddegeneration inanimalmodelsof chronichumandiseasesofdifferentetiology, includingmuscleatrophy,Alzheimer’sdiseaseandepilepsy.Consequently,selectiveHCblockerscouldbecomeanewgenerationofanti-inflammatoryagentstoreducetissuedysfunctionsinchronicdiseases.

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THE CIRCUITRY OF DOPAMINE SYSTEM REGULATION AND ITS DISRUPTION IN SCHIZOPHRENIA AND DEPRESSION

Grace, Anthony1.,1DepartmentsofNeuroscience,PsychiatryandPsychologyUniversityofPittsburgh,PA,USA.

Thedopamine-containingneuronsofthemidbrainhavebeenimplicatedinabroadarrayofpsychiatricdisorders,rangingfromschizophreniatodrugabuseanddepression.However,studiesseemtoindicatethatitisnotthedopamineneuronsthemselvesthat are responsible for thesepathological states,but instead thedisorders appear toarisedue toadisruptionofdopamineneuron regulation by afferent inputs. Dopamine neurons recorded in vivo are known to exhibit multiple functional activitystates,includingbaselinetonicfiringandphasicactivationinresponsetosalientstimuli.Phasicburstfiringisbelievedtobethebehaviorallyrelevant“signal”ofthedopamineneuron,whereastheleveloftonicdischargerepresentsthe“gain”orthelevelofamplificationofthissignal. Thistonicgainisdifferentiallyregulatedbymultiplebrainregions,includingthehippocampus,theamygdala,andtheprefrontalcortex.Disruptionsintheseregionscaninterferewiththenormaltonic/phasicbalancewithinthedopaminesystem.Electrophysiologicalandbehavioralstudiesinanimalmodelsofpsychiatricdisorders,aswellasandhumanimaging studies in patients, suggest that this disruptionmay underlie the pathological state of the dopamine system that ispresentinpsychiatricdisorders. Specifically,wefoundthathippocampalhyperactivityinschizophreniamayberesponsibleforthehyperdopaminergicstateofpsychosis,whereasprefrontalcortical-amygdalaoverdrivediminishesreward-relateddopamineneuronactivityleadingtoanhedoniaindepression.Thistypeofinformationcancontributebothtoabetterunderstandingofthepathophysiologyofmajorpsychiatricdisorders,aswellasgleaninsightsintonovelavenuesoftreatmentandpotentiallyinpreventingtheemergenceofthesedisorders.

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ROUND TABLE

ALCOHOL Y CEREBRO ADOLESCENTE, UNA MIRADA BIO-PSICO-SOCIAL

FINANCIA: ChileanChapteroftheSocietyforNeuroscienceUSACoordina: MaríaEstelaAndrés

Participantes: KatiaGysling,FacultaddeCienciasBiológicas,P.UniversidadCatólicadeChile. PauloEgenau,DirectorEjecutivodelaFundaciónParéntesis. CarlosIbáñez,PsiquiátradelaUnidaddeAdicciones,ClínicaPsiquiátricaUniversitaria, UniversidaddeChile.

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SYMPOSIA

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SYMPOSIUM PHYSIOLOGY AND PATHOPHYSIOLOGY OF THE

RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM IN THE KIDNEY:FROM CELLS AND ANIMAL MODELS TO HUMAN DISEASE

Chair:AlexisGonzálezREGULATION OF RENIN IN THE RENAL COLLECTING DUCT CELLS

Gonzalez, Alexis A1., 1InstitutodeQuimica, FacultaddeCiencias, PontificiaUniversidadCatólicaDeValparaíso. (SponsoredbyFONDECYT11121217)

Renin-angiotensinsystem(RAS)playsamajorroleinthephysiologicalcontrolofbloodpressureandfluidvolume.RASdysregulationisalsoresponsibleforthedevelopmentofhypertension.BesidethesystemicRAS,whichismainlycontrolledbytheproductionandreleaseofreninfromthejuxtaglomerular(JG)cellsinthekidney,theRASisalsopresentintubularsegmentsofthenephron.Inparticular,reninhasbeendetectedinrenalprincipalcollectingduct(CD)cells.AlthoughtheregulationofJGreniniswellknown,themechanismbywhichreninisregulatedintheCDanditspotentialrole incontributingtoRASactivationanddistalsodiumreabsorptionhavenotbeeninvestigated.InM-1CDcellline,AngIIincreasedcAMPlevels,reninmRNAexpression,proreninandreninproteinabundanceandreninactivityinculturemedia.ThiseffectwaspreventedbyPKCinhibitorcalphostinC,PKC-alphadominantnegativeandbyPKAinhibition.Forskolin-inducedincreaseincAMPandreninexpressionwaspreventedbycalphostinC.PKCinhibitionandCa2+depletionimpairedAngII-mediatedCREBphosphorylationandreninupregulation.Adenylatecyclase6(AC)siRNAremarkablyattenuatedtheAngII-dependentupregulationofreninmRNA.PhysiologicalactivationofACwithvasopressinincreasedreninexpressioninM-1cells.TheresultssuggestthattheAngII-dependentupregulationofrenininCDcellsdependsonPKCα,whichallowstheaugmentationofcAMPproductionprobablyviaAC6andactivationofcAMP/PKA/CREBpathway.ThisisanovelmechanismresponsiblefortheregulationoflocalRASinthedistalnephron.

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ROLE OF 11B-HYDROXYSTEROID DEHYDROGENASE-2 IN ENDOCRINE ARTERIAL HYPERTENSION

Carvajal, Cristian1.,1Endocrinology,Medicine,PontificiaUniversidadCatólicaDeChile.(SponsoredbyThisWorkWasSupportedBy Chilean Grants: FONDECYT 1130427 (CFB) And 1150427 (CCM), CORFO 13CTI-21526-P1 And TheMillennium Nucleus OnImmunologyAndImmunotherapyP09/016-F(ICM).)

ArterialHypertension(AH)isamultifactorialconditionthatmostlyaffectsrenal,cardiacandvasculartissues.Herealdosteroneishighlightedaskeyplayerinbothphysiologicalandpatho-physiologicalconditions.Similartoaldosterone,cortisolcanbindtomineralocorticoidreceptor (MR) inepithelialtissuesandnon-epithelialtissueswithsimilaraffinityandcouldtriggerthesamehypertensiveeffects.Thisisparticularlyrelevantinhumanphysiologysinceconcentrationsofcirculatingcortisolare1,000-2,000timeshigher than aldosterone, thenatural agonist ofMR. In normal conditions, activationofMRby cortisol does not occur,because action of 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) enzyme, which inactivates efficiently cortisol (F) tocortisone(E),avoidingthebindingofFtoMR.In11βHSD2deficiency,AHoccursduetoagonistactionofcortisoloverMR,whichproducesinrenaltissueanincreaseinsodiumre-absorption,andavascularresponsethatfinally increasethebloodpressure.WehaveshownthatAHassociatedtopartialorsevere11βHSD2deficitcouldbemorefrequentthanexpected.Westudiedthe11βHSD2deficiencybyasensitiveLC-MS/MSdeterminationofbothFandE,andwefoundahighFtoEratio,withnormallevelsof cortisol tetrahydrometabolites. It suggestedahighprevalenceof11βHSD2enzymedisorders thatareundiagnosed,whichcouldreachupto15%ofthegeneralhypertensivepopulation.ThisprevalenceassociatedtolowfrequenciesofHSD11B2geneticalterations,ledustopostulatethatotherfactorsandepigeneticmodifications,suchaspromoterCpGmethylationandspecificmiRNAinHSD11B2gene,couldinfluencedynamicallytheonsetandprogressionofthehypertensivedisease.Inthisrespect,wehaverecentlyproposedandstudiedtheassociationof11BHSD2withaging,saltintakeandepigeneticmodifications.

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ROLE OF THE IMMUNE SYSTEM IN ANGIOTENSIN AND ALDOSTERONE DEPENDENT HYPERTENSION

Michea, L1.,1ICBM,ProgramadeFisiologíayBiofísica,IMII,FacultaddeMedicina,UniversidadDeChile.(SponsoredbyCONICYT/FONDECYT/RegularN°1130550,IMIIP09-016-F)

Hypertensionisamajorcardiovascularriskfactorthatinducestissuedamage,morbidityandmortality.Intheclinicalcontext,inthevastmajorityofhypertensivepatientsthecauseforthedevelopmentofhighbloodpressureisunknown.However,itisknownthattheinadequateactivationoftheRenin-Angiotensin–AldosteroneSystem(RAAS)causehypertensionandtargetorgandamage,characterizedbyoxidativestress, low-gradechronicinflammation,hypertrophyandfibrosis inkidneyandinthecardiovasculartissue. Furthermore, thepharmacological blockadeof theRAAS is effective to lowerbloodpressure inhypertensivepatients.Previousdatahavesuggestedthattheactivationoftheadaptiveimmuneresponsesinthedevelopmentofhypertension/tissuedamage.Thestateoftheartconcerningtheroleoftheimmunesysteminthedevelopmentofhypertensionandtargetorgandamagewillbepresented.ThedatasupportingtheactivationofadaptiveimmuneresponseduetoexperimentalRAAS-dependenthypertensionwillbepresented. Theavailableevidence indicate theactivationof lymphocyteThelper1 (Th1),Th17 immuneresponsesandthedownregulationofTreginRAAS-dependenthypertension.Morerecently,theuseofgeneticallymodifiedmicehasallowedtoinvestigatetheroleofofDendriticCells(DC),myeloidcellsthatorchestratetheimmuneresponse,intheinitiationofhypertensionandtheestablishmentoflow-gradeinflammationandfibrosis.TheresultsindicatethatDCs(CD11cHicells)arenecessary forhypertensiondevelopment/maintenanceandtissuedamagedueto thehyperactivityof theRAAS.Theseeffectspossiblyareindependentofadaptiveimmunityactivation.

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ROLE OF THE PRORENIN RECEPTOR IN THE MODULATION OF THE INTRATUBULAR RAS

PRIETO, M1., Gonzalez, AA2.,1Physiology, Medicine, Tulane University.2Instituto de Quimica, Facultad de Ciencias, PontificiaUniversidadCatólicaDeValparaíso.

AugmentationofreninsynthesisandactivityinthecollectingductsprovidesanovelpathwayforintrarenalandintratubularAngIIformationduetothepresenceofangiotensinogensubstrateandangiotensinconvertingenzymeinthenephron.Thebindingoftheproreninreceptor(PRR)toreninorproreninenhancesreninactivityandfullyactivatesthebiologicallyinactiveprorenin,respectively.ThePRRareaugmentedintherenal innermedullaandurineofAngIIhypertensiverats.ReninandthePRRmayinteracttoincreaseintratubularAngIIformationinthedistalnephron.ThelocalinteractionofproreninandPRRalsostimulatesintracellularsignalsthatmaycontributetothedevelopmentandprogressionofhypertension.However,thedemonstrationthatthePRRisalsoabletoup-regulateCOX-2in innermedullarycollectingduct(IMCD)cells,raisesthequestionwhetherthePRRplaysadualroleintheregulationofbloodpressure.AngIIincreasessolublePRR(sPRR)secretionthroughanAT1receptorandfurin-mediatedmechanisminIMCDcells.InAngII-infusedratsfor2wkduringtheearlyphaseofAngII-dependenthypertension,thePRRstimulatesCOX-2andsubsequentlyPGE2intherenalmedulla,whichattenuatesthevasoconstrictoreffectsofAngIIonrenalhemodynamics.Incontrast,after2wofAngIIinfusion,increasedsPRRdistalluminalsecretionoccursalongwithdecreasedPRRabundanceoncollectingductplasmamembranes.TheevidencereflectsthatthecomplexitybetweenshortandlongtermmodulatoryeffectsofthePRRdependonthestatusoftheRASactivationinthecollectingduct.

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NEW ROLES FOR MU-OPIOID RECEPTORS IN THE PHARMACOLOGICAL EFFECTS OF ETHANOL

Rivera-Meza, Mario1,2., Urra, Jonathan1., Berríos/Cárcamo, Pablo1., Herrera-Marschitz, Mario1,2., Quintanilla, María Elena1.,1Programa de Farmacología Molecular y Clínica, Facultad de Medicina, Universidad De Chile. 2Millenium Scientific InitiativeBiomedicalNeuroscienceInstitute.(Supportedby:FONDECYT#11130241:BNIP09-015-F.)

Relapsetoalcoholuse isthemajorprobleminthetreatmentofalcoholism,whiletheanti-relapsemedicationsavailablehaveonlylimitedclinicalefficacy.Animalsthathavechronicallyconsumedethanolandaredeprivedofitforlongperiodsshowagreatincreaseintheiralcoholintake(binge)whenitsaccessisre-allowed.Thisbinge-drinkingbehavior,termedthealcoholdeprivationeffect(ADE),constitutesanacceptedanimalmodelofalcoholrelapse.ThebiologicalandneurochemicalbasesofADEarenotknown;howeveranimalstudieshaveshowedthatADEbinge-drinkingresultsfromanenhancementoftherewardingpropertiesofethanol.Ithasbeenreportedthatanimalsdisplayingahigherexpressionofopioidreceptorsshowincreasedlevelsofalcoholintake.However,recentreportsindicatethatchronicethanolintakecaninduceadesensitizationofmu-opioidreceptors,likelybyreceptorsynthesisinhibitionorbyreceptoruncoupling-internalizationprocess.Asubsequentreboundinopiatereceptorlevelsormembranere-externalizationcouldconceivablybeinvolvedinthedevelopmentoftheADEinwhichethanoliswithdrawnforprolongedperiods.Animalstudieshaveshowedthatacetaldehyde,thefirstmetaboliteofethanol,isamotivationalandreinforcingmolecule.Inthebrain,ethanol-derivedacetaldehydecancondensewithdopaminetogeneratesalsolinol.Thiscompoundisself-administered intracranially by animals, suggesting that salsolinol is the resultingmoleculemediating the rewarding effects ofacetaldehydeandethanol.Recentevidenceindirectlysuggeststhatsalsolinolmayexertitsactionthroughanopiatemechanism,howevertherearenostudiesshowingthatacetaldehydeorsalsolinolactsdirectlyonopioidreceptors.Inthispresentationwewill report in vivo and in vitro studies aimedatdetermining (i) whetheranupregulationofmu-opioid receptor is involved inthedevelopmentoftheADEbinge-drinkinginUChBalcohol-preferringratsand(ii)whetherethanol-derivedsalsolinolmediatesits effects through an opioid receptormechanism. These studieswill help to determine the role of the opiate system in thedevelopmentofADEbinge-drinkingandmaybeofvalueinthedevelopmentofnewtherapeuticstrategiesagainstalcoholism.

SYMPOSIUMNEW MOLECULAR TARGETS FOR THE TREATMENT OF ALCOHOLISM

Chair:MarioRivera

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SALSOLINOL, AN ETHANOL METABOLITE, EXERTS MOTIVATIONAL EFFECTS LEADING TO INCREASES IN ETHANOL INTAKE

Quintanilla, M1,2,3., Rivera-Meza, Mario3., Berríos, Pablo3., Bustamante, Diego3., Morales, Paola3., Cassels, Bruce4., Herrera-Marschitz,Mario3., Israel,Yedy3., 1ProgramadeFarmacologíaMolecularyClínica,FacultaddeMedicina,UniversidadDeChile.2ProgramnadeFarmacologíaMolecularyClínica,FacultaddeMedicina,UNiversidaddeChile.3DepartamentodeFarrmacologíaMolecularyClínica,FacultaddeMedicina,UniversidaddeChile.4DepartamentodeQuímica,FacultaddeCiencia,UniversidaddeChile.In the brain, racemic salsolinol [(RS)-Sal] is generated by the condensation of acetaldehyde (generated from ethanol) withdopamine.Previous reportshaveshownthat (RS)SAL is self-infusedby rats into theposteriorventral tegmentalarea (VTA)atsignificantly lowerconcentrationsthanthoseofethanolandacetaldehyde,suggestingthat(RS)SAL isamostactiveproductofethanolmetabolism. InthepresentstudieswithUChBratsbredfortheiralcoholpreferenceweinitiallyassessedtheeffectof(RS)SALinjectedrepeatedlyintra-VTA(30.0pmol/0.2µl)or intraperitoneally(10mg/kg)on:(i)placepreference,(ii) locomotoractivityand(iii)ethanolintake.Resultsshowedthat(RS)SALinjectedeitherintotheVTAoradministeredintraperitoneallyledto(i)conditionedplacepreference,(ii)locomotorsensitizationand(iii)amarkedincreaseinethanolintake,whichwassuppressedbynaltrexone.Salsolinolwasdetectedinthebrainextracellularfluidfollowingthesystemic(i.p.)administrationof10mg/kgof(RS)SAL, suggesting that (RS)SAL isable tocross thebloodbrainbarrier. Insubsequentstudiesweseparatedand injected theenantiomers(R)SALor(S)SAL(30pmol/1.0μl)intotheVTAofnaïveratstostudywhetheroneorbothSALenantiomersis/areresponsibleforthemotivatedbehavioraleffects,sensitizationandincreaseinvoluntaryethanolintake.Thesestudiesshowedthatrepeatedadministrationof(R)SALledto(i)conditionedplacepreference,(ii)locomotorsensitizationand(iii)markedincreasesin binge-like ethanol intake. Conversely, (S)SAL did not influence any of these parameters. Overall, data indicate that (R)SALadministrationtoratsstereospecificallyinducesmotivationalandbehavioralsensitizationeffects,andleadstomarkedincreasesinintoxicatingethanolintake.

SupportedbyFONDECYTgrants#1130012,#11130241,and#1120079andtheMillenniumScientificInitiativeBNIP09-015-F

23

PERINATAL LEAD EXPOSURE AND INCREASED ETHANOL INTAKE: THE ACETALDEHYDE CONNECTION

Virgolini, M1.,1Farmacología,FacultaddeCienciasQuímicas,UniversidadNacionaldeCórdoba.

Emergingdatarevealthatdevelopmentalexposuretoenvironmentalneurotoxicantsinducesdifferentialreactivitytochallengingeventslaterinlife,includingdrugaddiction.Wehavedemonstratedthatlowlevellead(Pb)exposure(220ppmindrinkingwater)duringgestationandlactationenhancesthemotivationalandstimulantsresponsestoethanolinperiadolescentmalepups.Inanattempttounraveltheneurobiologicalbasisofthesedifferences,andbasedonourownandotherauthors’evidences,wehaveattributedacriticalroletobrainethanolmetabolismasaprobablemechanismforitsreinforcingeffects.ItisnoteworthythatgiventhereportedADH lowactivity inthebrain,catalasecatalysescentralethanoloxidationtoacetaldehyde,whereasALDHfavorsacetaldehydeoxidationtoacetate.Wethusfirstdemonstratedthatcatalaseactivitymodificationsbypharmacologicalactivation(3nitropropionicacid,20mg/kgs.c.)or inhibition (1,2,4aminotriazole,250mg/kg i.p.) resulted inparallelchanges inchronicethanolintakeandself-administrationinthePb-exposedanimals.Moreover,ashRNAanticatalaselentiviralvectormicroinfusedinventraltegmentalareareducedethanolintakeselectivelyinPb-exposedanimalsboth,atthebeginningofthefree-choicetestaswellasafterstableethanolintakelevelswerereached.Ontheotherside,brainacetaldehydeaccumulationbypharmacologicalALDHinhibition(cyanamide,0.3mgi.c.v.)enhancedethanolintakeandresultantlocomotion,althoughpredominantlyincontrolanimals,whileatrendwasevidentinthePb-exposedgroup.Theseresultscontrastwithperipheral acetaldehydeaccumulationbysystemiccyanamideadministration(25mg/kgi.p.),apharmacologicalapproachemployedinclinicalpracticetodissuadeethanolconsumptionthatresultedinadecreaseinboth,ethanolintakeandlocomotionpredominantlyinPb-exposedanimals.Overall,thesedatarevealtheimportanceofbrainacetaldehydeaccumulationintheenhancedethanolresponsesexhibitedinanimalsexposedtoenvironmentalPb levels.Theyalsoprovidefurtherevidenceandopenupnewavenues in the implicationofbrainmetabolisminethanolreinforcingandstimulanteffects.Supportedby:FONCyT,SECyTandCONICET.

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IS SALSOLINOL THE FINAL EFFECTOR OF ETHANOL REINFORCEMENT?

Berríos-Cárcamo, Pablo2,1.,Rivera-Meza,Mario2,1.,Quiroz,Gabriel3.,Zapata-Torres,Gerald4.,Iturriaga-Vásquez,Patricio3.,Reyes-Parada,Miguel5.,Herrera-Marschitz,Mario2,1.,Israel,Yedy2.,Quintanilla,MariaElena2.,1InstituteofBiomedicalSciences,MilleniumInstitute BNI, Faculty of Medicine, University of Chile.2Molecular and Clinical Pharmacology Program, Faculty of Medicine,UniversityofChile.3DepartamentodeCienciasQuímicasyRecursosNaturales,FacultaddeIngenieríayCiencias,UniversidaddelaFrontera,Temuco,Chile.4MolecularGraphicsSuite,DepartmentofInorganicandAnalyticalChemistry,FacultyofChemicalandPharmaceutical Sciences,University of Chile.5Escuela deMedicina, Facultad de CienciasMédicas,Universidad de Santiago deChile,Santiago,Chile.(SponsoredbyAcknowledgments:SupportedByCONICYT-PCHA/DoctoradoNacional/2013-21130865,AndFONDECYT(1120079,11130241,1130012,1110263).)

Thereinforcingeffectofethanol,definedasthepropertyofadrugtopromotebehaviorsthatfavorsitsconsumption,isamajorcontributor of ethanol dependence. Ethanol is reinforcing via local activation of the brain reward system. The formation ofacetaldehyde,theprimaryethanolmetabolite,hasbeenshowntoberequiredforethanoltobereinforcing,activatingdopaminergicneuronsintheventraltegmentalarea(VTA).Acetaldehydecancondensewithdopaminetoformracemic(R/S)salsolinol,whichhasbeenproposedtobethesubstancethatexertstheethanolreinforcingeffect.Forsalsolinoltobeacceptedasthefinaleffectorofethanol reinforcement itneeds: (i) tobe formed in theVTAafteranacuteethanolexposure; (ii) to reproduce theethanolreinforcingeffect;(iii)tobeapotentreinforcer,overtheefficacyofethanoloracetaldehyde;(iv)toimpairethanolreinforcementifsalsolinolsynthesisisprevented;and(v)tobereinforcingbyadirectmechanismofaction(notbefurthermetabolized).Evidenceshowsthat:(i)onlyanon-enzymaticpathwayforsalsolinolsynthesishasbeenthoroughlycharacterised.Asalsolinolsynthesisratefromtheconcentrationsofsynapticdopamineandacetaldehydederivedfromethanol,canbeestimatedtogeneratesalsolinol10-8to10-7M.(ii)&(iii)SalsolinoladministrationintotheVTApromotesthereleaseofdopamineinthenucleusaccumbens,inducesaconditionedplacepreference,andisself-administeredbyratsintotheVTAatlowerconcentrations(10-7M)thanacetaldehyde(10-5M)orethanol(10-2M).(iv)Currently,theeffectofsalsolinolcannotbepreventedduetolackofaspecificinhibitor.Nevertheless,whendopaminergicneuronsobtainedfromtheVTAhavebeenpharmacologicallydeprivedofdopamine(oneofthesalsolinolsubstrates)ethanolandacetaldehydeareunabletoactivatetheseneurons,whilesalsolinolcan.(v)(R)-salsolinoladministrationintheVTAenantioselectivelyinducesaconditionedplacepreference,whichimpliesaninteractionwithaproteintarget.Salsolinolisanagonistofµ-opioidreceptorsandbindstothedopaminetransporter,bothmechanismscouldgenerateitsreinforcingeffect.Insummary,whilethereisnoconclusiveproofthatsalsolinolisresponsiblefortheethanolreinforcingeffecttheevidencediscussedheresuggestthatsalsolinolisinvolved.

25

SYMPOSIUMPHARMACOLOGICAL MODULATION OF NEURONAL AND MUSCULAR

NICOTINIC RECEPTOR: IMPACT ON SYNAPTIC FUNCTIONChair:JorgeFuentealba

CHOLINERGIC REGULATION OF NEUROINFLAMMATION AND NEUROPROTECTION: IMPLICATION OF THE SIGNALING PATHWAY Α7NACHR/NRF2/HO-1

Lopez, Manuela1.,Parada,Esther1.,Navarro,Elisa1.,Buendia,Izaskun1.,Egea,Javier1.,1InstitutoTeofiloHernando-DepartmentofPharmacology,Medicine,UniversidadAutonomadeMadrid.(SponsoredbySpanishMinistryOfEconomyAndCompetenceRef.SAF2012-32223)nAChRsareshowntoaffordneuroprotectionandtoregulateinflammation,inparticularviatheα7nAChRactivationinmacrophageswhichregulatesthe‘cholinergicanti-inflammatorypathway’.ThetranscriptionfactorNrf2(nuclearfactor-erythroid2-relatedfactor2)isamasterregulatorofredoxhomeostasis,itcontrolstheexpressionofphase2enzymesthatactinacytoprotectivemanneragainstoxidativestress,includinghemeoxygenase-1(HO-1).Theaimofthisstudywastoevaluatetheimplicationtheα7nAChR/Nrf2/HO-1pathwayintwodiseasemodels:(i)a neurodegenerative model basedonsubchronicoxidativestress(rathippocampalorganotipiccultures–OHCs-exposedtolipopolysaccharideandantimycin-A;LPS/AA)and(ii)brain ischemia models(OHCsexposedtooxygenandglucosedeprivationandin vivostrokemodel).

OHCsexposedfor4daystoantimycin-A(0.1µM)andlipopolysaccharide(1ng/ml)causedlowneurotoxicityontheirown,measuredaspropidiumiodidefluorescenceinCA1andCA3regions.However,theircombinationcausedagreaterdetrimentaleffect,inadditiontomitochondrialdepolarization,overproductionofreactiveoxygenspeciesandNox4overexpression.Subtoxicantimycin-AperseincreasedROSandmitochondrialdepolarization,althoughtheseeffectsweresignificantlyhigherwhencombinedwithsubtoxicLPS.MoreinterestingwasthefindingthatexposureofOHCstothecombinationofsubtoxicconcentrationsofLPS/AAtriggeredaberrant protein aggregation,measured as thioflavin S immunofluorescence. The α7 nicotinic receptor agonist, PNU282987,preventedtheneurotoxicityandthepathologicalhallmarksobservedintheLPS/AAsubchronictoxicitymodel(oxidativestressandproteinaggregates);theseeffectswereblockedbyα-bungarotoxinandtinprotoporphyrin,indicatingtheparticipationofα7nAChRsandHO-1induction.

OHCsexposedtooxygenandglucosedeprivation(OGD),elicitedcelldeath,measuredbypropidium iodideandMTTstaining.PNU282987,afterOGD,reducedcelldeath,ROSproductionandTNFrelease.ThiswasassociatedwithinductionofHO-1expression;aneffect reversedby theα-bungarotoxinandbytinprotoporhyrin IX (SnPP). TheprotectiveeffectofPNU282987was lost inmicroglia-depletedOHCsaswellasinOHCsfromNrf2deficientvs.wildtypemice,aneffectassociatedwithsuppressionofHO-1expressioninmicroglia.AdministrationofPNU2829871hafterinductionofphotothromboticstrokein vivoreducedinfarctsizeandimprovedmotorskillsinHmox1lox/loxmice,thatexpressnormallevelsofHO-1butnotinLysMCreHmox1∆/∆inwhichHO-1expressionisinhibitedinmyeloidcells,includingthemicroglia.

Inconclusion,activationof theα7nAChR inducesNrf2andHO-1 to regulateneuroinflammationandoxidativestressaffordingneuroprotection.

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MODULATORS OF ACETYLCHOLINE RECEPTOR CLUSTERING IN THE MATURATION OF THE NEUROMUSCULAR JUNCTION: A PHARMACOLOGICAL APPROACH.

Mella,Jessica1.,Henriquez, Juan Pablo1.,1CellBiology,BiologicalSciences,UniversidaddeConcepcion.(SponsoredbyFONDECYT1130321AndMINREBRC120003)

Introduction:Clusteringofacetylcholinereceptors(AChRs)indiscreteregionsofthemusclesarcolemmaisahallmarkofpostsynapticdifferentiationattheneuromuscularjunction(NMJ).Duringpostnatalmaturationofthissynapse,pro-andanti-clusteringsignalsarebelievedtosculptthepostsynapticmorphology.ConsideringthatsignalingpathwaystriggeredbyACh/Cdk5andWntligandsmodulatetheembryonicformationoftheNMJ,wehavefocusedinstudyingweathertheycouldregulateNMJmaturationthroughapharmacologicalapproach.Materials and Methods:C2C12myoblastsweredifferentiatedontolamininmatricestoinducetheformationofcomplexpostsynapticstructures,whichwerestainedwithα-bungarotoxinaftersingleorcombinedtreatmentswiththeAChagonistCarbachol,theCdk5inhibitorRoscovitine,andtheWntactivatorLithium.Theintracellulareffectorsß-catenin,p35andnestinweredetectedbyimmunocytochemistryandWesternblot.Results:WhereasCarbacholandlithiuminducedatotaldisaggregationofAChRs,Roscovitineinducedandincreasedclusteringarea,evidencedbythepresenceoffragmentedpostsynapticstructures,acharacteristicfeatureofagedNMJs.RoscovitinewasunabletorescuethelossofAChRclusterstriggeredbylithium.Inturn,RoscovitinetreatmentincreasedtheACh/Cdk5effectorp35incytosolicandmembrane-associatedfractions,whereaslithiumdecreasedthetotalamountofp35,eveninthepresenceofRoscovitine.Discussion:TheresultsofourpharmacologicalapproachsuggestthatACh/Cdk5andWntpathwayscouldfunctionallyinteracttoregulateAChRclusteringand,thus,thematurationoftheNMJ.

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EFFECTS OF NICOTINIC MODULATORS ON THE NEUROTRANSMITTER RELEASE, IMPACT ON SYNAPTIC FUNCTION

GANDIA, L1., NANCLARES, CARMEN1., COLMENA, INES1., 1DEPARTAMENTO DE FARMACOLOGIA Y TERAPEUTICA UNIVERSIDADAUTONOMADEMADRID.(SponsoredbyMinisterioDeEconomíaYCompetitividad(MINECO,SAF2013-44108-P)AndFUNDACIONEUGENIORODRIGUEZPASCUAL)

Neuronalnicotinicacetylcholinereceptors(nAChRs)areligand-gatedchannelsexpressedinthecentralandperipheralnervoussystems,wheretheyplayimportantregulatoryneuralfunctions,suchasneurotransmitterreleaseandareimpliedinavarietyofphysiologicalandpathologicalresponsessuchasanxiety,pain,nicotineaddiction,andcognitivefunctions.nAChRsarepentamericcomplexesandtheirdiversityarisesfromthemultiplecombinationsofsubunitassembly.AlargenumberofnAChRsubunitshasbeenidentified:therearetenαsubunits(α1-α10),fourβsubunits(β1-β4),oneγ,oneδandoneεsubunit.ThemusclenAChRiscomposedoftwoα1subunitsandoneβ,δandε(orγintheembryonicreceptor)subunits.InneuronalnAChRs,twoαandthreeβsubunits,invariouscombinations,canproducedifferentreceptorsubtypes;Atthecentralnervoussystem,theα4β2*receptorsubtype form themajor nAChR subtype,while at the peripheral nervous system, nAChRs formedbyα3, β4 andα5 subunitsappear tobepredominant.Theα7*nAChR isexpressedonneuronsandnon-neuronal cells, aswell as in immunecells, thussuggestingitspossibleroleinbrainimmunity,inflammation,andneuroprotectioninadditiontoitswellknownroleinlearningandmemory.Furthermore,thesereceptorsarehighlypermeabletocalcium,implicatingthemassignificantmodulatorsofintracellularsignalingandneurotransmitterreleasefromneurons.Inthispresentationwewillfocusonthepharmacologicalmodulationofα7nAChRswithnovelpositiveallostericmodulators(PAM)thatimprovetheendogenouscholinergicneurotransmissionwithoutdirectlyactivatingα7nAChRs.DifferentcompoundshavebeenreportedasPAMofα7nAChR,includingivermectin,galantamine,5-hydroxyindole(5-HI)orPNU-120596.Accordingtotheirpharmacologicalprofiles(effectsoncurrentresponses,reactivationofdesensitizedα7nAChRs,augmentationofAChwindowcurrent,andagonistconcentration-responsecharacteristics)theexistenceofatleasttwosubtypesofα7nAChRPAMshasbeenproposed.WehavetestedtheeffectsofatypeI(5-HI)andatypeII(PNU-120596)PAMonneurotransmitterrelease inhippocampalneurons inculture.Wehavefoundthatbothcompounds increasedGABAreleaseinresponsetotheselectiveactivationofα7nAChRs,withnoeffectsonglutamaterelease.Theseresultssuggestthatthesecompoundsmightbetherapeuticallyusefulinprocessessuchasschizophreniainwhichadecreaseinthenumberofα7*nAChRs(particularlyinthehippocampus)hasbeendescribed.α7nAChRsagonistsandPAMsarebeingalsotestedforthetreatmentofprocessessuchasAlzheimer’sdisease,autism,orpai

28

EFFECTS OF NEW ALKALOIDS FROM NATURAL SOURCES THAT MODULATE THE NICOTINIC RECEPTOR IN NEURONAL AD MODELS.

Fuentealba, J1., Araya, Juan1.,Silva-Grecchi, Tiare1.,1Fisiologia, Ciencias Biologicas, Universidad De Concepción. (Sponsored byFondecytIniciacion1109091;Fondecyt1130747)EffectsofnewalkaloidsfromnaturalsourcesthatmodulatethenicotinicreceptorinneuronalADmodels.

JorgeFuentealba1,2,JuanAraya1,TiareSilva-Grecchi1

1Laboratory of Neuroactive Compound Screening, 2Center for Advanced Research in Biomedicine (CIAB-UdeC); PhysiologyDepartment,FacultyofBiologicalSciences,UniversityofConcepción,Concepción,Chile.

Alzheimer´sdisease(AD)isaprogressiveanddeadlyneurodegenerativedisorderthathasnocure.Ithasbeenestablishedthatsolubleoligomers(~56kDa)ofthebetaamyloidpeptidehaveafundamentalroleinthedevelopmentofthispathology,whichhasbeenstronglyassociatedwithdeteriorationincholinergicneuronaltransmission(cholinergictheory).Furthermore,somecurrentlyusedtreatmentsmodulatethecholinergictoneinthecentralnervoussystem,buttheyhavenotbeeneffectiveatstoppingthedisease.NeuroprotectivepropertiesofnicotineinADtreatmenthavebeendescribedthroughitseffectonα7subtypenicotinicreceptors (nAChR) and the further activationof thePI3K/Akt/Bcl-2 survivalpathway. This evidencepresents thepossibility toexamineα7agonistsasanewpossibletherapyforAD,whichdoesnotpossesssecondaryandadverseeffectsthatcomefromnicotineuse.

NewquinolizidinicalkaloidsfromtheFabaceaefamilywereobtainedtoevaluatetheirpossibleactiononα7nAChRandpossibleneuroprotectiveeffectsinaneuronalADmodel.Weusedcellularviability,Ca2+microfluorimetry,electrophysiologyandwesternblot techniques inneuronalprimary culturesandcell lines toevaluate theeffectsof theextractsonAβ-induced toxicity. Theprotectiveeffectofthesealkaloidswasobservedinratcorticalneurons,butnotincellsthatdidnotexpressα7nAChR,suchasHEK293T.Additionally,arecoveryintheAβ-induceddecreaseinsynapticactivityfrequencyintheneuronalnetworkwasobserved.OurresultssuggestthatquinolizidinicalkaloidsexertneuroprotectiveeffectsagainstAβtoxicitythroughmodulationofα7nAChR,whichcouldrepresentthestartingpointforcharacterizationofnewmoleculesfromnaturalsourceswithanti-ADactivity.

Fondecytiniciacion1109091;Fondecyt1130747

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SYMPOSIUMMULTIDISCIPLINARY APPROACHES IN THE STUDY OF THE BRAIN:

FROM GENES TO CLINICSChairs:JimenaSierralta-PedroMaldonado

THE ENDOPLASMIC RETICULUM AND PROTEIN TRAFFICKING IN AXONS

Couve, A1,2., 1ProgramofPhysiologyandBiophysics, InstituteofBiomedicalSciences(ICBM),UniversidaddeChile. 2BiomedicalNeuroscienceInstitute(BNI)UniversidaddeChile.

Neuronsare responsible for thegenerationandpropagationof actionpotentials that constitute the fundamentalmechanismforinformationtransfer.Theyarelargeandpolarizedcellswithdendritesandaxonsconstitutingtheirmajorfunctionaldomains.Axonsarethinandlongspecializationsthatmediatetheconductionofelectricalimpulses.Regulationoftheaxonalproteomeiskeytogenerateandmaintainneuralfunction.Althoughfastandslowproteintransportshavebeenknownfordecades,newlyidentifiedmechanismstocontroltheabundanceofaxonalproteinsbasedonlocalbiosynthesisandprocessinghaveemergedinrecentyears.Wespeculatethatearlysecretoryorganellesintheaxonprovideafunctionalroutefortraffickingand,incombinationwith localprotein synthesisofmembraneand secretedproteins, analternativemechanism to regulate theaxonalproteome.UsingcomplementaryinvitroandinvivoexperimentalmodelsinratsandDrosophilaweexplorethepresenceandfunctionoftheendoplasmicreticulumandotherearlylocalsecretoryorganellesintheaxon.InDrosophilaweinvestigatetheroleofneuronalatlastininlocomotionandaxonalproteintrafficking.Inmammalianperipheralneuronsweevaluatetheroleofearlysecretoryorganellesinthedeliveryofsodiumchannels.FundedbyCONICYTUSA2013-020,Fondecyt1140617andICMP-09-015F.

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CHEMOKINE SIGNALING PAVES THE WAY FOR THE INITIAL TRAJECTORY OF HABENULAR AXONS.

Guerrero, Nestor2,1.,Meynard,Margarita2,1., Armijo, Lorena2,1., Rojas-Rivera, Diego2,1., Palma, Karina2,1., Colombo, Alicia2.,Reig,German2,1.,Hetz,Claudio2,1.,Concha,Miguel2,1.,1BNIBiomedicalNeuroscienceInstitute.2ICBM,FacultaddeMedicina,UniversidaddeChile.(SponsoredbyGrantSponsors:FONDECYT(1120558)AndTheScientificMillenniumInitiative(P09-015-F).)

Onekeystepinneuraldevelopmentistheestablishmentofconnectivitybetweenneuronalgroups,aprocessthatisbasedontheinteractionbetweentheaxonalgrowthconeanditsenvironment.Besidestheclassicalaxonguidancesystemsthatcomprisesemaphorin-,netrin-,slit-andephrin-basedenvironmentalligands,signalsthatareessentialforcellularchemotaxissuchasthechemotacticcytokines(chemokines)alsoparticipateinaxonguidance.Forinstance,thestromalcell-derivedfactor-1(Cxcl12),achemokineknowntocontrolproliferationandmigrationofprogenitorcellsthroughthereceptorCxcr4,alsoworksasanaxonguidancecue invarious contexts. In theembryonic zebrafish forebrain,cxcr4b transcriptsaredetected in thehabenulae (Hb)atthetimeofefferentaxonalprojectionsuggestingaroleofCxcr4/Cxcl12signaling inguidingHbaxons.Duringdevelopment,theseaxonsorganizeasabilateralpairoffasciclesknownasfasciculiretroflexusthatextendventrocaudallyindirectionoftheventralmidbrain.Inthisstudy,weaskedwhetherCxcr4/Cxcl12signalingplayedanin vivoroleinguidingHbaxonstotheventralmidbraininzebrafish,andifthisroleinvolvedtheinteractionwithotheraxonguidancecues.WevisualizedHb-IPNconnectivityinlarvaeofthedoublemutant-transgeniccxcr4-/-;puo4f1-hsp70:GFPandcxcl12-/-;puo4f1-hsp70:GFP,usingin vivoimaging,anti-acetylatedtubulinimmunostaining,lipophilicdyetracing,focalelectroporation,andmorpholinooligonucleotideknock-downofrobo3a/b.We found thatHbaxonsdevoidofCxcr4a/Cxcl12b signaling fail toexit theHb from itsnormal ventralpoint, showconvoluted trajectories, excessive branching, and project ectopically to the contralateral Hb and the antero-dorsal forebrain.Importantly,thesedefectsarecompletelyrescuedbyfunctionalabrogationofrobo3a,which isexpressedathigh levelsbyHbneurons,suggestingthatCxcr4a/Cxcl12bantagonizesRobo3a-dependentrepulsivecuesnormallypresentposteriorandventraltotheHb.WeobservedthatCxcr4bbiochemicallyinteractswithRobo3,andthatbothreceptorsbecomeinternalizedfromthecellmembraneinthepresenceofCxcl12.WethusproposethatCxcr4/Cxcl12antagonizesRobo3-mediatedrepulsionthroughremovalof repulsiveprotein complexes from the surfaceof thegrowth cone.Our resultsprovidenovel insightsofhowgrowth conesintegratepotentiallyconflictingcuesandnavigatethroughrepulsiveenvironments.

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STUDY OF THE LOCALIZATION, TRAFFIC AND FUNCTION OF SYNAPTIC PROTEINS USING DROSOPHILA NEUROMUSCULAR JUNCTION AS MODEL SYSTEM.

Sierralta, Jimena1.,Astorga,César1.,DeGregorio,Cristian1.,Delgado,Ricardo1.,Andrés,Couve1.,1BiomedicalNeuroscienceInstitute,FacultaddeMedicina,UniversidadDeChile.(SponsoredbyICM-P-09-015F/FONDECYT1120684/CONICYT-USA20130020)

Drosophilaisapowerfulsystemtostudyneuronalfunctionduetothediversityofmolecularandgenetictoolsandthefeasibilitytodoin vivostudies.InparticularthestudyofthesynapticfunctioninDrosophilaisfacilitatedbytheuseofthelarvalneuromuscularjunction(NMJ)asamodelsystem.TheNMJisanaccessibleglutamatergicsynapsethatsharesseveralsimilaritieswithvertebrateglutamatergicsynapses,suchassimilarcomplementofreceptorsandscaffoldproteins,andaregulatedglutamatereleasesimilartocentral synapses.Evenmore,accessibleaxonsallowthe in vivo studyof the trafficof synapticproteinsalong theaxonsbytheuseofGFP-labeledproteins.Wewillpresentworkontheanalysisofthefunctionoftwohighlyconservedgenes.atlastin,the secondmost common genemutated in hereditary spastic paraplegias (HSP) a genetic disorder characterized by length-dependentaxonopathyofthecorticospinaltracts.Atlastin,islocalizedtothetubularER,whereitcatalyzesthehomotypicfusionofERmembranes.GeneticmanipulationofAtlastinexpressioninDrosophilamotorneuronassociatestolocomotordefectsofthelarvaeandadultflies.InthisHSPmodeltheorganizationoftheERandGolgiapparatusinmotorneuron`saxonsshowalteredmorphologiesaswellasthesynapticfunctionevaluatedbytheevokedcurrentsinthemuscle.Thesecondgenewehavestudiedisthegendlgascaffoldproteinthatinvertebrateshasbeenassociatedtotheanchoringoftheglutamatereceptors(GLUR)inthepostsynapticmembraneandtheiractivitydependentinsertionorremovalatthesynapse.Inadditiontothepostsynapticdefectsfoundindlgmutantsthatassociatenotonlytheclusteroftheglutamatereceptorbutalsothecomposition,ourresultsstronglysupportaroleofDLGproteinsintheefficiencyofthesynapticvesiclesrelease.Moreexactlyinthelocalizationandabundanceofvoltagedependentcalciumchannels.

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EYE MOVEMENT DURING FREE VIEWING OF NATURAL IMAGES AS MARKERS OF SCHIZOPHRENIA.

Maldonado, P1.,Mayol,Rocio2.,Egaña,Jose2.,Gaspar,Pablo3.,Silva,Hernan3.,1PDFB,BNI,FacultaddeMedicina,UniversidadDeChile.2BiomedicalNeuroscience Institute,Medicina,UniversidadDeChile.3ClinicaPsiquiatrica,Medicina,UniversidadDeChile.(SponsoredbySupportedByIniciativaCientificaMilenioBNIICMP09-015-F)

Inschizophrenia,patientsdisplaydysfunctionsduringtheexecutionofsimplevisualtaskssuchasantisaccadeorsmoothpursuit.Inmoreecologicalscenarios,suchasfreeviewingofnaturalimages,patientsappeartomakefewerandlongervisualfixationsanddisplayshorterscanpaths.Itisnotclearwhetherthesemeasurementsreflectalterationsintheirproficiencytoperformbasiceyemovements,suchassaccadesandfixations,orarerelatedtohigh-levelmechanisms,suchasexplorationorattention.Weutilizedfreeexplorationofnaturalimagesofdifferentcomplexitiesasamodelofanecologicalcontextwherenormallyoperativemechanismsofvisualcontrolcanbeaccuratelymeasured.Wehavefoundthatpatientsexhibitsimilarscanpathsaswellassaccadesfrequencyandfixationsduration.Newweareexploringdifferencesinsaliencyofstimulitoexplorepotentialimpairmentinthemagno-cellularandparvo-cellularpathways.Theseresultswillhelpelucidatethemechanismsofvisualmotorcontrolthatareaffectedinschizophreniaandcontributetothefindingofadequatemarkersfordiagnosisandtreatmentforthiscondition.

33

INNERVATION TARGET SPECIFICITY AND CELL-CELL INTERACTIONS DURING PERIPHERAL NERVE REGENERATIONCECI, M1.,MARDONES-KRSULOVIC,C1.,SANCHEZ,M1.,VALDIVIA,L1.,GUTIERREZ,D1.,ALLENDE,M1.,1DEVELOPMENTALBIOLOGYLABORATORY,FACULTADDECIENCIAS , FONDAPCENTERFORGENOMEREGULATION -UNIVERSIDADDECHILE. (SponsoredbyFunding:FONDECYT3120073(MLC),1110275AndFONDAP15090007(MLA).

Introduction:Damage toperipheral axonsusually leads toabetteroutcomecompared to injuries to centralnervous system;however it is currentlyunknownhowneurons re-establish their target innervations to recover functionafter injury, andhowaccessory cells contribute to this task. To answer this,we used the zebrafish posterior lateral line (pLL) system. The sensoryorgansare located in thebodysurfaceandare innervatedbyafferentneurons,whosesomasare located in thepLLganglion.TheperipheralprojectionsoftheseneuronsareenterelycoveredbySchwanncells,whereastheircentralprojectionsgenerateasomatotopicsensorymap inthehindbrain.Materials and methods:ByelectroablationwesectionedthepLLnerve.GeneticlabelingofsingleaxonsandtheuseoftransgenicmarkersofSchwanncellsallowedustoindividualizedifferentcomponentsinthissystemandtocharacterizetheirbehaviorsduringtheregenerativeprocess.Results:Afteraxonaldamageinthelaterallinenerve,theoverallanteroposteriorsensorymapisreproducedafteritsregeneration.However,thereisadegreeofpromiscuityandoriginaltargetorgansarenot reacquiredwithabsolutefidelity.Furthermore,Schwanncellsarerequired fordirectionalextensionandfasciculationoftheregeneratingnerve.Conclusion:TheaccessibilityofthepLLnerveandtheavailabilityoftransgeniclinesthatlabeltheirsynaptictargetsprovidesanoutstandinginvivomodeltostudythedifferenteventsassociatedwithaxonalextension,targetreinnervation,andthecomplexcellularinteractionsbetweenglialcellsandinjuredaxonsduringnerveregeneration.

SYMPOSIUMYOUNG NEUROSCIENTISTS SYMPOSIUM

Chair:AdriánPalacios

34

MODULATION OF BK CHANNEL VOLTAGE GATING BY DIFFERENT AUXILIARY Β SUBUNITS

Castillo, Karen1.,Contreras,Gustavo1.,Lorenzo,Yenisleidy1,2.,Pupo,A2,1.,Neely,Alan1.,Gonzalez,Carlos1.,Latorre,Ramon1.,1CentroInterdisciplinariodeNeuriociencia,Ciencias,UniversidadDeValparaíso.2ProgramadeDoctoradoenNeurocienciaUniversidadDeValparaíso.(SponsoredbyThisWorkIsSupportedByFONDECYTGrants1110430(R.L.),1120802(C.G);ANILLOGrantACT1104(C.G.);FONDECYTPostdoctoralFellowship3140590(G.F.C.);CONICYTGraduateFellowship(YLAndAP).TheCINVIsAMillenniumInstitute)

BKαchannelsarekeycomponentsforthephysiologyinmostmammaliantissuesandaremodulatedbyßsubunits(ß1-ß4),whichconferparticularfunctionalpropertiestothechannel,forspecificcellularneeds.MinimalalterationsinBKchannelfunctionmaycontributetothepathophysiologyofseveraldiseasesincludinghypertension,asthma,cancer,epilepsyanddiabetes.Auxiliaryß1andß2subunitsareable tostabilize theBKvoltagesensorequilibrium in theactiveconformation,whilstß3hasnoeffectonvoltagesensorbalance.DespitethefactthattheBKphenotypeproducedbyeachoftheßsubunitshasbeenwellcharacterized,controversiesexistregardingthemolecularmechanismsbymeansofwhichtheseauxiliarysubunitscanmodifythegatingpropertiesofBKchannel.Byconstructingchimericßproteinsbetweenß1andß3subunitsandbymeasuringgatingcurrents,wewereabletoidentifythatthecytoplasmicelementsofß1areresponsibleforthemodulationofthevoltagesensorofBKchannelinabsenceofCa2+.Particularly,lysineresiduesK3andK4arenecessaryandsufficienttorecoverthegatingchargemovementfeaturesofα/ß1channelphenotype.Moreover,weobservealeftshiftinmorethan100mVinthechargeversusvoltagerelationshipsinBKαandBKα/ß1channelsinpresenceofCa2+.TheseresultssuggestthatthevoltageandcalciumsensorsinBKchannelarestronglycoupled,allowingfastresponsestobothphysiologicalstimulus.

35

RYANODINE RECEPTOR AND ATLASTIN-2 SHAPE CALCIUM SIGNALS IN SINGLE RAT HIPPOCAMPAL NEURON DENDRITES

Cordova, John 1.,Ramirez,Omar1.,Cerda,Mauricio1,2.,Lobos,Pedro1.,Paula-Lima,Andrea1,3.,Couve,Andres1,5.,Härtel,Steffen1,4.,Hidalgo,C5,1,6,7.,1BiomedicalNeuroscienceInstitute,FacultyofMedicine,UniversityofChile.2AnatomyanddevelopmentalBiologyProgram,FacultyofMedicine,UniveristyofChile. 3Institute for research inDental sciences, FacultyofDentistry,UniversityofChile.4AnatomyanddevelopmentalBiologyProgram,FacultyofMedicine,UniversityofChile.5PhysiologyandBiophysicsProgram,FacultyofMedicine,UniversityofChile. 6Centrodeestudiosmolecularesde lacélula,FacultyofMedicine,UniversityofChile.7PhysiologyandBiophysicsProgram,FacultyofMedicine,UniversityofChle.(SponsoredbyFunding,CONICYT21120689(JAC),Fondecyt3110157(OR),1150756(APL),1151029(SH),1140545(CH),ICMP09-015-F(JAC,OR,APL,AC,SH,CH).))

Voltagegatedpotassiumchannelsappeartobemachinespreciselybuildtotransportpotassiumionswithremarkableefficiency.Bothselectivityandvoltagesensitivityappeartobehighlyoptimized.Tostudyionconductionandtheextentoftheconformationalchanges leading to activation by voltage,we used sucrose as a tool, both viscous and osmotic effects, to estimate the poredimensions and the volumetric changes in the voltage sensor during activation. Sucrose increased viscosity, allowing for anexperimentalconditioninwhichthepotassiumcurrentarelimitedbythediffusionalaccessofpotassiumionstothepore.Thus,makinguseofthetheoryofdiffusioncontrolledreactionsispossibletoestimatethechannelporeopeningdimensionsandexplainhowthisdimensionspecify loworhighconductance inK-channels.Sucrose increasedosmolarity favorordisfavorthevoltagedependentactivation.Thus,byaddingsucrosetoeithertheexternalortheinternalsideofthechannelwemeasuredtheosmoticworkdevelopedbythevoltagesensorduringactivation.Weproposethattheoverallosmoticworkofthevoltagesensorissmall,butitiscomposedbyverydissimilarworksbythedifferentcomponentsofthevoltagesensor.Thus,weconcludedthattheenergyrouteforeachmovingsensingchargemustbedifferent.

36

SWEET EXPERIMENTS: WHAT SUCROSE HAS TAUGHT US ABOUT POTASSIUM CHANNELS

Diaz, Ignacio1., Naranjo, David2.,1Centro Interdisciplinario de Neurociencias, Facultad de Ciencias, Universidad de Valparaíso,CINV.2CentroInterdisciplinariodeNeurociencias,FacultaddeCiencias,UniversidadDeValparaíso.(SponsoredbyThisWorkWasSupportedByFondecytGrants1120819And1131003)

Voltagegatedpotassiumchannelsappeartobemachinespreciselybuildtotransportpotassiumionswithremarkableefficiency.Bothselectivityandvoltagesensitivityappeartobehighlyoptimized.Tostudyionconductionandtheextentoftheconformationalchanges leading to activation by voltage,we used sucrose as a tool, both viscous and osmotic effects, to estimate the poredimensions and the volumetric changes in the voltage sensor during activation. Sucrose increased viscosity, allowing for anexperimentalconditioninwhichthepotassiumcurrentarelimitedbythediffusionalaccessofpotassiumionstothepore.Thus,makinguseofthetheoryofdiffusioncontrolledreactionsispossibletoestimatethechannelporeopeningdimensionsandexplainhowthisdimensionspecify loworhighconductance inK-channels.Sucrose increasedosmolarity favorordisfavorthevoltagedependentactivation.Thus,byaddingsucrosetoeithertheexternalortheinternalsideofthechannelwemeasuredtheosmoticworkdevelopedbythevoltagesensorduringactivation.Weproposethattheoverallosmoticworkofthevoltagesensorissmall,butitiscomposedbyverydissimilarworksbythedifferentcomponentsofthevoltagesensor.Thus,weconcludedthattheenergyrouteforeachmovingsensingchargemustbedifferent.

37

SYMPOSIUMEXTRINSIC AND INTRINSIC SIGNALS THAT MODULATE BRAIN

FETAL/NEONATAL PROGRAMMINGChair:PaolaHaeger

NEUROIMMUNE AND NEUROVASCULAR EFFECTS OF 3RD TRIMESTER ETHANOL EXPOSURE

Valenzuela, Carlos1., Topper, Lauren1.,Welch, Jason1.,Mayfield, Jacob1., 1Neurosciences,Medicine, University of NewMexicoHealthSciencesCenter.(SponsoredbyNationalInstitutesOfHealthGrants:R37-AA015614,P50-AA022534AndT32-AA014127)

Fetalalcoholexposureisaleadingcauseofpreventablebirthdefects.TherangeofeffectsthatcanbeproducedbyfetalalcoholexposureisdenotedasFetalAlcoholSpectrumDisorder(FASD).Inthisstudy,weinvestigatedtheroleoftheneuroimmuneandvascularsystemsinthepathophysiologyofFASD.Tomodelbinge-likealcoholexposureduringaportionofthe3rdtrimesterofhumanpregnancy,weexposedratstoalcoholvaporinhalation.AnimalprocedureswereapprovedbytheUNM-HSCInstitutionalAnimalCareandUseCommittee. Pregnant Sprague-Dawley ratswereobtained fromHarlan (Indianapolis, IN) andarrivedatgestationalday14-16.Subsequently,weexposeddamswiththeirpupstoairorethanolonpostnataldays3,4,and5for3-4hoursdailyinthevaporchambers.Wefoundthatexposuretohighlevelsofethanol(bloodethanolconcentration(BEC)=0.4-0.5g/dl)causesPurkinjecelldegenerationinthecerebellarvermisatP6andP45;however,thiseffectwasnotobservedinthehippocampalformation.SignificantincreasesinmRNAlevelsforpro-inflammatorycytokines(interleukin-1βandtumornecrosisfactor-α)wereobservedinboththecerebellumandhippocampusduringalcoholwithdrawalperiods.Importantly,markedastrocyteactivationwasobservedinboththehippocampusandcerebellarvermis,whereasmicroglialactivationwasobservedonlyinthecerebellarvermis.InthecerebralcortexofP6-7rats,wedetectedsomespontaneousmicro-bleedsinthesurfaceofthecerebralcortexofairexposedcontrolpups.Inbrainsfromethanolexposedpups,thenumberandsizeofbleedsperbrainweresignificantlyincreased.Coronalbrainsectionsshowedthatmostofthemicro-bleedswerelocatedinthemotorandsomatosensoryregionsofthecerebralcortex.Micro-bleedswerealsoobservedinratsexposedtolowerlevelsofethanolvapor(BEC=0.08g/dl).Activationofmicrogliaandastrocyteswasevidentinthebleedarea.Atthebehaviorallevel,ratsexhibitedalterationsingait(assessedwithusingtheCatwalktest)andspatialmemory(assessedusingcontextpre-exposurecontextualfearconditioning)atP45-50.Thesefindingssuggestthat3rdtrimesteralcoholexposurehastime-andbrainregion-dependent,andthatnotonlymicrogliabutalsoastrocytesandvascularalterationsplayanimportantroleinthemechanismofactionofethanol.Collectively,theseeffectscouldcontributetothebehavioraldeficitsassociatedwithFASD.

38

ROLE OF REACTIVE OXYGEN SPECIES IN THE ALCOHOL-DEPENDENT COGNITIVE DEFICIT IN RATS EXPOSED TO ETHANOL IN UTERO.

Haeger, Paola1., Contreras, Marcela1., Cortés, Paulina1., Muñoz, Daniela1., De La Fuente, Erwin1., 1DEPARTAMENTO CIENCIASBIOMEDICAS,FACULTADDEMEDICINA,UNIVERSIDADCATOLICADELNORTE.(SponsoredbyFONDECYT1140855)

Alcohol intakeduringpregnancymaygeneratesevereeffectsinthecognitivedevelopmentofoffspring.Childrenwithprenatalalcoholexposuremayexhibitmorphologicalandfunctionalalterations,whicharecalledbythegenerictermFetalalcoholspectrumdisorders (FASD). Particularly, prenatal alcohol exposure in both human and animal models produces learning and memoryalterations,andincreasesvulnerabilitytoalcoholandothersubstanceabuse.

Ethanolexposureincreasesoxidativestressindevelopingorgans,includingthebrain.Indeed,evenabriefexposuretoethanolduringgestationcanproduceperdurableimbalancebetweenthelevelsofintracellularreactiveoxygenspecies(ROS)andbrainantioxidants, which can be correlated with cognitive deficits. Interestingly, antioxidant treatment, during maternal ethanolingestion,hasbeenrepeatedlyshowntoimprovebehavioraldeficitsinrodentmodelsofFASD.However,theimpactofthegeneralantioxidanttreatmentintheadultageoftheoffspring,andthespecificROS-dependentmechanism,hasnotbeenfullystudied.

WedevelopedaratmodelofFASD,whichhasbeenevaluatedatadultage(p70).Theseanimalsconsumedaround25%moreethanol thancontrol rats,duringonedayofa freechoicealcohol test; theyspentsignificantlymoretime in thealcoholpairedcompartment in theplacepreference conditioning test; finally, they showeda significantdelay in spatialmemory acquisitioncomparedwithcontrolrats.TheneurobehavioralstatusfoundinthismodelresemblesFASDcharacteristics,previouslydescribed.We are currently performing studies to identify themolecular sources of ROS generation aswell as themechanism of theirinvolvementinthedeficitofmemoryacquisition,andincreasedvulnerabilitytoalcoholdependence.FundedbyFondecytgrantN°1140855

39

GESTATIONAL CHRONODISRUPTION IMPAIRS HIPPOCAMPAL EXPRESSION OF NMDA RECEPTOR SUBUNITS AND SPATIAL MEMORY IN THE ADULT OFFSPRING

Torres-Farfan, C1.,Vilches,Nelson2.,Spichiger ,Carlos1.,Mendez,Natalia1.,Richter,Hans1., 1InstitutodeAnatomía,HistologíayPatologia, Medicina, Universidad Austral De Chile. 2Fisiopatologia, Medicina, Universidad de Chile. (Sponsored by ProyectoFondecyt1120938(CTF)ANILLOACT1116(CTF-HGR))

Epidemiologicalandexperimentalevidencecorrelatesadverseintrauterineconditionswiththeonsetofdiseaselaterinlife.Forafetustoachieveasuccessfultransitiontoextrauterinelife,amyriadoftemporallyintegratedhumoral/biophysicalsignalsmustbeaccuratelyprovidedbythemother.Weandothershaveshowntheexistenceofdailyrhythmsinthefetus,withperipheralclocksbeingentrainedbymaternalcues,suchastransplacentalmelatoninsignaling.Amongdevelopingtissues,thefetalhippocampusisakeystructureforlearningandmemoryprocessingthatmaybeanticipatedasasensitivetargetofgestationalchronodisruption.Here,weusedpregnantratsexposedtoconstantlighttreatedwithorwithoutmelatoninasamodelofgestationalchronodisruption,toinvestigateeffectsontheputativefetalhippocampusclock,aswellasonadultoffspring’srhythms,endocrineandspatialmemoryoutcomes.Thehippocampusoffetusesgestatedunderlight:darkphotoperiod(12:12LD)displayeddailyoscillatoryexpressionoftheclockgenesBmal1andPer2,clock-controlledgenesMtnr1b,Slc2a4,Nr3c1andNMDAreceptorsubunits1B-3A-3B.Incontrast,in thehippocampusof fetusesgestatedunderconstant light (LL), theseoscillationsweresuppressed. In theadultLLoffspring(rearedinLDduringpostpartum),weobservedcompletelackofday/nightdifferencesinplasmamelatoninanddecreasedday/nightdifferencesinplasmacorticosterone.IntheadultLLoffspring,overallhippocampalday/nightdifferenceofgeneexpressionwas decreased,whichwas accompanied by a significant deficit of spatialmemory.Notably,maternalmelatonin replacementto dams subjected to gestational chronodisruptionprevented the effects observed in both, LL fetuses and adult LL offspring.Collectively, thepresentdatapoint toadverseeffectsofgestational chronodisruptionon long-termcognitive function; raisingchallengingquestionsabouttheconsequencesofshiftworkduringpregnancy.Thepresentstudyalsosupportsthatdevelopmentalplasticityinresponsetophotoperiodiccuesmaybemodulatedbymaternalmelatonin.

40

EFFECTS OF METABOLIC INSULT ON THE POSTNATAL NEUROGENESIS AND BEHAVIOR.

Morales, Paola1,2., Esmar, Daniela1., Tapia-Bustos, Andrea1., Espina-Marchant, Pablo1., Gutiérrez-Hernández, Manuel1., Rubio,Mariana1., Rojas-Mancilla , Edgardo1., Pérez-Lobos, Ronald1., Palacios, Esteban1., Serth,Marcos1., Muñoz, Valentina1., Lespay,Carolyne1., Bustamante, Diego1., Herrera-Marschitz,Mario1., 1BNI, Programme of Clinical&Molecular Pharmacology,MedicalFaculty,ICBM,UniversityofChile.2ProgrammeofClinical&MolecularPharmacology,MedicalFaculty,ICBM,UniversityofChile.(SponsoredbySupportedByInstituteMillennium-BNI(BNIP09-015-F)AndFONDECYT(No1120079;1110263)Grants)

Perinatalasphyxia(PA)primesthedevelopmentoftheCNS.Severeasphyxiahasbeenlinkedtocerebralpalsy,mentalretardation,andepilepsy,whilemild-severeasphyxiahasbeenassociatedwithattentiondeficitsandhyperactivityinchildrenandadolescents,andincreasedriskforlowintelligencequotientscore.Obstetriccomplicationsarealsocentralriskfactorsofpsychiatricdiseasesincludingschizophrenia,bipolardisordersandvulnerabilitytodrugabuse,inparticulartococaineaddiction.

PAaffectsmainlyneurocircuitriesofmesencephalonandhippocampusgeneratingdeficitsinneuro-behaviouraldomains,includingearlyneurological reflexes, locomotoractivities, spatialmemory,non-spatialworkingmemory, reactivity tostressandanxiousbehavior.

Severalcompensatorymechanisms,includingneurogenesis,havebeenproposedasmediatorsofendogenouslytriggeredprotectionagainstcelldeathinducedbyPA.Indeed,increasedneurogenesishasbeenobservedinDG,CA1andSVZafterPA.Neurogenesiscanberegulatedbyalargenumberofmolecules,includinggrowthfactors,neurotransmitters,suchasdopamineandserotoninandotherfactorsstillundercharacterisation.Theexpressionofbasicfibroblastgrowthfactor(bFGF)isupregulatedinDGandSVZfollowingPA.WehavereportedevidencesuggestingthatbFGF,throughactivationoftheMAPK/ERKpathwaycouldbeinvolvedintheneurogenesisinducedbyPA.Also,modulatorproteinsofbFGFpathway,Spry(Sprouty),FRLT3(leucine-richrepeattrans-membraneprotein)andSef(similarexpressiontoFGF)areupregulatedafterPAsuggestingafineregulationoftheneurogenicprocesssinceSpryandSefprovideinhibitoryregulation,whileFRLT3stimulatestheactivationofbFGFtransductionpathwaywithspecifictemporalandregionalpatterns.

Dopaminergic system isvulnerable toPA.Recently,usingorganotypic cultures fromDG,was found thatdopamine (DA)fibresoriginatedinsubstantianigra(SN)/ventraltegmental(VTA)areatargetingthehippocampusandSVZ,establishinganatomicalandfunctional contactswith neuronal progenitors anddiferentiallymodulates the neurogenesis. Recent studies have suggestedthatstimulationofD2receptorpromotesneuronalproliferation,providingperhapsatargetforneuroprotection.Thesereceptorsarealsolocatedonastrocytes,releasingneurotrophicfactorslikebFGF,drivingneurogenesis.

Therefore,furtherprogress isneededinunderstandingthesubjacentmechanismsinvolvedinthemodulationofneurogenesisafterbraininsults,inordertodevelopnoveltherapeuticstrategiesforrestoringthedamagedneurocircuitry.

41

SYMPOSIUMPHYSIOLOGICAL AND STRUCTURAL INSIGHTS OF

ION CHANNELS AND MEMBRANE RECEPTORSChair:ClaudioCoddou

INTRACELLULAR ATP REGULATION OF THE EXTRACELLULAR ATP GATED P2X2 RECEPTOR CHANNEL

Coddou, C1.,1DepartamentodeCienciasBiomédicas,FacultadMedicina,UniversidadCatólicaDelNorte.(SponsoredbyFONDECYT11121302)

ActivationofP2X2receptorchannels(P2X2Rs)ischaracterizedbyarapidcurrentgrowthaccompaniedwithadecayofcurrentduringsustainedATPapplication,aphenomenonknownasreceptordesensitization.Suchfacilitationofreceptordesensitization,termeduse-dependent desensitization (UDD), is observed inwhole-cell recordings and is determinedby intracellular domaincalcium.ExperimentswithvariousreceptorchimerasalsoindicatethatthetransmembraneandintracellulardomainsofP2X2Rare required for development of calcium-dependent desensitization and that decrease in current amplitude slows receptordesensitization. Further experiments inhibiting several protein kinases and phosphatases demonstrate an allosteric nature ofcalciumactionneartheP2X2Rpore.WefurthershowthatUDDispracticallyabolishedintheperforated-cellandtwo-electrodevoltageclampconfigurationsinHEK293cellsandXenopusoocytes,respectively.AdditionofATPbutnotGTPinthepipettesolutionalsoabolishesUDDinwhole-cellrecordings,suggestingthataleakofATPfromcytosoltopipettepermitstheactionofcalcium.Incontrasttocalcium,intracellularATPdoesnotmodulateP2X2Rgatingallosterically,asindicatedinexperimentswithATP-γ-Sintheintracellularsolution.Concordantly,intracellularinjectionofapyrase,anectoATPase,oralkalinephosphataseinoocytesfacilitatedP2X2Rdesensitization.TheadditionofstaurosporineandATPtothepipettesolutionalsoabolishesUDDbutthedesensitizationratesarefaster.MutagenesisstudiesofN-andC-terminalaminoacidsthatarepotentialphosphorylationsitesidentifiedacriticalroleoftheS363residueinATPintracellularaction.ThesefindingsindicatethatATPnotonlyactivatesP2X2Rextracellularlybutalso determines the desensitization kinetics intracellularly through phosphorylation of P2X2R, which prevents developmentof intracellularallostericcalciumaction.Therefore,themetabolicstateofthecellexpressingP2X2Rcould influencethegatingpropertiesofthisreceptor.

42

NEW INSIGHTS ON TRPV1 CHANNELS AS MODULATORS OF SYNAPTIC FUNCTION

Chávez, A,E.InstitutodeNeurociencias,FacultaddeCiencias,UniversidaddeValparaíso,CentroInterdisciplinariodeNeurocienciadeValparaíso.

Theequilibriumbetweenexcitationandinhibitionisessentialforproperbraincircuitfunction.Changesinexcitatoryorinhibitorysynapticefficacycouldhaveprofoundinfluenceoverthisbalanceandarecommonlylinkedtocertaincognitivedeficits,includingautismandepilepsy.Increasingevidencesuggeststhatendocannabinoid(eCBs)playanimportantroleinregulatingthisbalance,buttheprecisemechanismsofthisregulationarenotyetfullyunderstood.Inadditiontobindingtocannabinoidreceptors(CB1Rs),certaineCBssuchasanandamide(AEA)canalsobindandactivatethetransientpotentialreceptorofvanilloidtype1(TRPV1).WhileTRPV1receptorsaremainlyfoundintheperipheralnervoussystem,thesereceptorshavealsobeenfoundinthebrain,wheretheirlocalizationandfunctionalroleisfarlessunderstood.Recently,wereportedthattheeCBanandamideactsonpostsynapticTRPV1tosuppresssynaptictransmissionpresumablybyreducingthenumberofAMPA-typeglutamatereceptors (AMPARs)atexcitatorysynapsesaswellasthenumberofGABAA-typereceptorsatspecificinhibitorysynapses,stronglysuggestingthateCBandTRPV1mightplayanimportantroleinregulatingtheexcitatory/inhibitorybalancerequireforproperbrainfunction.Moreover,usingselectivepharmacologyforserotonin(5-HT)receptorinacuterathippocampalslices,wefoundthatbathapplicationof5-HTdepressesexcitatorysynaptictransmissioninaninput-specificmannerinratandmousedentategyrusviaactivationof5-HT2a/cRs.Notably,thisdepressionalsorequiresactivationofTRPV1channels.Thisfindingrevealsanovelformof5-HT-TRPV1mediatedregulationofexcitatorysynaptic transmissionatcentral synapses.ByelucidatinghoweCBscanmodulatesynapticefficacyviaTRPV1channelswithinaneuronalcircuit,theexactTRPV1localization,theroleofeCB-signalinginmediatingTRPV1activation,andthefunctionalimpactofsuchmodulationiscrucialnotonlyforamorerealisticrepresentationofneuralfunction,butalsoforthedevelopmentofnoveltherapeuticapproachestargetingtheeCBsystem.Inaddition,unmaskingthefunctionalroleofbrainTRPV1wouldallowforthedevelopmentofnovelanalgesicstrategiestocontrolpainwithoutaffectingnormalbrainfunction.

43

PHARMACODYNAMIC INSIGHTS OF Β-ADRENERGIC MECHANISMS: FROM CRYSTALS TO FUNCTION, A TALE FOR ALL CELLS/RECEPTORS

Garcia-Huidobro, J1.,1Biologia,QuímicayBiología,UniversidadDeSantiagoDeChile.(SponsoredbyFondecytGrant114-1132AndCEDENNA,PFB087ProvidedPartialFunding)

Recently,pharmacologicalexperimentsidentifiedtheexpressionandfunctionofβ1,β2andβ3-adrenergicreceptorsinendothelialcellsfromtheratarterialmesentericbedandin-vivoprotocols(AmJPhysiol297:H134,2009).Thisstudyraisedthenovelconceptthatatleastthreeβ-adrenoceptorsparticipateintheregulationofthevascularwallbyactingonendothelialcells,sincealltheseβ-adrenoceptors are coupled to the net synthesis of endothelial NO. This finding, in addition to thewell-known vasodilatorrole of theβ-2-adrenoceptor in the smoothmuscle, allowedproposing that adrenaline and related ligandsplay a role in theregulation of vascular resistance and in blood flow, particularly during exercise, via targets localized in both endothelial andvascular smoothmuscle cells. Theβ-adrenergic receptor is a “firstofakind” transductionprotein.During thefifties, thefirstadrenergic“pharmacophore”wasdesigned;although,bythen,nofirmnotionofthestructural implicationsofthishypothesiswasaccessible.Latter,thecouplingofthereceptortoproteinGswasdecipheredfollowedthereafterbyitssequencingandtheprompt identificationof itsmain structuraldeterminants in accordancewith theputativepharmacophoreunraveled30yearsbefore.TheadventofitscrystalstructurelaidthefoundationtounderstandingitsfunctioningattheatomiclevelandpavedtheroadtoscrutinizetheworkingsofotherGproteincoupledreceptors(GPCR).Infact,forabout7-8yearstherhodopsinandtheβ-adrenergicreceptorsweretheroutineparadigmsforGPCRpharmacodynamics.Thefindingthatthecrystalstructureofβ1andβ2-adrenoceptorscontaincholesterolmolecules imbibed in theprotein structure is compatiblewith thenotion thatallostericmodulationisinherenttothereceptorpharmacodynamics.ThisfindingrecognizedopportunitiesforthediscoveryofallostericmodulatorsinmostGPCRs.Theelucidationofthemolecularstructureoftheβ1theβ2-adrenoceptorshasallowedthedetailedunderstandingoftheirbindingselectivityattheorthostericsiteofeachreceptorsubtype,accountinginadditionforthedifferencesbetweenagonistsandantagonistsateachpharmacophoreofeachβ-adrenoceptorsubtype.Thisinformationwillproveusefultodiscovernovel“tailoredligands”forthesereceptorsindirectbenefitfrompatientsrequiringbetterdrugs.Onefieldofattentionisofcourse,β2-agonistsfortreatmentofasthmaandrelateddiseasesaswellassaferβ-1antagonistsforcardiovasculardrugsforthetreatmentofthemanyvasculardiseasesthatcurrentlyuseadrenergicdrugsfortherapy.

44

TRP CHANNELS IN NEURONAL PHYSIOLOGY AND PATHOLOGY

Leiva, E1.,1DepartamentodeBiología,FacultaddeQuímicayBiología,UniversidaddeSantiagodeChile.(SponsoredbyFONDECYTGrant11140731AndPAI79140059FundedThisWork.)

Thetransientreceptorpotential(TRP)familyofchannelscomprisesmorethan50cationchannels;theyarepresentfromalgaetohumans.Thesechannelsarecomposedofsevenmainsubfamilies;TRPA,TRPC,TRPM,TRPML,TRPN,TRPPandTRPV; theyare expressed in a variety of tissues participating in different cellular functions like intracellular calcium oscillation, immuneresponse,cardiacpacemaking,cellmigration,vasculartone,etc.Newevidenceshowedthatsomeofthesechannelsareimplicatedin importantneuronal functions like transductionof sensorystimulation,neuronalproliferation,differentiation,nervegrowth,excitabilitycontrol,synaptictransmission,neuronaldamageandneuronaldeath.Inthiscontext,TRPCchannelshavebeeninvolvedintheregulationofburstfiringandintrinsicneuronalexcitabilitythroughitssensitivitytomembranelipids.Additionally,TRPVchannelsregulatesynapticstrengththroughendocannabinoidincentralsynapses.Moreover,TRPMchannelshasbeenimplicatedinphysiologicalandpathologicalconditions,forexample,TRPM7isphysiologicallydescribedasanentrywayoftracemetalslikeMg2+,howeverduringischemicepisodesitdrivesneuronalCa2+overload.Furthermore,TRPM4channelsmodulatetheexcitabilitybydrivingtheafterdepolarizationcurrent,whichincreasestheneuronalabilitytofirecontinuousactionpotentials;alsoduringischemia-reperfusion injury,theirnon-regulatedactivationproducesNa+overloadcausingneuronaldeath.AdditionallyTRPM4impairedfunctionhasbeenassociatedwithaxonaldamageinanimalmodelsofmultiplesclerosis.Importantly,TRPC3,TRPM2,TRPM4andTRPM7showdifferentdegreesofredoxsensitivitysuggestingthatitsredoxmodificationcoulddrivesomeoftheirfunctions. In this context, TRP channels areemergingas essential cellular switches allowing tounderstand the control of theneuronalhomeostasisandhowtheirimpairedactivityplayakeyroleinpathologicalconditionsandpotentiallybringingnewdrugtargetagainstneuronaldisorders.

45

CALCIUM DYSREGULATION IN A RODENT MODEL OF ALZHEIMER´S DISEAS

Paula-Lima, A1,2.,More,J2.,Barattini,P2.,Adasme,T2,3.,Hidalgo,C2,3.,Valdes,JL4.,1InstituteforResearchinDentalSciences,Facultyof Dentistry, Universidad De Chile.2Biomedical Neuroscience Institute , Faculty ofMedicine, Universidad De Chile.3Center forMolecularStudiesoftheCell,InstituteofBiomedicalSciences,FacultyofMedicine,UniversidadDeChile.4CenterforNeuroscienceof Memory, Institute of Biomedical Sciences, Faculty of Medicine, Universidad de Chile. (Sponsored by Support: FONDECYT(1150756,1100052,11140580);BNI(P-09-015F).)

Introduction:Currentevidenceindicatesthatsolublebeta-amyloidoligomers(AβOs)arelikelycausativeagentsofmemorylossinage-relatedAlzheimer´sdisease.Recently,wereportedinratprimaryhippocampalneuronsthatAβOsgenerate longlastingCa2+-signals. These signals,which arise fromCa2+-inducedCa2+-release from the endoplasmic reticulum (ER), aremediatedbyryanodinereceptors(RyR)andaresensitivetocellularredoxstate.TheERisinphysicalcontactwithmitochondria;thus,prolongedCa2+release from the ERpromotes sustainedmitochondrial Ca2+-uptake, leading to excessive reactiveoxygen/nitrogen species(ROS/RNS)generationandoxidativestress.WehavereportedthatpersistentRyR-mediatedCa2+-signals invokedbysolubleAβoligomers(AβOs)preventthespineremodelinginvokedbybrain-derivedneurotrophicfactor,decreaseRyR2mRNAandproteincontentsandprovokemitochondrialfragmentation(Paula-Limaetal,2011).Moreover,pre-incubationwithN-acetylcysteine,aneffectiveantioxidantglutathioneprecursor,abolishesthecytoplasmicCa2+increasesandthemitochondrialfragmentationinducedbyAβOs(SanMartinetal,2012).Objectives: Here,weinvestigatedwhethermitochondrialROS/RNSgenerationmodulatestheRyR2 expression changes induced by AβOs, and if decreased hippocampal RyR2 expression affects hippocampal-dependentspatial learning andmemory.Materials and Methods: Primary hippocampal neuronswere transfectedwithmito-Pericam orHyPerMitotodetectmitochondrialCa2+andhydrogenperoxideproduction,respectively,orwereincubatedwithMitoSoxtosensemitochondrialsuperoxidegeneration.MitochondrialfragmentationwasdetectedinneuronsloadedwithMitoTracker.Malerats,bilaterallyinjectedintra-hippocampuswithAβOsorantisenseoligonucleotideanti-RyR2(O-RyR2),weretrainedintheOasisMazetask toevaluatehippocampal-dependent spatial learningandmemory. Fluorescence imagesacquiredby confocalor spinningdiskmicroscopywereanalyzedwith ImageJsoftware;RyR2mRNAexpressionwasevaluatedbyq-PCRandproteincontentbyimmunofluorescence.Results: Intra-hippocampal injections of AβOs or O-RyR2 significantly decreased RyR2 protein content,withoutalterations inRyR3protein levels,and impairedspatial learning.Thedecrease inRyR2mRNA levels inducedbyAβOsrequiredmitochondrialROS/RNSgeneration. Conclusions: Ourresultssuggestthatredox-sensitiveRyR2-mediatedCa2+-releaseis crucial for spatialmemory processes, and suggest that deficient RyR2-mediatedCa2+signaling contributes toAβOs-inducedlearningandmemorydeficits.Support: FONDECYT(1150756,1100052,11140580);BNI(P-09-015F).

SYMPOSIUMAging and Neurodegeneration

Chair:CeciliaHidalgo

46

WNT SIGNALING STIMULATES NEURONAL GLUCOSE METABOLISM AND ENHANCES NEUROPROTECTION AGAINST AΒ OLIGOMERS

Inestrosa, N1.,1CellandMolecularBiology,BiologicalSciences,PontificiaUniversidadCatólicaDeChile.

TheWntsignalingpathway is critical foranumberof functions in thecentralnervous system, including the regulationof thesynapticstructureandfunctionandtheneuroprotectionagainstseveralinsults,includingtheamyloid-bpeptide(Ab1-42)oligomersofAlzheimer´sdisease.ThelossofWntsignalinghasbeenassociatedwithseveralbrainpathologies,includingAlzheimer`sandParkinson`sdisease. In recentyears,anewrolehasbeensuggested for theWntpathwayasacentral integratorofmetabolicsignalsfromperipheralorganstothebrain.Mostneurologicaldisordershavebeenlinkedwithadecreaseinthebrain’scapacitytoutilizeglucose;however,despitetheimportanceofthisissue,littleisknownabouttherelationshipbetweenWntsignalingandbrainglucosemetabolismandhowthisinteractionmightplayaroleinneuroprotection.Herewewillpresentastudy,wheretheactivationofthecanonicalandthenon-canonicalWntsignalingpathways inducesastrongincreaseinglucoseuptakeandtheglycolyticrateincorticalneurons.TheeffectofthecanonicalWntsignalingwasindependentofthetranscriptionofWnttargetgenes.However, theeffectof thenon-canonicalWntsignaling required the intracellulargenerationofnitricoxide.Finally,wedemonstratethattheprotectiveeffectofWntsignalingagainstneurotoxicityinducedbytheAb1-42oligomersispartiallydependentonthestimulationofglucosemetabolism.Together,ourdatasuggestthatWntsignalingstimulatesenergymetabolisminneuronsandthatthiseffectcouldbeakeyfactortopreventneuronalcelldeath.

47

THE WNT EFFECTOR ß-CATENIN IN MODELS OF AMYOTROPHIC LATERAL SCLEROSIS: ALLY OR FOE?

Pinto,Cristina1.,Henriquez, Juan Pablo1.,1CellBiology,BiologicalSciences,UniversidaddeConcepcion.(SponsoredbyFONDECYT1130321AndMINREBRC120003)

Introduction:Wnt pathways are associated to neurodegeneration. Inmicemodels of amyotrophic lateral sclerosis (ALS), theexpressionofWntligandsisalteredinmotoneurons.OurpreviousfindingsshowedthatculturedALS-likemotoneuronsdisplayreducedWntactivity.Here,weanalyzedthelocalizationofß-catenininmotoneuron-likeNSC34cellsexpressingwild-type(NSC-hSOD1WT)ormutated(NSC-hSOD1G93A)superoxidedismutase-1(hSOD1),aswellasinspinalcordandneuromuscularjunctions(NMJs)ofmutanthSOD1mutantmice.Materials and Methods:hSOD1-expressingNSC34cellsweredoublestainedforß-cateninalongwithMAP1B,vimentin,ubiquitin,LC3andγ-tubulin.ß-cateninstainingwasalsoperformedinvibrosectionsofhSOD1G93AmicespinalcordsfromP21toP130andcryosectionsofskeletalmuscles.WesternblotswereperformedincytosolicandmembranefractionsofcontrolandALS-likeNSC34cells.Results:NSC-hSOD1G93Acellsdisplaylargeaccumulationsofß-catenininthecellcortex.These structuresarenotamyloid,andareneitherassociatedwithaggresomes,norwithhSOD1aggregates.Y489andY654 phosphorylated ß-catenin are not incorporated into these structures. In control NSC-hSOD1WT cells, lithium treatmenttranslocatesß-catenintothenucleus.Inturn,inNSC-hSOD1G93Acellslithiumdecreasedß-cateninaccumulations,intheabsenceofnuclearimport.Westernblotanalysesshowedthatß-cateninisaccumulatedinmembranefractionsofNSC-hSOD1G93Acells.Motoneurons of presymptomaticALSmice display ß-catenin structures,whereas controlmice show strong nuclear ß-cateninstaining.Eventhoughß-cateninlabelstheNMJofcontrolmice,thisstainingislostinsymptomaticstagesofALSmice.Discussion:ß-catenin is accumulated in ALSmotoneurons as in other neurodegenerative conditions; but, different to them, it does notaggregateinamyloidstructures.ThesestructurescouldberelatedtotheobserveddecreaseinWntpathwayactivation.Ournovelfindingsalsorevealapotentialroleforß-catenininthemaintenanceoftheNMJinthecontextofALS.

48

AGING AND NEURODEGENERATION: THE MITOCHONDRIAL CONNECTION

Nuñez, Marco T.1.,Urrutia,PamelaJ.1.,Aguirre,Pabla1.,Garcia-Beltran,Olimpo2.,Cassels,BruceK.2.,Tapia,Victoria1.,Mena,NataliaP.1.,1Biology,Sciences,UniversidaddeChile.2Chemistry,Sciences,UniversidaddeChile.(SponsoredbyThisWorkWasSupportedByGrantsFondecyt1130068AndACT-1114FromPIA,CONICYT.)

Mitochondriaare intrinsic sourcesofROS thatplayavital role in the regulationof intracellularCa2+andapoptoticprocesses.Inrecentyears,increasingattentionhasbeengiventotheroleofmitochondrialdysfunctioninthepathogenicdevelopmentofvariousneurodegenerativedisorders.NeuronsareparticularlysensitivetoROSandATPimbalancesderivedfrommitochondrialdysfunctionbecauseoftheiruniqueelongatedmorphologyandtheirdependenceonATPtopropagateelectricalsignals,maintainionicgradients,andfacilitateanterogradeandretrogradeaxonaltransport.Ironaccumulateswithageinthehealthyhumanbrain.Several recent reportshavedescribedbrainmitochondrialdysfunctionwith ironaccumulation inneurodegenerativedisorderssuchasPD,AD,HDandalessinvestigatedgroupofdisordersknownasNBIA(NeurodegenerationwithBrainIronAccumulation),characterizedbythepresenceofhighbrainiron,particularlywithinthebasalganglia.Herewewillreviewthemostprominentevidencethatlinksmitochondrialdysfunctionandironaccumulation,andpresentrecentevidenceontheinterventionofaviciouscycle mitochondrial dysfunction–iron accumulation by the use of novel iron chelators with mitochondria targeting and BBBpermeability,andbysilencingofthecellularironhomeostasisregulatorIRP1,whichisspuriouslyactivatedduringmitochondrialdysfunction.

49

INFLUENCE OF MATERNAL EXPERIENCE ON BEHAVIORAL RESPONSE TO THE MATERNAL SEPARATION STRESS IN MOTHER RATS

Rivarola, M1.,1Fisiología,FacultaddeCienciasExactasFísicasyNaturales,UniversidadNacionaldeCórdoba.(SponsoredbySECyT-CONICET)

Transition to motherhood induces a number of extraordinary physiological, neuroendocrine and behavioral modifications.Thereforepregnancyandpostpartumareperiodsofmaximumneuronalandbehavioralplasticityinafemalelife.Inlaboratoryrodents, thechroniceffectsofpostnatalmanipulationof the infant-mother relationshiphavebeenstudiedexperimentally formorethan50years.Whilemostofthestudiesinwhichtheparadigmofmaternalseparationisusedhavefocusedtheirattentionontheeffectsonoffspring,fewhavefocusedontheeffectsonmaternalbehaviorandphysiology.Althoughlimited,recentworkhasbeguntodocumenttheeffectofmaternalseparationstressondams.Wehypothesizedthatlengthyseparationfromtheirpupswouldaffectemotion,maternalbehaviorandneuroendocrineparameters.Ourworkdemonstratedthatthestressofmaternalseparationduringthepostpartuminducealterationsinthedam\’spup-retrievalbehavior,increasesanxiety-likebehavior,andhasadetrimentaleffectoncognitiveprocesseswhichispreventedbytherewardingaspectsofphysicalcontactwithpups.Additionallywe investigatedwhether reproductiveexperience (numberofpregnanciesandparturition) affects thebehavioral response toanenvironmentalstressastheearlymother-pupseparationbystudyingparametersknowntobeaffectedbylong-termstress.Primiparous(1reproductiveexperience)andmultiparous(MP)(2reproductiveexperiences)age-matchedfemaleWistarratsweresubjectedtoeitheranimalfacilityconditionsordaily4,5hofseparationfrompups(MS)frompostpartumday(PPD)1-21.Maternalbehaviorwasevaluatedduringearlypostpartum.Afterweaning, anxiety and spatialmemorywere assessed. ThepreliminaryresultssuggestthatMPfemalesshowagreaterefficiencyinmaternalcare.Duringearlypostpartum,multiparityandseparationfrompupsinducesanincreaseofactivematernalbehaviors.MPratsshowatrendtowardsbetterperformanceinspatialmemory.Contrary toexpected,MP females showed increasedanxiety-likebehaviors.Althoughpreliminary, thepresent results supporttheconclusionthatreproductiveexperienceinfluencesthematernalresponsetostress.Understandinghowadisturbedmother-infantrelationshipaffecttheneurochemical,physiologicalandbehavioralprofilesindamswillcontributetoabetterknowledgeofpostpartumpsychiatricdisordersandthedetrimentaloutcomeofseparationsforboththemotherandchild.

SYMPOSIUMNEUROPHARMACOLOGY OF STRESS, ANXIETY AND DEPRESSION

Chair:JavierBravo

50

CROSS-TALKS BETWEEN STRESS AND POLYUNSATURATED FATTY ACIDS: ROLE ON DEPRESSIVE DISORDERS

Dagnino-Subiabre, Alexies1., Pérez, Miguel2., Peñaloza-Sancho, Valentín1., Fuenzalida, Marco2., 1Laboratory of BehavioralNeurobiology, Center for Neurobiology and Brain Plasticity, Institute of Physiology, Faculty of Sciences, Universidad deValparaíso. 2Laboratory of Neural Plasticity, Center for Neurobiology and Brain Plasticity, Institute of Physiology, Faculty ofSciences,UniversidaddeValparaíso. (SponsoredbyThisWorkWasSupportedByFONDECYT1141276Grant(AlexiesDagnino).Labsite:www.stress.cl.)

Whilechronicstressinducesdendriticatrophyinthehippocampusandimpairslearningandmemory,supplementationwithn-3polyunsaturatedfattyacid(PUFA)isknowntoimprovesmemoryofunstressedrats.Whethern-3PUFAsupplementationcouldimprovedendriticmorphologyandmemoryofstressedratsremainunknow.MaleSprague-Dawleyratswererandomlyassignedtounstressedandstressed(chronicrestraintstress)experimentalgroups.Afterward,animalsweresupplementedwithn-3PUFAs(DHAandEPAmix)orvehicle.DendriticmorphologyandsynaptictransmissioninthehippocampuswereevaluatedbyGolgistainandpatch-clamptools,respectively.TheY-mazeandMorriswatermazewereusedtoanalyzetheeffectsofchronicstressonmemoryconsolidation.Supplementationwithn-3improveddendriticarchitectureandrestoredthefrequencyofinhibitorypostsynapticcurrentsofhippocampalpyramidal neuronsof stressed rats. In addition,n-3 supplementation improved spatialmemory.Ourresultsdemonstratethatn-3supplementationhadthreebeneficialeffectsonstressedrats:preventsdendriticatrophyinCA3,restorestheGABAreleaseprobabilityinCA1andimprovesspatialmemory.Wespeculatethatn-3supplementationcouldbeusedinthetreatmentofstress-relatedpsychiatricdisorderssuchasdepressivedisorders.

51

EARLY-LIFE INTESTINAL DYSBIOSIS AND ITS IMPACT ON STRESS-RELATED BEHAVIOURS IN YOUNG RATS

Bravo, Javier1., 1GRUPO DE NEUROGASTROBIOQUÍMICA, LABORATORIO DE QUÍMICA BIOLÓGICA, INSTITUTO DE QUÍMICA,FACULTADDE CIENCIAS, PONTIFICIAUNIVERSIDADCATÓLICADE VALPARAÍSO. (SPONSOREDBY FUNDEDBY: VRIEA PUCV #DI037.386/2014,FONDECYT#1130213&FONDECYT#1140776.)

Early-lifeexposuretomicroorganismsiskeytothedevelopmentofthestressresponseinanimals.Forexample,germ-freemicehave reduced anxiety-like behaviours despite producing an exaggerated release of corticosterone upon stressful stimuli. Thissuggests that thepresenceofmicrobesduringearly-life is necessary toadequate couplebehavioural andphysiological stressresponses.

Thecommunicationbetween thegastrointestinal tract (GIT)and thecentralnervoussystem(CNS) is thebasis toexplainwhyinterventions in gut bacteria affect stress-related behaviours, corticosterone levels and brain gene expression. Moreover,alterationsinthisconversationbetweentheintestinalmicrobiome,GITandCNSduringearly-lifecouldbethebiologicalsubstratetogastrointestinaldysfunctionandstress-relatedpsychiatricdisordersthatmightappearthroughoutlife.

The infant gut begins to get colonized at birth by cross-contaminationwith themother’s vaginal, faecal and skinmicrobiota.Inhumans,bytheageof2years,thegutmicrobiotaresemblesthatofanadult.However,alterationsinthebalancebetweenputativespeciesof“protective”bacteriaversus“harmful”ones inearly-lifecanoccur,affecting themicrobiome-gut-brainaxisinawaythataffectshowtheindividualcopeswithaneverchangingenvironmentlaterinlife.Theseepisodesofdysbiosiscanoccurinmanyforms,fromnutritionalalterations,toexposuretowidespectrumantibiotics.Interestingly,theeffectsofearly-lifedysbiosisonCNSfunctionandphysiologicalresponsestostressareevidentininfantilerats.Thispresentationwillreviewempiricalevidenceontwoanimalmodelsofearly-lifedysbiosis:1)dietarysupplementationofinfantileratswithaLactobacillus(aprobiotic),inulin(aprebiotic)andthemixtureofbothand2)perinatalexposuretobroad-spectrumantibiotics.Inbothmodels,theeffectofdysbiosisonstress-relatedbehavioursandrelevantmolecularmarkersassociatedwiththestressresponseand/orrelevanttotheneuropharmacologyofstresswasevaluatedatayoungage.Allofthesefindingsareobservableduringinfancy,suggestingthatfurtherinterventionscanbemadeinordertopreventtheoccurrenceofstress-relatedpsychiatricdisorderslaterinlife.

52

NEUROPHARMACOLOGY OF THE BRAIN-GUT-MICROBIOME AXIS: FOCUS ON SEROTONIN AND TRYPTOPHAN METABOLISM

Clarke, G1.,1Psychiatry/APCMicrobiomeInstituteUniversityCollegeCork.

The brain-gut axis is a bidirectional communication system between the central nervous system and the gastrointestinaltract.Accumulatingevidencepoints toacritical role for thegutmicrobiome in regulating thenormal functioningof thisaxis.Dysregulation of this reciprocal communication network impacts substantially on physiology, brain and behaviour across thelifespan.Inparticular,earlylifeperturbationsofthedevelopinggutmicrobiotacanimpactonbraindevelopmentandpotentiallyleadtoadversementalhealthoutcomeslaterinlife.Thisabilityofthegutmicrobiotatoinfluencebrainfunctionissupportedbybothclinicalandpreclinicalstudiesandextendstocomplexbehavioursrelevanttoanxiety,depression,painandcognition.Serotoninfunctionsasakeyneurotransmitteratbothterminalsofthebrain-gutaxisanditisbecomingclearthatthemicrobialinfluenceontheserotonergicsystemmaybeanimportantneurobiologicalconsequenceofperturbationstothegutmicrobiome.Thereisalsosubstantialoverlapbetweenbehavioursinfluencedbythegutmicrobiota,thosewhichrelyonintactserotonergicneurotransmission and the prominent features of stress-related microbiome-gut-brain axis disorders such as irritable bowelsyndrome.Themechanismsunderpinningthiscrosstalkrequirefurtherelaborationbutmayberelatedtotheabilityofthegutmicrobiotatocontrolhosttryptophanmetabolismalongthekynureninepathway,therebysimultaneouslyreducingthefractionavailableforserotoninsynthesisandincreasingtheproductionofneuroactivemetabolites.Thisdual impact inadditiontotheabilitytoregulaterelevantCNSreceptorexpressiongivesthegutmicrobiomeabroadneuropharmacologicalprofile.Inaddition,thereareneuralprocessesinthegastrointestinaltractwhichcanbeinfluencedbylocalalterationsinserotoninconcentrationswithsubsequent relayof signalsalong thescaffoldingof thebrain-gutaxis to further influenceCNSneurotransmission.Takentogether,thesefindingsfirmlyestablishthegutmicrobiotaasacriticalnodeinthebrain-gutaxisandonewhichmaybeamenableto therapeutic interventions. An improved appreciation of these dynamic interactions can bring benefits across diagnostic,preventativeandtreatmentdomains.

53

SYMPOSIUMPHARMACOLOGICAL APPROACHES FOR PATHOPHYSIOLOGICAL

CONDITIONS ASSOCIATED WITH HYPOXIA AND OXIDATIVE STRESSChair:RodrigoCastillo

CARDIAC HYPOXIC INJURY AND OXIDATIVE STRESS: PROTECTIVE STRATEGIES AND POTENTIAL CLINICAL APLICATIONS.

Castillo, R1.,Farías,Jorge2.,Herrera,Emilio3.,Carrasco-Pozo,Catalina4.,Sepúlveda,Nestor5.,1ProgramadeFisiopatología,InstitutodeCsBiomédicas,FacultaddeMedicina,Universidaddechile. 2Departamentode IngenieríaQuímica,Facultadde IngenieríayCiencias,UniversidaddelaFrontera,UniversidadDeLaFrontera.3ProgramadeFisiopatologíaOriente,ICBM,FacultaddeMedicina,UniversidaddeChile,Universidaddechile.4DepartamentodeNutrición,FacultaddeMedicina,UniversidadDeChile.5Laboratoriode ProducciónAnimal, Facultad de Ciencias Agropecuarias y Forestales,UniversidadDe La Frontera. (Sponsored by Fondecyt11110246(RLC.);1151119(EHV.);1130232(CCP.))Hypoxia is a pathophysiological condition associatedwith several responses at cardiovascular, pulmonary and vascular levels,whichmayderiveinchronicdiseases.Thisisrelevantinhumanpopulationsexposedtohighaltitude,ineitherchroniccontinuous(permanentinhabitants)orintermittenthypoxia(IH)(highaltitudeworkers,touristsandmountaineers).InChile,itisestimatedthat1.000.000peopleliveathighlandsandmorethan55.000workinhigh-altitudeshifts.IHisassociatedwiththedevelopmentofsystemichypertensionandleftventriculardysfunction.Atpresent,however,ourunderstandingofthebasicmechanismslinkingIHandcardiovasculardysfunctionislimitedbythepathophysiologicalheterogeneityofhypoxicpatientsandthepresenceofmultipleconfoundandcomorbidconditions,includingobesityandpreviouscardiacimpairments.Moreover,thegreatvarietyofresponsesranges fromno clinical effects to strongpulmonaryhypertensionandvital risk.Consequently, there is a seriousneed for thedevelopmentofexperimentalmodelstostudythemechanismsinvolvedinthecardiovascularresponsestoIHandthepotentialdeleteriouseffects.

Acute exposure to high altitude has been shown to induce oxidative stress in healthy human lowlanders, as indicated by anincreaseinfreeradicalformation.However,IHmayinduceanischemicpreconditioning-likeinsomeanimalmodels.Therefore,pharmacologicalpreconditioningstrategieshaveacquired importance inthedevelopmentofnovel therapies.Compellingdatashowcardiovascularbeneficialeffectsinconsumingfattyacidshighlypresentinfish,suchasomega3(Ω3),docosahexanoicacid(DHA22:6Ω3),andeicosapentanoicacid(EPA20:5Ω3).Thesefattyacidsregulatecellmembranephysicochemicalproperties(i.e.,fluidity,organizationandpermeability) thataffect signalingpathways,withprobableantioxidantandantiinflammatoryeffectsoncardiactissue.Also,dietaryΩ3wouldinduceaformofpreconditioning,nutritionalpreconditioning,limitinghypoxiccardiacinjury,andmyocardialinfarctionandendowscardioprotectionaspowerfulasischemicpreconditioning.Thesemechanismshavebeenshowedinsomeclinicaltrialsincoronaryandheartfailurepatients.Consequently,itisdesirableexploretheeffectsofΩ3supplementationinhumanpopulationsexposedtoIH.Grant:Fondecyt11110246(RLC.);1151119(EHV.);1130232(CCP.)

54

PERINATAL HYPOXIA AND OXIDATIVE STRESS: MECHANISMS AND POTENTIAL NEW THERAPIES.

Herrera, Emilio A1,2.,Ebensperger,German1.,Reyes,Roberto1.,Llanos,Anibal1,2.,1ProgramadeFisiopatología,ICBM,FacultaddeMedicina,UniversidaddeChile.2InternationalCenterforAndeanStudies(INCAS)UniversidaddeChile.(SponsoredbyFondecyt1110595,1120605,1130424,1140647&1151119.)

The etiology of several non-communicable diseases has been linked with adverse perinatal conditions, such as hypoxia andoxidativestress.Bothconditionsareseeninchronichypobarichypoxiaathigh-altitude(>2,500m).Gestationandbirthathighlandsinduceintrauterinegrowthrestrictionandpulmonaryhypertensionoftheneonate.Neonatalpulmonaryhypertensionmayinducecardiopulmonaryremodelingandrightheartfailureifsustainedintime.Thisisapathophysiologicconditionwithmultifactorialetiologies, due to an imbalance between vasodilator and vasoconstrictor mechanisms and maladaptive vascular remodelingofpulmonaryarteries. Several studieshavedefined themechanisms involved in theelevatedpulmonaryvascular resistanceandremodeling,deriving innovel therapeuticproposals tested inanimalmodelsandsomeof themhavereachedtheclinicalpractice.Theseapproachesincludetreatmentsaimingtoenhancevasodilatorpathwayssuchasnitricoxide-guanylylcyclase-GMP,prostaciclyn-adenylatecyclase-AMP;andtodecreasevasoconstrictorpathwayssuchasendothelin-1andcalciumchannels,amongothers.Further,inthelastyears,antioxidantsandantiproliferativeagentshaspositionedasimportanttherapeuticadjuvantstopreventand/ortreatpulmonaryhypertensionoftheneonate,suchasmelatoninandcarbonmonoxide.NewtreatmentsarebadlyneededsinceNOadministrationfails in40%oftheneonateswithpulmonaryhypertension.Therefore,wewillreviewsomeofthesenoveltreatmentstoattenuatehigh-altitudeperinatalcomplications.

55

OCCUPATIONAL EXPOSURE TO CHRONIC INTERMITTENT HYPOXIA: BETWEEN ACCLIMATIZATION, INTOLERANCE AND PROTECTION.

Jimenez, D1.,1PublicHealth,Medicine,UniversidadDeChile.Nativesealevelworkers,occupationallyexposedtochronicintermittenthypobarichypoxia,CIH,maybeaffectedintheirhealthandsafetybyenvironmentalandergonomicfactors,atmosphericandO2pressure,temperature,humidity,solarradiation,climateandunusualphysical-mentalexigencies.Hypobarichypoxiaisthemostimportantfactor.From3000meters,thoseexposedshowsignsintheirventilatoryresponse,elevatedEPO,Hb,pH,bloodpressure,pulse,coronaryandcerebralbloodflow,andpulmonarypressure,whileremainingstablecardiacoutputandstrokevolume;butduringsleeptheyhaveperiodicbreathing,dropSatO2andrisecentralapnea/hypopnea.

Thetypeofexposuretoaltitude,acute,chronicorintermittent,determinestheintensityandtimingofthesesigns.InCIHthatismorediverseandcomplex,becauseatcyclingdaysinnormobariaandhypobariathesubjectgoesthroughchronic,acuteanddeacclimationexposure.Inhomogeneouslaborgroupsbyage,healthstatus,altitudeandergonomicneeds,theanswertotheCIH has been heterogeneous, since excellent tolerance to proper acclimatization even intolerance. At higher altitudes moreheterogeneity.

Inoccupationalhealth isstrategicachievebetteracclimatizationandpreventthetwotypesof intolerance,aggravationofpre-existingconditions,oroccurrenceofdisablingaltitudeillnessesasacutemountainsickness,pulmonaryedema,cerebraledema,pulmonary hypertension, sleep disturbances, reactive hypertension, etc. Also interested occupational physical-muscularworktolerance,duetophysiologicalVO2maxdecline,withriskoffatiguefacingintenseeffort.

Adequatetoleranceandacclimatizationisprioritized,withstaffnotcarryinghypoxiaincompatibleillnesses,controllingthequalityof sleep, screeningand treatmentof altitude illnesses,promotinghealthy lifestyle, anddesignappropriateworksites.Besidesmitigationmeasures,suchweatherizationdormitories,useofacetazolamideandsupplementaloxygenincaseofcentralapneas.

Recentbiomedical information is confirming somecardioprotectiveeffectsof chronichypoxia,which fordecadeshadalreadybeenpointedoutininhabitantsofthePeruvianaltiplano.EvaluationsincontrolleddosesofHICindicatepositiveeffectsoncardiachemodynamics,homeostasisofredoxregulationandcalcium,modulationofapoptoicssignalsandneovascularization.ThisopensnewhorizonsfortheepidemiologytoleranceofworkerstooccupationalexposuretoCIH.

56

PHARMACOLOGICAL STRATEGIES FOR THE PREVENTION OF THE EFFECT OF INTERMITTENT HYPOBARIC HYPOXIA IN RAT TESTIS.

Farias, Jorge1., Castillo, Rodrigo2., Figueroa, Elias1., Short, Stefania1., Zepeda,Andrea1., Figueroa,Carolina1., 1DepartamentodeIngenieríaQuímica,FacultaddeIngenieríayCiencias,UniversidaddeLaFrontera.2ProgramadeFisiopatología,InstitutodeCienciasBiomédicas,FacultaddeMedicina,UniversidaddeChile.(SponsoredbyFondecyt1151315(JGF);11110246(RLC))

InChile,duetotheintensiveactivitydevelopedinconfiningareasoftheAndesMountainsranginginaltitudeover4000asl,therehasbeenanincreasingintermittentmigrationofhumanresourcestohighaltitude(HA)lands.Thisunusualcondition,definedashypobarichypoxia,affectsnotoriouslyinanylivingorganismandthereshowsaseriesofpathophysiologicalresponses.Studiesperformed in rats under chronic hypobaric hypoxia and intermittent hypobaric hypoxia have registered changes in testicularmorphology togetherwith lossof spermatogenic cells inall stagesof spermatogenic cycle. Furthermore, recentexperimentalevidencesreinforcedtheexistenceofanoxidativemetabolisminepididymisofratssubjectedtohypobarichypoxiaduetotheincreaseintheregulatorenzymeexpressionofreactiveoxygenspecies(ROS),ThisincreaseintheproductionofROSinducedariseinapoptosisatgerminalcelllevel,leadingtoastateofhypo-spermatogenesisthatmayjeopardisemasculinefertility.Therefore,theeventualdevelopmentofoxidativestressinspermatogeniccellsandconsequentlythespermatozoidsofworkerssubjectedtoHA,eitherchronicorintermittent,turnsouttobecriticalwhenitposesasanimminentrisktotheviabilityandqualityofthereproductivecellsofworkerssubjectedtointermittenthypobarichypoxia.Forseveralyears,thestudyoftheresponsestohypoxicinsultsandpharmacologicaltargetshasbeenthemotivationofourgroup. Inthiscase,theantioxidantsupplementationwithascorbate,melatoninorblueberrieshavebeenused toattenuate thisprooxidant imbalanceand theirderived functional andstructuralconsequencesinreproductivecells.Thisworkdescribessomeofthemechanismsunderlyinghypoxicresponsesandthenovelpotentialtherapeuticapproachesinratmodelofintermittenthypoxia.

57

A GABAERGIC SIGNALING WITHIN THE BASOLATERAL AMYGDALA COMPLEX MODULATES THE INFLUENCE OF STRESS ON FEAR MEMORY

Molina, V1.,RodriguezManzanares,P.1.,Ortiz,V1.,Espejo,P.1.,Giachero,M.1.,Calfa,G.1.,Calfa,G.1.,Isoardi,N.1.,Carrer,H.1.,Carrer,H.1.,Martijena, I.1.,Maldonado,N.1., 1DepartamentodeFarmacologia,FacultaddeCienciasQuímicas,UniversidadNacionaldeCórdoba.

Thereisconsensusthatstressfulexperiencesenhancefearmemoryformation.Thisinfluenceishighlyadaptivesinceitisextremelyrelevantfortheanimal’ssurvivaltoanticipateandavoidpotentialdangerousstimuli.TheAmygdalaBasolateralComplex(BLA)iscruciallyimplicatedinfearmemoryformation.ItiswellrecognizedthatGABAergicinterneuronswithintheBLAareresponsibleforcontrollingtheactivityofprojectingglutamatergiccellsthroughfeedforwardandfeedbackinhibition.FindingsfromourlaboratoryhaverevealedthatasinglerestraintexperienceelicitedBLAneuronhyperexcitability,whichresultedfromthereductionofrecurrentGABAergicinhibition.Thisviewwasfurthersupportedbypharmacologicalevidence,sinceintra-BLAinfusionofmidazolam(MDZ),apositivemodulatorofGABAasites,priortostresspreventedtheenhancedfearmemoryofstressedanimals.Incontrast,blockadeofGABAareceptorsintheBLA,butnotintheadjacentCeA,facilitatedfearmemory,similartotheamelioratinginfluenceinducedbystress.Moreover, thestress-inducedpromotingeffectontheemergenceofassociative fearmemory iscoincidentwith thefacilitatinginfluenceofstressonLTPgenerationinBLA,aneuroplasticityprocesslinkedtofearmemoryformation.Accumulatingevidence suggests that the dorsal hippocampus (DH) is a downstream target of BLA neurons in contextual fearmemory andhippocampalstructuralplasticityisproposedtoprovideasubstrateforthestorageoflong-termmemories.Recentdatashowedthat prior stressful experience promoted contextual fearmemory and enhanced dendritic spine density in theDH. Intra-BLAinfusionofMDZpreventedthefacilitatinginfluenceofstressonstructuralremodelinginDH.Similarlytothestress-inducedeffects,theblockadeofGABAasiteswithintheBLAamelioratedfearmemoryandinducedstructuralremodelingintheDH.Overall,thesefindingssuggestthatstress,byreducingthisinhibitoryGABAergiccontrol,wouldresultinanunmaskedactivationofprojectingneuronsoftheBLA,facilitatingfearmemoryandpromotingstructuralchangesintheDHassociatedtocontextualfearmemory.

SYMPOSIUMMEMORY AND STRESSChair:JimmyStehberg

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LONG TERM MEMORIES THAT ARE REACTIVATED WITHOUT BEING BEHAVIORALLY EXPRESSED

Delorenzi, A1., Maza, FJ1.,Ojea, A1.,Molina, VA2.,Stehberg, J3.,1Laboratorio de Neurobiología de la Memoria, DepartamentoFisiología,BiologíaMolecular yCelular, FacultaddeCienciasExactas yNaturales,UniversidaddeBuenosAires.2Departamentode Farmacologia, Facultad de CienciasQuímicas,Universidad nacional de Córdoba.3Laboratorio deNeurobiologíaUniversidadNacionalAndrésBello.

Duringtheconsolidationprocessthememorytracesarestabilizedand,consequently,memorieswereconceivedasfixedafterbeingconsolidated.Thisfixedmemorytraceparadigmwaswithdrawn;long-termmemoriesarenotinvariable.Activatedbyremindercues, thememory trace canbeunstabilizedagainduring the reconsolidationprocess. Thewithdrawalof theaforementionedparadigmhas led tonewperspectives about severalmnemonicprocesses. In this context,wewill discussour studieson themodulationofmemoryexpressionduringreconsolidationasabiologicalmechanism-conservedthroughoutevolution-thatenablescontinuousupdatingofmemory. Weproposethatduringbothmemoryconsolidationandreconsolidation,neuromodulatorscandetermine theprobabilityof thememory trace toguidebehavior, i.e. theycaneither increaseordecrease itsbehavioralexpressibilitywithoutaffectingthepotentialofpersistentmemoriestobeactivatedandbecomelabile.Moreover,thepositivemodulationofmemoryexpressionduringreconsolidationcanoccurevenifmemoryisunexpressed.Thisviewoffersanewviewregardingbothweaktrainings,whichdonotgeneratelongtermmemories,andexperimentalamnesia:memoryretrievalcannotbeconceivedasthesaurusofmemoryexpressionduringthetestingsessions.Thisnewviewshowthateveninhumandeclarativememorytheperiodsinwhichlong-termmemorycanbeactivatedandbecomelabileduringreconsolidationexceedstheperiodsinwhichthatmemoryisexpressed,providingdirectevidencethatconsciousaccesstomemoryisnotneededforreconsolidation.Inthehypothesispresented,memoryexpressibility-theoutcomeofexperience-dependentchangesinthepotentialtobehave-isconsideredasaflexibleandmodulableattributeoflong-termmemories.Expressionseemstobejustoneofthepossiblefatesofre-activatedmemories.

59

A ROLE FOR THE INSULAR CORTEX IN FEAR OF THE NEW AND ANXIETY

Stehberg, J1.,Moraga-Amaro,R1.,Díaz-Galarce,R1.,Quintana-Donoso,D1., Rojas, S1.,T amburini,G1.,Méndez, L1., Escorza, T1.,Pacheco, R2., 1Laboratorio deNeurobiología, Centro de Investigaciones Biomédicas, Universidad Andrés Bello. 2Laboratorio ofNeuroinmunologíaFundaciónCiencia&Vida.

TheInsularcortexisburieddeepwithinthetemporallobeandisacomplexstructurethatreceivesvisceral,gustatory,somatosensory,pain,visualandauditoryinformation.TheInsulahasalsobeenassociatedtoemotionsanditsaugmentedactivityiscommontoallanxiety-relateddisorders.HereweshowthattheInsularegulatesgeneralanxiety,viamediatingwithinthebrain,thesystemiceffectsofstresshormonesadrenalineandglucocorticoids.Moreover,glucocorticoidsactingattheInsulashowparadoxicaleffects,giving new clues into the complexities of orchestrating behavior in response to stress and arousal. Thisworkwas funded byFONDECYTN°1130724.

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THE ROLE OF ASTROCYTE-DERIVED EXOSOMES IN THE STRESS RESPONSE

Wyneken, U1., 1CIB,Medicina, UniversidadDe Los Andes. (Sponsored by ProgramaDe Investigación Asociativa (Conicyt) AndFondecyt(Conicyt))Thesearchforanimalmodelsofmooddisordersandoftheirsubtypes,supportedbythepresenceofbiomarkers,isayetunmetgoalthatwouldbeusefulfortheirdiagnosis,treatmentandthemonitoringoftheirprogressionandtreatmentresponsiveness.Recently,exosomes, i.e.extracellularvesiclesof30-100nmthatoriginate intheendosomalpathway,haveemergedaspotentmediatorsofdiseasesandassuch,asmarkersofdiseaseprocesses.ExosomescontainproteinsandRNAspecies(bothmRNAandmiRNAs),affectingthefunctionoftargetcellsbyregulatingtheirgeneexpression.

We induceddepressive-likebehaviors in ratsbyexposure tochronic stress,attainedbyprocedures that restrictmovementofanimalseitherbyrestrictioninsmallcagesorbyimmobilizationinbags.Depressive-likebehaviorsinratswererevertedselectivelybyantidepressantdrugsactingeitheronserotoninornoradrenalinneurotransmission.Moreover,whensearchingforabiomarkerwefoundthattheglycolyticenzymealdolaseCwasdifferentiallypresentinexosomesderivedfromthecerebrospinalfluid(CSF)orfromthebloodplasmaofstressedanimals.ThecentraloriginofaldolaseCinplasmaexosomeswasdemonstratedbyin uteroelectroporationofforebrainastrocyteswithGFP-aldolaseC.Inaddition,exosomesinstressedanimalscontainadistinctmiRNAsignature.Theeffectofstress-derivedexosomesofastrocyteoriginandofstress-regulatedexosomalmiRNAsonneuronalfunctionandmorphology,aswellasoftheirtargetgenesinrecipientcells,iscurrentlyunderinvestigation.

We have found a novel form bywhich stressmay affect neuronal function by volume transmission and possibly,may affectperipheraltissuesaswell.

61

ORAL PRESENTATIONS I

62

ACUTE AND CHRONIC AMPHETAMINE TREATMENTS MODULATES DIFFERENTIALLY NURR1 AND NF-KB P65 EXPRESSION IN THE RAT VENTRAL TEGMENTAL AREA

Arredondo, Cristian1.,González,Marcela1.,Andrés,María1.,Gysling,Katia1.,1BiologíaCelularyMolecular,CienciasBiológicas,PontificiaUniversidadCatólicaDeChile.(SponsoredbyFundedByProjectFONDECYTN°3130683)

Amphetamineisapowerfulpsychoactivedrug.Therewardingandreinforcingpropertiesareduetoitscapacitytoelevateextracellulardopaminelevelsintheventralmidbrainareaanditstargetnucleithroughthereversalofthedopaminetransporter(DAT)cycleNurr1isanuclearreceptoressentialforthedevelopmentandsurviveofmidbraindopaminergicneurons.ThistranscriptionfactorregulatestheexpressionofkeygenesofthedopaminergicphenotypeasDATandtyrosinehydroxylase(TH),therate-limitingenzymeinthedopaminesynthesis.

Moreover,drugsofabuseareknownastriggersofinflammatoryprocessesanddamageinthebrain.ExposuretococaineandmethamphetaminealtersthetranscriptionfactorNF-kBlevelsinPC12cells.Interestingly,Nurr1andNF-kBp65interacttoregulateinflammatorygenesinresponsetolipopolysaccharide(LPS)inmicrogliaandastrocytes.Itiswellknownthatrecurrentdrugsconsumptioncauseslong-lastingchangesindopaminergicneurons.So,weareinterestedinrevealingthemolecularmechanismunderlyingdrugabuse.InthisworkwestudytheeffectsofacuteandchronicamphetamineexposureintheexpressionofNurr1,NF-kBp65andTHintheratmidbrainregion.MaleSprague-Dawleyratsweighingabout280gwereinjectedwithamphetamine(1.5mg/kg)acutelyoreverydayduringfourteendays(chronic).OurdatashowthatNurr1andNFkB-p65co-localize inTH-positivecells intheratVTA.Nurr1,THandNFkB-p65expressionwasassessedbyWesternblotsofventralmidbraintotalproteinextracts.AcuteamphetaminetreatmentinducedanincreaseinNurr1,THandNFkB-p65proteinlevels.ChronicamphetaminetreatmentdecreasedNurr1andNFkB-p65proteinlevels,butTHwasunchangedcomparedtosaline-treatedrats.OurresultsshowthatNurr1inmidbraindopaminergicneuronsrespondsinadifferentwaytoacuteorchronicamphetaminetreatmentandsuggestthatNurr1andNFkB-p65couldmediateacommonresponsetoamphetamine.

63

KOR-DEPENDENT POTENTIATION OF QUINPIROLE-INDUCED SENSITIZATION: NEUROCHEMICAL INTERACTION AND COHABITATION WITH D2 RECEPTORS.

Escobar, Angélica1., Noches, Verónica1.,Meza, Rodrigo2,3.,Henny, Pablo3.,Gysling, Katia1.,Fuentealba, José4.,España, Rodrigo5.,Andrés,Maria1.,1Biologia Celular yMolecular, Ciencias Biológicas, Pontificia Universidad Católica De Chile.2Ciencias Fisiológicas, Ciencias Biológicas,PontificiaUniversidadCatólicaDeChile.3Anatomía,Medicina,PontificiaUniversidadCatólicaDeChile.4Farmacia,Química,PontificiaUniversidadCatólicaDeChile.5NeurobiologyandAnatomy,SchoolofMedicine,DrexelUniversity.(SponsoredbyFondecytGrants#1110352And1150200)

Kappaopioidreceptors(KOR)arelocatedpresinapticallyondopaminergicterminalsofthenucleusAccumbens(NAc).SimilartoD2Rautoreceptors,acute activation of KOR decreases dopamine extracellular levels and locomotor activity. Interestingly, co-activation of KOR potentiates thelocomotor sensitization inducedby repeated administrationof theD2Ragonist, quinpirole.Ourpreviousmicrodialysis studies showed thatrepeatedtreatmentwithU69593(U69),aKORagonist,decreasestheinhibitoryD2Rautoreceptorcontrolontonicdopaminerelease.Therefore,wehavehypothesizedthatKOR-dependentpotentiationofQNP-inducedlocomotorsensitizationisduetoareducedinhibitoryD2Rautoreceptorfunction.Here,weshowtheneurochemicalandmolecularinteractionsbetweenD2RandKORintheNAcofrodents.MaleSprague-DawleyratswereadministeredwithamixofU69andQNPevery2-3daysuntil8injectionswerecompleted.Horizontallocomotoractivitywasmeasuredimmediatelyaftereachinjection.Forty-eighthoursafterthelastinjection,ratswereanesthetizedforMicrodialyisisorfastscancyclicvoltammetry(FSCV)intheNAc.Afterbaselinecollections,ratswereinjectedwithU69andthirtyminuteslaterwithQNP.MicrodialysisdatashowsthatKORdependentpotentiationoflocomotorsensitizationisaccompaniedbydecreaseddopamineandaconservedD2autoreceptorfunction.FSCVdatashowasimilar rapiddecreaseofphasicdopamine release inbothcontrolanddrug-treatedrats,when injectedwithQNPandU69.ToevaluatewhetherKORandD2Rcolocalizeinanyneuronalsub-structure,immunofluorescenceassayswerecarriedoutinsynaptosomesfreeofpost-synapticelements,micebrainslicesandcultureddopamineneurons.ImmunofluorescentassaysinNAcsynaptosomesshowedthat20%ofKORpositivelabelco-localizewithD2R.Inaddition,mesencephalicprimarycellcultureshowedthatD2R-likelabellingismainlylocatedonsomato-dendriticprofilesofTH-positiveneurons,whileKORislocatedmainlyonaxonprofiles.ImmunofluorescenceinmiceNActissueslicesshowed thatD2R-like labelling ismainlypostsynapticandpartially colocalizeswithKOR-like labelling,probably inGABAprojectingneurons.Together,ourdata indicatethatpresynaptic inhibitorycontroloverdopamine ismaintainedafterrepeatedKORandD2RactivationandthatU69potentiationofQNP-inducedlocomotorsensitizationisnotlinkedtoanincreaseofphasicdopaminerelease.Inaddition,histologicaldatasupportthepossibilityofapostsynapticmolecularinteractionbetweenKORandD2R.

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Hypothalamic synaptic plasticity disturbance caused by a chronic high fat diet feeding

Guzmán, Luis1., Hernández-Galaz, Sergio1.,Valdivia, Sharin1.,Gutiérrez, Noemí2.,Kerr, Bredford2.,1Laboratorio de Biología Centro de Estudios Científicos-CECs, Universidad Austral de Chile.2Laboratorio de Biología Centro de Estudios Científicos-CECs. (Sponsored by Fondecyt 1140162, PFB 01/2007.)

The hypothalamus is the main structure of the body in charge of energy balance homeostasis, since the hypothalamus senses the nutritional state of the body and orchestrates feeding behavior and energy expenditure in order to maintain energy homeostasis. This system is primarily controlled in the arcuate nucleus of the hypothalamus (ARC), which holds two major neuronal groups involved in sensing the peripheral signals, nutrient availability and regulate caloric intake. These neurons are the Pomc neurons which are activated by anorexigenic peripheral signals and release α-MSH to the various target nuclei. On the other hand are the Agrp neurons, which are activated by orexigenic peripheral signals, releasing AgRP and GABA. These two groups of neurons have opposing functions, decreasing caloric intake/increasing energy expenditure and increasing caloric intake/decreasing energy expenditure, respectively. As a cause of the pandemic health issue caused by obesity, many studies have focused on the effect of diet induced obesity, as an experimental model of the present globalized “western diets”, showing alterations in the brain, in cognition and also metabolic disturbances. Diet induced obesity causes alterations in energy balance control by altering the capability of neurons located in the hypothalamus to interconnect and integrate peripheral signals into this negative feedback loop. This cause altered health conditions such as leptin resistance, insulin resistance, metabolic disease, chronic inflammation among other alterations. In this work, mice where fed with a high fat/low carbohydrate diet to evaluate the effect of a ketogenic/ non-overweighting diet in the hypothalamic synaptic plasticity and to comprehend the molecular events that conform the metabolic and neuronal alterations caused by high fat diets in different groups of neurons. For this purpose various assays were performed including the analysis of mRNA expression levels, western blot analysis, immunofluorescence and also food intake experiments. Mice fed with a high fat diet showed alterations in the expression of genes and proteins involved in energy balance and synaptic plasticity as well as changes in hypothalamic morphology. In addition, mice fed with a high fat diet had similar food intake and body weight in relation to control mice. This study provides an insight of the molecular changes caused by chronic high fat diet consumption in murine hypothalamus and better understanding of the molecular mechanisms by which the metabolic disturbances occur during high fat diet feeding.

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EXPOSURE TO AN ENRICHED ENVIRONMENT DURING PREGNANCY AND LACTATION MODULATES FEEDING BEHAVIOR IN ADULT OFFSPRING

Ojeda-Provoste, Patricia1.,Hernández-Galaz,Sergio1.,Kerr,Bredford2.,1LaboratoriodeBiologíaCentrodeEstudiosCientíficos-CECs,UniversidadAustraldeChile.2LaboratoriodeBiologíaCentrodeEstudiosCientíficos-CECs.(Sponsoredby1140162.PFB01/2007.)Agrowingbodyofevidencesuggeststhatenvironmentalfactorsduringearlystagesofdevelopmentarecapableofmodifyingthemetabolicphenotype observed during adulthood. In addition, a previous study demonstrated that the postnatal exposure of mice to an enrichedenvironment,awidelyusedparadigmtoinducesynapticplasticity,increaseshypothalamicsensitivitytometabolicsignalsthatcontrolfeedingbehavior.Here,weevaluatedtheeffectofexposingmicetoanenrichedenvironmentduringpregnancyandlactationonadultoffspringfeedingbehavior andbodyweight. In order to address thephenotypic andbehavioral effects of this early stage- environmental intervention, C57/B6breedingpairsofmicewereexposedtoeitheranenrichedenvironmentorstandardconditions.Maleoffspringfrombothconditionswerehousedinstandardcagesfromweaningtoadulthood.Bodyweightwasrecordedweeklyandbothfoodintakeandlocomotoractivitywastestedinmetaboliccagesatthree,sevenandtwelveweeksofage.

Ourresultsshowthatmicefrombreedingpairsexposedtoenrichedenvironmentexhibitedanincreaseinbodyweightbetween3and6weeksofage.Afterthisperiodoftime,wedidnotobserveanydifference inthebodyweightofmicefrombreedingpairsexposedtoeachoftheenvironmentalconditions.Thereductioninbodyweightgainexhibitedbymicefrombreedingpairsexposedtoanenrichedenvironmentafter6weeksofagewasnotassociatedtoanincreasedlocomotoractivity,butratherwithareductioninfoodintakethatremaineduntillaterages.Toevaluatewhetherthisdifferenceinfeedingbehaviorwasassociatedtoanepigeneticmodificationduringexposuretoanenrichedenvironment,impactingthepost-weaninggeneexpressionpattern,wefirstevaluatedtheexpressionofhypothalamicgenesassociatedtoenergyhomeostasis.OurresultsshowadecreasedexpressionoftheanorexigenicgenePomc,whichmayexplainthedecreasedfoodintakeobservedinadultmicefrombreedingpairsexposedtoanenrichedenvironment.Currently,weareassessingmethylationanalysisofaCpGislandlocatedatthePomcpromoter,inordertoassociatethephenotypeobservedinadultmicewithepigeneticmodificationswhichoccurrduringthefetaldevelopment/lactationperiod.Insummary,ourresultsshowthatexposuretoanenrichedenvironmentduringpregnancyandlactationimpactsonfeedingbehaviorexhibitedbyoffspringduringadulthoodasaconsequenceofchangesintheexpressionoftheanorexigenicgenePomc

66

ASSESSING EXPOSURE TO ORGANOPHOSPHATE PESTICIDES, BIOMARKERS AND NEUROPSYCHOLOGICAL OUTCOMES IN RURAL POPULATIONS OF CHILE

Ramirez, M1., Zuñiga-Venegas, Liliana2.,Corral-Zavala, Sebastian3.,Pancetti Vaccari, Floria2.,Sandoval-Guzman, Rodrigo2.,1SALUD PUBLICA,MEDICINA,UniversidadCatólicaDelNorte.2BiomedicalSciences,Medicine,UniversidadCatólicaDelNorte.3Psychology,FACSO,UniversidaddeChile.(SponsoredbyThisResearchWasSupportedBy:FONDEF:FondoDeFomentoAlDesarrolloCientíficoYTecnológico,CONICYTChile.GrantNumberD09I1057)

The increased agricultural activity that Chile has experienced in recent years has resulted in increased use of pesticides. According to therecordsofthemonitoringsystemoftheMinistryofHealth,thepesticidefamilymainlyinvolvedinepisodesofacutepoisoningcorrespondstoorganophosphates,causinga39%ofacuteintoxications.Whileacutepoisoningsareeasilydiagnosed,chronicexposureoftengoesunnoticed.Themainproblemformonitoringchronicexposureisthelackofhighlysensitivebiochemicalbiomarkercapableofbeingmonitoredinabodyfluid.Biomarkersavailabletodayonlyservetodiagnoseacutepoisoning.Theprojectsoughttodeterminewhethertheactivityoftheerythrocyteenzyme calledacylpeptide hydrolase (APEH) serves as a biomarker for biomonitoring of humanpopulations exposed to different levels oforganophosphate pesticides; correlating its catalytic activitywith cognitive performance. The project recruited a total of 268 volunteers ofwhich87 were occupationally exposed (OE), 81 were environmentally exposed (EE) and 100 as a reference group (RG). Thepopulationwashomogeneousinagerange,alcoholintake,drugsconsumptionandsmokinghabits;howeverthereweredifferencesingenderandeducationallevel,beingtheindirectlyexposedgroupthosepresentingahighernumberofwomenandeducationallevelthantheothertwogroups.Theinterviewconsistedinaneuropsychologicalevaluationandabloodsamplingformeasuring erythrocyte acetylcholinesterase (AChE), plasma cholinesterase (BChE) and erythrocyte APEH activities.Neuropsychological assessment included: general mental state, memory, language, attention, praxis, executive function, motor coordination and mood.Theresultsofthebloodtestsindicatedthattheenzymeactivitiesfoundtobeinhibitedbyhighenvironmentalburdenoforganophosphates(duringfumigation)wereAPEHintheenvironmentallyexposed(EE)groupandplasmacholinesteraseintheoccupationalexposed(OE)group.Regardingtocognitiveperformance,bothexposedgroupsshowedabnormalresultsinmostoftheareasevaluated;being most affected memory, executive function and fine motor coordination.Sixpredictivemodelsweregeneratedtorelateenzymesactivitywithcognitiveoutcomesduringfumigationperiodusingrandomforestanalysis.Whenanalyzingdelta (Δ) of enzymatic activity,APHE showed to be a good predictive variable for memory. BChEandAChEshowedsignificantpowertopredictexecutivefunctionandmotorcontrolrespectively.Othervariableswithsignificantstrengthwerethenumberofstudyyearsandalcoholconsumption.

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ORAL PRESENTATIONS II

68

THE NEUROPROTECTIVE EFFECTS OF AN ERYTHROPOIETIN ISOFORM WITH LOW GLYCOSYLATION AGAINST THE ß AMYLOID STRESS.

Castillo, Carolina1,2.,Hidalgo,Angela1.,Silva-Grecchi,Tiare2.,Fuentealba,Jorge2.,Toledo,Jorge1.,1BiotechnologyandBiopharmaceuticalsLaboratory,Departamentof Pathophysiology., School ofBiological Sciences,Universityof Concepcion.2Screeningof neuroactive compounds Laboratory.DepartamentofPhysiology.,SchoolofBiologicalSciences.,UniversityofConcepción.

Erythropoietinisaglycoproteichormonethatweighs34KDa.Itsprincipalfunctionistoregulatetheerythropoiesis;howeversincethediscoveryofitsreceptor(EpoR)inothernon-erythroidtissues,likecentralnervoussystem(SNC)theirfunctionhasbeenrelatedwithotherpleiotropiceffects.IntheSNChasbeenobservedaneuroprotectiveeffectofthishormonethat’srelatedwiththeupregulationofantiapoptoticpathwaysanddirectly relatedwithactivationof its receptor. InpatientswithAlzheimer’sdiseaseexistsan increaseofEpoRexpressionversus controlpatients.Theobjectiveof thisproyect is toevaluate theneuroprotectiveeffect in vitro against thebetaamyloid stress (Alzheimer’s relatedprotein)ofanerythropoietin isoformwith lowglycosylationandwithouthematopoieticactivityproducedtroughadenoviral transductionofgoat’smammarygland (EpoL) and theirdependenceof theactivationof EpoR. For thispurposewepreincubated cortical neuronsorPC12cellswithEpoLforanhourandthenweincubatedthesecellswithβ-amyloidpeptideorwithanuncouplingofrespiratorychainofelectronsinmitochondria(FCCP),likeamodelofoxidativestress.ThecellspreincubatedwithEpoLshownanincreaseofthecellularviabilityof32%morethanthecontrolcells,andanincreaseof26%morethancellsincubatedwithaninhibitorofactivationofEpoR.PreincubatedcellswithEpoLpreventtheincreaseofcalciumtransientsinducedbybetaamyloidpeptide.TheseresultsagreewithdifferentexpressionofEpoRandwiththe50%ofupregulationofBCL2geneobservedinthesametreatmentsbyqRTPCRexperimentsandalsoagreewithanincreaseofimmunoreactivityofEpoRinimmunofluorescencedata.WiththeseresultswehaveshownthatEpoLhasaneuroprotectiveeffectagainstthestressinducedbyAβandalsoagainstoxidativestress.FurtherthiseffectismediatedbyEpoRactivationandalsotheanti-apoptoticpathwaysactivation.ThereforetheseresultssuggestedthatEpoLcanbeusedlikeaneuroprotectiveagentagainstneurodegenerativediseases.

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BASAL CILIARY ACTIVITY DEPENDS ON ATP RELEASE IN MOUSE TRACHEAL EPITHELIAL CELLS IN VITRO.

Droguett, Karla1., Ríos, Mariana1.,Navarrete, Camilo1.,Fuentes, Christian1.,Barrera, Nelson1.,Villalón, Manuel1.,1Department of PhysiologicalScience,FacultyofBiologicalScience,PontificiaUniversidadCatólicaDeChile.(SponsoredbyFondecytPostdoctorado3150652)

Inmucociliaryepitheliafromrespiratorytract,ciliarybeatingisdeterminantinthevelocityandeffectivenessofmucociliaryclearance(MCC).ATPisknowntoincreasesciliarybeatfrequency(CBF)inciliatedcells,effectthatismediatedbypurinergicreceptoractivationandthesubsequentincreaseinintracellularCa2+.Inrespiratoryepithelialcells,ATPcanbereleasedconstitutivelyorfollowingmechanicalstimulation(MS)througha possiblemechanismmediated by pannexin 1 (Panx1) or connexin 43 (Cx43) hemichannels (HCs), however, it unclear if ATP release is aregulatedmechanismthatcontributetobasalciliaryactivity.TheaimofthisstudywastodeterminewhetherextracellularATP(eATP)releasecontributestoregulatebasalciliaryfrequency.Weusedprimaryculturesfrommousetracheaepithelialciliatedcells(MTEC).CBFwasrecordedusingvideomicroscopy(SissonAmmonsVideoAnalysis)andAtomicForceMicroscopy(AFM)wasusedtocaractherizeciliaryfunction.eATPwasmeasuredbyluminometricassayusingluciferin/luciferaseand[Ca2+]iweremeasuredusingFURA2AM.Apyrase(50U/mL),anectonucleotidasethathydrolyzesATP,significantlyloweredeATPlevelscomparedtovehicle(3.8±1.4versus8.1±0.8pmol/cm2after1min.treatment,*p<0.05).ApyrasealsoreducedCBFina45.5±2.3%(n=4),effectthatcorrelateswitha[Ca2+]ireduction.Simultaneoustreatmentwithcarbenoxolone(CBX)(100µM),aHCsinhibitor,andoxidizedATP(oATP)(100µM),aP2X7-Rantagonist,producedareductionofCBFcomparedtobasalactivityof57.5±3.0%after5minofincubation,returningtobaselineafter20min.ThisreductionwasprominentcomparedwithoATPalone(6.8±1.7%,*p<0.05)andCBXalone(24.9±6.8%,n=12).Inaddition,concomitanttreatmentwithCBX,oATPandapyrasereducedthebasalCBFin85.2±4.8%(n=10),inconcordancewithareductiononeATPlevels(19.9±9.5pmol/cm2forvehicle,n=11,versus2.6±0.1pmol/cm2fortreatment,n=4,*p<0.05).Furthermore,usingAFMwetopologicallydetecteddeflection,amplitude,phaseandheightofMTCE.UsingAFM-singleforcemode,wemeasuredareaswithoscillatorydeflectionpatternswhosedominantfrequencywasbetween4to14Hz,asimilarfrequencyrangedetectedwithvideomicroscopy.TheseresultssuggestthatATPreleasefromepithelialciliatedcellsisrequiredtomaintainbasalciliaryactivityassociatedto[Ca2+]ihomeostasis.TheunderlyingmolecularmechanismmightinvolveHCsandP2X7-R,throughanautocrinemechanismthatregulatesbasalciliaryactivityinrespiratoryepithelium.FondecytPostdoctorado3150652.Fondecyt1120169.

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MODELING THE SENSITIVITY OF COLD THERMORECEPTOR NEURONS AND COLD NOCICEPTORS IN TERMS OF ITRPM8 AND IKD CUR-RENT EXPRESSION.

Herrera Pacheco, Gaspar1.,Olivares,Erick1.,Madrid,Rodolfo2.,Orio,Patricio1.,1CentroInterdisciplinariodeNeurocienciadeValparaíso,FacultaddeCiencias,UniversidadDeValparaíso.2DepartamentodeBiología,FacultaddeQuímicayBiología,UniversidadDeSantiagoDeChile.Coldthermoreceptorneurons(CTs)andcoldnociceptors(CNs),activeintheinnocuousandnoxiouscoldrangerespectively,areresponsibleforthedetectionofenvironmentalcoldtemperatures inthesomatosensorysystem.Molecular,cellularandbehavioralevidencesareconsistentwiththeviewthatTRPM8,acold-andmenthol-gatedcationchannel,isthemainmoleculardeterminantofcold-sensitivity,andsuggestthatthebroaddetectionrangeanddistributionofthermalthresholdsmainlyarisesfromthecounterbalanceinthefunctionalexpressionofTRPM8(ITRPM8)andKv1.1-1.2channels(IKD).Thesmalldiameterandlowdensityofperipheralreceptorsprecludestheopticalandelectrophysiologicalapproaches, so it isnotclear towhichextentobservationsobtained inculturedprimarysensoryneuronscanbeextrapolatedtoperipheralthermotransductionoccurringat thenerveendings.Hereweusea conductance-basedmathematicalmodelof a genericCT, andcomputersimulationsunderdifferenttemperatureregimes,tounderstandthecontributionsofdifferentconductancestocoldresponses.Inthismodel,thefinesensitivitytofasttemperaturedropsisduetoadepolarizingTRPM8-dependentcurrent,whichgenerateslargechangesinthefiringrateofthecoldreceptor.Acalcium-dependentdesensitizationcausesthisresponsetobetransient.ExplorationoftheparametersspacewasperformedtostudyhowtheTRPM8andKDconductancesdeterminethedetectionthresholdsandmaximumfiringrates.ThermalthresholdsofthemodelsarecorrelatedtoTRPM8andKDconductancedensities,inagreementwithfunctionalstudiesinculturedcold-sensitiveneurons.ModelswithlowthresholdandthecharacteristicfeaturesofthedynamicandstaticresponseofCTshavehighTRPM8andlowKDconductancelevels.Conversely,highdetectionthresholdsandfiringpropertiesofCNsareassociatedtohighKDandlowTRPM8conductancedensities.WeprovidethefirstcomprehensivemathematicalmodelofbothCTsandCNsincludingtheexcitabilitybrakpotassiumcurrentIKD.Makinguseofit,wealsoshowthatneuropathicpainfulhypersensitivitytocoldcanbeexplainedasaconsequenceofanimpairmentinthisbrakepotassiumcurrent.SupportedbyGrantsFondecyt1130862,1131064andACT1104,ACT1113.CINVissupportedbytheMillenniumScientificInitiativeoftheMinisteriodeEconomía(Chile).

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STUDIES ON THE ROLE OF RHOA-RHO KINASE ACTIVATION IN AN ANIMAL MODEL OF METABOLIC SYNDROME

Leguina-Ruzzi, Alberto1., Peñaloza, Estefania1.,Pereira, Jaime1.,Alarcon, Cecilia1.,Romero, Diego2.,Roa, Juan2.,Mezzano, Diego1.,Sáez,Claudia1.,Velarde, Victoria3.,1Hematology and Oncology, Faculty of Medicine, Pontificia Universidad Católica de Chile.2Anatomic Pathology,FacultyofMedicine,PontificiaUniversidadCatólicadeChile.3Physiology,FacultyofBiologicalScience,PontificiaUniversidadCatólicadeChile.(SponsoredbyFondecyt1141051,BecaDoctoralConicyt21140004,PuenteVRI-UC20/2013)

MetabolicSyndrome(MetS)isacollectionofcardio-metabolicriskfactors.Endothelialdysfunction(ED)andatherosclerosisarechronicconditionsthatarethefoundationbehindcardio-ischemiccomplications.RhoA/Rho-kinase(ROCK),whichregulatesthecellularactindynamicsandotherfunctions,hasbeenassociatedto thepathogenesisofcardiovasculardiseasesandaRhoA/ROCK inhibitor,Fasudil, iscurrentlyusedto treatpulmonaryhypertension.However,RhoA/ROCKover-activationinrelationtoMetShasnotbeencompletelyaddressed.

Aim:TostudyinaMetSanimalmodeltheparticipationofRhoA/ROCKactivityassociatedtoMetSphenotypeandEDsigns.Methods: Groupsof12maleSpragueDawleyrats(100-125gr)werefedwithhighfatdietorchowdietadlibidumfor14weeks. Atweek12thegroupsweresubdivided for Fasudil 100mg/kg/day or vehicle oral treatment.Weight, food consumption, triglycerides, glucose and blood pressureweremonitoredthroughouttheprotocol.

Oxidativestressmarkers (AOPPandTBARS),NitricOxide (NO) (Griessmethod), IL-6, sCD40L, siCAM-1 (ELISA)and insulinweremeasured inserum.AortaslysateswereusedtodetermineRhoA/Rho-kinaseactivity,pAkt/Akt,Nrf2/ERKandpeNOS/eNOSbyWesternBlot.TriglyceridescontentwasstudiedbySudanIVandlipidoilredstainingandMMPsactivitybyzymography.

Results: Ratsfedwithhighfatdietgainedsignificantweightstartingfromthesecondweek,withoutmodifyingthefoodrateconsumption.Theyshowedhigherglucoseandinsulinlevels,systolicbloodpressure,triglycerides,cholesterolandlargerabdominalcircumferencethananimalstreatedwithchowdiet.TheseMetScharacteristicswereassociatedtohigher levelsofTBARSandAOPP,uricacid, IL-6,sCD40LandEDsigns(siCAM-1andlowerlevelsofNO)comparedtocontrols.AortasfromMetSanimalsshowedover-activationofRhoA/ROCKandreducedlevelsofpAkt,peNOSandNrf2aswell.AnimalsresemblingaMetSconditionalsoshowedelevatedlevelsofE-Selectin,MMP2activity,engrossedmediaintimaeandlipidinfiltrationthanchowdietfedrats.TwoweeksofFasudiltreatmentrevertedsignificantlythealterationsduetohighfatdiet.

Conclusions:OurresultsshowthatratsfedwithhighfatdietshowedbiochemicalandpathologicalcharacteristicsofMetS.ROCKinhibitionwasassociatedwithasignificantimprovementoftheseabnormalities.ThesefindingssuggestthatactivationofRhoA-ROCKpathwaymayplayanimportantroleinthepathogenesisofMSanditsinhibitionmightbeconsideredasapotentialtherapeutictargetforthiscondition.

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KCA3.1-DEPENDENT HYPERPOLARIZATION ENHANCES INTRACELLULAR CA2+ SIGNALING INDUCED BY FMLF IN DIFFERENTIATED U937 CELLS

Penna, Antonello1., Stutzin, Andrés2.,1Departamento de Anestesiología, Facultad de Medicina, Universidad De Chile.2Instituto de CienciasBiomédicas,FacultaddeMedicina,UniversidadDeChile.(SponsoredbyFONDAP15010006)

Background:Formylatedpeptidesarechemotacticagentsgeneratedbypathogens.Themostrelevantpeptide is fMLF(formyl-Met-Leu-Phe)whichparticipatesinseveralimmunefunctions,suchaschemotaxis,phagocytosis,cytokinereleaseandgenerationofreactiveoxygenspecies.InmacrophagesfMLF-dependentresponsesaredependentonboth,anincreaseinintracellularcalciumconcentrationandonmembranepotentialhyperpolarization.However, themolecularentityunderlying thishyperpolarizationremainsunknownand it isnotclearwhetherchanges inmembranepotentialarelinkedtotheincreaseinthe[Ca2+]i.Methods:U937cellsweredifferentiatedusing1mMdibutyrylcAMPfor48htoobtainamacrophage-likecellmodel.fMLF-dependentresponseswerestudiedusingpatchclampandCa2+imagingtechniquesindifferentiatedU937cells.PharmacologicalandmolecularapproacheswereusedtostudythemolecularentityoftheionchannelunderlyingfMLF-dependentresponse.Results:DifferentiatedU937cellsexposedto100nMfMLFrespondedwitharapidincreaseinthe[Ca2+]iandhyperpolarizationreaching~-70mV.Both,theincreaseinthe[Ca2+]iwasdiminishedandthehyperpolarizationwassignificantlymoreshorterintheabsenceofexternalCa2+.AsaCa2+-dependentK+channelunderlaythehyperpolarizationtriggeredbyfMLF,pharmacologicalandknockdowntechniqueswereusedtodeterminethatKCa3.1wasthemolecularentityoftheK+channel.Finally,theactivityofKCa3.1enhancedfMLF-dependentincreaseinthe[Ca2+]i inthedifferentiatedU937cells.Discussion:WedemonstratebymeansofpharmacologicalandmolecularbiologytoolsthatfMLFinducesaCa2+-dependenthyperpolarizationviaactivationoftheK+channelKCa3.1andthus,enhancingfMLF-inducedintracellularCa2+increasethroughanamplificationofthedrivingforceforCa2+entry.Consequently,enhancedCa2+influxwouldinturnlengthenthehyperpolarization,operatingasapositivefeedbackmechanismforfMLF-inducedCa2+signaling.

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SOFARCHI Members IncorporationENDOTHELIAL CELLS DIFFERENTIATED FROM MESENCHYMAL CELLS ISOLATED FROM WHARTON’S JELLY PROMOTE TISSUE REGENERATION IN HYPERGLYCEMIC MOUSE.

Ormazábal,Valeska1.,Camila,Reyes5.,Aguilera,Valeria5.,Maura,Rafael2.,Toledo,JorgeRoberto2.,Zuñiga,Ferlipe5.,Radojkovic,Claudia5.,Escudero,Carlos3,4.,Aguayo, Claudio 5,4.,1DepartamentodeFarmacología,FacultaddeCienciasBiológicas,UniversidadDeConcepción.2DepartamentodeFisiopatología,FacultaddeCienciasBiológicas,UniversidadDeConcepción.3DepartamentodeCienciasBasicas,FacultaddeCiencias,UniversidaddelBio-Bio.4GroupofResearchandInnovationinVascularHealth(GRIVASHealth).5DepartamentodeBioquímicaClínicaeInmunología,FacultaddeFarmacia,UniversidadDeConcepción.(SponsoredbyThisStudyWasSupportedByINNOVACORFOChile(12IDL2-13351)AndINNOVABIOBIO,Chile(1245-EM.TES(12.21)),DirecciónDeInvestigación,UniversidadDeConcepción(DIUC211.072.034-1.0),ChileAndConvenioDeDesempeño,UniversidadDeConcepción,UCO1201)

Mesenchymal stemcells (MSCs)haveahighpotential fordifferentiation,proliferationandplasticityand low immunogenicity,making themanexcellentcellsourcefortissuerepair.However,oneofthemainsourcesofMSCsisbonemarrow,whichreducesMSCisolation,itispoorlyefficientandunpleasantforpatients.TheaimofthisstudywastodeterminewhetherMSCsisolatedfromWharton\’sjellyofhumanumbilicalcordscanfavortissuerepairinginvivoinhyperglycemiccondition.MSCswereisolatedfromhumanWharton\’sjellybydigestionwithcollagenasetypeI.Endothelialtransdifferentiationwasinducedfor14(hWMSC-End14d).Immunophenotypingwasperformedusingmesenchymal(CD90,CD73,CD105)andendothelial (Tie-2,KDR,eNOS,ICAM-1)markers.Endothelialtrans-differentiationwasdemonstratedbytheexpressionofendothelialmarkersandtheirabilitytosynthesizesnitricoxide(NO).hWMSCscanbedifferentiatedintoadipocytes,osteocytes,chondrocytesand endothelial cells. Moreover, these cells have a high expression of CD73, CD90 andCD105but lowexpression of endothelialmarkers.hWMSCs-Endexpresshighlevelsofendothelialmarkersat14and30daysofculture,andtheycansynthesizeNO.Aftertwomonthsoftreatment,themouseexhibitshighbloodglucose levels (2-fold)and insulin (5 fold). Injectionofconditionedmedia fromhWMSC-End30dcultures inamousemodelof skin injuryacceleratedwoundhealing comparedwithanimals injectedwithhWMSCnon-differentiatedor control injectedwithvehicle.TheseresultsdemonstratethatdifferentiatedhWMSC-Endpromotesneovascularizationinhyperglycemicanimalmodelsprobablythroughthesecretionofpro-angiogenicsolublefactors.

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INHIBITION OF ETHANOL POTENTIATION OF GLYCINE RECEPTOR BY SMALL MOLECULES. IN VITRO AND IN VIVO STUDIES

Guzman, Leonardo1., San Martín, Loreto1.,Aguayo, Luis1.,Jin, Chunyang2.,Jimenez, Verónica3.,Cerda, Fabián1.,1Fisiología, Ciencias Biológicas,UniversityofConcepcion.2Center forDrugDiscovery,ResearchTrianglePark,ResearchTriangle Institue.3DepartamentodeCienciasQuímicasUniversidadAndresBello.(SponsoredbyFONDECYT1131004,FONDEFCA12i10280.)

Introduction.Glycinereceptor(GlyR),aninhibitoryligandgatedionchannelispotentiatedbyethanolbymeanoftheinteractionofGβγwiththeintracellulardomainofthischannel.PreviousstudieshavedeterminedtheGβγinteractingregionandapeptide(C7)thatinhibittheethanolpotentiationofGlyR.BasedontheseresultsavirtualscreeningwasperformedtoidentifysmallmoleculesthatmimicstheC7effects.Objectives.Toanalyzetheeffectivenessofselectedmolecules in vitroand in vivoandtodeterminetheirspecificitytotheethanolpotentiationofGlyR.Materials and methods.HEKcellsoverexpressingGlyRwereregisteredonpatchclamexperimentsinwholecellconfiguration.Smallmoleculeswere intracellularly added in the register pipette solution. Spontaneous synaptic activitywas registered in hippocampal neurons. HL60 cellactivatedwithfMLPwereassayedintermsofintracellularCa2+levels.GIRKchannelswereactivatedthroughaGABABagonist.Miceintoxicatedwithethanolwereanalyzedtodeterminetherighting-reflexrecoverytime.Results.TheIC50oftwosmallmoleculesforethanolpotentiationofGlyRwas25±5and26±4μM.Thedecaytimeconstantofsynapticeventswasreducedto8and3%.PLCβactivitywasnotaffectedbythepresenceofmolecules,andGIRKchannelwas inhibited to30and22%respectively.Finally, todemonstrate theeffectivenessofmolecules inethanolintoxication,lossofrightingreflex(LORR)experimentswereperformedinmiceintoxicatedwithethanol.Foroneofthemoleculesrecoverywas10minutesfasterthancontrol.Conclusions.SmallmoleculeswereidentifiedtohaveinhibitoryeffectontheethanolpotentiationofGlyRactivity.Thateffectwasconfirmedinin vivoexperiments.TheseresultsconfirmthattheGβγ-GlyRinteractioncouldbepharmacologicallymodulatedinordertoreducetheeffectsofethanolintoxication.Miembropatrocinante:Dr.JorgeFuentealba

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CONFORMATION-SPECIFIC MODULATION OF SYNAPTIC Α3-CONTAINING GLYCINE RECEPTORS OF THE SPINAL DORSAL HORN ALLEVIATES CHRONIC INFLAMMATORY PAIN.

Yévenes, Gonzalo1., Acuña, Mario2.,Ralvenius, William2.,Di Lio, Alessandra2.,Lara, Cesar1.,Muñoz, Braulio1.,Burgos, Carlos1.,Moraga-Cid,Gustavo3.,Corringer, Pierre-Jean3.,Zeilhofer, Hanns2.,1Department of Physiology Universidad De Concepción.2Institute of Pharmacology andToxicologyUniversityofZurich.3InstitutePasteurInstitutePasteur.(SponsoredbySupportedByFONDECYT1140515,SchweizerischerNationalFonds,ForschungskreditOfTheUniversityOfZurich,FondationDeLaRechercheMédicale, InstitutPasteurAndTheAgenceNationaleDeLaRecherche.SponsoredByDr.JorgeFuentealbaA.)

Diminished inhibitory neurotransmission in superficial spinal dorsal horn contributes to chronic pain. A PGE2-mediated PKA-dependentphosphorylation of α3 glycine receptors (α3GlyR) at serine 346 appears to be particularly relevant for the generation of hyperalgesia ininflammatorypainstates.Restoringtheactivityofspinalα3GlyRthroughpositiveallostericmodulatorsmaythusconstitutearationalapproachagainstinflammatorypain.WehaveinvestigatedthemodulationofrecombinantandnativeGlyRsbythenon-anestheticpropofolanalog2,6-di-tert-butylphenol (2,6-DTBP)and itspotentialeffects inbehavioralpainmodels.Wefoundthat2,6-DTBP isapositiveallostericmodulatorofhomomericα3GlyR.Heteromericα3βGlyRsweremuch lesssensitivethanhomomericreceptors, indicatingthat inclusionofGlyRβsubunitsin the receptor complex reduces allosteric modulation. Experiments performed in α3GlyR carrying point mutations mimicking either thephosphorylatedorthenon-phosphorylatedstate(S346EorS346A)revealedthatphosphorylationatS346didnotinfluencethesensitivityofhomomericreceptors2,6-DTBP.However,heteromericS346Epointmutatedα3βGlyRsdisplayedahighersensitivityto2,6-DTBPthanS346Amutatedreceptors.Wethenwenttoinvestigatetheeffectsof2,6-DTBPonglycinergicIPSCsofsuperficialdorsalhornneuronsusingspinalcordslices.2,6-DTBPcausedonlyaminorpotentiationofgly-IPSCsrecordedinnaïveslices.However,2,6-DTBPsignificantlyprolongedgly-IPSCsafterpretreatmentof thesliceswithPGE2.Thesedatasuggestthat inflammation-inducedphosphorylation increasesthesusceptibilityofsynapticα3GlyRtomodulationby2,6-DTBP.Inbehavioralstudies,wefoundthat2,6-DTBPalleviatedhyperalgesiainthezymosanAandCFAmodelsofinflammatorypaininwild-typemicewithoutalteringmusclestrength,motorcoordinationorlocomotoractivity.Noteworthy,theanalgesiceffectof2,6-DTBPwasreducedtoabout30%inα3GlyR-deficientmice,suggestingamajorroleofα3GlyRinthemechanismofactionof2,6-DTBPin vivo.Insummary,ourdataestablishthatpotentiationofsynapticα3GlyRsisapromisingstrategyagainstchronicpainandthat2,6-DTBPhasauniquepharmacologicalprofilefavoringaninteractionwithα3GlyRmodifiedbyphosphorylationinducedbyperipheralinflammation.

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REDUCTION OF ACUTE ANTHRACYCLINE CARDIOTOXICITY THROUGH THE DECREASE OF THE OXIDATIVE INJURY IN PATIENTS WITH BREAST CANCER.

Carrasco, Rodrigo1.,Florenzano,Fernado2.,Rodrigo,Ramón3.,Gormaz,JuanGuillermo4.,1LaboratoriodeInvestigaciónBiomedica.DepartamentodeMedicinaInterna.SedeOriente.,FacultaddeMedicina,UniversidadDeChile.2DepartamentodeMedicinaInterna,SedeOriente.,FacultaddeMedicina,UniversidadDeChile.3ProgramadeFarmacologíaMolecularyClínica, InstitutodeCienciasBiomédicas. ,FacultaddeMedicina,UniversidadDeChile.4ProgramadeFarmacologíaMolecularyClínica,InstitutodeCienciasBiomédicas,,FacultaddeMedicina,UniversidadDeChile.

Anthracyclinesisafamilyofagentsusedincancerchemotherapywithalimitedusebythecardiotoxicityoccurrence,whichhasnotbeenadequatelyprevented yet. Cardiotoxicity can be presented in acute form, evidenced through different biomarkers. Pathophysiological cardiotoxicitymechanismsincludegenerationofreactiveoxygenspeciesandaredifferenttoantineoplasticmechanisms.Theaimofourstudywastoevaluatetheeffectintheacuteanthracyclinescardiotoxicityoftwopharmacologicalinterventionsbasedonstrengtheningofantioxidantdefensesystembyadministrationofcarvedilolandomega3fattyacidsinbreastcancerpatients.Aplacebo-controlled,randomized,double-blindclinicaltrialwasperformedin36femalepatientswithbreastcancer(50.2±9years)andindicationofanthracyclines.Patientswereassignedto3differentgroupstoreceiveinthefirstcycleofchemotherapysince7daysbeforeuntil7daysposttreatmentwithanthracyclines:a)omega-31gevery12hpluscarvedilolplaceboevery12h;b)carvedilol12.5mgevery12hplusomega-3placeboevery12h;c)carvedilolplaceboeveryandomega-3placeboevery12h.Duringthefirstcycleofchemotherapy,patientswerecontrolledwith3bloodsamples:pre-chemotherapy,+3and+5daysafterchemotherapy.Electrocardiographiccontrolswerealsoperformed:pre-chemotherapy,6hoursand+3dayafterchemotherapy.PrimaryendpointofacutecardiotoxicitywasNT-proBNP.Secondaryendpointswereothermarkersofacutecardiotoxicity:QTc(intervalanddispersion)andserumtroponinT.Inaddition,effectsontheredoxstatuswereevaluated:totalplasmaantioxidantcapacity,erythrocytethiolindex,plasmaF2-isoprostanesandactivitiesofantioxidantenzymesinerythrocyte(SOD,CATandGSH-Px).PatientsshowedasignificantincreaseofNT-proBNPlevelsat+3day,howevertherewerenodifferencesbetweeninterventiongroups.Secondaryendpointsofacutecardiotoxicityneithershoweddifferencesbetweengroups.Inoxidativestressbiomarkers,placebogroupshowedhigherlevelsoflipoperoxidationandaloweractivityofthethreeerythrocyteantioxidantsenzymesin+3and+5days,effectswhichwerepreventedbythetwointerventions.Thedecreasedactivityofantioxidantenzymes intheplacebogroupwasassociatedwithan increasedinthethiol index.The interventionswithcarvediloloromega-3inpatientswithbreast cancerdidnot achievea reduction in acute cardiotoxicitybiomarkers.However, the two interventionswereable toreducethesystemicoxidativeinjuryandpreventthedecreasedactivityoferythrocytesantioxidantenzymes.Perhapsthereductioninsystemicoxidativestresscouldcontributetoareductionoftheeffectsofoxidativestressontheheart,thusshouldbestudiedthelongtermeffectsoftheseinterventions.

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POSTERS I

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1)Role of PKC in amphetamine- and cocaine-induced increase in dopamine and glutamate extracellular levels in rat ventral tegmental area

Abarca, Jorge1.,Sotomayor-Zarate,Ramón2.,Gysling,Katia1.,1DepartamentodeBiologíaCelularyMolecular,FacultaddeCienciasBiológicas,PontificiaUniversidadCatólicaDeChile.2InstitutodeFisiología,FacultaddeCiencias,UniversidaddeValparaíso.(SponsoredbyFundedByFONDECYTGrantN°1150244)

Dopaminergictransmissionatsynapsesisterminatedprimarilybytheremovingofdopamine(DA)fromthesynapticcleftbytheDAtransporter(DAT).Amphetamineandcocaineincreaseextracellular levelsofDAbydifferential interactionswithDAT.ThereisevidencethatPKCactivity isnecessaryforamphetamine-inducedincreaseinDAleves innucleusaccumbens. Inthisworkwehavestudiedtheeffectof intraVentralTegmentalArea(VTA) infusionofRO-31-8220(PKC inhibitor)onextracellular levelsofDAandglutamate(GLU)inducedbyintraVTAinfusionofamphetamineorcocaineincontrolrats. IntraVTAinfusionof30uMamphetamineor100uMcocainesignificantlyincreasedDAandGLUextracellularlevels.Co-infusionof10uMRO-31-8220inhibitedamphetamine-butnotcocaine-inducedincreaseinVTADAandGLUextracellularlevels.Thus,ourdatashowthatPKCactivityintheVTAisnecessaryforamphetamine-butnotcocaine-inducedincreaseinVTADAandGLUextracellularlevels.

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2)Potential neuroprotective effects of a series of indole derivatives in a model of Alzheimer´s Disease

Perez, Nelson1.,Peters,Christian2.,Bascuñan,Denisse2.,Aguayo,Luis2.,Pessoa-Mahana,Hernan1.,1QuimicaOrganicayFisicoquimica,CQF,UniversidadDeChile.2Physiology,BiologicalSciences,UniversidadDeConcepción.(SponsoredbyThisResearchWasFundedByFondecyt1140473And1130347)

Introduction. Alzheimer´sdisease(AD)isoneofthemajorneurodegenerativedisordersaffectingtheelderlyandischaracterizedbyaprogressivelossincognitivefunctions,memoryandlearning.CurrentdrugtherapyforADisdesignedtomitigatethediseasewithoutbeingabletostoporreverseitduetothemanyassociatedmechanismsofthedisorder.Theamyloidbetapeptide(Aβ)isoneofthemostcriticalfactorsthatcontributestothediversemechanismsoftoxicity,suchasitscapacitytobindtothemembraneand formpores that increase the permeability to ions. Among the compounds that have been tested againstAβ toxicity arethosehavinganindolicring.Indolederivativescompoundsarecharacteristicofalargenumberofalkaloidsandmoleculeswiththerapeuticinterest,suchasMelatoninandIndomethacin,whichhavebeenshowntoinhibitaggregationandtoxicityofAβ.Inaddition,theseindole-derivativeshavedemonstratedgoodlipophilicityandcanpenetratereasonablywellthebloodbrainbarrier.Main Aim. Toobtainindole-derivedmoleculescapableofreducingtheneurotoxicityofAβbyinterferingwithitsabilitytobindtoneuronalmembranes.Materials and Methods. Aninitialscreeningwasdoneto>100moleculesofindolicnaturethatwerepreviouslysynthesizedinthelaboratory,andtheircapacitytocounteractAβneurotoxicitywasdeterminedinPC12cellsusingtheMTTassay.WealsoperformedturbidityassaysforaggregationandDotBlotassaystoevaluatethecapacityofthemoleculestoinhibitthebindingofAβtothemembrane.Inaddition,dockinganalysisweredonetoassessthemostprobableinteractionsofthemoleculeswiththeAβpeptide.Results. WefoundthatanumberofthemoleculestestedsignificantlyantagonizedthetoxicityofAβ.Forexample,5µMM88wasabletoreducethetoxicityoncellularviabilityassayscausedbyapplicationof1µMAβ(65vs.85%ofcontrol,n=3).StudiesofturbidityandDotBlotrevealedthatsomecompoundsinhibitedtheaggregationandAβassociationtomembranes.Forinstance,M55inhibitedaggregationby45±3%andmembraneassociationby60±4%(n=3).Interestingly,dockingstudies showed favorableπ-stacking interactionswithaminoacidsPhe19andPhe20ofAβ. Conclusions.OurdatashowthatsomeindoliccompoundshavetheabilitytoreducethetoxicityinducedbyAβ,inadditiontoinhibitingthebindingofAβtothemembrane.CurrentexperimentsusingimmunocytochemistryandpatchclamptechniqueswillfurtherevaluatethesemoleculesandtheircapacitytointerferewithAβporeformationinneuronalmembranes.

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3)Effects of amphetamine sensitization on the extra-hypothalamic vasopressinergic system of adult rats

Ahumada, Catalina1.,Bahamondes,Carolina1.,Silva,Roxana1.,Cruz,Gonzalo1.,Sotomayor-Zárate,Ramón1.,Renard,Georgina1.,1CentrodeNeurobiologíayPlasticidadCerebral-InstitutodeFisiología,FacultaddeCiencias,UniversidaddeValparaíso.(SponsoredbyFONDECYTN°11140065ToGMRAndCommitteeForAidAndEducationInNeurochemistry(CAEN),ISN)

Drugaddictionisadiseasecharacterizedbytheneedtousedrugsdespiteadverseeffects.Ithasbeenobservedthatwomenaremorevulnerabletotheseeffectsthanmen.Medialamygdala(MeA)sendsvasopressin(AVP)projectionstothelateralseptum(LS),nucleusinvolvedintheaddictiveprocess.TheseprojectionsregulateseveralbehaviorsalteredbydrugaddictionanditisknownthatthedensityoftheAVPfibersintheLSishigherinmaleratsthaninfemales.So,AVPcouldberelatedwithsex-dependenteffectsobservedintheaddictiveprocess.Theaimofthisworkwastostudytheeffectsofsensitizationtoamphetamine(AMPH)(behavioralparadigmofaddiction)ontheextrahypothalamicvasopressinergicsysteminmaleandfemalerats.MaleandfemaleSpragueDawleyrats(55-60daysold)weredividedin2groups:Controlrats(saline,n=9femalesand7males)andAMPH-treatedrats(AMPH1.5mg/Kgi.p.,n=11femalesand7males).Thestageoftheestrouscyclewasdetermineddailybyvaginalsmearsexamination.Attheinductionphaseofsensitization,animalsreceivedadailyinjectionduring5consecutivedaysandlocomotoractivitywasmeasured.Thecriterionforsensitizationwasa20%increaseinlocomotoractivityover5-dayinjectionperiod.Fivedaysafterthelastinjection,allratswereinjectedwithAMPH(challenged)andlocomotoractivitywasmeasured.AftertheprotocoltheanimalsweredecapitateandthebrainwasremovedtomicrodissectMeAusedtoquantifyAVPmRNAexpressionbyRT-Q-PCRandtomicrodissectLStoquantifyAVPlevelsbyELISA.Ourresultsshowedthat75%oftheAMPH-treatedanimals,femalesandmales,sensitizedtoAMPH.Attheexpressionofsensitizationday,controlfemaleratsshowedhigherlocomotoractivitythanmales.NodifferencesinLSAVPcontentofcontrolandsensitizedfemaleandmaleratswasobserved,butintheMeAthereisadecreaseinAVPgeneexpressioninsensitizedanimals.AMPHtreatmentinfemaleratsproducesdifferencesdependingonphaseoftheestrouscycleintheAVPgeneexpressionattheMeA.Inconclusion,sensitizationtoAMPHcausesadecreaseinMeAAVPmRNAinbothfemalesandmalesrats,andwedidnotfindsex-dependenteffectsinourresults.

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4)Neonatal programming with Estradiol Valerate a vulnerability factor for Alcohol intake in adolescent female rats

Venegas, Francisca1.,Sanguinetti,Nicole1.,Cruz,Gonzalo1.,Acevedo,María2.,Sotomayor-Zárate,Ramón1.,1InstituteofPhysiology,FacultyofSciences,UniversidadDeValparaíso.2LaboratoriodeAlcohol,OntogeniayAprendizajeInstitutodeInvestigaciónMédicaMercedesyMartínFerreyra(INIMEC-CONICET).(SponsoredbyThisWorkWasFundedByFONDECYTGrantN°111-21205ForRS-Z)

Neonatalprogrammingbysexualhormonesuchasestradiolvalerate(EV) isatypeofphysiologicalreprogramming inwhich ithasbeendisplayedanincreaseindopamine(DA)contentinmidbraindopaminergicneuronsinsubstantianigra(SN)andventraltegmental area (VTA) in adult rats. This increase of dopamine (DA) content has been associated to an increase in tyrosinehydroxylase(unpublisheddata)throughthedirectestrogeniceffectofEVorthroughthearomatizationoftestosteronetoestradiol.Nevertheless,therelationshipbetweentheseresultsandalcoholintakebehaviorhasnotbeenstudiedyet.

Inthiswork,EV(0.1mg/50µLs.c.)wasinjectedinfemaleandmaleSprague-Dawleyratswithinthefirst12hoursoflife.Inparallelsesameoil(50µLs.c.)wasinjectedtothecontrolgroup.Theprotocolofintermittentaccesstoethanolfrompost-natalday(PND)28to65wasused.Ratswereallowedtodrinkfrom2-bottlesunderthefreechoiceparadigm.Everyweektheratswereexposedto3sessionsofethanolaccess.Thefirst2weekstheanimalswereabletochoicebetweenwaterwithsucroseandethanol5%solutionwithsucrose.Sincethe3rdweekthesucrosewastakenoutofthesolutionsforchoice.Inthelastsessionat4thweek,theratswereforcedtoabstinencecondition,andfinallythesewereexposedtothechallengesessionfor24hourstoethanol.

Wehavefoundthataverageethanolconsumptioninfemaleratswas1.12g/kg/dayin4weeks,meanwhileinEVfemaleratswas6.59g/kg/day.Inthechallengesession,theethanolconsumptionwas1.60g/kg/dayincontrolfemalerats,whileinEVfemaleratswas9.15g/kg/day.Inaddition,itwasobservedthatwaterandsucrosesolutionpreferencewas94%and58%forcontrolandEVfemalerats,respectively,inthefirsttwoweeks.Furthermore,theEthanolpreferenceduringthesesessionswas6%and42%forcontrolandEVfemalerats,respectively.

Ourresultsshowthatearlyexposuretoestrogeniccompoundsisavulnerabilityfactorforethanolintakeinadolescentfemalerats.Newstudieswillbecarriedouttoexplainmolecularmechanismimplicatedinthisvulnerabilityfactor.

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5)ACTIVATION OF THE TRANSIENT RECEPTOR POTENTIAL VANILLOID TYPE 1 (TRPV1) REDUCES THE INFLUENCE OF ANXIETY IN VISUOSPATIAL LEARNING OF MICE.

Castillo, Amparo1,2.,BURGOS,Héctor3,4.,Cofré,Christian5.,Hernández,Alejandro6.,Morgan,Carlos7.,Sáez-Briones,Patricio8.,Burgos-Villaseca,Jorge9,8.,Klagges,Jorge10.,Morales,Bernardo11.,Madrid,Rodolfo11.,Zeise,Marc12.,1EscueladePsicología,CienciasSociales,UniversidadSantoTomás.2ProgramadePostgrado,DoctoradoenPsicología,FacultaddeCienciasSociales,UniversidadDeChile.3PSICOLOGIA,FACULTADDEHUMANIDADES,UniversidadDeSantiagoDeChile.4EscueladePsicología,CienciasSociales,UniversidadCentraldeChile.5EscueladePsicología,LaboratoriodeBiopsicología,FacultaddeHumanidades,UniversidadDeSantiagoDeChile.6LaboratoriodeNeurobiología,FacultaddeQuímicayBiología,UniversidadDeSantiagoDeChile.7UnidaddeNutriciónHumana,,INTA,UniversidadDeChile.8LaboratoriodeNeurofarmacologíayComportamiento,,FacultaddeCienciasMédicas,UniversidadDeSantiagoDeChile.9DepartamentodeBiología,FacultaddeCienciasBásicas,UniversidadMetropolitanaDeCienciasDeLaEducación.10LaboratoriodeNeurofarmacologíayComportamiento,FacultaddeCienciasMédicas,UniversidadDeSantiagoDeChile.11LaboratoriodeNeurociencias,FacultaddeQuímicayBiología,UniversidadDeSantiagoDeChile.12LaboratoriodeBiopsicología,EscueladePsicología,FacultaddeHumanidades,UniversidadDeSantiagoDeChile.(SponsoredbyACT-1113)

TheTRPV1wasfirstdescribedinperipheralpainpathways,beingsensitivetonoxiousstimulisuchascapsaicin,temperature,acidityandphysiologicaleffectsinvolvingincreasedATP.Itisalsopresentinneuronsofthecentralnervoussystem,suchashippocampus,cortex,cerebellum,olfactorybulb,midbrainandrhombencephalicareas,amongotherlimbicstructures,andhasbeensuggestedtobeinvolvedinlearningprocesses.TostudytheroleofTRPV1onvisuospatialmemoryperformance,twogroupsofeightC57/BLmicewereinjectedwitheither1.0mg/kgi.p.oftheTRPV1agonistcapsaicinorsaline,andthensubjectedfor15daystotheOltoneight-armradialmazetotestshortandlong-termmemory.Anxietywasassessedonday1andday15intheelevatedplusmaze.

Therewasno significantdifference in the totalnumberof cumulatederrors committedbetween thecapsaicinand the salinetreatedgroupduring thewhole15-dayperiodof testing.However,during thefirst3daysof testing, short-termmemorywassignificantlybetterinanimalsreceivingcapsaicin,whilelong-termmemoryperformancehadsignificantlyimprovedlater,ondays4-9.Concerninganxiety,therewasnodifferenceinscores(entrytoopenarms)betweenthecapsaicinandthecontrolgroupinthefirstsessionintheplusmaze.However,whilecontrolsdisplayedhigherscoresinthesessionafterday15,thecapsaicingroupmaintainedtheirscores.

ThedatasuggestthatactivationofTRPV1bycapsaicininitiallyimprovesworkingmemoryinmiceandsubsequentlyimproveslong-termmemory,togetherwithproducingananxiolyticeffect.

ThisstudyhasbeensupportedbyACT-1113.

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6)THE TRPV1 ANTAGONIST CAPSAZEPINE INHIBITS LONG-TERM POTENTIATION IN THE RAT PREFRONTAL CORTEX.

Hernández,Alejandro1.,BURGOS,Héctor2,3.,Cofré,Christian2.,Castillo, Amparo4,5.,Sáez-Briones,Patricio6.,Burgos-Villaseca,J7.,Klagger,Jorge7.,Morales,Bernardo8.,Madrid,Rodolfo8.,Morgan,Carlos9.,Zeise,Marc2.,1LaboratoriodeNeurobiología,FacultaddeQuímicayBiología,UniversidadDeSantiagoDeChile.2LABORATORIODEBIOPSICOLOGÍA,ESCUELADEPSICOLOGIA,FACULTADDEHUMANIDADES,UniversidadDeSantiagoDeChile.3EscueladePsicología,FacultaddeCienciasSociales,UniversidadCentralDeChile.4EscueladePsicología,FacultaddeCienciasSociales,UniversidadSantoTomás.5ProgramadePostgrado,DoctoradoenPsicología,FacultaddeCienciasSociales,UniversidadDeChile.6LaboratoriodeNeurofarmacologíayComportamiento,,FacultaddeCienciasMédicas,UniversidadDeSantiagoDeChile.7DepartamentodeBiología,FacultaddeCienciasBásicas,UniversidadMetropolitanaDeCienciasDeLaEducación.8LaboratoriodeNeurociencias,FacultaddeQuímicayBiología,UniversidadDeSantiagoDeChile.9UnidaddeNutrición,INTA,UniversidadDeChile.(SponsoredbyACT-1113)

TRPV1 receptors are known tobeexpressed inperipheral painpathways, but also in the spinal corddorsal hornwhere theyplayaroleinspinalcordlong-termpotentiation(LTP).Morerecently,ithasbeenfoundthatTRPV1arealsoexpressedinseveralbrainregions,includingthehippocampusandthecerebralcortex,suggestingthatthesechannelsbeinvolvedinneuroplasticitynecessary formemory-relatedprocesses. To study the role of TRPV1 in LTP induction in theprefrontal cortex, two groupsofSprague-Dawleyratswere injectedeither5mg/kg i.p.of theTRPV1receptorantagonistcapsazepineorsaline,andthereaftersubmittedtoaprotocolofinvivoprefrontalcortexLTPelicitedbytranscallosalelectricalstimulation.RatstreatedwithcapsazepineexhibiteddecreasedLTP,ascomparedtocontrols.ThisresultisconsistentwiththeinhibitionofspinalLTPbycapsazepineinratandthereductionofhippocampalLTPinTRPV1o/omice.Takentogether,theseresultssuggestthatactivationofTRPV1maybenecessaryforsynapticplasticityincentralsynapses,beinginlinewithpossiblesupportingeffectsofTRPV1activationinlearningtasks.

ThisstudywassupportedbyACT-1113.

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7)A series of brominated derivatives of the superpotent 5-HT2 agonist 25B-NBOMe elicit anxiolytic-like responses in male Sprague-Dawley rats

Burgos-Villaseca, Jorge1,2.,Klagges-Troncoso,Jorge1,3.,Benavente-Schonhaut,Sofia1,3.,Malhue-Olmos,Valeska1,3.,Hernández,Alejandro4.,Burgos,Héctor5.,Cassels,Bruce6.,Sáez-Briones,Patricio1,3.,1LaboratoryofNeuropharmacologyandBehavior,FacultyofMedicalSciences,UniversidadDeSantiagoDeChile.2DepartmentofBiology,FacultyofBasicSciences,UniversidadMetropolitanaDeCienciasDeLaEducación.3SchoolofMedicine,FacultyofMedicalSciences,UniversidadDeSantiagoDeChile.4LaboratoryofNeurobiology,FacultyofChemistryandBiology,UniversidadDeSantiagoDeChile.5SchoolofPsychology,FacultyofSocialSciences,UniversidadCentralDeChile.6DepartmentofChemistry,FacultyofSciences,UniversidadDeChile.(SponsoredbyFONDECYTGrant1150868,DICYT-USACHGrant021401SB)

The synthetic psychotropic amphetamine 2-(4-bromo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine, alsoknownas25B-NBOMe,isaN-methoxylatedderivativeofthehallucinogen2C-B(2,5-dimethoxy-4-bromoamphetamine).Athough25B-NBOMeexhibitshighaffinityforserotonergic5-HT2receptorsandveryhighinvivopotency,theunderlyingstructure-activityrelationshipsremainnot fullyunderstood.Moreover,reliabledataregarding itsbehavioraleffects inanimalmodelsarescarceandsomerelevantaspectsofitseffectsinvivo,suchaspossiblealterationsinanixety-relatedresponses,havenotbeendescribedin detail. In the present work, a series of eight N-2-hidroxy-, N-2-methoxybenzylated and N-benzylbrominated 25B-NBOMederivativeshasbeensynthesizedandbehaviorallyevaluatedattheelevatedplusmazeinmaleSpragueDawleyrats(doserange1–8mg/kg).Thepercentagesofentriesand thecorrespondingpercentagesoftimespent in theopenarmsmeasuredafteracute i.p.administrationofdosesrangingbetween1–4mg/kgareconsistentwithananxiolytic-likeresponse for the25B-N-2-hydroxy-andN-2-methoxybenzylatedderivatives. Incontrast, thenon-methoxylatedN-benzylbrominatedderivativesarenotabletoelicitanyanxiolytic-likeresponse.Interestingly,allcompoundsareindistinguishablefromcontrolsat8mg/kg.Therefore,theanxiolytic-likeeffectsobservedforthewholeseriesofderivativesseemnottofollowarigiddose-responsecorrelation.Thisbehavioral profile is similar to that of the classical hallucinogenDOI (2,5-dimethoxy-4-iodoamphetamine),whichwasused asreferencedrug.Takentogether,theseresultsareinagreementwithaffinityandefficacydataobtainedpreviouslyforthisseriesof compounds.Nevertheless, theymight indicatealso that theanxiolytic-like responseelicitedcouldnotbeeasilymodulatedby aromatic bromination, as already shown for other paradigmatic behavioral responses in the rat associatedwith a typicalhallucinogenicprofile.The latterhighlights thenotion that, regarlessof the fact that superpotent serotonergic5-HT2agonistsmaypossessdistinctivepharmacologicalproperties,theyalsoseemtoshareothereffectswiththoseofstructurallyrelatedclassichallucinogens.

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8)Methylphenidate amplifies LTP by activation of β-adrenergic and D1/D5 receptors and increasing the AMPA currents of pyramidal cells.

Contreras, D1.,Carvallo,Claudia1.,Farias,Ricardo1.,Ugarte,Gonzalo1.,Zeise,Marc2.,Delgado,Ricardo1.,Rozas,Carlos1.,Morales,Bernardo1.,1Biologia,QuimicayBiologia,UniversidadDeSantiagoDeChile.2Psicología,EscueladePsicología,UniversidadDeSantiagoDeChile.(SponsoredbyFONDECYT1120580,FONDECY11140430AndCONICYT,ACT1113.CONICYTFellowshipToC.C./D.C)

Methylphenidate(MPH)isapsychostimulantusedinthetherapyoftheAttentionDeficit/HyperactivityDisorderandcurrentlyused also as a drug of abuse. It is known thatMPH increases the TBS-dependent Long Term Potentiation (LTP) in the CA1area of the hippocampus. Nevertheless, the cellular andmolecular mechanisms involved in these synaptic mechanisms arestillunknown. In thiswork,usingelectrophysiological recordingsweshowthatMPH inducesan increaseofLTP involving theactivationofβ-adrenergicandD1/D5dopaminergicreceptorsandanincreaseofAMPA-dependentcurrentinCA1area.3weeksoldSprague-DawleyratsweredecapitatedunderIsofluranoanesthesiaandhippocampusslices(400μmthick)wereprepared.LTPwasinducedandrecordedinCA1byapplyingaThetaBurstStimulation(TBS,5trains,100Hz)attheSchaefferCollaterals.Superfusionofhippocampalslicesduring20minwithMPHincreasedinadose-dependentmannerthemagnitudeofLTPfrom143.3±3.1%(controls;n=5,6)to146.2±2.8%(3nM;n=3,3;p>0.05),165.5±6.6%(50nM;n=6,6;**p<0.001),194.3±5.8%(5μM;n=6,8,***p<0.001),and196.4±4.2%(50μm;n=4,4;***p<0.001).Thiseffectwasofnaturepostsynapticsincethepaired-pulsecurvesremainedunchangedafterperfusionwithMPH.WefoundthatTimolol(5μM),aβ-adrenergicreceptorblocker,inhibitedsignificantlytheincreaseofTBS-dependentLTPbyMPHfrom194.3±5.8%(TBS+MPH,n=5,7)to152.7±1.7%(TBS+MPH+TIM,n=4,4)(**p<0.01).Interestingly,LTPincreasewasalsoinhibitedby5μMofSCH23390,aD1/D5receptorblocker,from195.5±6.2%(TBS+MPH;n=5,7)to144.4±3.5%(TBS+MPH+SCH:n=5,7;***p<0.001).Botheffectsprobablyarepostsynapticbecausethepaired-pulsecurveremainsunchanged.Finally,usingwhole-cellrecordinginCA1pyramidalcellsweevaluatetheeffectofMPHontheAMPA-dependentcurrents.AMPA-dependentEPSCswererecordedat-65mVthroughashort-termplasticityprotocolinresponsetostimulustrainsof22pulsesto30Hz.WefoundthattheamplitudeofAMPAcurrentsofMPH-treatedneuronsweresignificantlyhighercomparedtothoserecordedinsaline-treatedneuronsforalltheresponsesevokedbythestimulationprotocol(n=5,5;**p<0.01).Theseresultssuggest thatMPH increasesTBS-dependentLTP inCA3-CA1synapses throughapolysynapticmechanisminvolvingactivationofβ-adrenergicandD1/D5dopaminergicreceptorsandincreasingtheAMPAcurrentbyinsertionofthesereceptorsintheplasmamembrane.

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9)Fasudil prevents depressive-like behavior and hippocampal dendritic spine loss promoted by stress in rats

García-Rojo, Gonzalo1.,Vilches,Natalia1.,Fiedler,Jenny1.,1LaboratoriodeNeuroplasticidadyNeurogenética,FacultaddeCienciasQuímicasyFarmacéuticas,UniversidaddeChile.(SponsoredbyCONICYT21120711)

Brainatrophyaccompaniedbydendriticarborsimplificationandreductioninspinedensityofthehippocampus,alimbicstruc-tureimplicatedinmooddisorders,arefactorsthatseemstocontributetothesymptomsofdepression.Itisplausiblethatthesemorphologicalchangesimplyalterationsinthedendriticcytoskeletondynamic.RhoAisanimportantregulatorofactindynam-icthrough itseffectorROCK.TransfectionofhippocampalpyramidalneuronswithconstitutivelyactiveROCKmutantproducesdendriticsimplificationanddendriticspineloss.WeproposethatROCKinhibitorasfasudil(alsoknownasHA1077),maypreventboththestressinduceddepressive-likebehaviorinratsandthespinedensityreductioninthehippocampus.AdultmaleSpragueDawleyratswereinjectedwithsalineorfasudil(10mg/kg)startingfourdayspriorrestraintstressprocedure,whichwasconducted2.5hrs/dayduring14consecutivedays.Controlanimalswereinjectedwithsalineorfasudilduring18days.Inordertoobservetheeffectoffasudilondepression-likebehaviorpromotedbystress,wecarriedouttheforcedswimmingtestandconditionedavoidancetest.Fasudilpreventsthestress-inducedimmobilityobservedinforcedswimmingtest.Inaddition,fasudilpreventsthestress-inducedreduction inconditionedavoidanceresponses.Ontheotherhand,controlanimalstreatedwithfasudilshowedsimilarbehavioralpatternsascontrolsalineanimals.Furthermore,fasudilpreventsthestressspinelossinthedorsalhippocampuspromotedbychronicstress.ThusweproposedthatFasudilmaypreventabnormalbehaviorpromotedbystressprobablyblockingtheROCKactivitywithaconcomitantpreventionofthespineloss.

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10)Molecular modeling approaches to investigate corticotropin releasing factor receptor system structure-activity relationships

Lagos,Carlos1.,Gutiérrez-Maldonado,Sebastián2.,Slater,Paula3.,Gysling, Katia3.,1DepartmentofEndocrinology,SchoolofMedicine,PontificiaUniversidadCatólicaDeChile.2ComputationalBiologyLab(DLab)FundaciónCienciayVida.3DepartmentofCellularandMolecularBiology,FacultyofBiologicalSciences,PontificiaUniversidadCatólicaDeChile.(SponsoredbySupportedByFONDECYTGrants1110392&1150244,ProgramaDeFinanciamientoBasalPFB16Grants,CONICYTPhDThesisResearchGrantsToPGSAndSEGM.ProjectNLHPCECM-02SupercomputingInfrastructure:Powered@NLHPC.)

Corticotropin-releasingfactor(CRF)neuropeptideisthekeymediatorofthemammalianresponsetostressors.CRFactsthroughtwosubtypesofclass-BGprotein-coupledreceptors,CRFR1andCRFR2.ThemammalianCRFsystemalsocomprisesaCRFbindingprotein(CRFBP)andfourdifferentnaturallyoccurringligands,CRFandtherelatedpeptidesurocortinsI-III.ClassBGPCRsrepresentasmallGPCRsubfamilyencompassing15members,forwhichtheactivationmechanismsremainsunexplored.Ontheotherhand,CRFBPisasecretedproteinwithoutsignificantsequencehomologytoCRFreceptorsortoanyotherknownclassofproteins.Inrecentyears,structure-functionrelationshipstudieshavedemonstratedthattheN-terminalectodomain(ECD)ofCRFRsplaysapivotalrole innatural ligandrecognition,andmorerecently interactionswithCRFBPhavebeenidentified.Therecentlysolvedcrystal structures of the transmembrane domains (TMD) of the human glucagon receptor and human corticotropin releasingfactorreceptor1(CRFR1)haveopenedupnewopportunitiestostudythestructureandfunctionofclassBGPCRs.InthisworkwereportthemodelingoftheentireCRFsystemaswellastheinteractionwithCRFBPandpeptideligands.ThecompletereceptormodelsincludingtheECDandtheTMDwerefurtherstudiedusingmoleculardynamicssimulationsandprotein-proteindockingwithtwomaingoals:Firstly,structural investigationsthathelptounderstandthereceptorfunctionandthecharacterizationofCRFBPbindingtotheCRFR2αreceptorECDdomain,andsecondlytheidentificationofnovelCRFRmodulatorsaspotentialdrugs.ThepresentstudyprovidesapreliminaryframeworkforfurtherinvestigationofCRFR2α-CRFBPinteractionsandforthestudyofCRFreceptoractivationandregulation.

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11)Overexpression of LOXIN protects endothelial progenitor cells from apoptosis induced by oxidized low density lipoprotein.

Carlos,Veas4.,Willis,Naomi1.,Gutierrez,Nicolas2.,Radojkovic,Claudia4.,Zuñiga,Felipe4.,Toledo,JorgeRoberto2.,Escudero,Carlos3,5.,Aguayo, Claudio 4,5.,1HumanNutritionResearchCentre,InstituteofCellularMedicine,NewcastleUniversity.2DepartamentodeFisiopatología,FacultaddeCienciasBiológicas,UniversidadDeConcepción.3DepartamentodeCienciasBásicas,FacultaddeCiencias,UniversidaddelBio-Bio.4DepartamentoBioquímicaClínicaeInmunología,FacultaddeFarmacia,UniversidadDeConcepción.5GroupofResearchandInnovationinVascularHealth(GRIVASHealth).(SponsoredbyThisStudyWasSupportedByINNOVACORFOChile(12IDL2-13351)AndINNOVABIOBIO,Chile(1245-EM.TES(12.21)),DirecciónDeInvestigación,UniversidadDeConcepción(DIUC211.072.034-1.0),ChileAndConvenioDeDesempeño,UniversidadDeConcepción,UCO1201)

Humanendothelialprogenitorcells(hEPC)areadultstemcellslocatedinthebonemarrowandperipheralblood.Studieshaveindicated that hEPCs play an important role in the recovery and repair of injured endothelium, however their quantity andfunctionalcapacityisreducedinseveraldiseasesincludinghypercholesterolemia.RecentlyithasbeendemonstratedthathEPCexpresslectin-likeoxidizedlow-densitylipoproteinreceptor-1(LOX-1)anditsactivationbyoxidizedlow-densitylipoproteins(oxLDL)inducescellulardysfunctionandapoptosis.ThisstudyaimedtoinvestigatewhetheroverexpressionofLOXIN,atruncatedisoformofLOX-1thatactsasadominantnegativeplaysaprotectiveroleagainstoxLDL-inducedapoptosis inhEPC.HumanendothelialprogenitorcellsexposedtooxLDLshowedasignificantincreaseinLOX-1expression,andapoptosisbeganatoxLDLconcentrationsabove50μg/mL.AllhEPCapoptosedat200μg/mLoxLDL.HighLOXINexpressionwasgeneratedusingadenoviralsystemsinhEPCandSiHacellstransducedwith100colony-formingunits/cell.TransducedLOXINlocalizedtotheplasmamembraneandblockedoxLDLuptakemediatedbyLOX-1.OverexpressionofLOXINprotectedhEPCfromoxLDL-inducedapoptosisandthereforemaybeanovelwayofimprovinghEPCfunctionandquantity.TheseresultssuggestthatadenoviralvectorsofLOXINmayprovideapossibletreatmentfordiseasesrelatedtooxLDLandvascularendotheliumdysfunction,includingatherosclerosis.

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12)Circulating endothelial cells from patients with sepsis are source of activated fibroblasts

Becerra, Alvaro1.,Tapia,Pablo2.,Gonzalez,Alvaro1.,Simon,Felipe1,3.,1CienciaBiológicas,CienciasBiológicas,UniversidadAndrésBello.2MedicinaIntensiva,Medicina,PontificiaUniversidadCatólicaDeChile.3IMIIMillenniumInstituteonImmunologyandImmunotherapy.(SponsoredbyFondecyt1121078AndMIIP09-016-F)

Introduction: Sepsisismediatedbyimmunesystemover-activationpromotinganincreasedsecretionofinflammatorymediators,whicharereleasedintothebloodstream.Impairedcirculatoryfunctionisanimportantfactorinsepsispathogenesis,inwhichtheendothelialcells(ECs)dysfunctioniscrucialtobloodvesselsdamage.Severalevidenceshaveshownthatinflammatorymediatorsinduce conversion of ECs into activated fibroblasts through a process known as endothelialtomesenchymal transition. Septicpatientsexhibit increasedcirculatingendothelialcells (CECs) levels,which includecirculatingmatureendothelialcells (CMECs)andcirculatingendothelialprogenitorcells(CEPCs).Thus,ouraimwastostudywhetherCECsfromsepticpatientsexhibitfibroticfeatures,whicharecharacterizedby theacquisitionoffibroticproteinsandby the lossofendothelialmarkers.Methods and Results:CMECsandCEPCswereobtainedfrombloodsamplesofsepticpatientsandhealthyvolunteersusing immunomagneticbeadcapture(IBC).Endothelialfibrosiswasdemonstratedbydetectingchangesintheexpressionpattern,localizationandcellulardistributionoftheendothelial/progenitorsandfibroticmarkers.OurresultsshowedadecreasedexpressionofendothelialandprogenitorsproteinincludingVE-Cadherin,CD-31,VEGFR-2,andanincreasedexpressionofthefibroblast-specificproteins,α-SMAandvimentin.Inaddition,theamountofCMECsandCEPCsandthetotalCECswereincreasedinsepticpatientssamplescomparedtohealthyvolunteers.Conclusion:OurdatademonstratedthattheexpressionofendothelialproteinsinCMECsandCEPCsfromsepticpatientsisdecreasedwhencomparedwithECsfromhealthyvolunteers.Furthermore,CMECsandCEPCsfromsepticpatientsacquireexpressionoffibroticmarkers.Theseresultssuggestthatendothelialfibrosismaybeacommonmechanismofvascularendothelialdysfunctionduringsepsis.Thesedatacouldbeusefulfordiagnosticandsepsistreatmentimprovement.

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13)IN VIVO EVALUATION OF ATRIAL NATRIURETIC PEPTIDE ANP: CARDIOVASCULAR MEASUREMENTS IN CHRONICALLY HYPOXIC NEONATES IN THE ALTO ANDINO.

Beñaldo, Felipe1.,Guzmán,Constanza1.,Araneda,Felipe1.,Araya,Claudio1.,Poblete,Daniel1.,Moraga,Fernando2.,Herrera,Emilio1.,Reyes,Víctor1.,Ebensperger,Germán1.,Llanos,Aníbal1.,1Fisiopatología,Medicina,UniversidadDeChile.2CienciasBiomédicas,Medicina,UniversidadCatólicaDelNorte.(SponsoredbyAcknowledgments:SupportedByFONDECYT1140647,1120605,1130424&1151119.)

1ProgramadeFisiopatología,ICBM,FacultaddeMedicina,UniversidaddeChile.2InternationalCenterforAndeanStudies(INCAS),UniversidaddeChile.3DepartamentodeCienciasBiomédicas,FacultaddeMedicina,UniversidadCatólicadelNorte. Background:Highaltitudechronichypoxiaduringdevelopmentinducespulmonaryhypertensionoftheneonate(1).Newbornsinhighaltitudehavea reduced lung sGCexpression (1), centralmolecule involved inpulmonaryvasodilatation. In contrast,no information isavailableregardingtheparticulateguanylylcyclase(pGC),amembraneguanylylcyclase,thereceptorforANP&BNP.ParticulateGCispresentinthelung(2)andcouldbethetargetfortheANPsecretedbytherightheart,elicitingpulmonaryvasodilatation.WehypothesizethatanacutedoseofANPreducespulmonaryhypertensioninnewbornlambsgestated,bornandraisedathighaltitude.WeaimtodeterminetheeffectsofANPonpulmonary(PAP)andsystemicarterialpressure(SAP),pulmonary(PVR)andsystemicvascularresistance(SVR),heartrate(HR)andcardiacoutput(CO). Methods:Fournewbornsheep,bornandraisedatPutreResearchStation, INCAS,UniversityofChile(3,600m),werecatheterized inpulmonaryartery(SwanGanzcatheter)andabdominalaortaatthefourdaysold.Onedayaftersurgery,theneonatesweresubmittedtoa85minprotocol(15minofbasal,10minIVinfusionofANP(5ugkg-1)and60minofrecovery),whilerecordingcardiovascularvariables.Results: Basalhemodynamicvariablesweresimilaraspreviouslydescribed(1).ANPinfusiondecreasedPAPandthisreductionwasmaintainedduringrecovery(Infusion: 17.6±5.8%, Recovery (R) 15: 21.5±2.9%, R30: 15.8±3.1%, R45:13.2±3.8, R60: 13.0±2.7 mmHg). However, PVR onlydecreaseduringtheinfusionperiod(13.5±3%).SAP,SVRandHRdidnotchangeduringthestudy,whereasCOdiddecreaseonlyatR15(13.2±1.7%). Conclusions:ANPdiddecreasePAPduringacuteinfusionwithnochangesinsystemicarterialpressure.Thesepreliminaryresultsofferapromisingpossibilitytotreatneonatalpulmonaryhypertension,sincenochangesinsystemicarterialpressurewereobserved. References:1)Herreraetal.Cardiovasc Res 77(1):197,2008.2)PandeyKN.Elsevier26:901,2005.

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14)CINACIGUAT (BAY-582667): A POTENTIAL TREATMENT FOR PULMONARY HYPERTENSION IN CHRONICALLY HYPOXIC NEONATES.

Beñaldo, Felipe1.,Araneda,Felipe1.,Guzmán,Constanza1.,Araya,Claudio1.,Castillo-Galán,Sebastián1.,Chen,Zhuoming1.,Moraga,Fernando2.,Herrera,Emilio1.,Reyes,Víctor1.,Ebensperger,Germán1.,Llanos,Aníbal1.,1Fisiopatología,Medicina,UniversidadDeChile.2CienciasBiomédicas,Medicina,UniversidadCatólicaDelNorte.(SponsoredbyAcknowledgments:SupportedByFONDECYT1140647,1120605,1130424&1151119.)

1ProgramadeFisiopatología,ICBM,FacultaddeMedicina,UniversidaddeChile.2InternationalCenterforAndeanStudies(INCAS),UniversidaddeChile.3DepartamentodeCienciasBiomédicas,FacultaddeMedicina,UniversidadCatólicadelNorte. Background:Gestationalchronichypoxiainducesneonatalpulmonaryhypertension(1).Inhalednitricoxide(iNO)istheonlyapprovedtreatment(iNO),whichiseffectiveonlyin60%ofthenewborns(2).Onereasonofthisfailure,amongothers,isthatguanylylcyclase(sGC)isdownregulatedand/orthehemeironoftheenzymeisoxidized,losingitsactivity,underchronichypoxia(1).Therefore,enhancingitsactivityorexpressioncanbeapotentialtreatment.CinaciguatisasGCactivator,evenwhenthehemeironisoxidized,capableof inducingNO-independent vasodilatation (3). Hence,we hypothesized that an acute dose of Cinaciguat reverts pulmonaryhypertensioninnewbornsheepgestated,bornandraisedathighaltitude.WeaimtodeterminatetheeffectsofCinaciguatonpulmonary(PAP)andsystemicarterialpressure(SAP),pulmonary(PVR)andsystemicvascularresistance(SVR),heartrate(HR)andcardiacoutput(CO). Methods:Fivenewbornsheep,bornandraisedatPutreResearchStation,INCAS,UniversityofChile(3,600m),werecatheterizedinpulmonaryartery(SwanGanzcatheter)andabdominalaortaatthefourdaysold.Onedayaftersurgery,theneonatesweresubmittedtoa78minprotocol(15minofbasal,3minIVinfusionofCinaciguat(35ugkg-1)and60minofrecovery),whilerecordingcardiovascularvariables.Results: Basalhemodynamicvariablesweresimilaraspreviouslydescribed(1).CinaciguatinfusionjustincreasedCOduringinfusion.Further,CinaciguatdecreasedPAP,SAP,PVRandSVRreachinganadirat30minpost-infusionwith34.7±5.5%,19.1±1.6%,36.6±8.0%and17±2.9%decreases,respectively.Incontrast,nochangeswereseeninHRalongtheprotocol. Conclusions:Cinaciguatdecreasespulmonaryarterialpressureandresistance,withaslightfallinsystemicpressure.CinaciguatcouldpotentiallyofferanewtherapeuticapproachforneonatalpulmonaryhypertensioneitherwheniNOfailsorisnotavailable. References:1)Herreraetal.Cardiovasc Res 77(1):197,2008.2)Walsh-Sukysetal. Pediatrics 105:14,2000.3)Chesteretal.Am J Physiol301:L7552011.

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15)NLRP3 inflammasome in cardiac fibroblast

Boza, P1.,Tapia,Felipe1.,Vivar,Raul1.,Humeres,Claudio1.,Díaz,Guillermo1.,1DepartamentodeFarmacologíayToxicología,CienciasQuímicasyFarmacéuticas,UniversidadDeChile.(SponsoredbyProjectFondecyt1130300Scholarship.CONICYTBecaDoctoradoNacional21120407.GrantCONICYTGastosOperacionales21120407)

IntroductionThesterileinflammationisanewtopicincardiovasculardisease.Afterastroke,residentheartcellsexpresshighlevelsofIL-1β.Thiscytokineisaninflammationmastercontroller,itssynthesisandsecretionisatwo-stepprocessthatconcludeswiththeinflammasomeactivationandIL-1βsecretion.WehypothesizethatNLRP3inflammasomeinneonatalratcardiacfibroblast(CF)canbeactivatedandisfunctional.MaterialsandMethodsCellswereseededuntilpassage1andfastedallnight.ThecellswerestimulatedwithLPS1μg/mland/orATP3mMfor24h.Pro-IL-1beta,NLRP3,ASCandpro-caspase-1weremeasuredusingWesternblot(WB).Cellspretreatedwith8hofLPSwerestimulatedwithATPfor16h.IL-1βsecretionwasmeasuredbyELISAandalsobyproteinprecipitationfollowedbyWB.Caspase-1activitywasmeasuredusingaFluorometricassay.ResultsCFexpressNLRP3,ASCandpro-caspase-1.LPSinducesNLRP3expression.Pro-IL-1βisinducedinatimedependingmanner.Bothproteinexpressionsaresignificantat8hofLPS.LPS8h+ATP16hgeneratethesynthesisandsecretionofIL-1βtoculturemedia(ELISA).However,proteinprecipitationfollowedbyWBindicatesthatthesecretedcytokineisamixbetweenIL-1βandpro-IL-1β.Caspase-1activityassayshowsanincreaseintheactivityinducedbyATPandLPS+ATP.ConclusionCFexpresstheproteinsthatconformstheNLPR3inflammasome.LPScanactlikeafirstsignal,inducingpro-IL-1βandNLRP3synthesis.ATPcanactlikeasecondsignal,inducingtheNLRP3inflammasomeassemblyandactivatingcaspase-1.Theassembledinflammasomeisfunctional,cancleavepro-IL-1βintoIL-1β.Bothcytokinescanbesecreted,howevertheinflammasomeisthepacemakerstep.

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16)Key role of connexin hemichannels and pannexin channels in the PAF-induced Ca2+ signaling in endothelial cells of post-capillary venules

Burboa, Pia1.,Poblete,Ines1.,Figueroa,Xavier1.,1Phisiology,CienciasBiológicas,PontificiaUniversidadCatólicaDeChile.(SponsoredbyFONDECYT1150530)

Endothelial cells constitute a permeability barrier between blood and tissue interstitium. Pro-inflammatory signals, such asplatelet-activatingfactor(PAF),induceaCa2+-dependentincreaseofendothelialpermeabilitytomacromoleculesinpost-capillaryvenules.ThisCa2+signalingdependsonCa2+releasefromtheendoplasmicreticulumandCa2+influxfromtheextracellularspace.However, themechanisms involved in theCa2+ influxhavenotbeenclearlydetermined.Weanalyzed theparticipationof theplasmamembranechannelsformedbyconnexin(Cx)proteins(i.e.hemichannels)orpannexinsinPAF-elicitedintracellularCa2+concentration ([Ca2+]i) increase.We used the intactmesenteric vascular bed and primary cultures ofmesenteric endothelialcells (EC)ofresistancearteries (EC-A)andvenules (EC-V).Changes in [Ca2+]iwererecordedby loadingECwiththefluorescentCa2+ indicatorFluo-4andactivityofconnexinhemichannelsorpannexinchannelswasevaluatedbyassessingethidiumuptakeinECandintactvessels.Stimulationwith10nMPAFdidnotaffect[Ca2+]iorethidiumuptakeinresistancearteriesandEC-A.Incontrast,PAFinducedanincreasein[Ca2+]iandethidiumuptakeinvenulesandEC-V.Boththeincreasein[Ca2+]iandethidiumuptakewereinhibitedbytreatmentfor15minwiththeconnexinblockingpeptide37,43Gap27orthepannexin-1blockingpeptide10Panx,suggestingthatconnexinhemichannelsandpannexin-1channelscontributetotheCa2+signal.Consistentwiththis,theexpressionofCx37,Cx40,Cx43andpannexin-1wasconfirmedinEC-Vbyimmunofluorescenceanalysis.ConnexinandpannexinchannelsarepermeabletoCa2+,buttheymayalsotriggerCa2+signalsthroughATPreleaseandblockadeofpurinergicreceptorswithPPADSbluntedthePAF-induced[Ca2+]iincrease.TheseresultssuggestthattheintracellularCa

2+signalingactivatedbyPAFismainlymediatedbyATPreleasethroughconnexinhemichannelsand/orpannexin-1channels,which,inturn,leadstoactivationofpurinergicreceptors.However,directCa2+influxviaconnexinhemichannelsorpannexin-1channelsmayalsocontributetotheresponse.

ProyectoFONDECYT1150530.

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17)Pulmonary artery remodeling and mitogens are reduced in 2-aminoethyldiphenylborinate treated lambs

Castillo-Galán, Sebastián1.,Quezada,Sebastián1.,Beñaldo,Felipe1.,Ebensperger,Renato1.,Ebensperger,Germán1.,Herrera,Emilio1,2.,Llanos,Anibal1,2.,Reyes,Roberto1.,1ProgramadeFisiologíayFisiopatologíadelDesarrollo,FacultaddeMedicinaOriente,ICBM,UniversidaddeChile,UniversidadDeChile.2InternationalCenterforAndeanStudies(INCAS)UniversidadDeChile.(SponsoredbySupportedByFONDECYT1120605,1151119,1140647,1130424)

Perinatalchronichypoxiainducesanimbalanceofvasoconstrictorandvasodilatorsmechanismsaswellasapathologicalpulmonaryartery remodeling. This remodeling is characterized by a thickening of themedial layer due to a proliferation of pulmonaryarterysmoothmusclecells(PASMC)amongotherprocesses.Thesefunctionalandstructuralchangesresultneonatalpulmonaryhypertension(NPH),adiseaseinwhichpersistenthighpulmonaryarteryresistance(PVR)andpressure(PAP)areobserved.Thestoreoperatedchannels (SOC),arecalcium-permeablecationicchannels, involved in the regulationof thepulmonaryarteriesreactivity and remodeling, particularly in response tohypoxia.Wepreviously demonstrated that a ten-dayblockadeof thesechannelswith2-aminoethyldiphenylborinate(2-APB)reducesthebasalPAPanditsincreaseinresponsetohypoxiaonnewbornlambswithNPHinducedbypartialgestationunderhypoxia.HerewestudiedifthistreatmentrevertsthepathologicpulmonaryarteryremodelinginnewbornlambswithNPH,andifthischangewasrelatedwithareductionintheexpressionofthemitogenVEGF-A.Tennewbornlambspartiallygestatedathighlands(3600m)andreturnedtolowlandsimmediatelyafterdeliveryweretreatedfortendayswith2-APB,aputativeSOCblocker,(10mg·kg-1·day-1)oritsvehicle(DMSO:saline1:10).Twodaysaftertheendofthetreatment,weevaluatedthepathologicpulmonaryarteryremodeling,bymeasuringtheareaofmediallayerthicknessandtheexpressionofthesmoothmusclemarkerα-actininthesmallpulmonaryarteries.WealsomeasuredthepulmonaryexpressionofVEGF-Aisoforms-188,-164and-120.Pulmonaryarteriesfromanimalstreatedfortendayswith2-APB,exhibitedathinnermediallayerandlowerexpressionofα-actin.Inaddition,2-APBtreatmentreducedthepulmonaryexpressionofVEGF-A-188and-164isoforms,whilst-120isoformexpressionremainedunchanged.Aten-daytreatmentwith2-APBreducesthepulmonaryarteryremodelinginnewbornlambswithNPHinducedbyperinatalchronichypoxia.Thiseffectof2-APBcouldberelatedwithitsabilitytoblockSOC,andthereforetoreducethelongtermproductionofmitogenslikeVEGF.

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18)Increased potassium in the diet intensifies ATP release from rat mesenteric endothelial cells elicited by mechanical stimulation.

Donoso, M. Veronica1.,Huidobro-Toro,Juan1.,1DepartamentodeBiología,FacultaddeQuímicayBiología,UniversidaddeSantiagodeChile.(SponsoredbyFundedByFONDECYTGrant1141132)

Diethasacriticalinfluenceincardiovascularfunctioning.Humansfeddietsrichinfreshfruitsandvegetableshaveconsistentlybeen demonstrated to decrease the risk of vascular pathology, compared to humans that consumemainly processed foods.Reducingsodiumintakeandincreasingconsumptionofpotassium(K)containingfoodspreventsandreducesthedevelopmentofhypertensionandothervasculardiseases.ToelucidatethebeneficialKeffectsinthecontrolofdiseases,westudiedthereleaseofATPelicitedbyamechanicalstimulusfromculturedendothelialcellsderivedfromtheratarterialmesentericbedofanimalfedacontrol(1%K)andadietsupplementedwith4%K,during4weeks.WehypothesizedthatmechanicalstimulationreleasedATPbyendothelial cellswhich acts on autocrinepurinoceptors, leading to an increasedNOproduction, apotent vasodilator.Endothelialcellfromcontrolratswereplaqued(3-4days)inmediacontainingeither5.3or10.6mMK.Inparallel,endothelialcellsfromanimalsfed4%K,wereplaquedin10.6mMK.Culturesweremechanicallystimulated;thesupernatantwascollectedandchemicallyderivatizedtoassesATP/metabolitesbyfluorimetricdetection.BasalATPvaluesfromcellsderivedfromanimalsingestingeitheracontroldietorhighKdietdidnotchangeovertime(0.5-15min)norshoweddifferenceswhencomparingvaluesfromcontrolcellsandthosemaintainedinmediawithhighK.MechanicalstimulationinducedtransientATPreleasethatpeaked1minafterstimulationandreturnedtobaselinevaluesafter15min.TheATPratioofthereleased/basal,1minaftermechanicalstimulation,incontrolcellswas5.8±0.98(n=16),avaluethatincreased2-fold(10.2±1.89(n=16),p<0.05)incellsfromthesameanimalmaintainedin10.6mMKduringthedaysofcellculture.Likewise,analmost2-foldincreaseintheATPratiowasobservedincellsofratsfedahighKdietandmaintainedin10.6mMK(10.4±1.8,n=16,p<0.05).ThesameincreaseintheratiowasobservedforADP,AMPandADOincellsmaintainedinhighK.RatstreatedwithadietsupplementedwithKfor4weeksshowedasignificantreductioninbodyweightcomparedtopairedcontrols(282±22vs406±16g,p<0.001,n=5each).Inconclusion,increasingKinthediet,orthecultureofcellsinhighK,increasedsignificantlytheATP/metabolitesreleasedfollowingmechanicalstimulation,providinganexplanationforthebeneficialeffectsofKinpreventinghypertensionandothervasculardiseases.

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19)Differential regulation of NO pathway in pulmonary circulation of neonatal sheep from low and high altitudes.

Ferrada,Javiera8.,Ebensperger,Renato8.,Cerda,Tania1.,Catriman,Danixsa2.,Araya,Claudio3.,Beñaldo,Felipe3.,Díaz,Marcela4.,Moraga,Fernando5.,Castillo,Sebastian8.,Herrera,Emilio8,6.,Reyes,Víctor7.,Llanos,Aníbal8,6.,Ebensperger, G8.,1ProgramaFisioipatologia,CampusOriente,ICBM,FacultaddeMedicina,UniversidaddeChile.2ProgramadeFisiopatologia,CampusOriente,ICBM,FacultaddeMdicina,UniversidaddeChile.3ProgramadeFisiopatologia,CampusOriente,ICBM,FacultaddeMedicina,UniversidaddeChile.4DepartamentodePromocióndelaSaluddelaMujeryelReciénNacido,FacultaddeMedicna,UniversidaddeChile.5CienciasBiomedicas,FacultaddeMedicina,UniversidadCatólicaDelNorte.6INCASUniversidaddeChile.7ProgramaFisiopatologia,CampusOriente,ICBM,FacultaddeMedicna,UniversidaddeChile.8ProgramaFisiopatologia,CampusOriente,ICBM,FacultaddeMedicina,UniversidaddeChile.

Background.Theneonatalpulmonarycirculationneedstoadapttoextrauterinelifeinfewhoursforneonatalsurvival.Forthisobjectivetheinductionofvasoactivepathwayssuchasnitricoxide(NO)isimperative,producingamarkeddecreaseinpulmonaryarterialpressure(PAP)andvascularresistance(PVR).Whenthebirthiscarriedoutinamilieuwithalowoxygen(suchaschronichypoxiaathighaltitudes,HA)thepulmonarytransitionisinefficient,resultinginneonatalpulmonaryhypertension.Neonatesofsealevel(SL)species,suchasthesheep,induceNOproductionwhenexposedtochronichypoxia(1,2).Wehypothesizedthathighaltitudenewbornsheep,notonlyinducesNOproduction,butaswelltheNOsignallingpathway(sGC&PKG-1).

Methods. Two weeks old newborn sheep from high altitudes (HANB, n=6; 3,600m) and sea level (SLNB, n=6; 580m) wereinstrumented in pulmonary arterywith a SwanGanz catheter for registering cardiopulmonary hemodynamic variables in thepresenceofeNOSinhibitor(L-NAME),inbasalandhypoxemicconditions.Thedayafterweextractedsmallresistancepulmonaryarteriestoassessedpulmonaryvasodilatorcapacityinawiremyograph.

Results.HANBshowedabasalpulmonaryhypertensionrelativetoSLNB(21±3vs12±1),asdescribedpreviously(2).Duringbasalconditions,LNAMEinduceincreaseinPAPinbothgroupofneonates,reachinghigherabsolutevaluesinHANB.However,duringsuperimposedhypoxemia,bothgroups reached similarPAP levels (42±8vs38±2). The isolatedvascular functionexperimentsshowedamarkedendothelialdysfunctioninHANBrelativetoSLNB,whichisnotrecoveredbythearginaseinhibitorBEC.Further,LNAMEfullyblockedendothelialvasodilatorfunctioninbothgroups.Inaddition,sGCfunctioninpulmonaryarteriesisdepressedinHANBrelativetoSLNB.However,thePKG-1andRhokinasefunctionsareincreasedinHANBrelativetoSLNB.

Conclusion.TheNOpathwayregulatesthepulmonarycirculationinnewbornsheepfromsealevelandhighaltitude.Highaltitudeexpositionduringdevelopmentexacerbate theNO,PKG-1andRhokinase functions, as a compensatory response to sGC lowexpressionandfunction.

SupportedbyFondecytRegular1130424,1110647,1120605,1151119.

(1)Herreraet al.CardiovascRes77(1):197,2008.

(2)Herreraet al.AmJPhysiol292(6):R2234,2007

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20)Effect of aging on calcium transients in rat cardiomyocytes: impact of NOX inhibition

Gonzalez, Daniel1.,Barrios,Guillermo1.,1DepartamentoCienciasBiomedicas,FacultaddeCienciasdelaSalud,UniversidadDeTalca.(SponsoredbyFundedByProyectoFondecyt1150662AndProgramaDeInvestigaciónDeExcelenciaInterdisciplinariaEnEnvejecimientoSaludableUniversidadDeTalca.)

Background. Cardiacagingischaracterizedbyalterationsincontractilityandcalciumhandling.Ithasbeensuggestedthatoxidativestressmaybeinvolvedinthisprocess.ThesuperoxidegeneratingsystemNADPHoxidase(NOX)isexpressedintheheart(isoformsNOX2and4).Weandotherhavepreviouslyobservedthat insomeformsofcardiacfailure,theNOX-derivedreactiveoxygenspeciesareincreased,withanegativeimpactoncalciumtransientsandcontractility,duetoredoxeffectsonthecalciumreleasechannelryanodinereceptor (RyR2)andthesarcoplasmicreticulumcalciumpumpSERCA2.Aims. Theaimof thisstudywastoanalyze calcium transients and contractility in aged rat cardiomyocytes and to evaluate the impact of NADPH oxidase (NOX)inhibition.Methods. Cardiacmyocyteswereobtainedformadult(5monthsold)andaged(20monthsold)Sprague-Dawleyrats.Agedmyocytesweretreatedwithapocynin(50μmol/L,30min).Cellswerefield-stimulatedfrom0.5to4Hz,usingTyrodesolutionat37˚C.Contractilitywasevaluatedassarcomereshorteningandintracellularcalciumwasevaluatedloadingthemyocyteswithfura-2.Results. Sarcomereshorteningwassimilarinadult,agedandagedtreatedmyocytes.Timetoreach50%ofpeakshorteningwasincreasedinagedmyocytes(p<0.05),andwasnotimprovedbyapocyinintreatment.Thesamewasobtainedforthetimetoreach50%ofrelaxation.[Ca2+]itransients(amplitudeandfractionalincrease)wereincreasedinagedcardiomyocytes(p<0.05)andwerefurtherincreasedbyapocynintreatment.Time50topeakCa2+wasincreasedinagedmyocytes(p<0.05),suggestingimpairmentintheryanodinereceptor,butwasimproved(reduced)bytheapocynintreatment.Time50topeakrelaxationwasincreased inagedmyocytes (p<0.05)andreducedtowardsnormalbyapocynin treatment.Usingthapsigargin toblockSERCAfunction,wesubmittedmyocytestotetanicstimulation(40Hz)toevaluatethemyofilamentsCa2+sensitivity.Bycomparingtheamplitudeofthetetaniccontractionachievedwiththelevelof[Ca2+]ievoked,wefoundthatmyofilamentsCa2+sensitivitywasreducedinagedmyoctes(p<0.05).Conclusions. Contractilitywaspreservedinagedmyocytes,butatahigher[Ca2+]ilevel,asaresultofdiminishedmyofilamentsCa2+sensitivity.NOXinhibitionwithapocyninincreasedCa2+transientsamplitudeandimprovedCa2+kinetics,withoutchangesincontractility.ThesechangessuggestaredoxeffectatthelevelofryanodinereceptorandSERCA.

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21)Chronic exercise reduces fibrosis and hypertrophy but not oxidative stress in diabetic cardiomyopathy

Novoa,Ulises1.,Arauna,Diego1.,Moran,Marisol1.,Gonzalez, Daniel1.,1DepartamentoCienciasBiomedicas,FacultaddeCienciasdelaSalud,UniversidadDeTalca.(SponsoredbySupportedByFondecyt1150662AndProgramaDeInvestigaciónDeExcelenciaInterdisciplinariaEnEnvejecimientoSaludableUniversidadDeTalca.)

Background:Diabeticcardiomyopathyreferstothecardiacmanifestationsobserved intheheartasaresultofalteredglucosehomeostasisthatisreflectedasfibrosis,cellularhypertrophy,increasedsourcesofoxidativestress,apoptosis,andfinallysystolicanddiastolicdysfunction.Exerciseisknowntoexertsalutaryeffectsoncardiovascularfunction,mainlythroughtheincreaseintheexpressionofnitricoxidesynthase,particularlyeNOS.

Aims:We tested the hypothesis that chronic exercise could reverse the cardiacmaladaptations and oxidative stress that areproducedbydiabetes.

Methods.Diabeteswasinducedinratsbyasingledoseofalloxan(200/mgkg, i.p).Diabeticratswererandomlyassignedtoasedentarygrouporsubmittedtoaprogramofexerciseonamotor-driventreadmill(80%ofmaximalaerobiccapacity)5days/weeks,for4weeks.Anothergroupofnormoglycemicratswasusedascontrol.CardiacfibrosiswasevaluatedbyPicrosiriusredstainingandthelevelsofNOXandNOSenzymeswereevaluatedbyreal-timePCRandWesternBlotting.TetrahydrobiopterinlevelswereanalyzedbyHPLC.

Results. Chronic exercise reduced cardiac fibrosis and cellular hypertrophy in diabetic rats (p<0.05,ANOVA).On the contrary,exerciseinducedtheexpressionoftheNADPHoxidasesNOX2andNOX4,bothatmRNAandproteinlevel(p<0.05,ANOVA).ExerciseinducednochangeinthelevelsoftotalanduncoupledeNOS.Furthermore,exercisewasunabletorestoretheintracardiaclevelsoftetrahydrobiopterin,anessentialcofactorforNOSactivity,thatwerereducedindiabeticrats.

Conclusions.Theseresultssuggestthatchronicexercisewasabletoreversecardiacremodelinginthediabeticheart,butwasunabletorestorethenitroso-redoximbalanceimposedbyoxidativestress.Thislatercouldbyrestoredbypharmacologicalmanipulations.

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22)Nitric oxide releasing aspirin affects morphogenesis and increases Candida albicans susceptibility to fluconazole in clinical isolated.

Madariaga, F1.,Urzua,Blanca1.,Fernandez,Roberto1.,Ramires,Ricardo1.,Jara,Jose1.,Herreros,Eduardo1.,Aguilera,Jocelyn1.,Molina,Alfredo1.,1institutodeinvestigaciónencienciasodontologicas,odontologia,UniversidadDeChile.(SponsoredbyProjectFONDECYT11140227,U-inicia195.CandidaSppStrainsWereKindlyGivenByDr.XimenaLeeAndLeylaGomez,FacultadDeOdontología,UniversidadDeChile.)

Introduction: Candidaalbicansisabletoformbiofilmsondentureprosthesisparticipatingindenturestomatitis,acommonoralpathologyinelderlypeople.C.albicansbiofilmsfunctionasaninfectionreservoirthatinsusceptibleindividualscouldleadtoinvasivecandidiasis.Biofilmsactsasmultipleresistancemechanismtoantifungals.BecauseofthehighresistanceofC.albicansbiofilmstoantifungals,newpharmacologicalstrategiestotreattheseinfectionsareneeded.Inthissense,non-steroidalantiinflamatorydrugs(NSAIDs),whichinhibitprostaglandinssynthesis,haveshowntoexertantibiofilmeffect.Amongthem,aspirinnotonlypreventsbutalsoreversebiofilmformation.Ontheotherhand,nitricoxide(NO)releasingdrugshaveshownsimilarresultstoaspirinandimproveconventionalantifungaleffectsonbiofilms.

Accordingtothisinformation,weevaluatedNO-releasingaspirin(NO-ASA)effectonC. albicansobtainedfromdenturestomatitispatientsaloneand incombinationwithfluconazole.Methods: Candidaspp.obtained fromoralmucosaofdenturestomatitispatients(n=60),wereidentifiedthroughCHROMagarCandidaMediumBDTMandbysequencingtheITS1-5.8SrDNA-ITS2regionusing theprimers ITS1and ITS4.Toevaluateantifungalsusceptibility, isolatedstrainswerestandardizedto0.5McFarlandandthengrownon sabouraudagarplates anddiskdiffusion testswereperformed inpresenceor absenceoffluconazol,NO-ASAandtheircombination.Candida albicansMorphogenesisisacrucialsteptobiofilmformationsoweevaluatedeffectofNO-ASAon hyphal induction. C. albicans hyphaewere induced in standardized overnight cultured cells in RPMI-1640medium (28°C,withoutagitation).Afteradjustingto1x106cells/mL(Neaubauerchamber)sampleswereincubatedat37°Cwithagitationfor3h.PercentageofHyphaewasobtainedcountingthecellsbylightmicroscopy.Results:fromthe60strains,55%(n=33)wereidentifiedasC.albicans.Indiskdiffusionassay,7strains(21%)wasclassifiedasresistanttofluconazole(diameter<16mm).NO-ASAhadnoeffectwhenwasaddedaloneinthedisks.However,inresistantstrainscombinationwithfluconazoleitwasobserved15-20%increaseininhibitionzonediameterrespecttofluconazolealone.Forhyphaeinductionexperiments,weselectedthe7strainsresistanttofluconazoletoevaluateNO-ASAeffect.ResultsshowthatNO-ASAisabletodecreaseyeasttohyphaemorphogenesisinallclinicalstrainstestedtoanextentof70-80%(p<0,05ANOVA). Conclusion: NO-ASAincreasessusceptibilitytofluconazoleinresistantstrainsandalsoaffectsabilitytoswitchfromyeasttohyphae.Wearecurrentlyinvestigatingpossiblemechanismswhichexplaintheseeffects.

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23)Cytotoxic effect of lipophilic cation derived from polyhydroxy-benzoic acids in oral squamous cell carcinoma

Aguilera, Jocelyn 1.,Molina,Alfredo1.,Fernández,Ricardo1.,Ferreira,Jorge2.,Castro,Vicente3.,Jara,José1.,1LaboratoriodeFarmacologíayFarmacogenómica,FacultaddeOdontología,UniversidadDeChile.2ProgramadeFarmacologíaMolecularyClínica,ICBM,FacultaddeMedicina,UniversidadDeChile.3DepartamentodeQuímica,FacultaddeCienciasBásicas,UniversidadMetropolitanadeCienciasdelaEducación.(SponsoredbyProyectoU-iniciaVIDN?196(JaraJ),FinanciadoPorElProgramaDeAyudaDeViajesParaElFortalecimientoDeLaInvestigación,FacultadDeOdontología(JaraJ).)

Oralcancerisadiseasewithhightimpactworldwide,infact,rankssixthintheworldinthecategoryofmostcommoncancersinmen.Assuch,cancerisadiseaseinwhichcellsundergoanimbalancebetweencelldivisionanddeath.TheuncontrolledgrowthofthesecellscomefromaseriesofgeneticalterationsthatallowthemtomultiplyoccursoutofcontextNormaltissuedevelopment.

These cancer cells have striking features asunlimited replicativepotential, sustainedangiogenesis, evasionof apoptosis, self-sufficiencyingrowthsignals,insensitivitytoanti-growthsignals,invasivenessandmetastasis.Allthesefeaturesprovideselectiveadvantagesendingintheprogressiondisease.Someofthesefunctionsareessentiallyrelevantinthesearchfornewtherapeutic,forexample,increasedmitochondrialtransmembranepotential,highlyglycolyticactivityandreducedmitochondrialmasstools.Thischaracteristicmakesmitochondrialanattractivedrugtargetforthesearchfornewmoleculesthatincreasetheefficiencyandselectivityofthetreatments.

It wasused as cytotoxic agents derived frommono and/or poly-hydroxybenzoic acids attached to the triphenylphosphoniumgroup,asthereisevidencethatthisgroupcanleadtopharmacophoretothemitochondria.Wetestedbromidesalts:Triphenyl(10 - ((3,4,5-trihydroxybenzoyl) oxy) decyl) phosphonium, 10- ((2-hydroxybenzoyl) oxy) decyl) triphenylphosphonium 10 -((2,5-dihydroxybenzoyl)oxy)decyl)triphenylphosphoniumand10-((2,3-dihydroxybenzoyl)oxy)decyl)triphenylphosphoniumontumor cell lines CAL-27 and normal cells.MTT viability assaywas used tomeasured cytotoxicity by calculating therespectiveIC50, ATP content through luminescence, mitochondrial transmembrane potential by fluorescence andinduction of apoptoticdeathwasdeterminedbyflowcytometry.Theresultsindicatethatthecompoundsexhibitenhancedcytotoxicityinthetumorcelllinethannormalcells.Furthermore,thecompoundsshowedanuncouplingeffect,andalsotriggereddecreasedtransmembranepotential,fallingATPlevelsandinduceapoptosisinamicromolarrangeofconcentration.Theresultsarepromisingintermsofcytotoxicityandselectivityandofferapharmacologicalopportunityoftreatmentinoralcancer.

101

24)Ar-c155858 decreases neutrophil extracellular traps formation and neutrophil adhesion onto endothelium induced by D-lactic acid

Alarcon, Pablo1.,Conejeros,Iván1.,Munoz-caro,Tamara2.,Taubert,Anja2.,Hermosilla,Carlos2.,Burgos,Rafael1.,1InstitutodefarmacologiaymorfofisiologiaUniversidadAustralDeChile,Valdivia,Chile.2InstituteofParasitologyJustus-LiebigUniversityGiessen,Giessen,Germany.(SponsoredbyFinancedByFONDECYT1151035,DID-UACh,MECESUPAUS1203)

InCattle,ruminalacidosisoccursmainlybyahighlyfermentablecarbohydrateoverload,whichleadstoincreasedlevelsofD-lacticacidintherumenandplasma.Also,rumenacidosishasbeencorrelatedwithanincreaseinacutephaseproteinsinplasmaandneutrophilsinfiltrationinseveraltissuescausing,rumenitis,laminitis,polysynovitis,suggestingtheinvolvementofanextentpro-inflammatory response in ruminal acidosis.Neutrophils represent the first line of defense against pathogens but recent dataalsoindicatesthatD-lacticacidinterfereswithneutrophilactivationtherebydecreasingtheROSproductionandthereleaseofmetalloproteinase9inbovineneutrophilsstimulatedwithplatelet-activatingfactor.Inaddition,D-lacticacidincreasedneutrophilextracellulartraps(NET)formationandadhesionofbovineneutrophilstoendotheliumunderflowconditions.Wedemonstratedthepresenceofmonocarboxylatetransporters(MCT-1,4)inbovineneutrophils.Inthiswork,weshowforthefirsttimeD-lacticacid-triggeredNETformation,colocalizedwithH4Citrullinated3andCD11b,throughimmunofluorescenceanalysis.FurtheraimofthisstudywastoevaluateifblockingtheMCT1transporterwithAr-c155858wouldbeabletoreducetheeffectofD-lacticacidonNETformationaswellasadhesionontoendothelium.WedemonstratedthatAr-c155858reducedtheNETformationandadhesionofneutrophilontoendotheliuminducedbyD-lacticacid.Indeed,neutrophilsadhesiontoendotheliuminducedbyD-lacticacidwasalsoreducedwhenDNAseIwasaddedandmeasuredunderflowconditions.Moreover,whenneutrophilstreatedwith5mMD-lacticacidplusanti-CD11bantibodies,andthenperfusedontoendothelium,therewasadecreaseofneutrophilsadhesionunderflowconditionswhichleadsustosuggestthattheendothelium-neutrophiladhesioninducedbyD-lacticacidCD11b-dependentprocess.Secondly,whentheendotheliumwasperfusedonlywithsupernatant fromneutrophils treatedwith5mMofD-lacticacid for10minutesand later,perfusedneutrophilswithout treatment,weobservedan increaseofneutrophil adhesionontoendotheliumunderflowconditions,andthussuggestingthattheremightbeasolublecomponentreleasedD-lacticacid-treatedneutrophilwhichmighthaveactivated thevascularendothelium.Finally,weconclude thatD-lacticacid isable to triggerNETformationandneutrophiladhesionandthusmightbeinvolvedintheactivationofthevascularbedwhichcouldcontributetoneutrophilinfiltrationintolocomotorapparatusduringacuteacidosisincattle.

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25)Evaluation of leukocyte extravasation inhibition by Ugni molinae genotypes

Avello, Zita1.,ArancibiaRadich,Jorge1.,Seguel,Ivette2.,Delporte,Carla1.,1QuímicaFarmacológicayToxicológica,CienciasQuímicasyFarmacéuticas,UniversidadDeChile.2InstitutodeInvestigaciónAgropecuarias,InstitutodeInvestigaciónAgropecuarias,INIA,Carrillanca,Temuco.(SponsoredbyAcknowledgments:WeAreGratefulToFONDECYT,Chile,GrantN°1130155,CONICYT,ChileGrantsN°21130672AndN°21120377.ThankYouToINIA(Carillanca,Temuco)ForTheGenotypes.)

Ugni molinae TurczisaChileanevergreenshrub,itbelongstotheMyrtaceaefamilyandisnativetocentralandsouthernChile.Amongitsdenominationsare“murtilla“,“murta“or“Ugni”.ResearchconductedintheLaboratoryofNaturalsProductsattheFacultyofChemicalandPharmaceuticalSciencesattheUniversityofChilehasrecognizedpentacyclictriterpenoidderivatesofursane,oleananeandlupaneas:ursolicacid,oleanolicacid,corosolicacid,alphitolicacid,madecassicacid,asiaticacidandmaslinicacid.Whichhaveanti-inflammatoryactivity. Inotherstudiesthepresenceofphenoliccompoundshasbeenreported,suchascatechin,epicatechin,myricetin,quercetin,kaempferol,myricetinglycoside,quercetinglycoside,rhamnoside,andgallicacid.Theaimofthisstudywastoevaluatecomparativelytheanti-inflammatoryeffect,throughtheinhibitionofleukocyteextravasationinamicemodel,ofethanolandethylacetateextractsfromleavesofgenotypesofUgni molinae growninthesamesoilandclimateconditions andwith the same agronomicmanagement. The evaluated extractswere selected due to their anti-inflammatoryactivityin vivointheTPA(phorbol12-myristate13-acetate)inducedinflammationinmiceedemaearmodel.Asampleof6mmdiameterwasremoved,fixedin4%paraformaldehydesolutionandtreatedwithmethanolsolutionsofincreasingconcentrationtodehydrateit.Afterwards,xylenewasaddedtothesampleandfinallyembeddedinparaffin.Afterthis,histologicalcutsweredone.Fordeparaffinizethesamplewashydratedwithmethanolsolutionsofdecreasingconcentration.Thesamplewasstainedwithhematoxylinandeosinthatallowedfortheobservationandcountofleukocytesintheopticmicroscope.Thenumberofleukocytesinthetreatedsamplesandcontrolssampleswasdetermined.Withthesedata,theinhibitionpercentageofextravasationintheinflamedsitewascalculated.Intheevaluationoftheanti-inflammatoryeffectthroughtheinhibitionofleukocyteextravasation,theresultsshowedsignificantdifferencesbetweentheextractsfromdifferentUgni molinae genotypes.

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26)Selective sympathetic plasticity in basal and evoked levels due to chronic sympathetic over-stimulation

Pezo, Rafael1.,Donoso,MariaVeronica2.,Huidobro-Toro,JuanPablo1.,1DeparmentofBiology,ChemistryandBiology,UniversidadDeSantiagoDeChile.2Biologydeparment,ChemistryandBiology,UniversityofSantiagoofChile.(SponsoredbyPostdoctoralFONDECYTToRB3130573AndFONDECYT1141132.)

Augmentedsympatheticactivityisassociatedwithseveralchronicdisorderslikeobesity,diabetes,etc.Nowadaysthereisalackofbasicinformationregardingtheneurochemicaladaptationsassociatedtoanalteredsympatheticnerveterminalsphysiology,likedescribedin.Oneofthecommontradesoftheresponsetostressistheaugmentofthesympatheticnervousactivitytobloodvessels,includingarteriesandveins.

Basedonthisbackground,wehypothesizedthatratchroniccoldstress(CS)generatesanincreasednoradrenergicsecretioncoupledto an altered sympathetic varicosity functioning.We subjected rats to CSwithout hypothermia, and assessed neurochemicalchangesinthesympatheticvaricositiesthatinnervatetheratarterialmesentericbed.Tothisend,weperfusedex-vivoratarterialmesentericbedofcontrolandratsexposedtoCSfor3hourseverymorningfor4weeks.Toevokethereleaseofco-transmitters,weelectricallystimulatedtheperivascularmesentericnerves(60Hz,60volts,1minute)andanalyzedthesecretionofsympatheticco-transmitters(ATP,NAandir-NPY).ATPandmetabolitesweremeasuredbyfluorescence;NAwasassessedbyelectrochemicaldetectionandir-NPYbyRIA.Simultaneously,theperfusionpressure(PP)ofthevascularbedwasrecordedviaagrasstransducer.

PresentresultsconsistentlyindicatethatATPandmetabolitesarelowerinthegroupofCSratscomparedtocontrols;basalATPlevelswere24.4±4.8(n=10)vs.119.6±39.8pmol(n=6),p≤0.05.Likewise,ADPlevelswere28.2±10.8(n=10)control136.8±51.8(n=6)pmolp≤0.05.AMP;27.6±6.3(n=10)vs.161.5±71.3pmol(n=6)p≤0.05.ADO;8.4±0.6(n=10)38.7±16.0pmol(n=6)p≤0.05.BasallevelsofNAweresimilar4.424±1.027N=6(CS)vs.2.6±0.9N=5(Control).Inaddition,wefoundthattheNAsecretedbyelectricalstimuliwashigherintheCSvs.control47.0±15.7(n=6)vs.9.4±4.6(n=5)p≤0.05),whileATPvalueswere144.5±50.4(n=10)vs.130.9±77.4(n=6),forCSandControlrespectively.Inagreementwiththeco-transmittersreleased,weobservedanincreaseintheelectricallyevokedriseinPPintheanimalssubmittedtoCScomparedtotherespectivecontrols(114.9±10.8(n=6)vs78.9±12.0vs.(n=7)p≤0.05).Insum,wediscoveredthatCSinducesneurochemicalchangesinthesympatheticvaricosities.WeinterpretthepresentdataindicatingthatCScausesaselectiveplasticityofthesympatheticnervesinsuchawaythatbasalreleaseofATPisloweredbutincreasedfollowingtheevokedreleaseofNA.Thisisthefirstreportofspecificco-transmitterchangesbetweenthebasalandevokedreleaselevels;themechanicsofthesechangesremainasyetunknown.

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27)Interferon beta (IFN-β) activates the JAK-STAT pathway in cardiac fibroblasts and produces anti-inflammatory and anti-fibrotic effects

Bolivar, Samir1.,Humeres,Claudio1.,Vivar,Raul1.,Boza,Pia1.,Muñoz,Claudia1.,Díaz-Araya,Guillermo1.,1FarmacologiayToxicologia,CienciasQuimicasyFarmaceuticas,UniversidadDeChile.(SponsoredbyFONDECYTProject1130300,CONICYTPhDScholarshipForForeigners,OperatingExpenses63130233CONICYTScholarship.)

Introduction:Interferonbeta(IFN-β)isacytokinethatactivatesthesignalingtransductionpathway,Januskinase(JAK)andthetransducerandactivatoroftranscription(STAT),andcausesavarietyofresponses.CardiacFibroblasts(CF)arethemostabundantresidentcellsintheheart,andregulatethemaintenanceofextracellularmatrix.TheCFplaysacentralroleincardiacremodelingagainstcertain injuries;however, it remainsunknown if thereareanypotentialanti-inflammatoryandanti-fibroticof IFN-β inCFactions.ThisworkproposesthatIFN-βandtheactivationofthecanonicalpathwayinCF,couldbenoveldrugtargetsinthecardiovasculararea.

Objective:DeterminewhetheractivationoftheJAK-STATpathwayinCF,producesanti-fibroticandanti-inflammatoryeffects.

Materials and Methods:PrimaryculturesofadultratFCinpassage2weremaintainedinserum-freemediumfor24hoursandstimulatedby IFN-β in thepresenceorabsenceofRuxolitinib (JAK inhibitor) for1hour.TheexpressionofSTATproteins,pro-interleukin1beta(IL-1βpro),alphasmoothmuscleactin(α-SMA)and1collagen(ColI)wasevaluatedbywesternblot.Immuno-precipitationbyforminghomoandheterodimersofSTATproteinsafterstimulationwithIFN-βwasdetermined.

Results:TheactivationofthesignaltransductionpathwayJAK/STATbyIFN-β,inducedformationofhomodimersp-Stat1/p-Stat1andp-Stat3/p-Stat3-pandheterodimerp-Stat1/p-Stat2.CFstimulationwithIFN-βdecreasedtheexpressionofpro-IL-1β,ColIandα-SMA.

Conclusion:TheseresultsdemonstratethatIFN-βproducesanti-fibroticandanti-inflammatoryeffectsinCF,therefore,itcouldreduceinjuriesandimprovecardiacremodeling.

105

28)Oil essential from Cryptocaria alba and Peumus boldus with anti- Helicobacter pylori activities

Bravo, Jessica1.,Touma,Jorge1.,Venegas,Alejandro1.,Navarro,Myriam1.,Delporte,Carla2.,Sepulveda,Betsabet3.,1CIB,Medicina,UniversidadDiegoPortales.2Farmacognosia,QuímicayFarmacia,UniversidaddeChile.3CEPEDEQUniversidaddeChile.

ResistanceofH. pylori strainstocommonantibioticshasbeendevelopedindifferentpartsoftheworldandcontinuestoincrease.Itisimportanttoinvestigatethenovelandefficientanti-H. pylori drugs,amongwhichtheplantswouldbesuitablesources.

Thestudyaimstothedetection,purificationandcharacterizationofoilsfromnativevegetablespecieslikePeumo(Cryptocaria alba)andBoldo(Peumus boldus),insearchofaneffectiveantimicrobialactivityagainstHelicobacter pylori,thecausativebacterialagentofgastritis,ulcersandgastriccancerinhumans.Thecharacterizationoftheoilsistodefinetheirstructureandeventuallytheirpossiblemechanismsofaction.Thecontributionoftheproject istofindnaturalalternativestoantibioticsandtoreducesignificantlytheiruseduetotheincreasingemergenceofantibioticresistantstrains.Weexpecttofindcompoundsthatalsohavealowleveloftoxicity,thereforemakinghumanadministrationsafeandeffective.

Inthisstudy,weevaluatedtheantibacterialactivityofCryptocaria albaandPeumus boldusessentialoilagainst3clinicalisolatesofHelicobacter pylori bydiscdiffusionandagardilutionmethods.Cryptocaria albaessentialoilshowedstrongantibacterialactivityagainstclinicalisolatesofH. pylori (MIC0.00124μg/ml).ThechemicalcompositionofessentialoilwasanalyzedbyGCandGC–MS.a-Terpineol(27.38%),eucalyptoL(23.27%)andfellandrene(16.28%)weretheprimaryconstituentsofoil.Terpineol,asthefirstmaincomponent,hadasignificantroleinthiseffect(MIC0.00093μg/mL).Therefore,Cryptocaria albaessentialoilcanbeappliedasanalternativeagentfortreatmentofH. pylori infections.MorestudieswouldberequiredtobetterclarifyitsmechanismofactiononH. pylori.

Reference

1.Montenegro,G.2002.Nuestrafloraútil.UniversidadCatólicadeChile.267pp.

2.MiyamotoT,OkimotoT,KuwanoM.2014.Chemicalcompositionoftheessentialoilofmasticgumandtheirantibacterialactivityagainstdrug-resistantHelicobacter pylori. Nat Prod Bioprospect.4:227–31.

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29)Activation of μU-opioid receptor by Salsolinol, a brain metabolite of alcohol

Bravo, Alex1.,Berríos-Cárcamo,Pablo2.,Herrera-Marschitz,Mario3.,Quintanilla,MaríaElena2.,Rivera-Meza,Mario.,1DepartamentodeCienciasFarmacéuticas,FacultaddelaSalud,UniversidadArturoPrat.2ProgramadefarmacologíaMolecularyClínica,FacultaddeMedicina,UniversidadDeChile.3IniciativaCientificaMilenioInstitutodeNeurocienciaBiomédica.(SponsoredbySupportedBy:FONDECYT#11130241:BNIP09-015-F)

Ethanolismetabolizedintheliverbyalcoholdehydrogenase(ADH)toacetaldehyde,whichisinturnmetabolizedtoacetatebymitochondrialaldehydedehydrogenase(ALDH).Inthebrain,thereisnoexpressionofADH,thereforeethanolismetabolizedbytheactionofcatalasetoacetaldehyde,whichisthenconvertedtoacetatebyALDH.

Inthebrain,ethanol-derivedacetaldehydecancondensewithdopaminetogeneratesalsolinol.Therearereportsshowingthatsalsolinolisareinforcingmolecule,sinceratsself-administerthissubstanceintheventraltegmentalarea(VTA),anareaofthebraininvolvedintherewardsystem(pleasure).TheVTAiscomposedbydopaminergicneuronsthatprojecttothenucleusaccumbensandtheprefrontalcortex.VTAdopaminergicneuronsarenegativelycontrolledbyGABAergicinter-neurons.Invitrostudiessuggestthat salsolinol can inhibit the inhibitoryeffectsofGABAneuronsbyactivatingμ-opioid receptors;however, there isnodirectevidenceofthisaction.

Ithasbeenfoundthattheactivationoftheµ-opioidreceptorbyagonistsuchasmorphineorDAMGOcaninduceaphosphorylationofspecificsitesinitsintracellulardomain(e.g.,serine375).Thesemodificationsareessentialforsubsequentreceptordesensitizationanditsrecyclingintothecell.Theobjectiveofthisstudyistodemonstratethehypothesisthatsalsolinolisableofactingasanagonistofµ-opioidreceptor,throughthestudyofthephosphorylationthatthismoleculecan induce intheµ-opioidreceptor.Toconfirmthishypothesiswepropose:(i)toclonetheratgen(cDNA)oftheµ-opiodreceptor;(ii)toexpresstheratmu-opioidreceptorinHEK-293Tcellsbytransienttransfection;(iii)toexposethetransfectedcellstodifferentconcentrationofsalsolinoland(iv)usingaspecificantibodyforthephosphorylatedmu-opioidreceptor(S375-P),todeterminebywesternblotifsalsolinolisabletoactasanagonistoftheµ-opioidreceptor.

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30)Agonists of HCA2 receptor induce calcium mobilization and increase chemotactic response in bovine neutrophils

Carretta, Daniella1.,Hidalgo,Maria2.,Burgos,Rafael2.,1LaboratoryofInflammationPharmacology,InstituteofPharmacologyandMorphophysiology,FacultyofVeterinaryScience,UniversidadAustralDeChile.2InstitutodeFarmacologíayMorfofisiología,FacultaddeCienciasVeterinarias,UACH.(SponsoredbyFondecyt1151035)

In dairy cattle elevated concentrations of the ketone body β-hydroxybutyrate (BHB) during lactation are associated with anincreasedincidenceof inflammatorydiseasessuchasmetritisandmastitis,howeveritremainsunclearhowBHBcouldaffectsthe inflammatory response in dairy cows. BHB and the short chain fatty acid butyratewas identified as endogenous ligandsoftheHydroxycarboxylicacidreceptor2(HCA2),aGprotein-coupledreceptor.HCA2also isactivatedbynicotinicacid(NA),alipidloweringdrug,thusthisreceptor isbetterknownfor itsantilipolyticeffect inadipocytes.However,recentstudiessuggestthatactivationofHCA2canmodulatetheinflammatoryresponseinhumanmacrophages,monocytesandneutrophils,wherethisreceptorishighlyexpressed.Neutrophilsareundoubtedlythemajoreffectorsofacuteinflammationandarecharacterizedbytheabilitytodirectedmigratetothesiteofinfectionorinflammationinaprocessescalledchemotaxis.Becausethecriticalfunctionofneutrophilchemotaxistomanyinflammatorydiseasesinhumansaswellincattlethechemoattractantsreceptorsaretargetsofintenseinvestigation.HCA2expressionandtheresponseofspecificligandsonbovineimmunecellshavenotbeendemonstratedyet. In thiswork,wehavepreliminarily observed themRNAexpressionofHCA2 receptor in bovineneutrophils. Besides, thetreatmentwithMK-1903,aselectivefullagonistofHCA2elicitatransientriseofintracellularCa2+levels,suggestingthatbovineneutrophils express a functionalHCA2 receptor.Also,weobserved thatMK-1903 increase thebovineneutrophil chemotacticresponse induced by PAF, a potent chemoattractant for bovine neutrophils. Sowe propose that activation of HCA2 receptorenhancethechemotacticresponseofbovineneutrophils.Firstwewillcharacterizethepharmacologyofthisreceptorusingtheendogenous (BHB)andsynthetic (NAandMK-1903)agonists.WewillestimatedeEC50ofeachoneby theirability to induceCa2+flux.WewillevaluateifHCA2agonistsinducethechemotaxisofbovineneutrophilsorincreasethechemotacticresponseelicitby chemoattractants suchasPAF.Weobserved that theadditionofBHB,NAandMK-1903 to fura-2-loadedneutrophilsledtorapidandtransientchangesinCa2+levelsthatwereconcentrationdependent.Also,weobservedthatactivationofHCA2receptor,increasesbovineneutrophilschemotaxisaloneorinducedbyPAF.Insummary,theseresultswillcontributetoimproveourknowledgeaboutthenovelmodulatorymechanismofHCA2intheinnateimmunesystemthatcouldbeinvolvedinvariouspathophysiologicsituationsincattle.

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31)Alkylhydroxy-benzoate derivatives as new compounds with cytotoxic effect in human colon cancer cells

Catalán, Mabel1,2.,Rojas,Diego2.,Guzmán,Daniela2.,Jara,José3.,Castro-Castillo,Vicente4.,Rebolledo,Solange4.,Madrid-Rojas,Matías4.,Pavani,Mario2.,Ferreira,Jorge2.,1ProgramadeFarmacologíaMolecularyClínica,Medicina,UniversidadDeChile.2ProgramadeFarmacologíaMolecularyClínica,Medicina,UniversidaddeChile.3ProgramadeFarmacologíayFarmacogenética,Odontología,UniversidadDeChile.4Química,Cienciasbásicas,UniversidadMetropolitanaDeCienciasDeLaEducación.(SponsoredbyFONDECYTRegularN°1130772(FerreiraJ),ProyectoInserciónEnLaAcademia(CatalánM)N°791220004)

Cancercellshaveinterestingfeatures,resistancetocelldeath,highinvasivenessandmetastasis,inductionofangiogenesisandmetabolicreprogramming.Somecharacteristicsareespeciallyrelevantinthesearchofnewtreatment:i)highertransmembranepotentialduetoaninnermembranemodifiedcomposition,highlyglycoliticactivityandii)reducedmitochondrialmass.Thesepropertiesmake themitochondria aconvincingpharmacology target in thesearchanddesignofnewmolecules targeting tobioenergeticsofcancercells.Dataexistabouttriphenylphosphoniumgroupapplicationtodrivepharmacophoremoietiestowardsmitochondria. Previously,wehave used 3,4,5- tri-hydroxybenzoic acid derivatives linked to triphenylphosphonium group as amitochondriotrophiccytotoxicagentwithsuccessfulresultsregardingtheircytotoxicactivityandselectivity.Itwasalsoestablishedthatthesemoleculesexerciseduncouplingeffect.Now,weassessmono-anddi-hydroxybenzoateslinkedbyanaliphaticchainof10carbonatomstothetriphenylphosphoniumgroup,totherebyestablishthecytotoxicactivityinrelationtothenumberandpositionofhydroxylgroupsinthebenzoicacidring.Wetestedbromidesalts:10-((2-hydroxybenzoyl)oxy)decyl)triphenylphosphonium,10-((2,3-dihydroxybenzoyl)oxy)decyl)triphenylphosphonium and 10-((2,5-dihydroxy benzoyl)oxy)decyl)triphenylphosphoniumoverpercentagesurvivalofHCT-15andCOLO-205tumorcellslinesbyMTTassay.Mitochondrialuncouplingeffectbypolarography,ATPcontentbyluminescenceandtransmembranepotentialbyspectrofluorographywerealsoevaluatedinintactcells.Thus,thefallingofNAD(P)H levelbyautofluorescence,and inductionofapoptoticdeaththroughflowcytometryweredetermined.Theresults indicatedcompoundsexhibited increasedcytotoxicity inboth cell line types,establishing IC50values. Thecompoundsshoweduncouplingeffect,alsotheytriggeredthedecreaseofboththeNAD(P)Hlevelandthetransmembranepotential,fallingATPlevels,andtheinductionofapoptosisinbothcelltypes.

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32)Comparative study of Inhibitory activity of the protein tyrosine phosphatase 1B of Ugni molinae leaves genotypes

Bugueño,Italo2.,Arancibia-Radich,Jorge2.,Peña-Cerda,Marcelo2.,Cortez,Giovanni2.,Seguel,Ivette1.,Delporte, Carla2.,1InstitutodeInvestigacionesAgropecuarias,InstitutodeInvestigacionesAgropecuarias,INIA,Carillanca,Temuco,Chile.2QuímicaFarmacológicayToxicológica,CienciasQuímicasyFarmacéuticas,UniversidadDeChile.

Ugni molinae,Myrtaceae,isusedagainstdiabetesinfolkmedicine.InourpreviousUgni molinaestudieswithwildmurtillaleaves,bothethylacetateandethanolicextractshaveshownanti-inflammatoryandanalgesicproperties.Wehaveadditionallyidentifiedtriterpenoidacidsinmurtillaleaveshavinganti-inflammatoryactivity,namely,oleanolic-,ursolic-,betulinic-,alphitolic-,corosolic-,maslinic-,asiatic-andmadecassicacids.Proteintyrosinephosphatase1B(PTP1B)isaneffectivetargetforthetreatmentofbothtype2diabetesandobesity.

TheaimofthisstudywastoassesscomparativelytheinhibitoryactivityofPTP1Boftheethylacetateandethanolextractsfromleavesoftengenotypesofthisspeciesatthesingleconcentration,growninthesamesoilandclimateconditionsandwiththesameagronomicmanagement.

Methodology:tenUgni molinaeleavesgenotypeculturesfromthegermplasmbankofINIAdeCarillanca.FivegenotypeswereselectedduetotheiragronomicpotentialinrespecttotheproductionoffruitsaccordingtopreviousstudiesmadebytheINIA,andanother5wereselectedduetotheircapacitytoproducealargenumberofleaves.

Driedandgroundleaves(2kg)ofeachgenotypeweresuccessivelyextractedbymacerationatroomtemperaturewithhexane,dichloromethane,ethylacetateandethanol(6Lofeachsolvent);afterremovingthesolvents,theextractswerecompletelydriedat30°C,toobtaindryextracts(HE,DME,EAEandETE,respectively)

Proteintyrosinephosphatase1B(PTP1B)assay.Theenzymeactivitywillbemeasured

usingp-nitro-phenylphosphate(pNPP)asdescribedYi-mingMaetal. (2011).Theproduct,p-nitrophenolwillbeestimatedbymeasuringtheabsorbanceat405nm.Thenonenzymatichydrolysisof2mMpNPPwillbecorrectedbymeasuringtheincreaseinabsorbanceat405nmobtainedintheabsenceofPTP1Benzyme.Sodiumorthovanadatewasthereferencedrug.StatisticalsignificancewasevaluatedusingANOVAfollowedbyTukeytests.

Results:Ugni molinaeleavesofdifferentgenotypesshowedsignificantdifferencesintheirinhibitoryactivityofPTP1B.TheETEs(1µg/mL)weremoreactivethanEAEs(2µg/mL).Bothextractsweremoreactivethansodiumorthovanadate(2µg/mL).

Acknowledgments:WearegratefultoFONDECYT,Chile,GrantN°1130155,CONICYT,ChileGrantsN°21130672andN°21120377.ThankyoutoINIA(Carillanca,Temuco)forthegenotypes.

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33)New isoxazole compounds: Activity on α7 nicotinic receptors and toxicity in endothelial cells

Espinoza, Hilda1.,Sepúlveda,Evelyn1.,Álvarez,Rocío1.,Vallejos,Gabriel2.,Cortés,Magdalena1.,1PharmacyFacultyUniversidadofValparaiso.2ChemistryInstituteAustralUniversityofChile.(SponsoredbyDIPUVProject59/2009.)

Introduction:Theisoxazolemolecules(ISO)exhibitnumerouspharmacologicalproperties,onethathasattractedincreasedinterestinrecenttimes;itisitseffectonthehomomericα7nicotinicacetylcholinereceptor(α7nAChR).Recentevidencessuggestthatα7nAChRparticipate inangiogenesisandwouldbeanewendothelial targetontherapeuticangiogenesis.Therefore, it’sofgreatintereststudynewmoleculesthatbehaveasα7nAChRagonists.PreviousstudieshaveshownthattwoISO,ABT-418andPNU-120596,behaveasagonistandpositiveallostericmodulatorofα7nAChRrespectively.Inadditiontothebioactivityevaluation,alwaysisnecessarytoruleoutthepossiblecytotoxiceffectsthatwouldmakenotviableitssubsequenttherapeuticuse.Inthissense,thetoxicityassessmentofthecompoundPNU-120596demonstratedthatitdoesn’tpresenttoxiceffectsonratcorticalneuronsandpheochromocytomacellsofratadrenalmedulla.Inthesameway,ABT-418doesn’tinducedcytotoxicityandevenexhibitedneuroprotectiveeffectsonratcorticalneuronsandneuroblastomacellsIMR32.Objetives:TheobjectivesofthestudyweredeterminatetheeffectsofthreeISO,ISO-1,ISO-2andISO-3onthecytosolicCa2+signal,determiningtheinvolvementofα7nAChR;andevaluatethepossiblecytotoxiceffectsofthesecompoundsinthreebiomarkersonHUVEC.Methodology:Theeffecton[Ca2+]iwereassessedusingepifluorescencemicroscopy.Theinvolvementofa7nAChRwasevaluatedusingwithnonselectivenAChRantagonists,mecamylamine(MECA)andhexamethonium(HEXA),andtheselectiveα7nAChRantagonistmethyllycaconitine(MLA).ThepossiblecytotoxiceffectswereevaluatedusingconcentrationsofISOfrom10-9to10-3,5Mandincubationtimesof6and24hoursonthelysosomalfunction(NRUassay),mitochondrialactivity(MTSassay)andtotalproteincontent(SRBassay)inHUVEC.Results:ThethreeISOinducedaconcentration-dependentincreaseonthe[Ca2+]iinHUVEC.ISO-1at10μMinducedthehighestincreaseonthe[Ca2+]i(6timeshigherthanat100nM;p<0,001).TheeffectofISO-1wassignificantlyinhibitedbyMLA(10nM;p<0,05)andin100%byMECA.Meanwhile,theeffectofISO-2andISO-3at10and100nMweren’tinhibitedbyMLA,howeverwerecompletelyinhibitedbyHEXA(100mM).Inrelationtothecytotoxicityevaluation,nostatisticallysignificanttoxiceffectswerefoundinanyofthebiomarkersinrelationtothenegativecontrolinHUVEC.Conclusions:TakentogethertheresultsindicatethatISO-1,ISO-2andISO-3increasethe[Ca2+]iinHUVECinaconcentration-dependentmanneranddoesn’texhibitcytotoxiceffectsinHUVEC.AccordingtotheresultsobtainedforISO-1,itwouldbehaveasα7nAChRagonist,andthereforeitwouldbeinterestingitsassessmentontheangiogenesisprocessinHUVEC.

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34)Increased intestinal permeability induced by diet modification: a novel animal model for pharmacological studies of the gut barrier

Eyzaguirre-Velásquez, Johana1.,Olavarría-Ramírez,Loreto1.,González-Arancibia,Camila1.,Escobar-Luna,Jorge1.,Díaz-Merino,Camila1.,Barrera-Bugueño,Camila1.,Bravo,Javier1.,Julio-Pieper,Marcela1.,1GrupodeNeuroGastroBioquímica,LaboratoriodeQuímicaBiológica.InstitutodeQuímica,FacultaddeCiencias,PontificiaUniversidadCatólicaDeValparaíso.(SponsoredbyFunding:PUCVDI037.470/2015,Fondecyt1130213)

Evaluatingdrugsdirectedtotheprotectionoftheintestinalbarrierrequiressuitableanimalmodels.Mostofthesemodelsinvolvestress,whichmaybeunfavorableifthedrugsunderstudyhavedifferentpharmacologicaltargets,beingoneofthemthecentralnervoussystem.

Theobjectiveofthisworkwastogenerateananimalmodelwithincreasedintestinalpermeability,bymeansofdietmodification.Adietlowinprotein(LP)where4%ofcaloriescomefromprotein,wasappliedto40daymaleSpragueDawleyrats.Controlani-malsreceivedadietwhere26%ofcaloriescomefromprotein.TheeffectofLPdietwasassessedat5,10or20days,afterwhichplasmasamples,colonandileumweretakentoanalyzeexvivo:1)thetissuepermeabilityto40and4.4kDafluorescentmacro-molecules,2)thetransepithelialelectricalresistance(TEER)and3)plasmacorticosteronelevelsasaphysiologicalmarkerofthestressresponse.

Inthecolonandileumofanimalstreatedfor5,10or20dayswithLPdiet,thetissuepermeabilitytomacromoleculeswasin-creasedcomparedtothatobservedinthecontrols,althoughitdidnotreachstatisticalsignificance.LPdietinducedasignificantdecreaseinTEERat10daysonlyincolonwhileat20daysadecreaseinTEERwasobservedinbothcolonandileum,suggestingageneralincreaseingutpermeabilitycomparedtocontrols.Finally,corticosteronelevelsofratsinalltreatmentgroupsshowednodifferencefromcontrols.

Theresultsshowthat20daysofLPdietresultinincreasedintestinalpermeabilitywithoutaffectingthephysiologicalresponsetostressintherat.Theanimalmodelproposedallowsforthestudyofdrugsatintestinallevel,independentofactionsinthecentralnervoussystem.

112

35) Cryopreservation induces alterations in the mitochondrial function of Atlantic salmon spermatozoa (Salmo salar).

Farias, Jorge1.,Figueroa,Elias1.,Valdebenito,Ivan2.,Risopatron,Jennie3.,Short,Stefania4.,Zepeda,Andrea1.,Figueroa,Carolina1.,1DepartamentodeIngenieríaQuímica,FacultaddeIngenieriayCiencias,UniversidaddeLaFrontera.2EscueladeAcuiculturaUniversidadCatolicadeTemuco.3BIOREN?CenterforBiotechnologyinReproductionUniversidaddeLaFrontera.4DepartamentodeIngenieríaQuímica,FacultaddeIngenieriayCiencia,UniversidaddeLaFrontera.(SponsoredbyFONDECYT1151315(F.JG);,FONDEFD10I1064(I.V).CONICYTDoctorateGrant(F.E;S.SE))

Todatetherearefewdataontheeffectofcryopreservationonthemitochondrialdynamicinfishspermatozoa.TheobjectiveofthisworkistoassesstheeffectofcryopreservationonthemitochondrialdynamicinAtlanticsalmonspermatozoa.ThespermwerefrozeninCortland®medium+1.3MDMSO+0.3Mglucose+2%BSAforthetreatment(T);freshsemenwasusedforthecontrol(C).Wedetermined[ATP]withtheCellTiter-Glo®kitand[O2]withtheMitoXpress®Xtrakit.Intheseanalysesweusedelectrontransportchaininhibitorsanduncouplers,namely:rotenone(R,10µM),antimycinA(A,10µM),cyanide(C,0.5µM)and2,4dinitrophenol(D,10µM).Inthecryopreservedspermatozoa(T),thebase[ATP]was5.7±1.2nmoles/109sppresentingsignificantdifferencesfromthecontrol(7.4±0.64nmoles/109sp,p<0.05);likewisethecellsincubatedwithR(2.9±0,78nmoles/109sp),A(3.98±0.92nmoles/109sp),C (1.37±0.66 nmoles/109sp) and D (1.59±0.48 nmoles/109sp) presented statistically significant differences during the first 10seconds of incubation as compared to the control (5.5±0.84 nmoles/109sp; 6.1±0.56 nmoles/109sp; 4.1±0.99 nmoles/109spand 4.9±0.79nmoles/109sp respectively, p<0.05). Thebase [O2] in control spermatozoawas 4230±520RFU/109sp, presentingsignificantdifferencesfromT(3040RFU/109sp);thetreatmentsincubatedwithR(3508±320RFU/109sp),A(3627±480RFU/109sp)andD(4290±429RFU/109sp)presentedsignificantdifferencesfromthecontrol(R:2704±298RFU/109sp;A:2852±570RFU/109spandD:3442±612RFU/109sprespectively,p<0.05).Thechangesinthe[O2]rateinspermatozoainthepresenceofinhibitorsanduncouplersoccurredafter10secondsofincubation.PreliminaryresultssuggestthatcryopreservationinducesalterationsinthemitochondrialfunctionofAtlanticsalmonspermatozoa.

113

36)Differential effect of NO-aspirin on susceptibility to fluconazole in C. glabrata and C. tropicalis obtained from denture stomatitis patients

Fernández, Roberto1.,Madariaga,Francisco1.,Urzúa,Blanca2.,Fernández-Ramires,Ricardo1.,Jara,José1.,Herreros,Eduardo1.,Molina-Berríos,Alfredo1.,1InstitutodeInvestigaciónenCienciasOdontológicas,LaboratoriodeFarmacologíayFarmacogenética,FacultaddeOdontología,UniversidadDeChile.2InstitutodeInvestigaciónenCienciasOdontológicas,LaboratoriodeBiologíayBioquímicaOral,FacultaddeOdontología,UniversidadDeChile.(SponsoredbyProjectFONDECYT11140227,U-inicia195.CandidaSppStrainsWereKindlyGivenByDr.XimenaLeeAndLeylaGomez,FacultadDeOdontología,UniversidadDeChile.)

Candida spp.biofilmshavebeenpointedasthemaincauseofdenturestomatitis,oneofthemostfrequentconditionsinelderlypeoplewearingdenture.AlthoughC. albicansisthemostfrequent,ithasbeendescribedanincreaseinprevalenceofC. glabrata and C. tropicalis inhumanoralflora,andtheirbiofilmsareassociatedwithhighresistancetoconventionalantifungaltreatment.BecauseofthehighresistanceofCandida spp. biofilmstoantifungals,newpharmacologicalstrategiestotreattheseinfectionsareneeded.Aspirin,anantiinflamatorydrugwithoutantimicrobialactivity,hasshowntoinhibitC. albicansbiofilmformation.Ontheotherhand,nitricoxide(NO)releasingmoleculeshaveshowntoinhibitC. albicansbiofilmsandtopotentiatetheeffectofconventionalantifungals.NOreleasingaspirin(NO-ASA)hasbeenproposedfortreatmentofdifferentcardiovascularaffectionsandinflammatoryconditions,sincemaintaintheanti-inflammatoryeffectsofaspirin,besidesthebeneficialeffectsofNOsuchasendothelialprotection.Accordingtothis,weevaluatedtheeffectofNO-ASAaloneandincombinationwithfluconazoleonC. glabrata and C. tropicalisclinicalisolatesobtainedfromdenturestomatitispatients.Methods: Candida spp. obtainedfromoralmucosaofdenturestomatitispatients (n=60),were identified throughCHROMagarCandidaMediumBDTM andbysequencingthe ITS1-5.8S rDNA-ITS2 region using the primers ITS1 and ITS4. To evaluate antifungal susceptibility, isolated strains werestandardizedto0.5McFarlandandthengrownonsabouraudagarplatesanddiskdiffusiontestswereperformedinpresenceorabsenceoffluconazole(SigmaAldrich,USA),NO-ASA(Sigma-Aldrich,USA)andtheircombination.Results: fromthe60strains27%correspondedtoC. tropicalis (n=16)andremaining18%wasidentifiedasC. glabrata(n=18).C. glabratastrainswereallclassifiedas resistant to fluconazole,with inhibition zone diameters , C. tropicalis strainswere all classified as fluconazole susceptible,withinhibitionzonediametersrangingfrom16mmto27mm.CombinationofNO-ASAwithfluconazoleproducedasignificantincreaseofatleast20%ofinhibitionzonediametersascomparedwithfluconazolealone(p<0,05,ANOVA)forC. tropicalisstrains.Conclusion: AlthoughNO-aspirinhasnoantifungaleffectbyitself,itcanpotentiatetheeffectoffluconazoleinC. tropicalisstrains.However,thiseffectappearstobedependentofthecharacteristicsofthestrainassayed.Wearecurrentlyworkingtoelucidatethepossiblemechanismsassociatedwiththeseeffects.

114

37) Synthetic coumarins able to inhibit α-glucosidase and exhibiting antioxidant activity

Figueroa, Catalina1.,Rivera,Constanza1.,Olea,Claudio2.,Delporte,Carla1.,1QuímicaFarmacológicayToxicológica,CienciasQuímicasyFarmacéuticas,UniversidadDeChile.2QuímicaInorgánicayAnalítica,CienciasQuímicasyFarmacéuticas,UniversidadDeChile.(SponsoredbyGrant,BecaDoctoradoNacionalCONICYTNº21141172AndFONDECYTN°1130155)

INTRODUCTION: The diabetes mellitus type 2 (T2DM) is a metabolic disease that is characterized by a chronicincrease of glycemia and oxidative imbalance leading to the appearance of pathophysiological complications.Theα-glucosidaseisanenzymethatcatalyzesthehydrolysisofdisaccharidestoabsorbablemonosaccharides,thusitsinhibitionsuppressestheinfluxofglucosefromtheintestinetothebloodvesselsandtherefore,isconsideredasanimportanttargetforhandling the hyperglycemia linked to T2DM.METHODOLOGY: From19 synthetic coumarins, a preliminary study of inhibitionagainstα-glucosidasewasperformed,toasingledose.Accordingtotheobtainedinhibitorycapacity,10compoundswereselectedtodeterminetheinhibitorypotency(calculationofhalfmaximal inhibitoryconcentration[IC50])andalsoantioxidantcapacity,determinedby theassayofoxygen radicalabsorbanceapacity (ORAC-FL). INHIBITIONOFα-GLUCOSIDASE:AbufferedsodiumphosphatesolutionatpH6.8,ρ-nitrophenyl-α-D-glucopyranoside(substrate)andtheenzymeto0.1U/mL,wasused.Absorbanceat400nmwasdeterminedbyamicroplatereader.Acarbosewasconsideredasstandardinhibitor(Lordanetal.,2013).ORAC-FL:Thefluorescenceemittedbyfluorescein(FL)wasreadevery1mininamicroplatereaderatanexcitationwavelengthof485/20nmandanemissionfilterof528/20nm.ThereactionwasperformedinphosphatebufferatpH7.0andwasadded2,2-azobis(2-methylpropionamidine) dihydrochloride (AAPH) as peroxyl radical source. The standard molecule was 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylicacid(Trolox).TheantioxidantcapacitywasquantifiedbyintegratingtheareaunderthecurveofFLdecay(Perezetal.,2013).RESULTSANDDISCUSSION:Fromtencoumarinsstudied,sixwereabletoinhibitα-glucosidase,allofthemshowingagreatinhibitorypotency,evenhigherthanthestandardinhibitoracarbose.Asregardstotheantioxidantcapacity,nocoumarinexcelled,givingORACindexeslowerthanthoseobtainedbyotherstudies.However,mostofcoumarinsORACindexespresentedwerehigherthantroloxindex.Byanalyzingbothassays,coumarin18highlights,becauseithasanIC50of5.96g/mLagainstα-glucosidaseand2.06ORAC.CONCLUSION:Severalcoumarinsthatareabletoinhibitα-glucosidaseexhibitedantioxidantcapacitydeterminedbyORAC-FL.This isusefulsinceitcouldreducethehyperglycemiaassociatedwithDMT2andsecondly, itcouldreduceoxidativestressthatacceleratestheprogressofthedisease.Inthisregard,coumarin18couldbeconsideredasamoleculewithpotentialcapacitytotreatT2DM.Lordanetal.(2013).Theα-amylaseandα-glucosidaseinhibitoryeffectsofIrishseaweedextracts.FoodChemistry.

115

38)Protective effect of ascorbic acid on recombinant Pichia pastoris.

Figueroa, Carolina1,2.,Zepeda,Andrea1,2.,Figueroa,Elías1.,Abdalla,Dulcineia3.,Pessoa,Adalberto2.,Farías,Jorge1.,1DepartmentofChemicalEngineering,FacultyofEngineeringandSciences,UniversityofLaFrontera.2DepartamentodeTecnologiaBioquímico-Farmacêutica,FaculdadedeCiênciasFarmacêuticas,UniversidadedeSaoPaulo.3DepartamentodeAnálisesClínicaseToxicológicas,FaculdadedeCiênciasFarmacêuticas,UniversidadedeSaoPaulo.(SponsoredbyPhDScholarshipN°21110913,CONICYT.CoordinationOfSuperiorLevelStaffImprovement(CAPES,Brazil),NationalCouncilForScientificAndTechnologicalDevelopment(CNPq,Brazil)AndSãoPauloResearchFoundation(FAPESP,Brazil).)

Pichia pastorisisamethylotrophicmicroorganismusedasanexpressionsystemfortheproductionofheterologousproteins.Itusesamethanol inducible-promoter,alcoholoxidase.Methanoloxidationhasbeenrelatedwithreactiveoxygenspecies(ROS)generationinyeastperoxisomesandthemainROSgeneratedduringmethanoloxidationishydrogenperoxide(H2O2).OtherwisetheincreasedculturetemperaturecouldalsogeneratehigherlevelsofROS.Themainaimofthisworkwastoexposetherecombinant Pichia pastoriscellstoascorbicacidandtoevaluatetheprotectiveeffectonROSgeneration.TheyeastcultureconditionswereControl:30°C-1%(v/v)methanolandAA:30°C-1%(v/v)methanoland6.7mMascorbicacid,bothcultureswereincubatedat250rpmfor72hoursinshakingincubator.IntracellularH2O2wasevaluatedusing2´,7´dichlorofluoresceindiacetate(DCFH-DA),antioxidantproteinsexpressionwereevaluatedusingindirectimmunofluorescenceandcellviabilityusingpropidiumiodidebyflowcytometry. IntracellularH2O2incontrolgroupat72hwashigherthan0h.At theendof incubationan increasedproteinexpressionofcatalaseandglutathioneperoxidasewasobservedinbothcultures.However,thesuperoxidedismutaseexpressiondidnotchangeinanyculture.Meanwhileat72hinthecontrolculturewasobserveddecreasedcellviabilitycomparedwith0h.InconclusiontheseresultssuggestthatascorbicacidsupplementationduringinductionphaseofrecombinantPichia pastoriscouldprotectcellsanditcouldpreventoxidativestress-inducedcellularsenescence.

116

39)Ulva Compressa, a chlorophyte algae releases extracellular ATP and metabolizes the nucleotide through multiple ATPases

Garcia-Huidobro, J Pablo1.,Donoso,MVerónica1.,Gómez,Melissa1.,Moenne,Alejandra1.,1DepartamentodeBiología,FacultaddeQuímicayBiología,UniversidaddeSantiagodeChile.(SponsoredbyFundedCEDENNA,FondecytGrants1141132,1130118)

RecentreportsindicatethatATPinducecoalescenceinsporesofredalgae,suggestingaroleofnucleotidesascellmessengersinthesecells,whichappearedalmost1.5billionyearsago.ATPhasbeenrecognizedasanearlysignalingmoleculepresentfromunicellulartomulticellularorganisms.ItfollowsthatifATPisrelevantasasignalingmolecule,theremustbeamechanism(s)toturnoffthesignal.Basedonthispremise,wehypothesizedthepresenceofectoATPasesintheseorganisms.Totestwhetherpurinesareextracellularsignalsingreenalgae,weassessedwhetherfreshculturesofUlva compressa(UC),acommonchlorophyte(greenalgaeofthecentralChileancoast)releaseandmetabolizeextracellularATP.WeassessedATP/metabolitesinUCcultures(1gofUCin30mlseawater).MediasamplesfromcontrolsorUCexposedtoextracellularATP/metaboliteswerechemicallyreactedwithchloroacetaldehyde toobtain thecorrespondingetheno-fluorescentderivatives.ThepurineswereseparatedbyHPLCcoupledtofluorometricdetection.StirringUCreleasedtothecellmedia1.3±0.6nMATP(n=4),avaluethatwasreducedbyadditionofexogenousapyraseto0.01±0.01nM(n=4,p<0.05).Samplesincubatedwith3mMorthovanadate(inhibitorofectoATPases)tripledseawaterATPto4.1±0.4nM(n=3,p<0.05).Applicationof1µMexogenousATPtoUCcultureswasrapidlymetabolized;30and60-minafterexogenousATPaddition,theATPcontentinthecellmediawasreducedto80.8±2.9and96.8±0.9%,respectively,(n=7).Likewise,exogenousapplicationsof1µMADP,or1µMAMPor1µMadenosineshowedasimilardecay,anindicationofmultipleectoenzymaticactivity.Wefurtherdemonstratenotonlythedecreaseoftheenzymesubstarte,buttheaccumulationofenzymeproducts,particularlyafter1-10mMorthovanadateaddition,afindingconsistentwithmultipleectoATPaseactivity.ToexaminewhetherectoATPaseissoluble,theUCsupernatantwasstirredfor30-mininseawater,thesupernatantwasfilteredfreeofalgaeandexaminedforATPaseactivity.ResultsconsistentlyshowedthatATPwasmetabolizedbysupernantenzymes;thisenzymeactivitywasinhibitedby3mMorthovanadate.Inconclusion,stirringUCresultsinextracellularATPrelease;thesignalisrapidlymetabolizedbymultiplesolubleecto-ATPases,suggestinganextracellularroleofATPinthisprimitiveorganism.Red,andbrownalgaewillbeexaminedtoassayforATPsignalingmechanism(s).

117

40)A comparative study of the in vitro antioxidant capacity of ethanolic extracts from leaves of different Ugni molinae genotypes.

Pérez-Arancibia, Rodrigo1.,Valenzuela-Bustamante,Paula1.,Peña-Cerda,Marcelo1.,Seguel,Ivette2.,Delporte,Carla1.,1DepartamentodeFarmacologíayToxicología,FacultaddeCienciasQuímicasyFarmacéuticas,UniversidadDeChile.2CentroRegionaldeInvestigaciónCarillancaInstitutodeInvestigacionesAgropecuarias(INIA).(SponsoredbyFONDECYTN°1130155,BecaCONICYTN°21120377,INIACarillanca,Chile.)

Murtilla (Ugni molinae Turcz,Myrtaceae) is awild shrub native from south-central Chile that contains different polyphenoliccompoundsonitsleaves,includingseveralflavonoidslikemirycetinandquercetinglycosylatedderivatives.Thepolarextractsofmurtilla’sleavesareasignificantantioxidantsourcewhichcouldpreventoxidativestress,generatedwhenthebalancebetweenfreeradicalsandtheendogenousantioxidantsystemsislost.Oxidativestressisimplicatedinthedevelopmentofdifferentpathologies,contributing to cellular aging, mutagenesis, carcinogenesis, and coronary heart disease through membrane disruption, DNAdamageand lowdensity lipoproteinoxidation.Duetothementionedaboveandbecauseofthe influenceofthegenotypeonthe chemical composition, theaimof thisworkwas to comparativelyevaluate theantioxidant capacityof10 serial ethanolicextracts(EET)frommurtillaleaves,cultivatedunderthesameconditionsbytheInstitutodeInvestigacionesAgropecuarias(INIA)inCarillanca,Temuco,assessingitsDPPHandperoxyl(ORAC-FL)radicalscavengingcapacity.ResultsshowedthattheZF-18genotypehadthelowestEC50ontheDPPHassay(9.3±0.6mgdryEET/L),andthe19-1genotypehadthehighestORACvalue(23.8±1.7μmolTroloxequivalents/mgdryEET).Furthermore,totalphenolicsweredeterminedbytheFolin-Ciocalteumethod,expressedasgallicacidequivalents(EAG),beingtheZF-18genotypetheonethatexhibitedthehighestphenoliccontent(260.6±3.7mgEAG/gdryEET).Finally,quercetin,myricitrin,andgallicacidwereidentifiedbyHPLC-MSandquantifiedbyHPLC-DADineverysample.TheseresultssuggestthatmurtillaleavesareagoodsourceofantioxidantsandthatthedifferentantioxidantcapacitiesoftheirEETs,mediatedbyfreeradicalscavenging,areinfluencedbythedifferencesonpolyphenolconcentrationduetothegenotype.

118

41) Chronic Ketamine treatment during adolescence induces long-term impairment of prefrontal cortex in adult rats.

MORALES, CAMILA1,2,3.,PÉREZ,MIGUEL1,2,3.,ARRIAGADA,JORGE1,2,3.,FUENZALIDA,MARCO3,1,2.,1NeuralPlasticityLabUniversidadDeValparaíso.2CentrodeNeurobiologíayPlasticidadCerebral,InstitutodeFisiologíaUniversidadDeValparaíso.3MillenniumNucleusinNeuropsychiatricDisordersNU-MINDUniversidadDeValparaíso.(SponsoredbyThisWorkWasSupportedByGrantsFromMillenniumNucleusNU-MINDNC-130011(M.F),FONDECYT1130614(M.F.)MasterFellowshipCONICYT20877(CMM).)

Ketamine(Ket),anNMDA-receptorantagonist,hasbeenwidelyusedasananimalmodelofschizophrenia(SZ),dueitscapabilitytoinducemolecular,cellular,synapticandbehavioralSZ-likeimpairment.However,thereisonlyfewevidencerelatedtoinhibitorysynapticdysfunctionandeven lessabout theeffectof systemicKet-treatmentover specificbrain structures.UsingBehavioraltasksandelectrophysiologicalrecordingswestudiedthecognitiveperformanceandGABAergicsynaptictransmissionoftwobrainstructuresinvolvedinSZ,thehippocampus(HPC)andprefrontalcortex(PFC)ofadultrats,whichwerechronicallytreated(i.p.)withKet(30mg/kg)orVehicle(Veh,NaCl0.9%)duringadolescence.First,weevaluatedtheexecutivefunctionthroughworkingmemory(WM)assays,whichdifferentiallyassessedHPCandPFC.WeappliedthespontaneousalternationtaskinaY-mazewithvisualcluestotestHPCfunction,foundingnodifferencesbetweengroups.Furthermore,toevaluatePFCdependentWM,weusedthedelayednon-matchtosampletask,whereKet-treatedratsdisplayedaworseperformancecomparedtoVeh. Interestingly,ourbehavioral resultswereaccordingtothe intracellularrecordingsofpyramidalneurons frombothstructures.Wefoundnodifferences in theevokedandspontaneous inhibitorypost-synapticcurrents (eIPSCandsIPSC, respectively)betweenKet-andVeh-HPCslices,whereasinPFCtheeIPSCandsIPSCwerechanged.Theanalysisofpairedpulseratio(PPR)allowedustorecognizetwogroupsofsynapsesinKet-PFCslices;agroupinwhichthePPRwasreducedcomparedtoVeh,andanothergroupthatshowedanincreaseinthePPR.Additionally,weobservedthat,unliketomIPSC,thefrequencyofsIPSCinKet-PFCsliceswasdiminished.Nevertheless,theamplitudeofboth,sIPSCandmIPSCwasincreased.OurresultsindicatethatchronicKet-treatmentinadolescentrats isable to induce specificPFC impairmentand suggest that change inPFC inhibitory transmissiondisrupts theexcitatory/inhibitorybalance,triggeringthecognitivedysfunction.

119

42)A failure in ascorbic acid recycling and release from striatal astrocytes is responsible for the metabolic impairment in Huntington’s disease.

Beltran, Felipe1.,Troncoso-Escudero,Paulina1.,Valverde-Porras,Naizmi2.,Rojas,Patricio3.,Li,Xiao-Jiang4.,Li,ShiHua4.,Castro,Maite1.,1InstitutodeBioquímicayMicrobiología,FacultaddeCiencias,UniversidadAustralDeChile.2BiotecnologíaTEC.3DepartamentodeBiologíaUniversidadDeSantiagoDeChile.4DepartmentofHumanGeneticsEMORYUniversity.

Introduction: Huntington’sdisease(HD)isaneurodegenerativedisordercharacterizedbyanimpairmentinglucosemetabolisminthebasalgangliaandinthecerebralcortexofsymptomaticHDpatients.WhenHDanimalmodelsbecomebehaviorallyactive,thelevelsofascorbicacidinstriatalextracellularfluidisabnormallylowinrelationwiththelevelsfoundintheirlittermatecontrols.Wehavedemonstratedthatintracellularascorbicacidinhibitsglucosetransportandstimulateslactatetransportinsynapticallyactiveneurons.Inthiswork,westudiedtheabilityofascorbicacidtomodulateneuronalglucoseconsumptioninYAC128mice.Additionally,weevaluatedtheabilityofstriatalastrocytesofYAC128micetorecycleandreleaseascorbicacid.Finally,toevaluatethecontributionofastrocytestotheimpairmentinascorbicacidmetabolicmodulation,weusedtransgenicmicethatexpressN-terminalmutanthuntingtinonlyinastrocytes.Materials and Methods: Synapticactivitywasmeasuredasrecordingsfromstriatalmediumspinyneurons.Excitatorypostsynapticpotentials(EPSPs)wereevokedbystimulatingthecortico-striatalpathway.UsingqPCRandWesternblotanalyses,weexploredproteinandmRNAlevelsofproteinsinvolvedinascorbicacidrecyclinginastrocytes.AscorbicacidreleaseandrecyclingwasevaluatedbyHPLC.Results: AscorbicacidwasnotabletomodulatetheabilityofglucosetofunctionasanenergeticfuelsustainingglutamatergicsynapticactivityinpresymptomaticYAC128mice.WeobservedafailureinascorbicacidrecyclingandreleasefromstriatalastrocytesfromYAC128mice.Finally,similarresultswereobserved inmicethatexpressmutanthuntingtinonlyastrocytes.Discussion:Abnormalitiesobservedintheascorbicacid-dependentmodulationofneuronalmetabolismofpresymptomaticHDmice,suggeststhatascorbicacidhomeostasisfailurecouldbeimportantintheprogressionofHD.OurfindingsdemonstratethatastrocytemutanthuntingtincancontributetotheneuronalmetabolicfailureandglialfunctioncouldbeaneffectiveroutetotherapiesforHD.FONDECYT1151206;FONDECYT1110571;MECESUPAUS1204;BecaCONICYTDoctorado;GastosOperacionalesCONICYT;DID-UAChandCISNE-UACh.

120

43)Iron Regulatory Protein 1 (IRP1) dysregulation mediates neuroblastoma cell death induced by mitochondrial complex I inhibition

Aguirre, Pabla1,2.,Urrutia,Pamela1,2.,Mena,Natalia1.,Tapia,Victoria1,2.,Esparza,Andrés1.,Núñez,Marco1,2.,1Biología,FacultaddeCiencias,UniversidaddeChile.2Biología,FacultaddeCiencias,ResearchRingonOxidativeStressintheNervousSystem.

InhibitionofmitochondrialcomplexIresultsindecreasediron-sulfurcluster(ISC)synthesis,whichmayleadtoactivationofIRP1, a key regulator of cellular ironhomeostasis. This processmaybe relevant for theunderstanding of Parkinson’s disease(PD)neuropathology,wheremitochondrialdysfunction,ironaccumulationandoxidativestressarepathognomonicsigns.Here,wereporttheeffectsofmitochondrialdysfunctiononIRP1activityand ironhomeostasis,and itsrole inthedeathofSH-SY5Ydopaminergicneuroblastomacells.MitochondrialcomplexIwasinhibitedwithrotenoneandirondyshomeostasiswasevaluatedbychangesinTransferrinReceptor1(TfR1),DivalentMetalTransporter1(DMT1),Ferroportin1(FPN1),ferritin,55Feuptakeandoxidativemodificationofproteins.ResistancetoapoptoticcelldeathinducedbyoxidativeinsultsandComplexI inhibitionwasevaluatedinIRP1knockdowncelllines.ComplexIinhibitionassociatedwithincreasedIRP1IREbindingactivity,increasedlevelsofTfR1andDMT1,anddecreasedlevelsofFPN1,togetherwithincreased55Feuptakeactivity.SilencingofIRP1abolishedtherotenone-inducedincreaseinironuptakeactivityandprotectedcellsfromdeathinducedbycomplexIinhibition.IRP1knockdowncellsdemonstrated increased resistance tocysteineoxidationanddecreased lossof cell viability inducedbyoxidative stimuli.These results support the idea that IRP1 isanoxidativestressbiosensor thatwhenderegulatedbymitochondrialdysfunctionmediates ironaccumulationandcelldeath. IRP1activation,secondaryto inhibitionof ISCsynthesis,mayunderlinetheeventsleadingtotheaccumulationofironobservedinPD.

FinancedbyPIAgrantACT1114andFONDECYTgrant1130068.

121

44)CHANGES ON PURINERGIC RECEPTOR P2X2 AND PROTEIN FE65 EXPRESSION, AND ITS EFFECT ON MITOCHONDRIAL FUNCTIONS

Barra, Karen1.,Silva-Grecchi,Tiare2.,Godoy,Pamela1.,Celis,Teresa1.,Panes,Yessica1.,Fuentealba,Jorge2.,1Fisiología,CienciasBiológicas,UniversidadDeConcepción.2FisiologíaUniversidadDeConcepción.(SponsoredbyThisWorkHasBeenFundedByThe1130747FONDECYTProject)

Alzheimer’sDisease(AD)isaneurodegenerativedisorderandtherearedifferenthypothesisforexplainitspathogenicmechanism.Theamyloidogenictheoryhasbeendeeplystudied,andplacetheAmyloid-βpeptide(Aβ)asoneofmostimportantresponsibleofAD,thatisgeneratedfromtheproteoliticprocessingoftheAmyloidprecursorprotein(APP);whichalsogeneratestheAmyloidintracellulardomain(AICD).TheAβpeptidehasmanytoxiceffectssuchasthealterationoffunctionandexpressionofdifferentproteinsanddishomeostasisofintracellularCa+2,whichaltersthemitochondrialactivityandfinallyproducestheneuronaldeath.Additionally,changesonthepurinergicreceptorsP2X(P2XR)andFe65(amultidomainadaptorprotein)levelshavebeenreportedbyusandothers. Inourgroup,wehaveobservedthattreatmentswithAβincreasetheP2X2expression,whichcouldsuggestahigher interactionwithFe65 thatcouldaffect the functionoravailabilityof thisprotein.On theother side, thePeroxisomeproliferator-activated receptor-γ coactivator 1α (PGC-1α) expression, key molecule in the cellular energy metabolism andmitochondrialbiogenesis,isregulatedbydifferentsignalsastheinteractionbetweenFe65andAICD,sincetheseproteinsformacomplexwhichactivatesthiscofactorexpression.Meanwhile,PGC-1αinteractswithvariedfactors,whichallowthetranscriptionofseveralgenesthatparticipatesinthemitochondrialbiogenesis.

TheaimofthisworkwastoevaluatethechangesonP2X2RandFe65levelsinmousehippocampalslices,afteracuteandsubchronictreatments(1and5hoursrespectively)withAβpeptide.Usingimmunohistochemistrytechniqueswehaveobservedchangesinimmunoreactivityanddistributionoftheseproteins,shownvariationsdependingonthezonethatwasevaluated(dentategyrus,CA1andCA3hippocampalzones)andthesechangesshowntobedependentoftreatmenttime.Besides,westudiedchangesonPGC-1αandobservedadecreaseonitsimmunoreactivity.TheseresultssuggestthatahigherinteractionbetweenP2X2RandFe65couldbeplayingakeyroleintheexpressionofthecoactivatorPGC-1α;furthermore,theycouldparticipateinthepotentiationofAβtoxicityinducingdeepalterationsonmitochondrialmetabolismandbiogenesis.

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45)Characterization of neuroprotective peptidomimetics based on the C terminal region of the β amyloid peptide (Aβ)

Bascuñán, D1.,Peters,Christian1.,Perez,Nelson1.,Burgos,Felipe1.,Aguayo,Luis1.,1DepartamentodeFisiología,CienciasBiológicas,UniversidadDeConcepción.(SponsoredbyWorkSupportedByFondecytGrant1140473.)

Alzheimer´sdisease(AD)isaneurodegenerativedisorderofhighprevalencethatmainlyaffectselderlyindividuals.Theprincipalneurotoxicagentcorrespondstooligomersoftheβamyloidpeptide(Aβ)thatassociatetotheneuronalmembraneandform“pore-like”structures.Thesemembraneperforationsaltercalciumhomeostasisandultimatelyleadtosynapticfailure.Currenttherapiesarenoteffectiveatcuringordeterringdiseaseprogression,thusfurtherresearchisneededtofindmoreeffectivetherapeutictargetstorevertAD.WerecentlydemonstratedtheimportanceoftheCterminalregionofAβintheassociationandperforationoftheneuronalmembrane.Specifically,weblockedAβaggregation,association,membraneinsertion,andsynaptotoxicitywiththepentapeptideG33LMVG37derived fromtheAβpeptide.Wearecurrentlyexamininga libraryof syntheticmoleculesas thebasisfor in silicoscreeninginordertofindsmallpeptidomimeticmoleculesthathavesimilarneuroprotectiveeffects,butwithhigherpharmacologicalpotential.AgroupofmoleculeshavingahigherinteractionwithAbwereselectedandanalyzedfortheireffectsonAbaggregation,association,mitochondrialfunctionandmembraneperforationinPC12cellsand/orrathippocampalneurons. The results showed that a benzopirolic derivative atmicromolar concentrationswas able to inhibit Ab aggregation(70±5%ofcontrol)andsubsequentmembraneassociation(55±3%ofcontrol)withoutproducinganintrinsictoxiceffect(97±3%ofcontrol).ThesepreliminaryresultsindicatethatitispossibletodeveloppeptidomimeticmoleculesthatexertprotectiveactionsagainstAbsimilartothethoseobservedwiththeG33LMVG37pentapeptide.

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46)Ventilatory arrest is the primary event that leads to sudden death after heat-induced seizures in a Dravet mouse model.

Bravo, E1.,YuJaung,Kim1.,Richerson,George1.,1NeurologyUniversityofIowa.

InpatientswithDravetSyndrome(DS)andintractableepilepsy,SuddenUnexpectedDeathinEpilepsy(SUDEP)isthemostcommoncauseofdeath.Comparedtothegeneralpopulation,DSpatientsareupto40timesmorelikelytodiefromprematuredeath.Several typesofpostictal arrhythmiahavebeenassociatedwith seizures, leading some investigators to conclude that cardiacarrestistheprincipalcauseofSUDEP.TherecentMORTalityinEpilepsyMonitoringUnitStudy(MORTEMUS)reportedthelargestseriesofSUDEPcasesinepilepsymonitoringunits(EMUs),andincludedvideo,EEG,andEKG.Respiratoryactivitywasassessedindirectlybyobservingthevideorecording,butventilationwasnotmeasureddirectly.InamousemodelofDS(Kalumeetal,JClinInvest,2013),heatinducedseizuresarefollowedbyprogressivebradycardiaanddeath,whichhasbeenproposedtoreproducetheeventsthatoccurinhumanDSpatientswithSUDEP.However,postictalbreathinghasnotbeenmeasuredinthesemice.HerewestudiedamousemodelofDStodeterminetherelationshipbetweencardiacactivity,respiratoryoutputanddeathafterheat-inducedseizures.

AmouseEMUwasusedtocontinuously recordEEG,EMG,EKG,whole-bodyplethysmography (breathing),bodytemperature,roomtemperature,humidityandvideo.Heterozygousmicewithaknockinmutation(R1407X)ofSCN1A(Scn1aR1407X/+)andwild-type(WT)littermatesonaC3Hbackgroundwerebredandgenotypedaspreviouslydescribed(Auerbachetal,PLoSONE,2013).WTandScn1aR1407X/+micewereexposedtoaheatlamptocauseacontinuousincreaseinbodytemperaturefrom37°Cto43°C.Forthosemicethatdidnotdieonthefirsttrial,asecondtrialwasperformedtwodayslater.

Inresponsetoanincreaseinbodytemperatureto43°C,100%ofScn1aR1407X/+micehadatleastoneseizureonthefirsttrial,with84%ofthesemicehavingatleastoneconvulsiveseizure.78%ofScn1aR1407X/+micedied,andthisalwaysoccurredafteraconvulsiveseizure. Twodays later,asecondtrial induceddeath in100%oftheremaining Scn1aR1407X/+mice. Incontrast,noneoftheWTmicehadanyseizuresand100%survivedthetwoheatingtrials.InScn1aR1407X/+mice,whendeathoccurredthefirstabnormalitywasalwayscompletecessationofallrespiratoryeffortthatdidnotreturn.Heartrateremainednormalfor20seconds,andthensubsequentlybegantodecreaseprogressivelyoverthenext3minutesuntilasystoleoccurred.

TheseresultsindicatethatpostictaldeathinScn1aR1407X/+miceisduetorespiratoryarrest,andthatthesubsequentbradycardiaislikelysecondarytoanincreaseinparasympatheticoutputduetohypoxia.

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47)The transcription factor Nuclear receptor related 1 (Nurr1) is down-regulated by iron and mitochondrial complex I inhibition.

Carrasco, Carlos M.2,1.,Aguirre,Pabla2,1.,Gonzalez-Billault,Christian1,2.,Nuñez,Marco1,2.,1Santiago,ChileResearchRingonOxidativeStressintheNervousSystem.2Biology,Science,UniversidadDeChile.

Parkinson´sdisease(PD)ischaracterizedbythelossofmotorcontrolasaconsequenceofdenervationinthestriatumofdopaminergicneuronsofthesubstantia nigra pars compacta.HallmarkofPDthatleadstoneurodegenerationareinflammation,elevatedironlevelsandoxidativestress.RecentdatafromourlaboratoryshowthattreatmentswitheitherironchelatorsorantioxidantsprotectdopaminergicneuronsfromMPTP-induceddegeneration,bothinvitroandinvivo,suggestingthatreactiveoxygenspecies(ROS)resultingfromironaccumulationarecriticalinthisdegenerativeprocess.Nurr1,atranscriptionfactorinvolvedindevelopmentandmaintenanceofmidbraindopaminergicneurons,decrease itsactivityunderoxidativeconditions. Inthiswork,westudiedtheinterplaybetweenironandinhibitionofmitochondrialactivity(1-methyl-4-phenylpyridinium(MPP+))indeterminingNurr1transcriptionalactivity.WeusedmesencephaliccellsincultureandSH-SY5Ycells.Wetestedtheeffectofiron,MPP+,rotenoneandironchelatorsoni)dopaminergicneurondeath,ii)thenucleus/cytoplasmdistributionofNurr1andiii)Nurr1transcriptionalactivity.Inaddition,weanalyzedthemRNAexpressionofproto-oncogeneRET,atranscriptionaltargetofNurr1,upontreatmentwithMPP+oriron,intheabsenceorpresenceofironchelatorsM30,DFO,orthenovelchelatorQ11.Wefoundthatco-treatmentwithchelatorsprotecteddopaminergicneuronsfromMPP+-induceddeath.TreatmentswithironorMPP+resultedindecreasedlevelsofNurr1inthenucleianddecreasedRETexpression.Furthermore,ironchelation,bothasapre-orasapost-treatment,revertedRETexpressiontocontrollevels.WeconcludethatintheMPP+PDmodel,ironaccumulationiscrucialforMPP+-induceddeath,andthatNurr1exclusionfromnucleicouldbeadownstreamconsequenceofironaccumulation.

FinancedbyPIAgrantACT1114,FONDECYTgrant1130068andCONICYTFellowship21130574.

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48)Amyloid-β peptide induces an increase in P2X2 receptor levels and changes mitochondrial dynamic-related protein expression.”

Celis, Teresa1.,Barra,Karen1.,Godoy,Pamela1.,Panes,Jessica1.,Fuentealba,Jorge1.,1Physiology,BiologicalSciences,UniversidadDeConcepción.(SponsoredbyThisWorkHasBeenFundedByFONDECYTProject1130747)

Mitochondrialdysfunctionhasbeenassociatedwithneurodegenerativedisorders,suchasAlzheimer’sdisease(AD).Thishighlydynamicorganellecontrolsseveralcellularprocessesthatinvolveenergeticsupply,metabolicpathwayregulationandintracellularcalciumsignaling,entailinggrandrepercussionsonneurons.Allofthesefeaturesrelyonfissionandfusionforcesthatcontrolshape,size,distributionandmaintenanceofthisorganelle.Fissionandfusionbalance,ismediatedbyspecificGTPases,likeDrp1(dynamin-relatedprotein)whichcontrolsfissionevents,inducingmitochondrialfragmentationviaoutermembraneconstriction.DisturbedmitochondrialdynamicshasbeenobservedonADbrains,where increased intracellularconcentrationofAmyloidβpeptide(Aβ)appearstoberesponsibleoffissionandfusionimbalance.Ontheotherhand,amyloidintracellulardomain(AICD)generatedfromamyloidprecursorprotein(APP)proteolyticprocessing(alongwithAβ)displaysanucleustranslocationalongwithFe65,amultidomainadaptorprotein,enhancingPeroxisomeproliferator-activatedreceptor-γcoactivator1α(PGC-1α)expression,arenownedmitochondrialtranscriptionalcoactivator.Fe65interactswiththepurinergicreceptorP2X2whoseexpressionafterAβtreatmentsincreasesaccordingtopreviousresultsfromourwork;nonetheless,Fe65expressionremainsstablebutdecreasesitspresenceinthenucleus.TheaimofthisworkwastostudymitochondrialdynamicsrelatedtoAβtoxicityandP2X2Roverexpression,throughPGC1-αandDrp1expressionquantificationonPC12cellsemployingwesternblotandimmunofluorescencetechniques.ImmunoreactivityforPGC1-αshoweddecreasedexpressionlevelsafterchronicaltreatmentswithAβ(0,5µM),PPADS(10µM)-aP2Xreceptorantagonist-andPPADS+Aβ(C:100±4%;Aβ:57±7%;PPADS+Aβ:47±5%).Additionally,weobservedasignificantdecreaseonDrp1expressionafterP2X2RtransfectionandAβtreatments;furthermore,simultaneousconditionsrevealedadditiveeffects(C:100%;Aβ:76±9%;P2X2:85±5%;Aβ+P2X2:46±11%).Takentogether,theseresultssuggestthatenhancedP2X2Rexpression could be sequestrating Fe65 protein, reducing its availability to interact with AICD along with their capability tomodulatePGC1-αexpression levels,entailingmitochondrial consequences, suchasmitochondrialdynamicsperturbationonacalcium-independentmechanism.Thisobservationrepresentsapotentialtargetfornewbiomarkerdevelopment.

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49)Study of locomotor activity in Octodon degus: a potential natural model for neurodegeneration Parkinson type

Gajardo, Ivana1.,Palacios,Adrián1.,1CentroInterdisciplinariodeNeurocienciadeValparaíso,FacultaddeCiencias,UniversidadDeValparaíso.(SponsoredbyMillenniumInstituteICM-P09-022-F)

WehaverecentlyshownthatOctodon degusdevelopsduringagingthemainsignsofaneurodegenerationAlzheimerdiseasetype(deguAD),includingtheaccumulationofAβ(amyloid)solubleandphosphorylatedTauprotein;decreasedofpostsynapticproteins,aswellreducedspatialmemoryandobjectrecognition,andadecreaseinlocomotorsactivity.Parkinson’sdisease(PD)representsaprogressiveneurologicaldisorderwith lossofdopaminergicneurons (substantianigra), leading toa reduction indopamine,whichisrelatedtotheonsetofmotorrigidity,tremor,slownessordecreasedmovement.WehavepreviouslyshownindeguadecreaseoflocomotorexplorationduringagingandpromptustoaskifdeguwouldbeapotentialanimalmodeltostudyPD.Inalargegroupofdeguswefirstcharacterizedthelocomotoractivitythroughtheopenfieldtest(OF).Ourpreliminaryresultsshow,independentofages,thatindividualdegusshowhighandlowerlocomotoractivity.Fromthis,weevaluatedtheeffectoflevodopaadministrationonlocomotoractivity,intwoexperiments,todifferentconcentrationsofthedrugandtodifferenttime’spost-injectionlevodopathroughofthetestOF.Resultsofbothexperimentsshowthatoldanimalswithlowlocomotoractivityhaveapositiveresponsetothedrugincreasingitsactivity,unliketheotheranimals.Basedonourresultswecanpredictthatthedecreaseoflocomotoractivityofsomeoldanimalsmaybeduetoadeficitofthenigrostriataldopaminergicpathway.

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50)Single unit activity in the dorsolateral striatum of amphetamine treated rats: preliminary results

Gatica, R.I.1.,Aguilar-Rivera,M.I.2.,Fuentealba,J.A.1.,1LaboratoriodeNeuroquímica,DepartamentodeFarmacia,FacultaddeQuímica,PontificiaUniversidadCatólicaDeChile.2DepartmentofBioengineeringUniversityofCalifornia,SanDiego.(SponsoredbySponsoredByFONDECYTN°1141088)

Drug addiction is a chronic disease associatedwith physiological, cognitive and behavioral impairments. The transition fromvoluntarydrugusetoacompulsivehabitbyrepetitivedrugintakecharacterizesthedevelopedofthispathology.Thedorsolateralstriatum(DLS)hasakeyroleinthehabitslearning.IthasbeenreportedthattheincreaseofdopaminelevelsintheDLS,afterpsychostimulantsadministration,iscorrelatedwiththeestablishmentofhabitualdrugseeking.However,datawithregardchangesintheelectrophysiologicalpropertiesofDLSneurons,afterpsychostimulantadministration,arestilllacking.Inthiswork,westudiedtheeffectofrepeatedamphetamine(AMPH)administrationonthefiringrateofDLSsingleunits.RatsreceivedrepeatedinjectionsofAMPH(1.0mg/kg)beforethesingleunitsrecordingexperiment.AnarrayofeighttetrodeswasloweredtotheDLSinurethane-anesthetizedrats.Neuralactivitywasrecordedusingthefollowingprotocol:20minofbasalactivity,20minaftersalineinjectionand40minafteranacuteinjectionofAMPH(1.0mg/kg).Preliminaryresultshowsanon-significantdecreaseofbasalfiringrateinAMPHgrouprelativetocontrolgroup(0.088+0.04486HzAMPHgroupv/s0.479+0.195Hzcontrolgroup).WeobservedatrendintheDLSneuronsofAMPHtreatedratstoincreasetheirfiringrateinresponsetoanacutedoseofAMPHincomparisontocontrolrats(33%v/s17%respectively).ThesepreliminarydatasuggestthatanacuteexposureofAMPHwouldchangeDLSneuralactivity.

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51)Use of deep transcranial magnetic stimulation (deep TMS) as add-on treatment for Parkinson’s disease.

Linsambarth, Sergio1.,Villalon,Esteban2.,Moraga-Amaro,Rodrigo1.,Zangen,Abraham3.,Poblete,Patricio3.,Stehberg,Jimmy1.,1LaboratorioNeurobiologiaUniversidadAndrésBello.2NeuroclinicNeuroMagnetics.3NeuroscienceLaboratoryUniversityoftheNegev.

ThestandardpharmacologicaltreatmentforParkinson’sdisease(PD)istheadministrationoflevodopa,whichisveryeffective,butrequiresprogressiveincrementsinthedosageleadingtoconcomitantincreasesinadverseeffects.ThedevelopmentofnoveltherapiesthatcanimprovePDsymptomsiscritical.Thenon-invasiverepetitivetranscranialmagneticstimulation(rTMS)hasbeenproposedasanadd-onforthetreatmentofParkinson´sdisease.Recently,aspecialcoil(DeepTMSordTMS)capableofstimulatingdeeperbrainareasincludingthecompletecorticalthicknesshasbeendeveloped.Theobjectiveofthepresentstudyistoevaluatethesafetyandefficacyofcombined1Hzprimarymotorcortexand10HzprefrontalcortexstimulationusingthedTMSH-coilasanadd-ontreatmentforPD.45patientsweretreatedwithdTMSandshowedsignificant improvements inUPDRS,gaitspeed,depressivesymptoms,balance,autonomicsymptomsanda73%increaseindailyONtimeinresponsetolevodopa.Inthecohort,dTMSwaswelltoleratedwithonlyminoradverseeffects.ThedTMSinducedsignificant improvements inmotor,postural,andmotivationalsymptomsofPDpatientsandmaypotentiateconcurrentlevodopatreatment,demonstratethatdTMSmayhaveamuchwiderspectrumofbeneficialeffectsthanpreviouslyreportedforTMS.ThepresentresultssuggestthatfutureclinicaltrialswithdTMSshouldincludeabroaderrangeofsymptommeasurements.

129

52)Role of CaV1.2 calcium channel as gene regulation in a depression-like model

Moreno, C1,2.,Hardy,Paulina1.,Hermosilla,Tamara2.,Varela,Diego2.,Rojas,Patricio2.,1Biología,QuímicayBiología,UniversidadDeSantiagoDeChile.2InstitutoCienciasBiomédicas,Medicina,UniversidadDeChile.(SponsoredbyFondecytRegular1130904And1120240;BecaDoctoradoNacional21130549.)

Mayordepressionhasahighprevalenceand isseriouspublichealthproblem inmostcountries.Thestudyofdepressiontoabiological level has elucidated partially the development of this condition. Previous studies demonstrate that neurons fromdepressivepatientsshowareductionindendriticarborizationandnumberofdendriticspines,however,themechanisms,atthemolecularlevel,responsibleforthesechangesarestilltobedefined.CaV1.2calciumchannelsaretheprincipalpathwayforcalciuminfluxinneuronalsoma,andhavebeenassociatedtoseveralcellularprocessessuchaschangesinneuronalmorphology,activationofCalmodulin,nucleartranslocationofNuclearFactorActivatedcellulesT(NFAT)andnucleartranslocationofC-terminusregionoftheCaV1.2.Theselastthreeprocessesinvolvechangesingeneexpression.

OurworkinghypothesisisthatchangesinmorphologyandfunctioninhippocampalneuronsfromanimalmodelsofdepressionareduetoregulationingeneexpressiondependentonsignalingthroughCaV1.2.Inordertotestthishypothesisweusechronicrestraintstresstogenerateamodelofchronicdepression.Behavioralparameterssuchasanhedonia,changes insocial interactionandbehavioraldespair,demonstratethattheseanimalssharecharacteristicofmayordepression,ashasbeenreportedforthismodelpreviously.Byusingelectrophysiologicalandmolecularbiologytechniques,wearecurrentlystudyingchangesinCaV1.2calciumchannelexpressionandsubcellularlocalizationthatcouldberelatedtothedescribedpathwaysthatmodulategeneexpression.

Ifourhypothesisistrue,wewouldbeabletoestablishthatCaV1.2isakeyplayerinthedevelopmentofdepression.

130

53)Evaluation of Obsessive Compulsive Disorder- related behaviors in a mouse model with altered Eaat3 expression in GABAergic neurons

Delgado, Claudia3.,Martinez,Jonathan3.,Henriquez,Francisca3,1.,Gonzalez,Luis2.,Moya,Pablo3.,1BioquimicaUniversidadCatolicadeValparaiso.2QuimicayFarmacia,Farmacia,UniversidadDeValparaíso.3Fisiologia,Ciencias,UniversidadDeValparaíso.(SponsoredbyThisWorkWasFundedByMillenniumNucleusNU-MINDNC130011(PRM)AndFONDECYTGrants1141272(PRM))

Obsessive-CompulsiveDisorder(OCD)isaneuropsychiatricdisordercharacterizedbypersistent,intrusivethoughts(obsessions)andrepetitive,intentionalbehaviors(compulsions).Extensiveevidencesuggestsalteredglutamatergicsynapsesincortico-striato-thalamo-cortical(CSTC)circuitry.GlutamatergicsystemgenesemergethenasgoodcandidatesforstudiesinOCD,particularlytheSLC1A1geneencodingEAAT3,theneuronalglutamatetransporter.EAAT3isexpressedinsomaanddendritesofglutamatergicandGABergicneurons,andprominentlyintheCSTCcircuit.EAAT3playsmultiplerolesinregulatingneuronalfunction,removesglutamate from the extracellular space limiting the activation of extrasynaptic neurotransmitter receptors and consequentexcitotoxicity,andregulatescysteinetransport-anessentialrate-limitingstepintheproductionoftheendogenousantioxidantglutathione.Moreover,whenpresentinGABAergicneurons,EAAT3providestheprecursorforthesynthesisofGABA.Therefore,itisofgreatinteresttofindoutwhetheralteredEAAT3expressionisrelatedtoOCD-likebehaviors.Thus,restrictedEAAT3expressionmanipulations in specificneuronal types,particularly inGABAergicneurons, areneeded toevaluate thepotential roleof thistransporter.Toaccomplishthisaim,conditionalKnockout(KO)andconditionaloverexpressing(OE)miceweregenerated,aimingtomanipulateEaat3inacell-specificmannerusingtheCre/loxPsystem.Here,wedescribethecurrentstatusofvalidationandcharacterizationofGAD65-Cre-mediatedmousemodelswithalteredEaat3expression. Eaat3-OEmatedwithGAD65-Cremicewere generated and genotyped by PCR. Anxiety and OCD-related behaviors are currently underway using several paradigmsincludingopenfieldtest,elevatedplusmaze,andmarbleburyingtests.WeexpectthisstudywillcontributetohelpunderstandthepotentialroleofEaat3inneuropsychiatricdisordersandparticularlyinOCD.

131

54)* Strategies to identify neuroprotective molecules in the diet: From genome to behavior.

Prado, L1.,1CentrodeGenómica,FacultaddeCIencias,Universidadmayor.

Diet impacts thephysiologyoforganismsatmolecular,metabolicandsystemic levels.However, the individualcomponentsoffood thatdetermine individual changes inphenotypeandbehavior areunknown. ThebacterivorenematodeC. elegans is anexcellentmodel to study the effect of diet onphenotype andbehavior becauseboth the animal and its food are geneticallytractable.We have previously shown that different types of bacterial diet change the degeneration rate ofmechanosensoryneuronsthathavebeentriggeredtodiebytheconstitutiveexpressionofthedegenerinsMEC-4dandDEG-1.AnimalsfeedingonE.coliOP50degeneratetheirneuronsin72hours,frombirthtoadulthood.However,whenanimalsfeedonE.coliHT115,neuronsarelargelyprotectedeventhreedaysafteradulthood.Todissecttheindividualcomponentsoftheprotectivediet,wefirstsequencedthegenomesandtranscriptomesofbothE.colistrainsandgeneratedalistofuniqueanddifferentiallyexpressedgenes.SubsequentlywegeneratedbacterialvectorsexpressingtheuniquegenesfromE.coliHT115underinduciblepromotersandintroducedthemintoE.coliOP50.FinallyE.coliOP50expressinguniquegenesfromE.coliHT115isfedtomec-4danimalsandthreedayslater,themorphologyoftheneuronsistestedunderafluorescentmicroscopeandafunctionalbehavioraltestis performed to test the integrity of the touch circuit. Thiswork establishes a powerful strategy to discover the contributionof individual components of the bacterial diet to neuroprotection, by functional supplementation of each gene product.

132

55)Role of chloride co-transporters in animal models of Schizophrenia

Lorca, Enrique1.,Lara,Marcelo1.,Rojas,Patricio1.,1Biología,QuímicayBiología,UniversidadDeSantiagoDeChile.

Schizophreniaisaneuropsychiatricdiseasethatprimarilyaffectsthesocialandcognitivecharacteristicsofthesubjectthroughanalteredperceptionoftherealityandadisruptionofformalthought.Themainhypothesisaboutthecauseofthediseaseisanalterationofdopamineneurotransmission.However,severalstudieshaveshownthattheneurotransmitterGABAwouldbedirectlyinvolvedinthedisease,resultinginadiminutionofinhibition.Recently,ithasbeenshownthatchlorideco-transportersNKCC1andKCC2,which regulate the intracellular concentrationof this anion, and thus theexcitationand inhibitionbalance,exhibitalteredexpression.Theaimofthisworkistostudytheroleoftheseco-transportersinthehippocampalcircuits,oneoftheareasofthebrainaffectedbythedisease.Westudiedthechangesinexcitabilityofthehippocampalcircuitsintwoanimalmodelsofschizophrenia,Ketamineadministrationandmaternaldeprivation.WedemonstratedthatthereisadecreaseintheglutamatergicexcitabilityintheCA3-CA1circuitandanincreaseindentategyrus.Otherwise,theGABAergicresponsewasonlyfoundalteredindentategyrusinbothmodels.PharmacologicallyblockofNKCC1withitsselectiveinhibitorBumetanide,producesadecreaseinGABAergicresponseonlyindeprivationmodel.Ontheotherhand,aftertheBumetanideadministrationinketamineanimalmodelandsubsequentbehavioralstudies,adecreasewasobservedinthepositivesymptomsofthedisease,sothisdrugcouldbeapotentialantipsychotic.WecanconcludethatNKCC1functionisalteredinonemodelofschizophrenia,beingresponsibleofchangesinGABAergicresponse.

133

56)Identity and Characteristics of Nitric Oxide synthesizing Bipolar Cells in the Retina

Agurto, Adolfo1.,Vielma,Alex1.,Schmachtenberg,Oliver1.,1Neuroscience,Science,UniversidadDeValparaíso.(SponsoredbyFondecyt1120513AndMilleniumInstituteCINV)

Nitric oxide (NO) is aneuromodulator involved inbothphysiological andpathological processes in themammalian retina. Itssynthesisisestablishedforcertainamacrinecellswhichmodulateinnerretinalsynapses,butbipolarcells,theonlyconnectionbetweentheinnerandouterretina,havealsobeenreportedtoproduceNO.However,identificationandcharacterizationofthisalternativeNOsourcearenotcomplete.WeincubatedratretinalsliceswiththeNO-fluorescentprobeDAF-FMandmorphologicallyidentified labeledbipolar cellsby injectionofan intracellulardye.Underdarkand lightadaptation, immunohistochemistryofneuronalNOsynthase(nNOS)wasperformedinretinaswithpreviouslyidentifiedNO-positivebipolarcellstoestablishtheirNOsourceandtoassessaputativenNOSexpressiondependenceonlightconditions.Bipolarcelltype8wasfoundtobethemostfrequentNO-positivecell.Ontheotherhand,immunohistochemistryshowedthatatleastoneONtypebipolarcellandoneOFFtypebipolarcellexpressnNOS.OneoftheONbipolarcellscorrespondstoBCtype9,amorphologicallydistinguishablenNOSexpressingbipolarcell.MostotherbipolarcelltypesdonotcontainnNOS,andtheoverallexpressionpatternofnNOSdidnotchangedependingondarkor lightadaptation.Finally,NOfluorescencewasmost frequentlypresent inaxonlessbipolarcells,suggestingthatNOisalsosynthesizedinresponsetoinjuryorpathologicalconditions,possiblybytheinducibleisoformofNOS.

134

57)Carotid chemosensory responses to acute hypoxia are reduced by chronic phenytoin treatment

Alcayaga, Julio1.,Oyarce,María2.,DelRIo,Rodrigo3.,1Biología,Ciencias,UniversidadDeChile.2CienciasFisiológicas,CienciasBiológicas,PontificiaUniversidadCatólicaDeChile.3UnidaddeControlCardiorrespiratorio,CentrodeInvestigaciónBiomédica,UniversidadAutónomaDeChile.(SponsoredbySupportedByGrantFONDECYT1130177)

Carotidbody(CB)chemoafferentsarethemainperipheralsignalthatparticipatesinventilatorycontrol.Intherat,thesensoryneuronsthatinnervateCBreceptorcellspresentapersistentNa+current(INaP),whichblockadereducesbothnormoxicafferentactivityandincreasesinfrequencydischargeinducedbyacutehypoxia.BecausethereisnodataavailableontheeffectsofprolongedINaPblockadeonchemoafferentactivity,werecordedcarotidnervedischargesandresponsestoacutehypoxiafromanimalstreatedwithphenytoinfortwotofourweeks.MaleSprague-Dawleyrats(204±6g;n=18),underisofluraneanesthesia,wereimplantedsubcutaneouslywith anosmoticpulpfilledwith vehicle (control) orwithphenytoin (10mgdailydose); theanimals receivedantibioticsandanti-inflammatoryaftersurgery.Aftersixteento28days,theratswereanesthetizedwithsodiumpentobarbitone(60mg/Kg)placed ina thermoregulatedpad.Theneckwasopened through themidline, the tracheacannulated, thecarotidbifurcationexposedandthecarotidnerveseveredatitsoriginintheglossopharyngealnerve.ThenervewasplacedinpairedPt/Irelectrodes,connectedinturntoanACpreamplifier,andcoveredwithmineraloil.Therecordedsignalwasamplified,band-passfiltered(10-1000Hz)anddigitallycountedtoassessthechemoafferentdischarge(ƒx),inHz.Basalchemoafferentdischargesinnormoxia(fractionofinspiredoxygen,FIO2=21%)aswellaswellasthechangesinchemoafferentdischarges(Δƒx)inducedby30schangesinFIO2(0–100%range)wererecorded.Theanimalsweresacrificedwithananestheticoverdoseattheendoftherecording.Thebasalchemoafferentdischargeswerenotsignificantlydifferent(P>0.3;Student´st-test)betweencontrols(76.5±8.6Hz;n=10)andphenytointreated(93.4±14.5Hz;n=8)animals.However,responsesinducedbychangingFIO2weresignificantly(P<0.02;2wayANOVA)reducedbyphenytointreatment.Moreover,responsesinducedbythelowestvaluesofFIO2intherange,weresignificantlylower(P<0.05;Bonferrronitestafter2WayANOVA)intreatedanimals(FIO25%,Δƒx=96.5±22.4Hz;FIO20%,Δƒx=92.0±14.4Hz)thanincontrolones(FIO25%,Δƒx=161.2±17.3Hz;FIO20%,Δƒx=167.3±20.0Hz).Thus,afferentdischargesinnormoxiaarenotmodifiedbyphenytointreatment,whileresponsestoshortacutehypoxicchallengesaresignificantlyreducedbyphenytointreatment.Thisreductioninhypoxicsensibilitymayalterventilatoryresponsesafterchronicphenytointreatment.

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58)STUDYING THE ROLE OF AMINERGIC RECEPTORS EXPRESSED IN DROSOPHILA MUSHROOM BODIES IN BEHAVIORAL RESPONSES TO AN AVERSIVE STIMULUS

Fuenzalida-Uribe, Nicolás1.,Campusano,Jorge1.,1BiologíaCelularyMolecular,FacultaddeCienciasBiológicas,PontificiaUniversidadCatólicaDeChile.(SponsoredbySupportedByFondecyt1141233)

TheMushroomBody(MB)isabrainintegrationcenterkeyinprocessingseveralsensorystimuliandindefininglocomotorbehaviorin insects. Aminergic systems innervate andmodulate the activity of neurons in theMB. Through the regulation of differentneuronalpopulationsinDrosophila MB,thesesystemsbecomerelevantinthemodulationoflocomotorbehaviorininsects.Herewehavestudiedtheroleofdifferentaminergicreceptorsonlocomotorbehaviorinresponseanaversivestimulus(Benzaldehyde,Bz).WedirectedtheexpressionofspecificRNAifordifferentDopaminergicandOctopaminergicreceptorstoDrosophilaMB,byusingtheGal4-UAStechnique.Werecordedandanalyzedflybehaviorsinacirculararena,whenfliesareexposedornottoanaversiveodorant,Benzaldehyde(Bz).OurresultsshowthatoneDopaminereceptoranalyzed(DAMB-R)contributestolocomotorbehaviorinresponsetoBzinDrosophilawhile,Octopaminereceptorsdonothavemajoreffectsoninnatelocomotorbehavior.Interestinglythough,oneofthesereceptors(Octb3-R)hasamarkedeffectoncentrophobism,aparameterreflectingthewayfliesexploretherecordingarena.ThisdatasuggeststhatcomplexbehaviorsassociatedtoanxietyaremodulatedbythisreceptorinMB.OurresultsshowadifferentialcontributionofaminergicreceptorsexpressedinMBindifferentbehaviors.

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59)On the spatial extension of the correlations on a retinal ganglion cells population: dependence on the stimuli.

Herzog, Rubén1.,Palacios,Adrian2.,Escobar,Maria-Jose3.,1Biología,Ciencias,UniversidadDeChile.2CINV,ciencias,UniversidadDeValparaíso.3ElectrónicaUniversidadTécnicaFedericoSantaMaría.(SponsoredbyFondecyt#1150638,MillenniumInstituteICM-P09-022-F.,ANR_CONICYTANR-47ECOS-ConicytC13E06Fondecyt#1140403,BasalProjectFB0008)

Theretinaisanextensionofthebrain,aneuralnetworkworkingasavisualsensoryinterfacebetweenthenervoussystemandtheworld.Theretinaisorganizedincellularandplexiformlayers,andcomposedbydifferentcelltypes.Oneofthetypesformingtheretinaisretinalganglioncells(RGC),whichreceivesinputsfromtheothercelltypesandsendsretinaactivitytothebrainthroughtheopticnerve.Nowadays,thedevelopmentofMulti-electrodeArraysystemsallowsthesimultaneousrecordingandanalysisofhundredsofRGCs,shiftingthefocusofthequestionsandobservationstowardsthenetwork(population)level,insteadofstudyingsingleneuronsactivity.Inthiscontext,werecordedseveralin vitro retinaspatchesfromtheChileandiurnalrodentOctodon degus underdifferentnaturalandartificialstimuliconditions,obtainingtheresponseofmorethan700retinalganglioncells.

TakingintoaccountthenetworknatureoftheretinaweaskedwhethertheconcertedspikingactivityofRGCsdependsonthevisualstimuliandthelocalconnectivityoftheseveralrecordedsties:So,whatisthespatialextensionofthecorrelationsontheRGCs?Dothiscorrelationsanditsspatialextensiondependonthestimuli?Toaddressthesequestions,wecharacterizethosecellsusingspike-triggeredaverage,whichallowustoestimatethelinearreceptivefieldanditsspatialpositionintheretina.Addition-ally,wecomputedthecross-correlationfunctionbetweenallpairsofcellsandthen,withthepositionofeachcell,wecomputedthedistancebetweenallthepairsofneurons,obtaining,attheend,afunctionthatrelateshowthecorrelationdependsonthephysicaldistancebetweentworecordedneurons.Usually,weobserveanexponentialasymptoticdecayofthecorrelationswiththedistancebetweencells.

Usingdatarecordedfromnaturalstimulusweobservedthatboth,baselinecorrelationsandspatialextentof thiscorrelationsreachthehighestvalues,whencomparedtootherexperimentalconditions.Thespatialextensioninthecaseofnaturalstimulusextendsbeyondthemerereceptivefieldoverlapbetweenneurons,suggestingthepresenceofalateralmechanismontheretinathatcouldbecoordinatingtheRGCactivityonaconstrainedlocality.

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60)EFFECT OF 4-METHYL-THIOAMPHETAMINE ON OLFACTORY RESPONSES IN DROSOPHILA IS EXPLAINED BY AN INCREASED SEROTONIN RELEASE IN THE FLY BRAIN.

Hidalgo, Sergio1.,Molina,Daniela1.,Fuenzalida-Uribe,Nicolás1.,Campusano,Jorge1.,1Biologíacelularymolecular,Cienciasbiológicas,PontificiaUniversidadCatólicaDeChile.(SponsoredbyAcknowledgementToDr.Iturriaga-Vasquez.SupportedByFondecyt1141233)

4-MTA (4-Methylthioamphetamine) is a “designer drug” which induces prolonged stimulation and euphoria, and has beenassociatedwithneurotoxicityandevendeath.Itwasdesignedtospecificallyblocktheserotonergicplasmamembranetransporter(SerT), leadingtoan increase intheextracellularcontentof thisamine.However, ithasbeenalsoshowntoactonothertwomoleculartargets:thedopaminetransporterandMAO-A.Thus,thebehavioralconsequencesof4-MTAexposure inananimaldependontheeffectsinducedbythisdrugonallthesetargets.Invertebratesexposedtodrugsofabusedisplayasetofbehaviorsthatdependontheactivationofaminergicsystems,whicharehighlyconservedwhencomparedtovertebratecounterparts.Inourlab,weareusingsomeofthebehavioral,physiologicalandgenetictoolsavailableintheflyDrosophila melanogastertodissectoutthecontributionofdifferentaminesystemstothebehavioraleffectsinducedby4-MTA.

Our data show that 4-MTA at different concentrations induce differential effects on fly olfaction and motor responses.Chronoamperometrystudiesindicatethat4-MTAinducesthereleaseofendogenousBAsintheflybrain,withaslowkineticsascomparedtotheeffectsobservedfornicotine.Experimentsinmutantfliessuggestthattheaminewhosereleaseisbeingmodifiedby4-MTAisserotonin.WefurthershowthattheeffectsonolfactionarenotobservedinanimalsexpressingamutationforSerT.Altogether,thisdatasupportsthepropositionthat4-MTAinducesthereleaseofserotonintomodulateolfactoryresponses inDrosophila.

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61)Synchrony of neural oscillations in the olfactory system of the rainbow trout

Olivares, Jesús1.,Herzog,Rubén1.,Orio,Patricio1.,Schmachtenberg,Oliver1.,1Neurociencia,Science,UniversidadDeValparaíso.(SponsoredbyFONDECYT1120513,InstitutoMilenioCINV,BecaDeEstudiosDeDoctoradoCONICYT)

The olfactory system is evolutionarily conserved and shows a similar general operating scheme across invertebrates andvertebrates. Two regions of the telecephalon, the ventral nucleus of the ventral region of the telencephalon (Vv) and dorsalposteriorregionoftelencephalon(Dp),arerelatedtotheanalysisofolfactoryinformationintherainbowtrout.Neuraloscillationscanbeobservedinthelocalfieldpotential(LFP)inspecificfrequencybandswithintheseareas.Fourieranalysisshowsthattheolfactorybulb,VvandDposcillatewithfrequenciesaround9-10Hzduringtheolfactoryresponse.Byperformingthecalculationofcoherencebetweenthesetwozones,ahighdegreeofcoherenceisobservedatfrequenciesbelongingtobandsknownasTheta(t)(9-12Hz)Beta(b)(about15Hz)andGamma(g)(about25Hz).Neuraloscillationsinthesefrequencybandscanbefoundintheolfactorysystemsofmanyspeciesandarethoughttoberelatedtoolfactorylearningandalertness,buttheirmeaningatthelevelofolfactory informationprocessingremainstobeinvestigated. Inordertodeterminewhetherresponses inthetelencephalonshowdifferencesdependingonthetypeofodorant,westimulatewithdifferentconcentrationsofartificialmixturesofaminoacids(AA)andbilesalts(BS)andwithtwonaturalodors,humanskinrinse(HSR)andtroutskinextract(TSE).Equipotentconcentrationsoftheseodorswereestablishedbasedontheelectroolfactogramamplitude.Theseexperimentsallowthecomparativeanalysisofdifferences inamplitude,delay,duration, frequency,andenvelope shape foreachodoramongotherparametersofneuraloscillations.

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62)Inatentively viewing a bistable stimulus induces simultaneous processing of both alternative percepts

Rodriguez, Eugenio1.,Campos,German1.,Artigas,Claudio1.,Morales,Ricardo1.,1EscueladePsicologia,FacultaddeCienciasSociales,PontificiaUniversidadCatólicaDeChile.(SponsoredbyProyectoFondecyt1120752)

Inbistableperceptionasinglesensorystimulusgiverisetotwomutuallyexclusivealternatingperceptualstates.Acasemodelofself-organizingpropertiesofbrainactivity,bistableperceptionhasbeenextensivelystudied.Howevermanyaspectsofbistableperceptionarestillpoorlyunderstood.Oneofsuchis‘whathappenswhenattentionisdivertedawayfromthebistablestimulus?’Doestheperceptualalternationgoeson?Doesitstayfixedinonestate?Orthereisnoperceptionwhatsoever?Wehypothesizethatwhenattentionisdivertedfromthebistablestimulus,thebrainsimultaneouslyprocessesbothperceptualstates.InordertotestthishipothesiswepresentedsubjectswiththeRubinVaseillusion(vase-facepicture).Vaseandfaceareasweretaggedbyflashingthemat12and15Hzrespectively.TheEEGactivityof4subjectswasrecordedwhiletheywerelookingattheRubinvasebuteitherattendingtoorattendingawayfromthestimulus.Resultssuggestthatduringinatentivewievingofthestimulusbothperceptualstatesaresimultaneouslyprocesedtohighercognitivelevelsasshownbytaggedactivityuptofrontalbrainareas.Bycontrastwhenattentionisturnedtothestimulusthetaggingfrequencieschangewithoneofthembeingenhancedwhiletheotherisatenuatedinaparalelwaytoperceptionwerethecorrespondingperceptsisselectedwhiletheotheronevanishes.

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63)Serotoninergic modulation of synaptic strength in rat dentate gyrus

Chavez, Andres1,2.,Castillo,Pablo2.,1InstitutodeNeurociencias,Ciencias,UniversidaddeValparaiso,CentroInterdisciplinariodeNeurociencias.2DominickP.PurpuraDepartmentofNeuroscience,Neuroscience,AlbertEinsteinCollegeofMedicine.(SponsoredbyThisWorkWasSupportedByNIHGrantsMH081935AndDA017392(toP.E.C.)AndByNucleoMilenioNu-MINDNC130011(toA.E.C).)

Severallinesofevidenceindicatethatthecomplexactionofserotonin(5-HT)insynapticfunctionisdeterminedbydiverse5-HTreceptor(5-HTR)subtypes.However,theprecisecontributionofeachreceptorsubtypetobrainfunctionregulationisnotfullyunderstood. Inthe inferiorolive, ithasbeenreportedthattheGq/11-coupled5-HT2Rcanindirectlysuppressexcitatorysynaptictransmissionbymobilizingendocannabinoids(eCB)thatactivatepresynapticcannabinoidreceptors(CB1Rs)tosuppressglutamaterelease.Morerecently,wereportedthattheeCBanandamideactsonpostsynaptictransientreceptorpotentialvanilloid1(TRPV1)tosuppresssynaptictransmissionpresumablybyreducingthenumberofAMPA-typeglutamatereceptors(AMPARs)atthesynapse.Giventhat5-HT2Rs,CB1RsandTRPV1channelsareexpressedinthedentategyrus(DG),wesoughttoexaminewhether5-HT2Rs,viaeCBproductionandactivationofpresynapticCB1Rand/orpostsynapticTRPV1channels,couldregulateexcitatorysynaptictransmissionindentategranulecells(DGCs).Tothisend,wemonitoredtwodistinctglutamatergicinputsondentategranulecells(DGCs)inacutehippocampalslices:mossycellfiber(MCFs)inputs,whicharemodulatedbyretrogradeeCBsignalingandCB1Rs,andmedialperforantpathway(MPP)inputs,whichareknowntobemodulatedbynon-retrogradeeCBsignalingandTRPV1channels.Usingselectivepharmacologyfor5-HTRsinacuterathippocampalslices,wefoundthatbathapplicationof5-HT(50µM-10min)depressesMPP-EPSCs,butnotMCF-EPSCsviaactivationof5-HT2a/cRs.ThiseffectwasobservedatAMPAR-EPSCs,butnotNMDAR-EPSCs,indicatingthatchangesinsynapticefficacywerenotduetoadirectmodulationoftransmitterrelease.Moreover,the5-HT-mediateddepressionrequiredpostsynapticCa2+riseandinternalizationofAMPARs,stronglysuggestingapostsynapticmechanismofaction.Consistentwiththis ideaandthepostsynapticlocalizationofTRPV1channelsattheMPPsynapse,pretreatingslices(10–20min)withtwodifferentTRPV1antagonists,capsazepine(CPZ,10µM)orAMG9810(AMG,3µM)eliminated5-HT-mediateddepressionofMPP-EPSCs.Furthermore,5-HT-mediateddepressionwasstillpresentinCB1Rsknockoutmice,butwasabolishedinTRPV1knockoutmice.Takentogether,ourfindingsrevealanovelformof5-HT-TRPV1mediatedregulationofexcitatorysynaptictransmissionatcentralsynapses.ThepotentialcontributionofeCBssignalinginthisnovelformof5-HT-mediateddepressioniscurrentlyunderinvestigation.

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64)Prostaglandin E2 decrease inhibitory post-synaptic current in CA1 pyramidal neurons of hippocampus

Ahumada, Juan1.,Bonansco,Christian1.,Fuenzalida,Marco1.,1Fisiología,Ciencias,UniversidadDeValparaíso.(SponsoredbyThisWorkWasSupportedByGrantsFromMillenniumNucleusNU-MINDNC-130011(MF),FONDECYT1130614(M.F.)And1130491(CB),CONICYTPhDFellowNº21130547(JA).)

Thenervoussystemoperates inabalancedregimeofexcitationand inhibition.Severalneurotransmitterandneuromodulatorsystemsareessentialtomaintainingtheexcitationandinhibition(E/I)ratio,whichiscriticalforcognitiveprocessessuchaslearningandmemory.Increasingevidenceindicatesthatcentralcomponentoftheimmuneresponse,suchascyclooxygenase-2(COX-2),canregulatethephysiologicalfunctioninthebrain.COX-2mediateschangesinneurotransmissionandsynapticplasticityintheCNSthroughitschemicalproducts,principallybyprostaglandinE2(PGE2),whichactivatesfoursubtypesofseventransmembraneG-proteincoupledreceptors(EP1-EP4).AlthoughseveralstudieshasdescribedtheeffectsofPGE2activationonglutamatergictransmissionandtheirpossibleconsequencesintheexcitatorylong-termpotentiation(LTP),thecellularmechanismthroughwhichthePGE2canregulatethesynapticefficacyofGABAergicsynapsesandE/Ibalanceispoorlyknown.UsingelectrophysiologicaltoolsweinvestigatedwhethertheactivationoftheEP3and/orEP4receptorscanmodulatedtheGABAergicsynapsesonpyramidalneuronsofCA1region.Accordingto literature,weshowedthatPGE2 inducesLTPoffieldexcitatorypost-synapticpotential inCA3-CA1synapses,interestingly,withoutblockGABAergicreceptors.Remarkably,wefoundthattheactivationofEP3and/orEP4receptorscandecreasetheamplitudeofevokedinhibitorypost-synapticcurrent(eIPSC)onCA1pyramidalneurons.Takentogether,theseresultssuggest thatPGE2couldbean importantsignalingpathway inmodulationofbothglutamatergicandGABAergictransmissionandsynapticplasticityaswellasthemaintenanceofE/IbalanceintheCNS.Furthermore,PGE2signallingcouldbeanimportantcomponentofcognitivedeficitsobservedindifferentneuropathologiessuchastraumaticbraininjury,epilepsyandschizophrenia,whenithasbeendescribedthatCOX-2activityisimpaired.

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65)Brevican and reelin regulate neuronal refinement in hippocampal neurons.

Ampuero, Estibaliz1.,Jury,Nur1.,Marzolo,MaríaPaz2.,Jaramillo,Karen1.,Montecino,Martín3.,VanZundert,Brigitte1.,1LaboratoriodePlasticidadNeuronal,CentrodeInvestigacionesBiomédicas,UniversidadAndrésBello.2LaboratoriodeTráficoIntracelularySeñalización,FacultaddeCienciasBiológicas,PontificiaUniversidadCatólicaDeChile.3LaboratoriodeRegulaciónGénica,CentrodeInvestigacionesBiomédicas,UniversidadAndrésBello.(SponsoredbyFondecyt-3130582(E.A.),Fondecyt-1140301(B.v.Z.),AnilloACT1114(B.v.Z.).)

Introduction:Theextracellularmatrixproteins(ECM)brevicanandreelinaresecretedbyastrocytesandGABAregiccells,respectively.Bothproteinsarefoundsurroundingtheexcitatorysynapseswheretheycontributetosynapsematurationandrestrictingreceptormobility.IthasbeenreportedthatreelinbindstothelipoproteinsreceptorsApoER2andVLDLR,herebyinducinganintracellularsignaling pathway thatmodulates neuronal plasticity. It is believed that brevican restrict synapses by forming a perineuronalnetworks. Herewe studiedhow interferenceof reelin-mediated signalingordegradationofbrevican inmaturehippocampalneuronsimpactsdendritogenesisandthedistributionofthesynapticproteinslikeNMDAreceptorsubunits(NR2BandNR2A),thescaffoldingproteinpostsynapticdensity95(PSD95)andthepresynapticproteinsbassoonandsynapsin-I.Wehypothesizedthatinterferingthepresence/signalingofreelinandbrevicancanreactivateneuronalplasticityinmatureneuronsbyincreasingthesynapticexpressionofNR2BandPSD95thatallowspineremodelingconcomitantwithdendritogenesis.MaterialandMethods:Expressionofbrevicanandreelinwasanalyzedbyimmunostainingsusingpermeablizationandnon-permeablizationconditionsduringdevelopmentof culturedhippocampal neurons (0-20days in vitro (DIV)).Maturehippocampal neurons (15DIV)weretransfectedwithGFPbymagnetofectionandchronicallytreatedduring5dayswithGST-RAPorCR-50todecreasereelinsignaling,orchondroitinaseABC(ChABC)forbrevicandegradation.At20DIVweanalyzedthedendriticarchitectureandtheexpressionofsynapticproteinsusingdoubleimmunostainings.ToevaluateifdegradationofECMalsoregulatesthearchitectureofhippocampalneurons in vivo, CR50orChABCwere injected into thedentategyrusofadultmice.HSV-GFPwasco-injected tovisualize themorphologyofneurons.Results:Withhippocampalmaturationin vitrograduallymoreneurons(MAP2+)showedanincrementin extracellularbrevicanand reelin immunoreactivity.We found that interferenceof reelin-mediated signalingordegradationof brevican resulted into a significant increase in dendritogenesis relative to control neurons. Increased dendritogenesiswasassociatedwithanexchangeofsynapticproteins,withan increment in theclusteringof theNR2Bsubunit,concurrentwithareductionofPSD95clusters.Discussion:OurresultsshowthatthemodificationofcriticalECMproteinsinmatureneuronsleadstoanimmaturephenotypeoftheneurons,potentiallyfavoringformationofnewcontact.

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66)Methylphenidate amplifies LTP in hippocampus CA1 area involving the insertion of AMPA receptors by activation of β-adrenergic and D1/D5 receptors

Carvallo, Claudia1.,Contreras,Darwin1.,Farias,Ricardo2.,Rozas,Carlos2.,Zeise,Marc2.,Morales,Bernardo2.,1Biología,Quimica-Biología,UniversidadDeSantiagoDeChile.2Biología,QuímicayBiología,UniversidadDeSantiagoDeChile.(SponsoredbySupportedByFONDECYT1120580,AnilloACT-1113AndCONICYTFellowshipToC.C./D.C)

Methylphenidate(MPH,Ritalin©)iswidelyusedinthetreatmentofAttentionDeficitHyperactivityDisorderandrecentlyasadrugofabuse.TheeffectofMPHinhippocampushasreceivedrelativelylittleattentionconsideringthatitplaysaprominentroleinmemoryandlearningprocesses.ItisknownthatMPHincreasestheTBS-dependentLongTermPotentiation(LTP)intheCA1area.However,thecellularandmolecularmechanismsinvolvedinthisprocessarestillunknown.UsinganelectrophysiologicalapproachandWesternblotanalysiswestudiedthetransductionmechanisminvolvedintheeffectofMPHonLong-TermPotentiation(LTP)inrathippocampusslices.3-4weeksoldSprague-Dawleyratsweredecapitatedunderhalothaneanesthesia,andhippocampusslices(400µmthick)wereprepared.LTPswereinducedbyapplyingthetaburststimulation(TBS,5trains,100Hz)attheSchaeffercollateralsandrecordedinthestriatumradiatumoftheCA1area.Superfusionofhippocampusslicesduring20minwithMPHenhancesLTPinCA3-CA1synapsesinadose-dependentmannerwithanEC50of73.44±6.32nMandamaximumresponseof52.93±0.63%.Paired-pulsefacilitation(PPF)curvesremainedunchangedafterperfusionwithMPH,suggestingthattheeffectofMPHisatpostsynapticlevel.UsingspecificantagonistsandPPFprotocols,wefoundthattheMPH-dependentincreaseofLTPinvolvesnotonlyβ-adrenergicreceptorsactivationbutalsopost-synapticD1/D5dopaminereceptors.TheinhibitionofPKAwithPKI,suppressedthefacilitationofLTPinducedbyMPHconsistentwithaninvolvementofPKAdependentcascadedownstreamoftheactivationofD1/D5receptors.ToevaluatewhethertheincreaseofLTPinducedbyMPHinvolvestheinsertionofnewAMPAreceptorsinthepost-synapticmembrane,wecollectedCA1areasfromhippocampalslicesusedinLTPexperimentsandperformedWesternblotanalysisofthephosphorylationstateofSer845andSer831residuesinGluA1.SamplesofCA1areastakenfromslicespotentiatedwithMPHpresentedanincreaseinthephosphorylationoftheSer845residueoftheGluA1subunitofAMPAreceptorscomparedtocontrolslices.ThiseffectwasinhibitedbySCH23390,antagonistofD1/D5receptors.Moreover,usingcross-linkingessaywefoundanincreaseofAMPAreceptorsinthesurface.TheseresultssuggestthatMPHincreasesTBS-dependentLTPinCA3-CA1synapsesthroughapolysynapticmechanisminvolvingactivationofβ-adrenergicandD1/D5dopaminergicreceptorsandpromotingthetraffickingandinsertionofAMPAreceptorstotheplasmaticmembrane.

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67)Cannabinoid receptor activation modulate the temporal properties of scotopic visual signal in rat retina

Palacios-Muñoz, Angelina1.,Vielma,Alex2.,Palacios,Adrian3.,Chavez,Andres4.,1InstitutodeNeurociencias,FacultaddeCiencias,CentroInterdisciplinariodeNeurocienciadeValparaíso,UniversidaddeValparaíso.2InstitutodeNeurociencias,FacultaddeCiencias,UniversidaddeValparaíso,CentroInterdisciplinariodeNeurocienciadeValparaíso.3InstitutodeNeurociencias,FacultaddeCiencias,UniversidaddeValparaiso,CentroInterdisciplinariodeNeurociencias.4InstitutodeNeurociencias,FacultaddeCiencias,UniversidaddeValparaiso,CentroInterdisciplinariodeNeurociencia.(SponsoredbyThisWorkWasSupportedByFONDECYTGrant#1151091(AEC),IniciativaCientíficaMilenioICM-P09-022-F(AECAndAGP)AndNúcleoMilenioNu-MIND#NC130011(AEC).)

Extensivedistributionsoftype-1cannabinoidreceptor(CB1R)inmajorretinalneuronssuggestsaroleasmodulatorsofretinalnetwork,buthowactivationofthesereceptorscanmodulatesvisualactivityremainlargelyunexplored.Usingin vivoextracellularelectroretinogram(ERG) recordings,weexaminethe functionalconsequencesofCB1Ractivationonrat retinalcircuitryunderscotopic(dark)andphotopic(light)conditions. Intravitreal injectionoftheCB1RagonistWIN55,212-2(WIN,1μM)enhancedtheamplitudeofERGa-andb-waveunderscotopicconditions,withoutalteringthegainofERGa-andb-wavesunderphotopicconditions.Inaddition,WINalsoprolongedthedecaytimeofthescotopicERGb-wave,whichreflectsONbipolarcelldepolarization,butnotthephotopicERGb-wave,whichreflectsconeONbipolarcells.Importantly,WIN-mediatedeffectonERGa-andb-wavedependsonCB1RactivationasthechangesintheamplitudeandkineticswereeliminatedbyblockingCB1RswithAM251(5μM).Remarkably,WIN-mediatedeffectsonERGwaveswereoccludedbyblockinginhibitoryGABAergicsynapticreceptors(GABAAandGABACtype),stronglysuggestingthatactivationofCB1Rsmayplayaroleinmodulatingthesignaltransferthroughtherod/darkpathwaybyreducingGABAergicinhibitoryinputsontosecondorderONrodbipolarcell(RBC)terminals.Totestthispossibility,whole-cellrecordingsweremadefromONRBCsinacuteretinalslices,whileGABAergicfeedbackIPSCsmediatedbyA17amacrinecellswereelicitedbystepdepolarizationsintheRBCs.WIN(5µM)significantlyreducedvoltagestep-evokedIPSCsrecordedinRBCs,aneffectthatwaslargelyblockedbytheCB1RantagonistAM251(5μM).Altogethertheseresultssuggestthatcannabinoidreceptor activation modulate signal transfer through the rod pathway by reducing GABAergic inhibitory feedback onto RBCterminals.TheprecisemechanismunderlyingthiseffectonGABAergicfeedbackinhibitioniscurrentlyunderinvestigation.

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68)Role of NADPH oxidase (NOX) in spatial memory formation and synaptic function in rat hippocampus

Contreras, Marcela1.,Chávez,Andrés2.,Sanchez,Gina3.,Hidalgo,Cecilia4.,Haeger,Paola1.,1DepartamentoCienciasBiomédicas,FacultaddeMedicina,UniversidadCatólicaDelNorte.2CentroInterdisciplinariodeNeurocienciasdeValparaíso,FacultaddeCiencias,UniversidadDeValparaíso.3ProgramadeFisiopatologia,ICBM,FacultaddeMedicina,UniversidadDeChile.4BNI,CEMC&ICBM,FacultaddeMedicina,UniversidadDeChile.(SponsoredbyFundedByFondecyt#1140855(P.H),Fondecyt#1151091&MillenniumNucleusNU-MINDNC-130011(AEC),#Fondecyt1140545&BNIP-09-015F(CH))

TheNOXfamilyencompassesseveralenzymaticcomplexesthattransferelectronsacrossbiologicalmembranesandwhichareresponsible for superoxideproduction,acellular reactiveoxygenspecies (ROS). Increasingevidencesuggest thatactivationofNMDAreceptors(NMDARs)increasesROSlevelsthroughactivationoftheNOX2isoform,whereasNOXinhibitionreducesNMDAR-dependentERKactivation.Moreover,studiesinmaleknockoutmicedemonstratedthatNOX2activityisnecessaryfortheinductionoflong-termpotentiation(LTP)inthehippocampusaswellasforspatialmemoryformation.However,directinvolvementofNMDARfunctionintheseprocessesremainsunclear.Here,usingtheMorrisWaterMazeparadigm,weevaluatedtheroleofNOXactivityinhippocampalmemoryformationinadultrats.OurresultsindicatethatratsorallytreatedwiththeNOX2inhibitorapocynin(5mM),spentmoretimetoreachthehiddenplatforminthelastdaysoftrainingcomparedwithcontrols,withoutchangesintheswimmingspeed.Additionally,weinvestigatedtheroleofNOXonsynapticfunctionbyassessingexcitatorysynaptictransmissionattheCA3-CA1synapseinacuterathippocampalslices,measuringextracellularfieldpotentials(EPSP)andusingwhole-cellrecording(EPSC)techniques.Wefoundthatbathapplicationfor20minofVAS2870(VAS,10mM,aspecificNOXfamilyinhibitor)didnotaltertheslopeofAMPAreceptor-mediatedEPSP,butreducedthemagnitudeofNMDAR-dependentLTPcomparedwithcontrolslices.TotestwhetherthiseffectonLTPcouldbeduetoaVAS-dependentalterationofNMDAR-dependentbasaltransmission,weevaluatedtheeffectsofVASonisolatedNMDAR-mediatedcurrents.NodifferencewasfoundintheamplitudeofNMDAR-mediatedEPSCcomparedtobaseline.Takentogether,theseresultssuggestacuteNOXactivityinvolvementinhippocampalLTPandspatialmemoryformation,presumablyviamodulationofactivity-dependentNMDARfunction.TheprecisemechanismsunderlyingNOXeffectsonLTPandmemoryformationarecurrentlyunderinvestigation.

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69)ATP a probable mediator of the respiratory response at caudal medullary chemosensitive nuclei.

Gómez, Karina1.,Olivares,MaríaJosé1.,Beltran-Castillo,Sebastián1.,Donoso,MaríaVerónica1.,Huidobro-Toro,JuanPablo1.,Eugenin,Jaime1.,1Biología,QuímicayBiología,UniversidadDeSantiagoDeChile.(SponsoredbyFondecyt1130874YFondecyt1141132)

ATP, releasedbyastrocytesat the retrotrapezoidnucleus (RTN) in response tohypercapnia, increases the frequencyoffictiverespirationinrodents.However,inotherchemosensitivenucleus,liketheraphenucleus(RN)andthenucleustractussolitarius(NTS),theATProleasamediatoroftherespiratoryresponsetohypercapnia isquestioned. Inthiswork,wewantedtoknowwhetherATPcanbereleasedbyhypercapnicstimulationfromotherregionsofthemedullalocatedcaudallytotheRTN.

Eightmedullaryslices(700μmwidth)fromCF1miceatpostnataldays1-4(P1-P4)containingthecaudalportionoftheventralrespiratorycolumn (VRC),NTSandRN,butnotcontaining theRTNwereacutely incubated in30mlofartificial cerebrospinalfluid (aCSF) equilibratedwith10%CO2and90%O2 for10minutes (hypercapnia-conditionedmedium,h-cm). Similarprotocolwas followed toobtain anormocapnia-conditionedmedium (n-cm, control), that is, 8 sliceswere incubatedwith30ml aCSFequilibratedwith5%CO2,90%O2for10minutes.Conditionedmediawerecollectedandequilibratedwith5%CO2,95%O2,andsuperfusedtoanothermedullarysliceinwhichfictiverespirationwasrecordedatbasalconditionswithasuctionelectrodeplacedontheVRC.

H-cm, but not n-cm, increases the frequencyof inspiratory bursts up to 180%of the basal rate. This increasewas abolishedbyapyrase,enzymethatdegradesATP.ResultsareconsistentwiththeHPLCdetectionofaugmentedATPconcentrationintheconditionedmediumafterthehypercarbicacidosis.

ThesedatastronglysuggestthatATPisreleasednotonlybytheRTNbutalsobyothercaudalmedullarynucleiduringhypercarbia,anditwouldcontributeatRN,NTSorVRC,likelyasmediator,totherespiratoryresponsetohypercapnia.

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70)Adaptation of CA1 pyramidal neuron excitability to chronic inactivity: role of CaMKII

Karmelic, D1.,Palma,Verónica1.,Sanhueza,Magdalena1.,1DepartamentodeBiología,FacultaddeCiencias,UniversidadDeChile.(SponsoredbyFinancialSupport:CONICYTScholarship21110650AndFONDECYTGrants1140700And1140697.)

“Hebbian” synaptic plasticity processes, such as long-term potentiation, generate positive feedback loops that can resultdestabilizingforneuronalcircuits.Thisisthoughttobepreventedbyasetofslowerplasticityphenomena,termed‘homeostaticplasticity’,actinginoppositiontothechangesinneuralactivity.Thesemechanismsarenotentirelyunderstood,butcanincludesynaptic changes and changes in intrinsic neuronal excitability throughmodification of differentmembrane conductances. Inthepresentwork,westudytheadaptationtochronicinactivityoftheexcitabilityofCA1principalcellsinculturedslices.Usingwhole-cell recordingweevaluated,asafirststeptodescribesuchplasticity,thespikingfrequency inresponsetodepolarizingcurrentinjectioninCA1neuronsfromslicesincubatedin1µMTTXfor3dayscomparedtoneuronsfromcontrolslices.NeuronsfromTTX-treated slices show significantlyhigher excitability,with increasedfiring frequencies at lower stimulation levels.Wethenmeasuredthefiringthresholdusingacurrentrampandfoundnosignificantdifferencesinthisparameter,suggestingthatchanges involtage-gatedsodiumchannelsdonotexplaintheobservedrise inexcitability.Membraneresistance,ontheotherhand,increasedsignificantlywithchronicinactivity.Searchingforpossiblemechanismsfortheexpressionoftheseadaptationsin excitability triggered by chronic inactivity,we assessed the role of CaMKII activity by co-incubating TTX-treated sliceswith10µMKN-93,awell-characterized inhibitorofCaMKIIactivationbycalcium.Thestudiedexcitabilityvariables inCA1neuronsfromtheseslicesareindistinguishablefromTTX-treatedcells.Ontheotherhand,chronicincubationwithKN-93aloneinducesan important excitability increase in terms of firing frequency vs. injected current, alongwith a significant rise inmembraneresistance.SeverallinesofevidenceinmurinemodelsofepilepsylinkCaMKIIinhibitionwithincreasedseizureoccurrence.Thehigherneuronalexcitabilityobservedinthepresentworkisconsistentwiththoseepilepticphenomena.ThefactthattheeffectsofCaMKIIinhibitionandchronicinactivityhavevirtuallythesamemagnitudeandarenotadditive,pointstowardsanocclusionoftheeffectsduetothephysiologicalconstraintsofasharedmechanism.

148

71)Sorting determinants of Corticotrophin Releasing Factor Binding Protein towards the Regulated Secretory Pathway

Bastias, Cristian1.,Blanco,Elías1.,Gysling,Katia1.,1DepartamentoBiologíaCelularyMolecular,FacultaddeCienciasBiológicas,PontificiaUniversidadCatólicaDeChile.

CorticotrophinReleasingFactorBindingProtein(CRFBP)isa37kDaglycoproteinwithhighaffinityforbothCRFandurocortin-1peptides.Traditionally,ithasbeenacceptedthatCRFBPhasaninhibitoryrolebybindingCRFand/orurocortin-1impedingtheirbinding toCRF receptors. However, recently it is has been shown that CRFBP also facilitateCRF receptor actions. Previouslywe have shown that CRF-BP is secreted through the regulated secretory pathway, like a neuropeptide. However, the sortingdeterminantstotheregulatedsecretionpathwayarepresentlyunknown.ThepurposeofthepresentstudywastoidentifythesortingdeterminantsofCRFBPtothesecretorypathway.Weusedin silicotoolstodetermineputativesortingdomainsinCRFBP.Theanalysisusingsecondarystructurepredictionprograms (GORVandNPS@ConsensusSecondaryStructurePrediction)andstructuralmodelling(ROBETTA3Dmodellingwebserver)showedputativealphahelicaldomainsinCRFBP.Inaddition,thehelicalwheelprojectionprogram(PEPWHEEL)showedthatthe(50-74)CRFBPalphahelicaldomainhasanamphipathicconfiguration,signatureof other conserved sortingdomains. Thepredicted (50-74)CRFBP amphipathic alpha-helical domainwas capable ofsortingtotheregulatedsecretorypathwayamutantformofcocaineandamphetaminerelatedtranscriptpro-peptide(proCART)without its sortingdomain.The (1-53)ProCart/(50-74)CRFBP-EGFPmchimerasignficiantlycolocalizedwithsecretogranin IIandpresentedasubcellularlocalizationassociatedtothetransgolginetworkandsecretorygranulesinPC-12Cells.Preliminarydatashowedthatthischimeraisreadilysecretedupondepolarization.Thus,CRFBPhasanamphipathicalphahelixdeterminingitssortingtothesecretorypathway.

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72)The lack of a functional allele for Methyl CpG Binding Protein-2 alters reproductive lifespan and fertility in female mice.

Alarcon, Gloria1.,Camila,Navia1.,Kerr,Bredford2.,1LaboratoriodeBiologíaCentrodeEstudiosCientíficos-CECs,UniversidadAustralDeChile.2LaboratoriodeBiologíaCentrodeEstudiosCientíficos.(SponsoredbyFondecyt1140162,PFB01/2007.)

MethylCpGBindingProtein-2(Mecp2)isatranscriptionfactorthatbindsmethylatedCpGdinucleotidesinthepromoterofitstargetgenes,regulatinggeneexpressionthroughtheinteractionwithhistonedeacetylasecomplexorCreb1,andthereforeeithersilencingoractivatinggeneexpression.PreviousstudiesshowthatRettsyndromepatients,asevereneurologicalandprogressivedisordermainlycausedbymutations inMeCP2,exhibitalterations in theageofpubertyonsetandfirstmenarche.Moreover,observations from ourmouse facility suggest a decreased fertility in heterozygous femalemice carrying aMecp2-null allele(Mecp2+/-).However,themechanismunderlyingtheattenuatedfertilityobservedintheabsenceofafullyfunctionalMecp2allelehasnotyetbeencompletelyelucidated.Theaimof thisstudywastocharacterizethereproductivefunctionofMecp2+/-miceandunderstandthemechanismbywhichtheabsenceofafullyfunctionalMecp2allelealtersfemalereproductivelifespan.Toaccomplishouraim,wecomparedthereproductivephenotypeexhibitedbyMecp2+/-andwildtypefemalesassessingfertility,ovarianmorphometry,estrouscycleevaluationsandqRT-PCRforkeygenesrequiredforthepropercontrolofthereproductiveaxis.Our results showadecreasednumberof littersandpupsperdam inMecp2+/- in comparisonwithwild type females. Inaddition,Mecp2+/-exhibitedlongerestrouscyclesincomparisonwithwildtypefemales.Theestrouscycleisorchestratedbytheexpressionofseveralgenesatdifferentlevelsofthereproductiveaxis.InordertoevaluatewhethertheabsenceofafunctionalalleleforMecp2alterstheexpressionofsomeofthesekeygenes,weperformedqRT-PCR.OurresultsshowthattheexpressionoftheGnRHreceptormRNAisincreasedinovariantissue,whiletheKisspeptinreceptorGPR54mRNAisdecreasedinpituitary.Itisworthytonotethatnolitterswereobservedfrom6montholdMecp2+/-femalesinmating,timeatwhichwildtypefemalesarestillfertile.TodeterminewhetheranalterationinfolliculardevelopmentunderliestheprematureendingofreproductivelifespanobservedinMecp2+/-,weassessedamorphometricanalysisofovariesfrom8day-oldMecp2+/-andwildtypefemales.Weobservedasignificantdecreaseinprimordial,primaryandsecondaryfollicles;whichindicatesanalterationinfolliculardevelopmentandmaybeassociated to a reduction in thenumberof follicles in adulthood. In summary,ourdata indicates thatMecp2 is anepigeneticmodification-associatedtranscriptionfactoressentialfortheproperreproductivelifespanandfertilityinfemales.

150

73)Exposure to a high fat diet during pregnancy and nursing increases serum estradiol in the offspring through a decrease in its metabolism

Alvarez, Daniela1.,Reyes,Aldo1.,Ramírez,Luisa1.,Olguín,Sofía1.,Ambrosetti,Valery1.,Guerra,Marcelo1.,Fernandois,Daniela1.,Cerda,Tania1.,Cruz,Gonzalo1.,1LaboratoriodeAlteracionesReproductivasyMetabólicas.InstitutodeFisiología,FacultaddeCiencias,UniversidadDeValparaíso.(SponsoredbyFONDECYTINICIACION11130707(GC)AndCentroDeNeurobiologíaYPlasticidadCerebral(CNPC)OfUniversidadDeValparaíso.)

InChile2outof3peopleiseitheroverweightorobese,andobesityismoreprevalentinwomenthaninmales.Thisleadstothefactthat50%ofwomenhavemalnutritionbyexcessofnutrientsduringpregnancy.Maternalobesityhasanimpactonthenormaldevelopmentofpregnancyandalsocouldcontributetothedevelopmentofreproductiveandmetabolicdiseasesintheoffspring.Sinceexposuretoestrogeniccompoundscouldproducesomesimilaralterationsthanmaternalobesityweaimedtostudythemetabolismofestradiolanditsplasmaticconcentrationsintheoffspringofobesemothers.Spraguedawleyratswerefedwithahighfatdiet(60%Kcalfat)from1monthprevioustopregnancyuntilweaningoftheoffspring.Controlratsreceivedacontroldiet(12%Kcalfat).HepaticexpressionofCYP3A2wasdeterminedbywesternblotandplasmaticestradiolandestriolbyELISAintheoffspring.Analyseswereperformedat1,7,14,30y60posnataldays.Estradiollevelswereincreasedinratoffspringofobesemothersatallages.HepaticCYP3A2expressionwasdecreasedfromposnatalday1untilposnatalday60.Estriolplasmaticlevelswere reducedatposnatalday. Inconclusion,maternalobesitycausesan increase in serumestradiol fromchildhood toadulthood,probablydue toadecrease in theexpressionofhepaticCYP3A2.The fact that there is a reduced levelof estriol,estradiolmetabolite,confirmsalowerhepaticmetabolismofthishormone.

151

74)Cryopreservation induces alterations in the mitochondrial function of Atlantic salmon spermatozoa (Salmo salar).

Figueroa, E3.,Valdebenito,Iván1.,Risopatrón,Jennie2.,Short,S.E3.,Zepeda,A.B3.,Figueroa,C.A3.,Farias,J.G3.,1SchoolofAquacultureCatholicUniversityofTemuco,Temuco,Chile.2BIORENCenterforBiotechnologyinReproductionLaFronteraUniversity,Temuco,Chile.3DepartamentodeIngeniería,FacultaddeIngenieríayCiencias,UniversidadDeLaFrontera.(SponsoredbyFONDECYT1151315(F.JG);FONDEFD10I1064(I.V).CONICYTDoctorateGrant(F.E;S.SE))

Todatetherearefewdataontheeffectofcryopreservationonthemitochondrialdynamicinfishspermatozoa.TheobjectiveofthisworkistoassesstheeffectofcryopreservationonthemitochondrialdynamicinAtlanticsalmonspermatozoa.ThespermwerefrozeninCortland®medium+1.3MDMSO+0.3Mglucose+2%BSAforthetreatment(T);freshsemenwasusedforthecontrol(C).Wedetermined[ATP]withtheCellTiter-Glo®kitand[O2]withtheMitoXpress®Xtrakit.Intheseanalysesweusedelectrontransportchaininhibitorsanduncouplers,namely:rotenone(R,10µM),antimycinA(A,10µM),cyanide(C,0.5µM)and2,4dinitrophenol(D,10µM).Inthecryopreservedspermatozoa(T),thebase[ATP]was5.7±1.2nmoles/109sppresentingsignificantdifferencesfromthecontrol(7.4±0.64nmoles/109sp,p<0.05);likewisethecellsincubatedwithR(2.9±0,78nmoles/109sp),A(3.98±0.92nmoles/109sp),C (1.37±0.66 nmoles/109sp) and D (1.59±0.48 nmoles/109sp) presented statistically significant differences during the first 10seconds of incubation as compared to the control (5.5±0.84 nmoles/109sp; 6.1±0.56 nmoles/109sp; 4.1±0.99 nmoles/109spand 4.9±0.79nmoles/109sp respectively, p<0.05). Thebase [O2] in control spermatozoawas 4230±520RFU/109sp, presentingsignificantdifferencesfromT(3040RFU/109sp);thetreatmentsincubatedwithR(3508±320RFU/109sp),A(3627±480RFU/109sp)andD(4290±429RFU/109sp)presentedsignificantdifferencesfromthecontrol(R:2704±298RFU/109sp;A:2852±570RFU/109spandD:3442±612RFU/109sprespectively,p<0.05).Thechangesinthe[O2]rateinspermatozoainthepresenceofinhibitorsanduncouplersoccurredafter10secondsofincubation.PreliminaryresultssuggestthatcryopreservationinducesalterationsinthemitochondrialfunctionofAtlanticsalmonspermatozoa.

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75)The environmental toxicants induce sperm acrosome reaction (AR) through a protein Kinase A (PKA) pathway

Gallardo,Luz1.,Moreno, Ricardo1.,1Fisiología,CienciasBiológicas,PontificiaUniversidadCatólicaDeChile.(SponsoredbyFONDECYT1150352(RD.M),LMG:BecariaDoctoradoCONICYT)

Environmental toxicants, could affect the reproductive outcome at different levels, but there is no study about if they affectdirectlythedifferentstepsofthefertilizationprosseses.Inthiswork,weselecttwoenvironmentcontaminants,Endothall,awildelyusedpesticide,whichinhibitsthephosphatasePP2Aandnonylphenol,axenoestrogenthatmimictheestrogen.TheaimofourworkwastodeterminewhetherEndothallandnonylphenolinducetheAR,byregulatingactivationofPKA.Tothisend,mouseepididymalcaudalspermatozoawererecoveredwithorwithoutthePKAinhibitorH89and,incubatedwithEndothall,nonylphenol,orprogesteroneasapositivecontrol,incapacitatingornoncapacitatingconditions.TheroleofPKAwasdeterminatedbystudyingitsphosphorylated(pT197)formbyWesterblot.TheARpercentagewasquantifiedbyComassie-GBlueorLysoTrakerdyes.OurimmunofluorescentsresultsshowedthatPP2A,waslocatedinspermtailandacrosomeregions.EndothallinducestheARupto31%inarangeofconcentrationsallowedbytheUSEPAandnonylphenolinducetheARupto19%atconcentrationsfoundinhumanfluid,dependingonspermcapacitationstatus.Moreover,EndothallandnonylphenolpotentiatetheAR inductoreffectofprogesterone.Inaddition,wefoundthatH89preventedtheARinducedbyEndothallandnonylphenol.Finally,EndothallandnonylphenolincreasethelevelofpT197PKA4and2.5times,respectively,regardlessthecapacitationsatusofthespermatozoa.Inconclusion theseenvironmentalcontaminants induce theAR inmicespermatozoa inaPKA-dependentwayand theycoulddisturbedmammalianfertilization.

153

76)Organotypic culture as an in vitro spermatogenesis model: comparison between rat and mouse

Carmona-Rojas, E1.,Berrios-Amaro,C1.,Moreno,R2.,Reyes,J1.,1Chemistry,Science,PontificiaUniversidadCatólicadeValparaíso.2EndocrinologyandReproduction,BiologicalSciences,PontificiaUniversidadCatólicaDeChile.

Spermatogenesisisacomplexprocessthatoccursinsideofseminiferoustubules(ST)inthetestis.GermcellspresentintheSTincludespermatogonia,whichcanproliferateordifferentiate,meioticcellsknownasprimaryandsecondaryspermatocytes,andhaploidcells,spermatidsandsperm.Additionally,Sertolicells(SC)aresomaticepithelialcellslocatedinsidetheST,andLeydigcells(LC)locatedintheinterstitium.LCsynthesizetestosterone,andSCareincontactwithallgermcells,supportingthem.ThereisabloodtestisbarrierformedbytightjunctionsbetweenSC.Inthismanner,SCarecapableofcontrollingtheinternalmicroenvironmentoftheST.TheorganotypicculturemaintainsthestructuralintegrityoftheST,soitisabletoproducein vitrospermatogenesisinmouse.Thelipidrichalbumin,AlbuMAX,seemstobethedeterminantfactorintheculturemediaforcompletionoftheprocessin vitro.ThegoalofthisworkwastocharacterizeandtocomparethistypeofcultureinmouseandratusingmediasupplementedwithKnockoutSerumReplacement(KSR),fetalbovineserum(FBS)withAlbuMAX(AlbM),andFBSwithbovineserumalbuminfattyacidfree(AlbFF).Wecultured7daysBALB/cmiceandSprague-DawleyratsSTfor30and40days,respectively.Theprogressionofthefirstwaveofspermatogenesisineachconditionwasstudiedanalyzing(i)thehistologyofslidesofculturesbybrightfieldandtransmissionelectronmicroscopy,(ii)presenceandlocationofspecificcellmarkersbyimmunohistochemistry,and(iii)cellDNAcontentbyflowcytometry.Spermatogenesisprogressioninratcultureswasminimum,showingafewprimaryspermatocytesattheendinKSRandAlbM.Ontheotherhand,mouseculturesinKSRshowedformationofelongatedspermatidscells,andsomeroundspermatidscellswereseeninAlbM.Inboth,ratandmouse,therewasalittleornospermatogenesisprogressioninAlbFF.Weneverobserved spermatozoawhenculturesweredisaggregated inanycondition.CellDNAcontentanalysis indicated thepresenceofahaploidpopulationonlyinculturesofmouseinKSRandAlbM.Accordingwithit,somehaploidantigenswerepresentinthesecultures.Ourdatasuggestthattherearedifferencesintheregulationofspermatogenesisinmouseandrat.Ratculturesdidnotproceedin vitrobeyondprimaryspermatocyte,probablyduetotheabsenceofsomeregulatorymolecule(s)inthemedia,whichisnotnecessaryinthemouse.ThisworkwassupportedbyFONDECYTN°1140758.

154

77)Novel LC-MS/MS method for simultaneous determination of serum corticosteroids and the role of 11β-HSD enzymes in essential hypertension

Allende, Fidel1.,Benitez,Agustin1.,Silva,Patricio1.,Campino,Carmen2,3.,Vecchiola,Andrea2,3.,Valdivia-Pizarro,Carolina2.,Rojas,Maria4.,Lagos,Carlos2,3.,Baudrand,Rene2.,Carvajal,Cristian2,3.,Solari,Sandra1.,Fardella,Carlos2,3.,1DepartmentofClinicalLaboratories,SchoolofMedicine,PontificiaUniversidadCatolicadeChile.2DepartmentofEndocrinology,SchoolofMedicine,PontificiaUniversidadCatolicadeChile.3IMIIMillenniumInstituteonImmunologyandImmunotherapy.4DepartmentofFamilyMedicine,SchoolofMedicine,PontificiaUniversidadCatolicadeChile.(SponsoredbySupportedByFONDECYT1130427,FONDECYT1150437,CORFO13CTI-21526-P1&IMIIP09/016-FGrants.)

Background Cortisol(F)homeostasisisimportantinmaintainingbloodpressureanditsderegulationisimpliedinhypertension(HT)andmetabolicsyndrome(MetS).TheavailabilityofFisregulatedbytheenzyme11β-hydroxysteroiddehydrogenasetype1(11β-HSD1)whichpreferentiallyconvertstheinactivecortisone(E)toactiveFand11β-HSD2,whichtransformsFtoE,protectingthemineralocorticoidreceptorfromerroneousactivationbyF.Accordingly,theFtoEratioinserumispotentiallyusefulinevaluatingtheactivityof11β-HSDinpatientswithHTAand/orMetS.Therefore,thedetectionofFandEinserumbyliquidchromatographytandemmassspectrometry(LC-MS/MS)isaimedtobeareliable,highlysensitiveandselectivemethod.AimTodevelopandvalidateofaLC-MS/MSmethodforthesimultaneousdeterminationofFandEinserumtobeuseinaclinicallaboratoryforcontributingtoexplainthepathogenesisofhypertensionand/ormetabolicsyndromethrough11β-HSDactivity.MethodsSteroid-freeserumenrichedwithknownconcentrationsofeachanalytewereusedformethodoptimizationandvalidation.Steroidswereextractedfrom300µLof serumwithsolidphaseextraction,usingD4-CortisolandD2-Cortisoneas internal standards,beingafterwardsanalyzedinLC/MSMS,withachromatographiccolumnIntersil®ODS-3andtheionizationsourceinmodeESI+.Forclinicalvalidationofthemethodology,serumfromhypertensivepatients(HT)andcontrols(NT)wereanalyzed.ResultsThemethodquantificationrangewas1-200ng/mLwithaaveragecorrelationcoefficientof0.998forbothanalytes,an85%averagerecovery,matrixeffectbetween85-115%forFandE.LODandLLOQwere0.2and1.0ng/mLrespectively.Thecoefficientofvariation intraand interassaywereandtheaccuracybetween96-105%.ThemethodologyallowedquantifyingFandEinclinicalsamples,findingaveragevaluesforFandEinserumof150and23ng/mLrespectivelyandasignificantdifference(p<0.05)inF/EratiosbetweentheHTandNTevaluated.ConclusionThedevelopedLC-MS/MSmethodisaccurateandpreciseaccordingtoFDAandsuggestedacceptancecriteriaformethodvalidation.Italsoshowedagoodsensitivity,recoveryandmatrixeffectforthesimultaneousmeasurementofFandE.Themethodprovidesaninnovativetooltobeusedasaguidebyclinicalendocrinologisttodeterminetheetiologyofessentialhypertensionand/ormetabolicsyndromethrough11β-HSDactivity.

155

78)Evaluation of functional connectivity between the prefrontal cortex and hippocampus in freely moving mice

Negron, Ignacio1.,Aguilar,Marcelo1.,Espinosa,Nelson1.,Aboitiz,Francisco1.,Fuentealba,Pablo1.,1DepartamentodePsiquiatriaPontificiaUniversidadCatolicaDeChile.(SponsoredbyWeWereSupportedByMilleniumCenterForTheNeuroscienceOfMemory,NC10-001-F,FromTheMinistryForEconomics,FomentationAndTourism,Chile,FONDECYTForPostdoctoralGrantNº3140370ToI.N-O.)

Wehaverecentlyshownthatprenatalstressinducedpersistenceofspatialmemoryinanaversiveenvironmentinadultmice.Thiswasparalleledwithareductionoffiringrateintheprefrontalcortex(PFC),andalterationsoffunctionalconnectivitybetweenthePFCandhippocampus.ThiswasmeasuredwithLFPrecordings in vivo inanesthetizedmiceaftermemoryevaluation.Hence,iftheseneurophysiologicalalterationsarerelatedtoneuralprocessesassociatedtotheencodingofspace,goal,strategyoranotherbehavioralrelevantparameterremainsunknown.

Toevaluatethispossibility,weproposetoassesstheeffectofprenatalstressonneuralactivityinthePFCandhippocampusinfreelymovingmice.Duetothelackofcommercialdevicestorecordactivitysimultaneouslyinthesetwodifferentbrainstructuresinfreelymovingmice,wehavedesignedandconstructedamicrodrivefortetroderecording.

Withourmicrodrive,wehaveobtainedstablerecordingsofLFPandunitsforupto2months,bothinthePFCandhippocam-pus.Recordingsallowedoff-lineanalysisofspectralpowerbothinthePFCandhippocampus,spectralcoherencebetweenthesestructures,andspikesortinginthePFC.WerecordedtheimplantedmiceintheBarnesmazeforevaluationofspatialmemoryinanaversiveenvironment.PreliminaryresultsshowedanincreaseofspectralcoherencebetweenPFCandhippocampusatthebeta-frequencyband(20-30Hz)atthemomentwhentheanimalfoundtheescapebox,suggestingtheencodingofgoallocation.Spectralcoherenceincreasedastheanimalprogressedthroughthetrials,suggestingthatneuralrepresentationisdependentonorrelatedtolearning.Futurestudieswillallowustoevaluatetheeffectofprenatalstressongoalrepresentation,orotherneuro-physiologicalparameters.Finally,ourmicrodrivewillallowustoevaluateneuralactivityinfreelymovingtransgenicmice,includingfortheimplementationofoptogeneticmanipulations.

156

79) Behavioral and electrophysiological indices of a modified error monitoring in meditators.

Andreu, Catherine I.1.,Slagter,HeleenA.2.,Franken,IngmarH.A.3.,López,Vladimir1.,Cosmelli,Diego1.,1EscueladePsicologíaPontificiaUniversidadCatólicaDeChile.2DepartmentofPsychologyUniversityofAmsterdam.3InstituteofPsychologyErasmusUniversityRotterdam.(SponsoredbyThisWorkWasSupportedByTheNationalCommitteOfScienceAndTechnologyOfChile(CONICYT),ThroughADoctoralScholarshipGivenToCatherineAndreu(number21140175).)

Errormonitoringisdefinedastheabilitytomonitorongoingperformanceinordertodetectandcorrecterrors.Impairmentsincognitivecontrolprocesses,suchaserrormonitoring,havebeenassociatedwithseveralpsychiatricdisorders, includingADHDand substanceabuse.Recent research indicates that thepracticeofmeditationas amental training technique,may improvecognitivecontrol.Yet,ifandtowhatextentmeditationmayenhanceerrormonitoringiscurrentlyunknown.Thepresentstudyaddressedthisgapinknowledgeandexaminedeffectsofmeditationpracticeonbehavioralandelectrophysiologicalindicesoferrorprocessingandperformancemonitoring,specificallytheerror-relatednegativity(ERN).Twogroups(meditatorsandnon-meditators controls) performed an Eriksen-Flanker taskwhile their brain activitywas recorded using electroencephalography(EEG).Behaviorally,meditatorsshowedasignificantdecreaseinthenumberoferrorscomparedtocontrols.EEGanalysesrevealedanincreaseintheamplitudeoftheERNcomponentinmeditatorscomparedtocontrols.Thesefindings,whichareindicativeofenhancederrormonitoringinmeditators,suggestthatmeditationcouldbearecommendablepracticetotrainandimproveerrormonitoring.

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80)Gestational stress induces resilience to depressive-like behaviors in the post-weaning

Arriagada, M1.,Dagnino,A.1.,1LaboratoryofBehavioralNeurobiology,CenterforNeurobiologyandBrainPlasticity,InstituteofPhysiology,FacultyofSciences,UniversidadDeValparaíso.

Gestational stress can inducedepressive-like behaviors in rats during early postpartum.However, it’s unknown the effects ofgestationalstressonpost-weaning.Theaimofthisstudywastodeterminetheeffectsofgestationalstress,appliedatlastthirdofratpregnancy,ondepressive-likebehaviorsinthepost-weaning.Sprague-Dawleyratswerepregnantandafterwardsubjectedtodailyrestraintstress(45minutes/3timesperday).Pregnantratsundisturbedandonegroupofvirginratswereusedascontrols.After weaning, depressive-like behaviors were evaluated in the mothers by forced swimming and sucrose preference tests,respectively.Additionally,anxiety-likebehaviorsandlocomotoractivitywereanalyzedbyopenfieldandelevatedplusmazetests,respectively.Damsthatweresubjectedtogestationalstressspentmoretimeinclimbingduringforcedswimmingtestcomparedtocontrolanimals.Gestationalstressdidnotaffectlocomotoractivityanddamswithgestationalstressshowedmorenumberofentriesintotheopenarmsintheelevatedplus-maze.Wehavedevelopedananimalmodelofpostpartumresilience,whichcouldbecomparedwithwell stablishedanimalsmodelofpostpartumdepression.Thus,wecancontribute tounderstandingof theneurobiologicalbasisofpostpartumdepressionandresilience.

Acknowledgements:ThisworkwassupportedbyFONDECYT1141276grant(AlexiesDagnino).Labsite:www.stress.cl.

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81)Characterization of feedback error-related negativity for the study of adaptive behavior and the reward system.

Astudillo, Aland1.,Vinales,Laura2.,Orio,Patricio1.,Quilodran,Rene2.,1CentroInterdisciplinariodeNeurocienciadeValparaíso,FacultaddeCiencias,UniversidadDeValparaíso.2Medicina,FacultaddeMedicina,UniversidadDeValparaíso.(SponsoredbyFB0008FromConicyt(PO).TheCINVIsSupportedByTheMilleniumScienceInititative(MinisterioDeEconomía,Chile))

Prefrontalcortex(PFC)activityisrelatedtocognitivecontrol(e.g.planning,problemresolution,performancemonitoring),withanimportantfunctioninoptimalbehavioraladaptation(Miller&Cohen,2001).PFCactivityisanimportanttargetofdopaminergicafferences from themidbrain,and inneurodegenerativediseases suchasParkinsonDisease (PD), thisnetwork showsalteredactivitysecondarytodopaminedepletioninthebrain(Rodriguez-Orozetal.,2009).Therefore,itisimportanttounderstandhowthesealterationsaffectthepatternof theelectricalactivity.However, thefirststep is tounderstandtherelationshipbetweentheelectricalpatternandtheadaptivebehaviorandtherewardsystem.Hereweproposetoevaluatecognitiveimpairmentsandmeasurerelatedelectrophysiologicalmarkersincontrolsubjectsbyusingelectroencephalography(EEG)whiletheyperformatrial-and-errorproblemsolvingtask.Thisprotocolhasbeendesignedtoevaluateexecutivefunctionsandmodifiedfromeventrelatedpotentials(ERP)analizedinmonkey(Procyk&Joseph,1996;Quilodran,Rothé,&Procyk,2008).Fromthecharacterizationofthepatternofamid-frontalevokedpotential,theFeedbackError-RelatedNegativity(fERN),whichisobservedmainlyinerroneouslyperformancesinvariouscognitivetasks,inassociationtosensoryfeedbacksignalinganerror,wecanobtainaviablebiomarkertocharacterizethePFCactivity(Debeneretal.,2005).Currentresearchinvestigatesthispatternforitsuseinearlydiagnosisofneurodegenerativediseases (Willemssen,Müller, Schwarz,Falkenstein,&Beste,2009). In this studywecharacterize the fERNpatternsobtained fromsubjects,byusing temporaland frequencyanalysisand linking thischaracterizationwith theadaptivepattern.

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82)Determining the role of insular cortex in anxiety: a study of anxiety behavior in different zones of the Insula

Escorza, Tomás1.,Tamburini,Giovanni1.,Méndez,Luis1.,Rojas,Sebastián1.,Díaz-Galarce,Raul1.,Moraga-Amaro,Rodrigo1.,Stehberg,Jimmy1.,1LaboratoriodeNeurobiología,CentrodeInvestigacionesBiomédicas,UniversidadAndrésBello.

Anxietyisacommonsymptomassociatedtodisease,andstressinthegeneralpopulation,whichtogetherwithanxietydisorders,havebecomeahugeunresolvedprobleminpublichealthofemergenteconomies.Inspiteoftheenormousprevalenceofstress,anxiety and anxiety disorders, our current knowledgeon the brain areas andmechanisms associated to anxiety itself is onlybeginningtobeunveiled.Understandingthemechanismsandbrainpathwaysassociatedtoanxietymayleadtothedevelopmentofnoveltreatmentstrategiesanddrugtargets.TheInsulaorInsularcortexisacomplexcorticalstructureburieddeepwiththetemporallobe,whichhasbeenassociatedtomanybrainfunctions,includinganxietydisorders.WesetouttodetermineiftheInsulaisinvolvedinanxietyandtoidentifytheareawithintheinsulathatmediatesanxiety.Tothisend,weperformedintra-insularmicroinjectionofAMPAantagonistCNQXindifferentareaswiththeinsularcortexinrats(rostralanterior(RAIC),mediorostral(MRIC),gustatory(GIC)andtheSomatosensory(SIC))eitherwithoutpreviousstressorafter30minofacuteimmobilizationstress.AnxietywasmeasuredusingtheElevatedPlusMazeparadigm.Resultssuggestdifferentialroleofthemorerostralareascomparedtothecaudalones.Ourresultssuggestthatdifferentinsularcortexareasmayhavedifferentrolesonanxietyandpostulatetheinsulaasacriticalregulatorofanxiety.

ThisworkwasfundedbyProyectoFONDECYTN°1130724

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83)Effect of humor on decision making: a behavioral and electrophysiological report

Flores, Jorge1.,Rodríguez,Eugenio1.,Campos,German1.,1Psychology,SocialSciences,pontificiauniversidaddechile.(SponsoredbyBecaDoctoradoNacional2014)

Ithasbeenshownthatemotionalregulation,andespeciallylosssensitivity(negativeemotionalreactionafterbaddecisions)couldexplaindifferencesindecisionmakingperformanceinwomen,whencomparingwithmenduringtheIowaGamblingTask(IGT)(vandenBos,Homberg&Visser,2013).This“losssensitivityeffect”afterdisadvantageousdecisionscouldbemodulated,infactbehavioralresultssuggestsdownregulationofnegativeemotionsbyhumor(Samson&Gross,2012).SoweproposehumortoaffectIGTperformanceespeciallyduringitsfirststageswhicharesupposedbeguidedbyemotions(Bechara,2005).Inordertosubstantiatethisideatheparticipantswillbeshownhumorousornothumorousshortfilmsprevioustoeachdecisionmakingtrial,andafterdecisionistakenfRNpotentialwillbemeasured.WeexpectatendencytowardsignificantstatisticaldifferencesinIGTperformanceduringthefirst2blocks(trials1-41)obtainingmorelongtermadvantagedecisionsduringthehumorouscondition,especiallyinwomencomparedwithmen.Ontheotherhand,weexpectatendencytowardasignificantstatisticaldifferenceinfRNamplitudeduringthehumorouscondition,specificallyadecreasinginfRNamplitudeduringthefirst2blocksinwomencomparedwithmen.

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84)Effects of n-3 PUFAs supplementation on auditory attention of chronically stressed rats.

Gárate-Pérez, Macarena1.,Dagnino-Subiabre,Alexies1.,1LaboratoryofBehavioralNeurobiology,CenterforNeurobiologyandBrainPlasticity,InstituteofPhysiology,FacultyofSciences,UniversidadDeValparaíso.(SponsoredbyThisWorkWasSupportedByFONDECYT1141276Grant(AlexiesDagnino).Labsite:Www.stress.cl)

Chronicstressimpairsauditoryattentioninrats,whileomega-3polyunsaturatedfattyacids(n-3PUFAs)supplementationinducesanti-stresseffects.Inthiscontext,theaimofthisstudywastoanalyzetheeffectofn-3PUFAssupplementationonauditoryattentionofchronicallystressedrats.Sprague-Dawleyratsweretrainedinatwo-alternativechoicetask(2-ACT),abehavioralparadigmtostudyauditoryattentioninrats.Trainedanimalsthatreachedaperformanceover80%ofcorrecttrialsinthe2-ACTwererandomlyassignedtocontrolandstress(chronicrestraintstress)experimentalgroups.Afterward,adultanimalsweresupplementedwithn-3PUFAs(DHAandEPAmix)orwater.Toanalyzetheeffectsofchronicstressandn-3PUFAssupplementationontheauditoryattention,trainedratsofbothgroupsweresubjectedto502-ACTtrialsonedaybeforeandonedayafterofthestressperiod.Adifferencescorewasdeterminedbysubtractingthenumberofcorrecttrialsafterfromthosebeforethestressprotocol.Stressedratsthatweresupplementedwithn-3PUFAsshowedanincreasesofcorrecttrialsduringthe2-ACTthanthatofstressedanimalstreatedwithwater.Wespeculatethatn-3PUFAssupplementationcouldbeusedinthetreatmentofstress-relatedpsychiatricdisorderstoimprovecognitivefunctionslikeattention.

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85)Characterization of the role of Octopamine and Tyramine on locomotor, olfactory and anxiety-related behaviors in Drosophila melanogaster.

Herreros, Claudia1.,Fuenzalida-Uribe,Nicolás1.,Campusano,Jorge1.,1Biologiacelularymolecular,CienciasBiológicas,PontificiaUniversidadCatólicaDeChile.(SponsoredbySupportedByFondecyt1141233)

Ithasbeenshownthatbiogenicamines(BAs)playanimportantroleinthegenerationofcomplexbehaviors,rangingfromlocomotorcontroltoolfactorylearning.InrecentyearsithasbeenevidenttheevolutionaryconservationoftasksplayedbyDopamineinthegenerationandmodulationofseveralbehaviorsinvertebratesandinvertebrates,includingtheflyDrosophila melanogaster.Lessisknownonthecontributionofotheraminergicsystemstobehaviors.HerewestudiedtheroleofOctopamine(OA)andTyramine(TYR)neuralsystemsininnatebehaviorsinDrosophila.Usingvideorecordings,weanalyzedseveralbehaviorsinsinglemaleflies,inabsenceandpresenceofanaversiveodorant(Benzaldehyde,Bz).WeblockedOA/TYneurotransmissionbyexpressionoftetanustoxinpeptide (Tetx)using theGAL4/UASsystem,andassessedtheeffectof thismanipulation inanimalbehavior.ThisgeneticmanipulationinducedadecreaseinseverallocomotorparametersandalsoinCentrophobism,ananxiety-relatedparameter.Onotherhand,blockingOAand/orTYreleasehaveoppositeeffectsonolfactoryacuitytotheodorant.Furthermore,ourdatashowthatdifferentOArgicclustershaveoppositerolesintheaforementionedparameters.Insum,theseresultssuggestthatOAandTYplaycomplexrolesonolfaction,locomotorandanxiety-relatedbehaviors.

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86)Can you control your attention when you are stressed?

Palacios-Garcia, Ismael1.,Villena-Gonzalez,Mario1.,Artigas,Claudio1.,Jaramillo,Karina2.,Campos,German1.,Silva,Jaime2.,Rodriguez,Eugenio1.,1FacultaddePsicologiaPontificiaUniversidadCatólicaDeChile.2FacultaddeGobiernoUniversidadDelDesarrollo.(SponsoredbyThisWorkWasSupportedByTheNationalCommitteOfScienceAndTechnologyOfChile(CONICYT),ThroughADoctoralScholarshipGivenToIsmaelPalacios(number21140884)AndFundedByFondecyt1130810)

Everydaywehavetodivideourlimitedattentionalresourcesintodifferentexternalandinternaldemands, inaprocesswhichwecoulddefineasattentionalcontrol.ThePsychosocialstress isastatethatpromotestheallocationofattentional resourcesinternally,especiallytothreat-relatedstimulisuchasthesocialevaluation,theaimofthestudyis(1)toinvestigateifpsychosocialstressaffectsthebehavioralperformanceinanattentionalshiftingtaskand(2)tosearch,underanexploratoryapproach,someofitsneuralcorrelates.40healthyparticipantswereexposedtoeitheranelectroencephalogram-compatibleversionoftheTrierSocialStressTest(TSST)oracontrolprotocol.Additionally,immediatelybeforeandaftertheseprotocols,subjectparticipatedintheattentionalshiftingtask.Manipulationcheckswereverifiedthroughthechangesoftheheartrate,salivaryconcentrationofcortisolandthescoreintheanxietyscaleinthe“stress”conditionrespectthecontrol.Whenwecomparedthebehavioralperformanceintheattentionaltaskpriorandafterbothconditions,wefoundthatthecontrolgroupshowedaclearimprovementinperformance,characterizedbyarelativeincreaseofcorrecttrialsandadecreaseofomissions.Analogously,aftertheTSST,participantsdidnotshowedthesamesignificantincreaseofperformance,moreover,weshowedthatasfarasthescoresintheSTAI-StateincreasedaftertheTSST,thenumberofcorrectstrialsdecreasedandthenumberofomissionsincreased.Inadditiontobehavioralresults,wefoundthattheoscillatoryactivityinalpha(8-12Hz)andgammabands(30-50Hz)weredifferentinbothconditions.Behavioralandelectrophysiologicalresults,suggestthatpsychosocialstressdirectstheattentioninternally,affectingtheattentionalcontrolandlimitingtheattentionalresourcesforattendingtheexternaldemands,whichleadstocognitivefailures.

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87)Effects of prenatal stress on the development of depressive-like behaviors in infant rats.

Iturra-Mena, Ann 1.,Dagnino-Subiabre,Alexies1.,1LaboratoryofBehavioralNeurobiology,CenterforNeurobiologyandBrainPlasticity,InstituteofPhysiology,FacultaddeCiencias,UniversidadDeValparaíso.(SponsoredbyThisWorkWasFundedByFONDECYTGrantN°1141276ToAD-S.LabsiteWww.stress.cl)

Depression isamentaldisorderthathasbeenstudiedprimarily inadulthood.However, it iscurrentlyknownthatthisdiseasealsoaffectschildren.Recentresearchhaspointedtoprenatalstressasariskfactortodevelopdepressionandanxietydisordersinadulthood,butitisunknownwhetheritproducesthesameeffectonchildren.Theaimofthisstudywastodetermineifprenatalstressinducesdepressionandanxiety-likebehaviorsinprepuberalrats.Forthis,femaleSprague-Dawleyratsweresubjectedtoarestraintstressprotocolbetweengestationaldays14and21.Controlgroupincludedpregnantratsthatremainedundisturbed.Behavioraltestswereappliedtoallmaleandfemalepupsatpostnatalday24.Locomotoractivity,anxiety-anddepression-likebehaviors (anhedoniaand learnedhopelessness), social interactionandsocialplayweremeasured.While the followingstressmarkersweremeasured:bodyweightgain,adrenalweight,basalandacute-stress (swim inwaterat20°Cduring60seconds)evoked levelsofserumcorticosterone.Prenatalstress increasedthephysiologicalstressmarkers inprepuberalrats,aswellasdepression-likebehaviorsinbothmalesandfemales,althoughtheyshowedsomedifferencesaccordinggender.Inconclusion,wedevelopedanewanimalmodelofchildhooddepressionthatcanbeusefultostudytheneurobiologicalbasisofthisdiseaseatapre-clinicallevel.

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88)Low and high-level visual features modulate saccade-related EEG signals in humans.

Devia,Christ1,2.,José,Egaña4,1.,Montefusco-Siegmund,Rodrigo4,3.,Maldonado, Pedro4.,1DepartamentodeAnestesiologíayReanimación,FacultaddeMedicina,UniversidaddeChile.2DepartmentofBrainandCognitiveSciences,ThePicowerInstituteforLearningandMemory,MassachusettsInstituteofTechnology..3DepartmentofPsychology,CentreforVisionResearch,YorkUniversity.4PDFB,BNI,FacultaddeMedicina,UniversidaddeChile.(SponsoredbyThisWorkWasMadePossibleInPartByAGrantFromCONICYT,FONDECYT/Postdoctorado3140306ToCDAndByICM-P09-015F.)

Ournervoussystemisadaptedtoexploretheenvironmenttriggeringaself-pacedstimuliinput.Invisualprocessing,thisactivesensingofvisualscenesoccursthroughsaccadiceye-movements.Oneachsaccadetheeyereachesavisual location,engagingthebraininprocessingnewinformation.Detailsofthisprocesscomefromworkonanimalmodels,butlittleisknownabouthowthisoccurs inhumans.Here,weshowthatduring self-paced imageexploration, saccadeevent-relatedpotentials (sac-RP)aremodulatedbyscenesfeaturesandbytop-downeffects.Werecordedelectroencephalographicbrainactivityandeyemovementswhilehumansubjectsfreelyexplorednaturalandartificialscenes.Wefoundthatthesac-RPisthelargestbrainactivityrelatedtoself-pacedexploration,itisrestrictedtooccipitalsites,andismodulatedbylow-levelimagefeatures.Inaddition,ourevidencedemonstratesthatunderequallow-levelimageconditions,high-levelimagefeaturesalsomodulatetheamplitudeofsac-RP.Ourresultssuggestthatself-pacedvisualperceptioninhumansisdependentonbothlow-levelandhigh-levelfeaturesofthevisualscene,arguinginfavorofbottom-upandtop-downmodulationsoftheactivityintheprimaryvisualareas.

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89)Studying the neural correlates of Conscious Perception with a Low-Features visual stimulation: P3b as the earliest ERP NCC.

Boncompte, Gonzalo1.,Cosmelli,Diego1.,1PsicologíaPontificiaUniversidadCatólicaDeChile.(SponsoredbyWeWouldLikeToAcknowledgeCONICYTForItsFinancialHelpInTheFormOfAPhDGrantToGB)

Consciousperceptionisanveryfrequentprocessinoureverydaylife,yettheneuralsubstratesnecessaryforitsoccurrencearefarfrombeingelucidated.MostoftheworkonthistopicfocusesonwhathasbeencalledtheNeuralCorrelatesofConsciousness(NCC),neuralactivitythatdirectlycorrelateswithconsciousexperience,yetthereisaconsiderablediversityinwhathasbeenproposedasNCCs.InfluentialtheoriesofconsciousnesslikeGlobalNeuronalWorkspaceandIntegratedinformationTheoryemphasizetheimportanceofneuronal integrationasakeyaspectofperceptual consciousness. In this lineawidespreadbrainactivationofassociativecortices,liketheP3componentoftheEventRelatedPotential(ERP),hasbeenaconsistentcandidatefortheearliestERPNCC.However,severalstudiesreportdifferencesinthemagnitudeofearlyERPslikeP1asNCC,yetearlyERPcomponentslikeC1orP1arerelatedtotheprocessingofvisualfeaturesofwhatispresentedtosubjects,likecolor,shapeandtexture.Thisiswhywebelievethattheyarenotnecessarilylinkedtoconsciousperception,thuswehypothesizedthatifwereducedtheamountof featureprocessing requiredofwhatsubjectsperceive,butwemaintain the fact thatsubjectsconsciouslyperceive it,earlyERPcomponentsshouldceasetocorrelatewithconsciousperception.Totestthiswedesignedavisualdetectionexperimentalparadigmtocompareseenandunseentrials,e.i.thoseinwhichourreduced-featurestargetstimuluswasconsciouslyperceivedandwhenitwasmissed.Inthisparadigmthetargetstimuluscomplexity, intermsoftheamountofearlyneuronalprocessingneeded,wasmaximized.Our results showno ERP component before 200ms in the general ERPwaveform, independently ofwhethersubjectsdetectedthetarget.Thisiscoherentwithourlow-featurestimulation.SecondlyweseearobustP3b,andonlywhen subjects consciously perceived the target stimulus. It appeared consistently for every subject.Moreover, this P3bwasnotonlyamplitude-modulated,butwascompletelyabsentwhensubjectsmissed the target,andwitha typicalmagnitudeascomparedtoresultsobtainedwithcomplexstimulus.OurresultssupporttheideaofP3bastheearliestERPNCC,whichisinlinewithtwomajorintegrativetheoriesofconsciousness.

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90)Dendritic cells are necessary for the upregulation of the intrarenal RAS and renal sodium transporters in Angiotensin II and high salt

Araos, Patricio1.,Hevia,Daniel1.,Fuentes,Eugenia1.,Prado,Carolina2.,Pacheco,Rodrigo2.,Michea,Luis1.,1Fisiología,Medicina,MillenniumInstituteonImmunologyandImmunotherapy,CEMC,ICBM.2LaboratoryofNeuroimmunologyFundaciónCienciayVida.(SponsoredbyFONDECYT1130550,IMIIP09-016-F,BECACONICYT21130482)

ThedevelopmentofhypertensioncausedbyAngiotensin II (AngII) andahigh saltdiet ismediatedbya rapid increase in theexpressionsodiumtubuletransporters,whichleadstoapositivesodiumbalance.Inanormalconditions,theintrarenallevelsofAngIIare≈200timesdeplasmalevelsduetothepresenceoftheintrarenalrenin–angiotensinsystem(iRAS),thetissularexpressionof all the classical componentsof theRenin-Angiotensin–System.Themodulationof renal sodium transportdependson theactivityoftheiRAS.Moreover,recentstudieshaveshownthattheAngIIplushighsaltdiet(HS)treatmentcausetheupregulationoftheiRAS,suggestingthatthiseffectcouldbeamajorfactorcausinghypertensionand/orrenaldamage.OurpreviousstudiesshowedthattheablationofDendriticCells(DC)inmicepreventedthedevelopmentofHTinresponsetoAngII+HS.Inthepresentstudy,weevaluatediftheablationofDCsalterstheupregulationoftheiRASandtubularsodiumtransportersbyAngII+HS.

CD11c.DOGmice,forselectivelossofDCs(CD11cHi)cellsafterDiphteriaToxin(DT) injection,receivedvehicle,AngII+HS(AngII,osmoticminipump450μgKg/day+1%NaClindrinkingwater)orAngII+HS+DT(DT,8ng/g)during14days;PairedWTmicereceivedvehicle,AngII+HSorAngII+HS+DT.Wemeasuredbloodpressure(days0,4,8,14),andatday14weharvestedtissuestomeasuretheabundanceof renalDCs (MHC-II+ andCD11c+ by inmunofluoresce), the iRAS, the sodium-protonexchanger3 (NHE3), thesodium-chloridecotransporter(NCC)andtheEpithelialSodiumChannel(αENaC;qRT-PCRandWesternblot).

TheinjectionofDTpreventedthedevelopmentofHTinresponsetoAngII+HSonlyinCD11c.DOGmice.CD11c.DOGandWTmiceshowedincreasedabundanceofDCsinthecortex(peritubular);OnlytheCD11c.DOGmiceshowedasharpreductionofrenalDCsafterDTinjection(n=3).Both,inWTasinCD11c.DOGmicetheadministrationofAngII+HSincreasedtheiRAS(infoldofinduction:AGT,1.5;ACE,1.9;andAT1R,5),NHE3,NCCandαENaC(infoldofinduction:4.2;6;2),respectivelyvsvehicle–treatedmice(p<0.05;n=5-9).TheinjectionofDTconcomitanttoAngII+HSpreventedthechangesinsodiumtransportersandiRASinCD11c.DOGmice(p<0.05comparedtoAngII+HS;n=5-9).

WeconcludethatDCsarerequiredforthemodulationofiRASandtubularsodiumtransportersbyAngII+HS.

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91)Aldosterone downregulates the expression of Sodium Potassium ATPase β3 subunit in kidney and renal collecting duct cells.

Diaz, Pablo1.,DeGregorio,Cristián2.,Cutiño,Andrea2.,González,Magdalena2.,Michea,Luis2.,1Fisiología,Medicina,UniversidaddeChile.2FisiologíaUniversidaddeChile.(SponsoredbyCONICYT,FONDECYTRegularN°1130550;IMIIP09-016-FAndBECACONICYTN°21120658)

Introduction:Sodiumhomeostasisisregulatedbyaldosterone,inpartbymodulatingactivityoftheNa+-K+-ATPase(NKA)presentinthebasolateralmembraneofprincipalcells(PC)ofkidneycollectingducts(CD).TheNKAisaheterodimerwithacatalyticαsubunitandaregulatoryβsubunit.Theβsubunitsmaybedeterminantofsubcellularlocalizationandtraffickingoftheα-βheterodimers,andalsomayhavea role in thecell-celladhesionthatmayberelevant inparacellularpermeability.Theα1,β1andβ3are theonlysubunit isoformsexpressed in thekidney.Previousstudieshave focusedon themodulationofα1expression in responsetoaldosteroneviathemineralocorticoidreceptor(MR).However,theroleofaldosteroneintheisoform-specificregulationofβsubunitsinthekidneyremainspoorlyunderstoodandthepotentialmodulationoftheβ3subunitbyaldosteronehasnotbeenanalyzed.

Hypothesis:ActivationoftheMRdownregulatestheexpressionofNKAβ3subunitinCDincreasingNKAactivity.

Material and methods:C57Bl/6miceunderwentadrenalectomy(ADX)orshamsurgery(SHAM).TheADXmicereceivedhighsaltdietorhormonereplacementtherapywithdeoxycorticosterone(ADX+DOCA,20mg/mL).Inasecondsetofexperimentmicewereadministeredwithspironolactone(Spi,50mg/Kg/day)orvehicle(Control)treatment.After3dayskidneyswereharvested(cortexandmedulla)fortheanalysisofNKAα1,β1andβ3subunits(mRNAandproteinabundancebyqRT-PCRandWesternblot).Finally,westudiedtheeffectofaldosteroneinprimarycultureofinnermedullarycollectingductscells(IMCD,24hours).

Results:AdrenolectomyandspironolactonetreatmentincreasedtheNKAβ3-subunitexpressioninmouserenalmedullabutnotinkidneycortex(n=9P<0,001increaseof50%,SD0,6;andn=5P<0,05increase120%,SD=1).Incontrast,neithertheADXnorSpimodifiedtherenalabundanceofα1orβ1transcriptsandproteinsinmice.

TheincubationofIMCDcellsinthepresenceofAldosterone10nMfor24hoursdecreasedNKAβ3-subunitexpression(n=3,p<0,05,mean45%,SD0,01)andtheoverexpressionofNKAβ3subunitdecreasedNKAactivity.

Conclusions: TheresultsindicatethattheNKAβ3subunitdecreasestheNKAactivityandthatincrementinNKAactivitycausedbyaldosteronemayinvolvethedownregulationtheNKAβ3subunitexpression.

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92)Role of Angiotensin II and Vasopressin on the expression of Renin in renal collecting duct cells

Gonzalez-Vergara, Alex1.,Salinas-Parra,Nicolás1.,Henríquez,Ricardo2.,Gonzalez,AlexisA2.,1InstitutodeQuimica,FacultaddeCiencias,PontificiaUniversidadCatólicaDeValparaíso.2InstitutodeBiología,FacultaddeKinesiología,PontificiaUniversidadCatólicaDeValparaíso.(SponsoredbyFONDECYT11121217)

Thereninangiotensinsystem(RAS)playsakeyroleontheregulationofarterialbloodpressureandsaltandwaterhomeostasis.OneofthefinaleffectorsoftheRASisvasopressinhormonereleasedinresponsetoanincreaseinplasmaosmolalityandangio-tensinII(AngII)inthehypothalamus.Recently,ithasbeenshownthatallthecomponentsoftheRASareexpressedintherenalcollectingducts(CD),asegmentwithaprimaryroleinthereabsorptionofsaltandwater.Despitethesuppressedreninexpressioninjuxtaglomerular(JG)cellsmediatedbyAngII,reninexpressionisaugmentedinresponsetoAngIIintheCD,whichmaypromotesodiumandwaterreabsorption.WehypothesizethatactivationofV2RincreasesreninexpressionindependentofAT1receptorinCDcells.Inwaterdeprivedmice(48h)pro-reninandreninproteinabundancewasaugmentedinrenalmedullarytissues(freefromJGcells).ToseeifthiseffectwasindependentofRASactivationandonlyduetoosmolality,weusedtheAT1receptorblockerlosartan(30mg/kg)andangiotensin-converting-enzymeinhibitorcaptopril(40mg/kg).Pro-reninbandwasupregulated,eveninthepresenceofRASinhibition(̴1,5fold;P<0.05)suggestingavasopressindirecteffect.Usingintraperitonealinjectionsofmannitol(20%)asastrategytoincreaseplasmaosmolalityweobservedanincreaseinpro-reninandreninproteinabundancesinmedullarytissues(pro-renin̴1,4fold;renin̴1,5fold;P<0.05).Theseresultsdemonstratethatvasopressinstimulatesreninsynthesisindepen-dentofRASactivation.

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93)Angiotensin-(1-7) prevents the skeletal muscle atrophy induced by myostatin decreasing the Smad signaling pathway.

Aravena, J1.,Simon,Felipe2.,Cabello,Claudio1.,1DepartamentodeCienciasBiológicas,FacultaddeCienciasBiológicas,UniversidadAndrésBello.2DepartamentodeCienciasBiológicas,FacultadCienciasBiológicas,UniversidadAndrésBello.(SponsoredbyAssociation-FrancaiseContreLesMyopathiesAFM#16670;FONDECYT#1120380,1121078;IMII#P09-016-F;UNABDI-741-15/N.)

Skeletalmuscleatrophyischaracterizedbythelossofmusclemassandstrengthmainlyduetoanincreaseinthedegradationofmyofibrillar proteins suchasmyosinheavy chain (MHC).Myostatin (MSTN) is a soluble factor that regulatesmusclemass.Its levels are augmented in several typesofmuscle atrophy.Among theeffectsofmyostatin is the increaseof theubiquitin-proteasomesystem(UPS)activity,speciallyanincreaseofAtrogin-1andMuRF-1,twoE3ubiquitinligaseswhichresultinmuscleproteinbreakdown.MSTNpromotesskeletalmuscleatrophythroughSmadsignalingpathwaywhencoupledspecificallytoactivinreceptor.

Ang(1-7)isapeptidefromnon-classicaxisofrenin-angiotensinsystem(RAS).WehavepreviouslydemonstratedthatAng-(1-7)hasanti-atrophicactivityinskeletalmuscleinmodelsdependentonangiotensinIIandlipopolysaccharide,twomodelsinwhichwehavefoundhighlevelsofcirculatingMSTN.WeevaluatedtheeffectofAng(1-7)onthemuscleatrophyandSmadsignalinginducedbyMSTN.

Forthat,C2C12myotubesweretreatedwithMSTNinabsenceorpresenceofAng(1-7)andseveralparametersofatrophyweremeasured:myotubediameters,proteins levelsofMHC,Atrogin-1andMuRF-1. Smadsignalingpathwaywasevaluatedby thephosphorylationofSmad2(pSmad2).

OurresultsindicatethatAng-(1-7)hasapreventiveeffectuponatrophyparametersinducedbyMSTN.MyotubediametersandMHCproteinlevelswerediminishedinpresenceofMSTN,whereasAtrogin-1,MuRF-1andpSmad2wereincreased.Interestingly,Ang(1-7)preventedtheatrophiceffects inducedbymyostatinrestoring itsvaluetoasimilar levelsobserved intheuntreatedmyotubes.

Insummary,ourresultssuggestthatAng-(1-7)isapeptidecapabletopreventtheinductionofatrophicparametersandSmadsignalingpathwayinducedbyMSTN.

Funding:Association-FrancaiseContreLesMyopathiesAFM#16670;FONDECYT#1120380,1121078;IMII#P09-016-F;UNABDI-741-15/N.

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94)Hydrogen peroxide and nitrite increase in exhaled breath condensate after low-intensity aerobic exercise in non-trained active subjects

Tuesta, Marcelo1,2.,Araneda,Oscar3.,1UDACienciasdelaSalud,CarreradeKinesiología,FacultaddeMedicina,PontificiaUniversidadCatólicadeChile.2PhysiologyandImmunologyLaboratory,FacultyofBiology,UniversidaddeBarcelona.3IntegrativeLaboratoryofBiomechanicsandPhysiologyofEffort(LIBFE),KinesiologySchool,FacultyofMedicine,UniversidaddelosAndes.(SponsoredbyFundedByFondoDeAyudaALaInvestigación(FAI),UniversidadDeLosAndesProjectINOGTO2013,AndTheNationalFundForScientific&TechnologicalDevelopment(FONDECYT),ProjectNumber11130082.)

It is a well-established fact that exercise increases pro-oxidants and promotes oxidative stress; however, this phenomenon has been poorly studied at lung level. In non trained subjects, it has been observed an increase on pulmonary pro-oxidants after high intensity aerobic exercise for nearly an hour, but it has not been studied in low-intensity exercise and less in under-trained subjects. Pro-oxidative generation (H2O2, NO2

-), lipid peroxidation markers (MDA) and inflammation (pH) in exhaled breath condensate (EBC) were obtained from 11 active under-trained subjects. All subjects completed two sessions of cycloergometric exercise at low intensity (30-40% Heart Rate Reserve) and equal lung ventilation during 30 and 90 minutes respectively. Samples from both protocols were obtained immediately before, at 20 and 80 minutes post exertion. There were no differences in lung ventilation between both exercise tests, moreover heart rate remained within the established ranges for both protocols. On [H2O2]EBC an increasement was observed at 80 post in the 30 min protocol (Pre: 0.13±0.13 µmol·l-1 and Post80: 0.24±0.17 µmol·l-

1;p<0.05). This same finding was observed on the 90 min protocol (Pre: 0.08±0.08 µmol·l-1 and Post80: 0.15±0.01 µmol·l-1; p<0.05). [NO2

-]EBC showed a tendency towards an increase at 80 post in the 30 min protocol, while there was an increase in the 80 post on the 90 min protocol (Pre: 0.35±0.49 µmol·l-1and 0.92±1.66µmol·l-1;p<0.05). There were no differences in [MDA]EBC on both protocols. pHEBC values showed no variations in the 30 min protocol (p=0.35), while there was a tendency towards increase in the 90 minute protocol (p=0.086). In conclusion, low intensity exercise increases lung originated pro-oxidatives in under-trained subjects. There was no evidence of changes on lipid peroxidation or early inflammation.

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95)DIFFERENTIAL EXPRESSION OF CHOP AND GADD34 IN HUMAN FETAL ENDOTHELIUM FROM GESTATIONAL DIABETES.

Valdivia, Luz1.,Susana,Rojas1.,Andrea,Saavedra1.,Astrid,Haensgen1.,Marcela,Cid2.,Marcelo,Farías3.,Marcelo,Gonzalez1.,1Depart-mentofPhysiologyUniversidadDeConcepción.2Obstetrics&ChildcareUniversidadDeConcepción.3DivisionofObstetricsandGy-naecologyPontificiaUniversidadCatólicaDeChile.(SponsoredbySupportedByVRID-Associative213.A84.014-1.0AndFONDECYT1121145/11100192.)

Gestationaldiabetesmellitus (GDM)hasbeenassociatedwith fetoplacental vasculardysfunction,a conditionassociatedwithchangesinvascularresponsestodifferentagonistlikeU46619(thromboxaneA2analog)andinsulin,whichmodulatethevasculartoneinnormalplacentaindifferentways.TheC/EBPhomologousprotein10,CHOP,andDNA-damageinducibleprotein,GADD34,areassociatedwithendoplasmic reticulum (ER) stress. ThemechanismofGADD34 inductionduring cellular stress isnotwellunderstood.TheERstressisaconditionlinkedtomaternalobesityandGDM,bothpathologiesassociatedwithinsulinresistance(IR).

Objectives:TodeterminetheexpressionofCHOPandGADD34infetalendotheliumandvascularreactivityofplacentalcirculationfromGDMinthepresenceandabsenceofU46619andinsulin.

Methods:NormalandGDMsampleswereobtained fromHospitalGuillermoGrantBenavente,Concepción (ethics committeeapprovalandinformedpatientconsentwereobtained).Humanumbilicalveinendothelialcells(HUVEC)wereisolated(collagenasedigestion)andmaintainedinmedium199(M199)withsera(20%).TotalRNAwasisolatedandCHOP,GADD34and28SexpressionwasdeterminedbyRT-PCR.AsuitablefetalveinandarterypaironthesurfaceofthechorionicplateofnormalandGDM,leadingtoaperipheralcotyledon,wascannulatedandcontinuouslyperfusedwithaKrebssolution(95%O2/5%CO2,pH7.4,37°C).Afterstabilization,theeffectsofU46619(5nM)and/orinsulin(0.1nM)onperfusionpressureweredetermined.

Results:InGDM-HUVEC,CHOPandGADD35mRNAwereincreased(p<0.05)2.8-foldand2.5-foldrespecttocontrol,respectively.ThehighlevelofCHOPmRNAwasrevertedbyinsulin(0.1nM)butGADD34mRNAlevelswasnotalteredbythehormoneinGDM.U46619(10nM)increased(5-fold)perfusionpressureofisolatedcotyledonofplacentaandpre-incubationwithinsulinattenuated(53%)thisresponse.InGDM,U466619increasesthepressure(3-fold)butthereisnoeffectofpre-incubationwithinsulin.

Conclusions:ThereisanincreaseofcontractileresponseinGDMplacentaassociatedwithhighexpressionofCHOPandGADD34infetalendothelium.InsulinrelaxationisimpairedinGDM,linkedtonullregulationofGADD34expressionbythehormone.

SupportedbyVRID-Associative213.A84.014-1.0andFONDECYT1121145/11100192.

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96)Inducción de estrés oxidativo en el hígado graso experimental y suproyección sobre la funcionalidad hepática en ratones machos.

Lopez-Ortega,Aura1.,Marquez,Ysabel1.,Sanabria,Mariana2.,Plaza,Miguel3.,Murillo,MaríaDivina3.,1CienciasBásicas,CienciasVeterinarias,CentroccidentalLisandroAlvarado.2MedicinayCirugía,CienciasVeterinarias,CentroccidentalLisandroAlvarado.3FarmacologíayFisiología,CienciasVeterinarias,Zaragoza.

Eldañocelulardebidoalestrésoxidativo(EO)desempeñaunafunciónvitalenlapatogénesisdelasalteracioneshepáticas,entreellaselhígadograso(HG).Enmedicinaveterinaria,elroldelEOenlaproducciónyreproducciónanimalhaadquiridorelevanciadebidoaldeteriorodeambasfuncionesenanimalesconhepatoesteatosis.ElobjetivodeesteestudiofuedeterminarsielHGexperimental inducidopor etionina (7,5mg/20 g peso corporal), en ratonesNMRImachos adultos, provocaba EO y afectabala funciónhepática.Seutilizarondosgruposde10animales:unocontrolyotro tratadoconDL-etionina.ElHGseevaluópormétodoshistológicosyporcuantificacióndelostriglicéridoshepáticos,queindicaronhepatoesteatosisenlosmachosinyectadoscon etionina. Como parámetros de EO, se determinó por espectrofotometría la concentración hepática de malondialdehído(MDA) y de dienos conjugados (DC). Para evaluar la funcionalidad hepática se cuantificó la concentración plasmática delas aminotransferasasALT y AST a través de kits comerciales. La inducción deHG causó una elevación significativa delMDA:de364,91±17,73nmoles/mgproteínasa852,91±55,26 nmoles/mgproteínas(P<0,001),asícomode losDC:de231,18±15,53mmoles/mgproteínasa297,45±23,10mmoles/mgproteínas(P<0,027).EnelHG,laactividadplasmáticadelasaminotransferasasaumentósignificativamente:ALTde59,40±5,16U/la169,86±18,78U/l(P<0,001)yASTde158,35±13,54U/la241,93±10,14U/l(P<0,05).Estos resultadosmuestranqueenelHG inducidoporetioninaenratonesmachosseproduceunEOquepodríaserresponsabledelaalteraciónenlafuncionalidadhepática.

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97)Cannabinoid receptor type 1 modulates the effects of polyunsaturated fatty acids on memory consolidation of stressed rats

Jujihara, Germán1.,Dagnino-Subiabre,Alexies1.,PeñalozaSancho,Valentín1.,1LaboratoryofBehavioralNeurobiology,CenterforNeurobiologyandBrainPlasticity,InstituteofPhysiology,Sciences,UniversidadDeValparaíso.(SponsoredbySupport:ThisWorkWasFundedByFONDECYTGrantN°1141276ToAD-S.Labsite:Www.stress.cl.)

Ithasbeenshownthatomega-3polyunsaturatedfattyacids(n-3PUFAs)haveanti-stresseffectsinrats,whileendocannabinoidsregulate learning and memory. Thus, relationship between PUFAs and endocannabinoid system on stress responses remainunknown.Theaimof thisstudywastoevaluatewhethercannabinoidreceptortype1 (CB1) regulatestheeffectsofPUFAsonmemoryofstressedrats.MaleSprague-Dawleyratsweresubjectedtochronicrestraintstress.Inthecourseofthestressperiod,animalsweresupplementedwithn-3PUFAs(fishoil)orn-6PUFAs(primroseoil)aswellasvehicle(i.p.,physiologicserum)orAM251(CB1antagonist;i.p.,0.3mg/kg/day)orWIN55,212-2(CB1agonist;i.p.,0.5mg/kg/day).Afterward,CB1levelsweredeterminedby immunofluorescence and memory consolidation was evaluated in the Morris water maze. CB1 levels were up-regulatedinCA1andCA3of stressedcompared to thatof controls,whileAM251andn-3PUFAs improved thestress-inducedmemoryimpairments.Moreover,n-6PUFAsimpairedmemoryandthiseffectwaspreventedbyAM251,butnotbyWIN55,212-2.Theseresultssuggestthatchronicstressandn-6PUFAsincreasestheendocannabinoidsystemactivity,whichinturndecreasesmemoryofrats.Conversely,AM251andn-3PUFAswoulddeclinetheeffectsofstress-inducedendocannabinoidsystemoveractivityinthehippocampus.ThisstudyopensanewapproachtounderstandtheinteractionsbetweenstressandPUFAsbyendocannabinoidsystem.

175

98)Structural insights on the rP2X4 receptor channel allosteric activation by alfaxolone from electrophysiology to molecular dynamics simulations

Alveal, Natalia2.,García-HuidobroToro,JuanPablo1.,Navarrete,Camilo2.,Barrera,NelsonP.2.,1Biología,QuímicayBiología,UniversidadDeSantiagoDeChile.2Physiology,BiologicalSciences,PontificiaUniversidadCatólicaDeChile.(SponsoredbyFundedByICMP10-035F,Fondecyt1141132And1120169,AndAnilloACT-1108Grants.)

Danio rerioP2X4receptorcrystals(zfP2X4R)with(holo)andwithoutATP(apo)intheorthostericsiteofferedthepossibilityofunderstandatthestructural/atomiclevelthefunctionofthisreceptorchannel.Alfaxolone,aprototypeneurosteroid,activatesCa2+currentsinRattus novergicusP2X4receptor(rP2X4R)throughapositiveallostericmodulation.Largersteroidconcentrations,throughaninteractionoccurringlikelyinthetransmembrane(TM)rP2X4Rdomain,elicitedper seanioniccurrentsuggestingporeopeningintheabsenceofATP(Codocedoetal.,2009).OuraimistocharacterizethesteroidbindingsiteandunderstandhowthestructureoftheporechangesinresponsetoalfaxolonebindingtotheTMdomainofrP2X4RintheabsenceofATP.BasedonthecrystallizedzfP2X4Rintheapoandholostates,correspondingrP2X4RmodelsoftheextracellularandTMdomainsforbothstateswerebuilt,includingtheNandC-terminuscytoplasmictailswhichareabsentinthecrystallizedzfP2X4R.Thesestructureswereusedfortheconstructionofthreemodels:apo,apowith3dockedalfaxolonemolecules(apo-alfax),andholorP2X4R.All-atommoleculardynamics(MD)simulationsofthethreemodelsembeddedinalipidmembraneenvironmentandsolvatedwererunfor100nanoseconds.MDanalysisoftheapo-alfaxtrajectorymapsthesteroidbindingsitetotheTMdomainandshowhowtheneurosteroidinteractiontriggersinitialconformationalchangesintheTMdomainthatpermitwaterpassthroughthegate.Resultsrevealthat intheapostate,3alfaxolonemolecules interactwiththeTMdomainthroughoutthesimulation;however,onlyasinglesteroidformshydrogenbondsacrossthesubunitinterfaceoftheTM,whiletheothertwosteroidmoleculesonlybindtotheTMdomainofthebindingsubunit. It isalreadyknownthatP2X4Ractivation involvestheseparationbetweenTMhelicesofneighbouringsubunitsintheporearea(Hattori&Gouaux,2012).SinglealfaxolonebindingpartiallyopenstherP2X4Rpore,revealedbythe increases inthesolventaccessiblesurfaceareaforTMresidueswhencomparingtheapo-alfaxandapostates.ThisfeatureisalsoobservedbetweentheapoandholostructuresofbothrP2X4RandzfP2X4R.AlfaxolonebindingfurtherproducesastrongeffectonthegreaternumberofhydrogenbondsbetweentheTM1andTM2ofthesubunitthatinteractswithit,reminiscentoftheTMconformationintheholostate.ThesestudiesprovideinsightsofthemechanismforpositiveallostericactivationofrP2X4R,helpingtounderstandthemotionoftheTMdomainduringrP2X4Rchannelgating.

176

99) Molecular dynamics simulations of the dynamin-2 mutation R465W: impact on dynamin-2 monomer structure and dimer interactions

Hinostroza, Fernando1.,Maraboli,Vanessa2.,Cardenas,Ana3.,Gonzalez,Danilo2.,1CentroInterdisciplinariodeNeurocienciadeValparaíso,Ciencias,UniversidadDeValparaíso.2CentreforBioinformaticsandIntegrativeBiologyUniversidadAndrésBello.3CentroInterdisciplinariodeNeurocienciadeValparaísoUniversidadDeValparaíso.(SponsoredbyThisWorkHasBeenSupportedByProyectoAnilloACT-1121(CONICYT)AndCentroInterdisciplinarioDeNeurocienciaDeValparaíso(CINV).)

Dynamin-2isacytosolicGTPasethatplaysakeyroleinseveralcellularprocesseslikeendocytosis.Specifically,itcatalyzesmem-branefissionduringendocytosis.Thisproteinhasfivedifferentdomains:aGTPasedomain(Gdomain)thathydrolizeGTP,amiddledomainthatinteractswithotherdynamin,apleckstrinhomologydomain(PH)thatbindsphospholipids,aGTPaseeffectordomain,andaproline-argininerichdomain(PRD)thatinteractswithSH3proteincontainingdomain.Mutationsinthisenzymeproducecentronuclearmyopathy(CNM),acongenitaldiseasecharacterizedbyaprogressivemuscularweakness.Themostcommonmuta-tionisR465Winthemiddledomain.In vitro,thismutationformsmorestableoligomersandhydrolizesmoreGTPthanwildtypedynamin.Intheabsenceofcrystallographicstructure,astructuralmodelofdynamin-2hasbeenbuilt,usinghomologymodelingtechniques.UsingthisnewmodelandMolecularDynamics(MD)simulations,wecanelucidatestructuralfeaturesproducedbyR465WmutationinDynamin-2monomeranddimer.Weobservedpreliminarilythatexchanginganarginineforatryptophanintheposition465increasethenumberofinteractionsbetweendynaminmonomers.Ourresultsexplainatmolecularlevelwhythismutationformsmorestableoligomersin vitro.

177

100)The endogenous agonist dopamine at the D1 dopaminergic receptor. A molecular dynamics study

Hugo, Estefanía1.,Fierro,Angelica2.,Cassels,Bruce1.,1dequimica,deciencias,UniversidadDeChile.2deorganica,químicayfarmacia,PontificiaUniversidadCatólicaDeChile.(SponsoredbyGrantsFromFONDECYT,N°1110146,AndCONICYT,N°24121296.)

Dopamine(DA)ischemicalmessengerinbloodvesselsanddifferentorgans.Inthecentralnervoussystem(CNS)itmodulatesneuronal activity related tomany disorders such as addictions, schizophrenia, depression and Parkinson’s disease.However,themechanism involved in dopamine receptor (DR) function is still unknown. Some authors have proposed, on the basis ofmutagenesis studies andmolecular simulations, theways in which various residues in transmembrane (TM) segments III, VandVIImightparticipateincomplexesbetweenagonistsandtheD1receptortype(D1R),

1,2butthespecificrelationshipsamongthemandDAhasnotbeenexploredyet.Suchstructuralstudieshavegivenussomeinsightsaboutthegeneralarchitectureandconformational statesofG-protein coupled receptors (GPCR), although themolecularmodels usedhave reliedon the crystalstructuresofnon-DAreceptorsastemplates.UsingthecloselyrelatedD3Rcrystalstructureasatemplate,wehavebuilta3-DstructureoftheD1RbyhomologymodelinginordertodescribeitstransitionfromtheinactivetotheactivestateonbindingDA.OurmolecularsimulationresultsshowthatDAstaysinthebindingpocketproposedforcatecholaminereceptors,formingasaltbridgebetweentheprotonatedaminegroupandaspartateresidueAsp3.32.LesspredictableonthebasisofcurrentknowledgeisanevolvingnetworkofhydrogenbondsbetweenthecatecholmoietyandTMV,TMIIIandTMVIwhichseemstobecrucialforreceptoractivationandakeytothedesignofnewD1Ragonistdrugs.Importantly,thesaltbridgenearthecytoplasmicendsofTMIIIandTMIVbreaksasaconsequenceofDAbinding,ashadbeenproposedaspartoftheGPCRactivationmechanism.1. J. P. Cueva, A. Gallardo-Godoy, J. I. Juncosa, P. A. Vidi, M. A. Lill, V. J. Watts, D. E. Nichols, Journal of Medicinal Chemistry. 54, 5508 (2011)2. B. R. Chemel, L. A. Bonner, V. J. Watts, D. E. Nichols, Molecular Pharmacology, 2012, 81, 729 (2012)

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101)Pharmacophore and shape-based virtual screening identification of selective 11β-HSD1 inhibitors

Lagos, Carlos F.3,1.,Vecchiola,Andrea3,1.,Ortiz-Canales,David3.,Allende,Fidel2.,Fuentes-Ibacache,Nataly3.,Fuentes,Cristobal3.,Gonzalez-Gomez,LuisMartin3.,Solari,Sandra2.,Baudrand,Rene3.,Campino,Carmen3,1.,Cifuentes,Mariana4.,Carvajal,Cristian3,1.,Fardella,Carlos3,1.,1IMIIMillenniumInstituteonImmunologyandImmunotherapy.2Dept.ofClinicalLaboratories,SchoolofMedicine,PontificiaUniversidadCatolicadeChile.3DepartmentofEndocrinology,SchoolofMedicine,PontificiaUniversidadCatolicadeChile.4InstituteofNutritionandFoodTechnology(INTA)UniversidaddeChile.(SponsoredbySupportedByCORFO13CTI-21526-P1,FONDECYT1150437&1130427,AndIMIIP09/016-FGrants.CFLAcknowledgesOpenEyeScientificSoftwareAndInteligand:GmbHForAcademicLicenseOfTheirProducts,AndTheDTP/NCIForProvidingTheCompoundsScreenedInThi)

Background 11-betahydroxysteroiddehydrogenase type1 (11β-HSD1), regenerates cortisol (F) from inactive cortisone in keymetabolictissues,andisatherapeutictargetforcentralobesity,andmetabolicsyndrome.Hereinwereporttheidentificationofnovelselectivenon-steroidal11β-HSD1inhibitorsusingpharmacophoreandshape-basedvirtualscreeningandin vitro assaysinhumanadiposecell-line.MethodsCrystalstructuresofhuman11β-HSD1wereretrievedfromtheProteinDataBankdatabase.Proteinpreparation,structuralalignment,andligandclusteringwereperformedusingDiscoveryStudiov2.1(AccelrysInc).Structure-basedpharmacophoremodelsweregeneratedwithLigandScoutv3.1 (Inteligand),and ligand three-dimensionalalignment foreach clusterwasused to generate the shape-basedqueries. TheOpenNCIdatabase (~260,000 compounds) conformersweregeneratedbyOMEGAv2.5.1.4(OpenEye).VirtualscreeningofthedatabasewasperformedwithROCSv3.2(shape-queries)andfurtherscoredandrankedbyaccomplishmentwiththecorrespondingpharmacophoremodel.CompoundswereobtainedfromtheDevelopmentalTherapeuticProgram(DTP/NCI),and11β-HSD1activityassaysperformedindifferentiatedLS-14adiposecellline.Cortisone(E)andCortisol(F)productionwerequantifiedbyLC-MS/MS.ThecitotoxicityofthecompoundswasdeterminedusingtheMTTassay(Promega).ResultsAsetof2511β-HSD1-ligandcomplexeswasstructurallyaligned,andtheCα-RMSDforthesuperimpositionofallproteinswas less than2Å.Enrichment rates for theshape-queryandpharmacophoremodelswerehigherthan75%.Thetop1000hitsfromshape-basedqueryvirtualscreeningfilteredwiththepharmacophoremodelsrenderthebest100hits.Afinalselectionof40compoundswasobtainedfromtheDTP/NCIandbiologicallyassayed,showingnoeffectovercellularviability.Inhibitionassaysidentified2novelhitcompoundsdisplayingenzymeinhibitoryactivitiesinthelowmicromolarrange in cell-based assay.Conclusion Virtual screening and subsequent in vitro evaluation of promising hits revealed severalselectiveinhibitors.Efficientinhibitionofhuman11β-HSD1-mediatedcortisolproductioninLS-14adipocyteswasdemonstratedfor2compounds,whichdisplayselectivityforthereductaseactivityof11β-HSD1andover11β-HSD2isoform.

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102)New N-Arylsulfonylindoles based serotonin 5-HT6 antagonists. Synthesis and binding evaluation studies

Lagos, Carlos F.1,2.,Vera,Gonzalo3.,Almendras,Sebastian3.,Hebel,Dan3.,Flores,Francisco3.,Valle-Corvalan,Gissella4.,Pessoa-Mahana,CarlosDavid3.,Mella-Raipan,Jaime4.,Recabarren-Gajardo,Gonzalo3.,1DepartmentofEndocrinology,SchoolofMedicine,PontificiaUniversidadCatolicadeChile.2FacultaddeCienciaUniversidadSanSebastian.3DepartmentofPharmacy,FacultyofChemistry,PontificiaUniversidadCatolicadeChile.4DepartmentofChemistryandBiochemistry,FacultyofSciences,UniversidaddeValparaiso.(SponsoredbySupportedByProyectoFONDECYTINICIACION11121418.)

Background The5-HT6receptoristhemostrecentlyidentifiedandclonedmemberoftheserotonin(5-hydroxytriptamine,5-HT)receptorsfamily.Previousstudiesdemonstratedthat5-HT6receptorhasamajorroleinobesity,thusboostingthesearchfornovelselective5-HT6receptorantagonists.Allthesereportedantagonistsshareacommonpharmacophoreconsistingofasulfonamidemoietyseparatedfrombasicionizableaminefunctionalitybyanarylgroupandlinker.Basedonthisknownpharmacophoremodelfor5-HT6receptorantagonists,aseriesofnovelderivativesbasedontheindolescaffoldweredesignedandidentifiedasanewclassof5-HT6receptorligands.GoalsTodesign,synthezise,determinethepharmacologicalprofileandidentifystructure-activityrelationships for a novel series ofN-arylsulfonylindole compounds, targeted to 5-HT6 receptor.Methods The pharmacophoremodelforN-arylsulfonylindoleclassof5-HT6ligandswasexplored.Thedesignedcompoundsweresynthezisedbyclassicorganicchemistry and structures confirmed through spectroscopicmethods. Radioligand competitionbinding assayswereperformedagainst[125I]-SB-258585,inHEK293cellsexpressinghuman5-HT6receptorswithClozapineascontrol.Thepharmacologicalprofilewas assessed by intracellular calciummobilization assay using 2-Me-5HT as control. Results All compounds tested displayedinhibitionof[125I]-SB-258585bindingto5-HT6receptors.Compounds4b,4f,4g,4i,and4jwerethemostpotentcompoundswithKivaluesof13.6nM,369nM,18.4nM,149nMand14.6nMrespectively,andClozapinedisplayedaKivalueof11.9nM(IC50valueof12.4nM).Allligandsevaluatedshowedantagonistprofile,andreducetheeffectsofadditionof2-Me-5-HT.Inthisassaythemostpotentcompoundwas4jwhichIC50valuewas32nM.CoMFAcontourmapsanalysisindicatesthatmajorcontributiontoactivityisgivenbythestericpropertiesofthecompoundsratherthantheelectrostaticpotentials. ConclusionWepresentthedesign,chemicalsynthesis,biologicalevaluationandCoMFAbasedQSARstudiesofnovelantagonistsofthe5-HT6receptor.Aconvenientsynthesisoftheextendedarylpiperazinederivativeswasachievedtoreadilyaccessdiverselysubstitutedanalogues.Severalofthetestedcompoundsexhibitednanomolaraffinity for the5-HT6 receptor.Finally, twocompounds (4gand4j) showedstronginhibitionof2-Me-5HT inducedCa2+mobilization ina cell-basedassay, suggesting thepotent cellularactivitymaybe inducedthroughantagonismof5-HT6receptor.

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103)Kappa opioid control on dopamine basal levels in dorsal striatum: study of no-net flux microdialysis

Azócar, V.H.1.,Aguilera,C.1.,Fuentealba,J.A.1.,1LaboratoriodeNeuroquímica,DepartamentoFarmacia,FacultaddeQuímica,PontificiaUniversidadCatólicaDeChile.(SponsoredbySponsoredByFONDECYTN°1141088)

Thedopaminergicneurotransmissionintheventralanddorsalstriatumisinvolvedinseveralfunctionslikemotorcontrol,motivation,learningandreinforcement.Theextracellularlevelsofdopamine(DA)intheventralstriatumareregulatedbyacomplexinteractionbetweendopaminergicD2auto-receptors(D2R),kappaopioidreceptors(KOR)anddopaminetransporter(DAT)(Meiergerdeta.,1993;Cheferetal.,2005).However,thereisalackofevidenceregardinghowthesesystemscontrolDAextracellularlevelsinthedorsalstriatum.TheaimofthepresentstudyistoassesstheeffectofKORandD2receptorblockadeonbothDAextracellularlevelsasDATactivityintheratdorsolateralstriatum(DLS).Conventionalmicrodialysisexperimentswerecarriedouttostudytheeffectraclopride(D2antagonist)andnorBNI(KORantagonist)onDAextracellularbasallevels.TheDATactivitywasstudyusingtheno-netfluxmicrodialysistechnique(SmithandJustice,1994).Theresultsshowadose-dependentincreaseinDAextracellularbasallevelsafternorBNIperfusioninDLS(169.7±19.08%relativetoDAbaseline,p=0.0412).Alsonor-BNIperfusionsignificantlyincreasetheextractionfraction(Ed),anindirectmeasureofDAuptake(0.243±0.016versus0.311±0.005,p=0.0235).Surprisingly,theDAbasallevelsremainconstantaftertheperfusionofseveralconcentrationofracloprideinDLS.Moreover,anon-significantdecreasedinEdwasobservedafterracloprideperfusioninDLS(0.243±0.016v/s0.196±0.016,p=0.091).TheseevidencesdemonstratethatKORexertsatonicinhibitorycontrolonDAextracellularlevelsinDLSandsuggestapresynapticcontrolofdynorphinonDArelease.OurresultsalsodemonstratethatKORactivationdecreaseEdandsuggestthatdynorphindecreasesDAuptakeinDLS.Ontheotherhand,a tonicD2R inhibitorycontrolonDLSDAextracellular levelswasnotobservedwith raclopridedosesused in thiswork.RegardingtoEd,ourresultssuggestthatD2RactivationincreasestheDAuptake.Inconclusion,intheDLStheKORcontrolsbothDAextracellularbasallevelsasDATactivity.However,inDLSD2RappearstocontrolonlyDATactivity.Theseresultsshowthatdopaminergicneurotransmission inthedorsolateralstriatumdiffersfromthatobserved intheventralstriatum,whereanimportantD2Rmediatedpresynapticcontrolisobserved.

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104)GR2 and α1-receptor expression in the PVN and effects of their activation on the hypothalamic-pituitary-adrenal axis in fetal malnourished rats.

Pérez,Hernán1.,Urrutia,Patricia1.,Sánchez,Sussan1.,Aravena,Marcela1.,García,Carolina1.,Morgan,Carlos2.,Barra,Rafael3.,Hernández, Alejandro4.,1LaboratoriodeFisiologíaPatológica,FacultaddeMedicina,UniversidadPedroDeValdivia.2LaboratoriodeNutriciónyRegulaciónMetabólica,InstitutodeNutriciónyTecnologíadelosAlimentos(INTA),UniversidadDeChile.3UnidaddeFarmacología,FacultaddeCienciasMédicas,UniversidadDeSantiagoDeChile.4DepartamentodeBiología,FacultaddeQuímicayBiología,UniversidadDeSantiagoDeChile.(SponsoredbyFundedByFondecyt1080684,FondecytPostdoctoradoRB3130573AndAGrantFromPedroDeValdiviaUniversity.)

Inuterocalorie-malnourishedratsdevelophypertensionwhenadults.Inpreviousstudiesweshowedthat,inmalnourishedrats(i) basal neuronal activity in theparaventricularnucleus (PVN)of thehypothalamus is increased, (ii) excitatorynoradrenergicinputtothePVNfromthelocuscoeruleusisenhanced,and(iii)corticosteronefeedbackcontrolofthehypothalamic-pituitary-adrenalaxis isdecreased.TostudywhetherhyperactivityofPVNneuronsinprenatalmalnutrition-inducedhypertensionistheresultof increasednoradrenergicinputand/ordecreasedcorticosteronefeedbackcontroltothePVN,westudiedtheeffectofthe intra-PVNmicroinjectionof theα1-adrenergic agonist phenylephrineor theGR2agonist dexamethasoneon themultiunitneuronalactivityofthePVN,systolicpressureandheartrate,andplasmalevelsofcorticosteroneinratsundernourishedduringfetallifeandincontroleutrophicanimals.Fetalmalnutritionwasinducedbyrestrictingthedietofpregnantmothersto10gdaily.Inaddition,theBmax(Scatchardanalysis)andtheexpressionlevels(immunohistochemicalassay)ofGR2andα1-adrenoceptorsweremeasuredinthewholehypothalamusandthePVN,respectively.Atday40ofpostnatallife:(i)onedayafteradministration,dexamethasoneintra-PVNinducedsignificantlylowereffectsonPVNneurons,systolicpressure,heartrateandplasmalevelsofcorticosteroneinmalnourishedanimals,ascomparedtocontrols;(ii)thirtyminafterinjection,phenylephrineintra-PVNproducedsimilarincreasesofPVNneuronalactivity,systolicpressureandheartrateinmalnourishedandcontrolanimals;and(iii)BmaxandexpressionlevelforGR2wereenhancedinthehypothalamusandPVNofmalnourishedrats,whiletherewerenodifferencesfortheα1-adrenoceptor.Datasuggestthatthe increasedactivityofPVNneuronsandthesubsequenthypertensionmainlyresultsfromadecreasedsensitivityofPVNneuronstocorticosteronenegativefeedbackfoundinmalnourishedanimals,ratherthanfromincreasedsensitivitytocoerulearnoradrenergicinput.

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105)Purinergic Signaling differentially regulates the proliferation of Normal and Gastric Cancer cells through P2Y2 and P2X4 receptors

Hevia,MaríaJosé1.,Ramírez,Sebastían1.,Bernal,Giuliano1.,Coddou, C1.,1DepartamentodeCienciasBiomédicas,FacultaddeMedicina,UniversidadCatólicaDelNorte.(SponsoredbyFONDECYT11121302)

Gastric Cancer (GC) is the leading cause of cancer-induced deaths in our countrywith an estimatedmortality of 20/100000inhabitantsover the last ten years. Previously,wehave found that themRNA for thepurinergic receptor P2Y2 is significantlyincreasedinGCsamplesascomparedtoadjacenthealthymucosatakenfrompatientsdiagnosedwithGC.TheexpressionoftheP2Y2receptorisincreasedinothertypesofcanceranditsactivationpromotescellproliferationthroughtheGq-IP3/DAGsignalingpathway.InordertostudytheroleofpurinergicsignalinginGC,weusedAGScells,acelllinederivedfromagastrictumor,andperformedproliferationstudieswithMTT.ATPregulatedAGScellproliferationinabiphasicmanner,increasingcellproliferationat10and100μM,butat300μMATPcellproliferationwassignificantlyinhibited.Ontheotherhand1-300μMUTP,aselectiveP2Y2agonist,increasedcellproliferationinaconcentration-dependentmanner.TheeffectsofUTPwerepreventedbytheadditionofthewiderangepurinergicantagonistsuramin.Moreover,wefoundthatATPandUTPcanelicitincreasesinintracellularcalciuminAGScells,confirmingthefunctionalexpressionofpurinergicreceptors.ThesedifferencesbetweenATPandUTPsuggeststhatthereareotherpurinergicreceptorsexpressedinAGScells.TosearchforotherP2receptorsweperformedreal-timePCRandfoundthatbesidesP2Y2,AGScellsalsoexpresstheP2X4receptor,a ligand-gated ionicchannel.Western-blotanalysisconfirmedthepresenceofbothP2Y2andP2X4.Finally,westudiedtheexpressionofthesereceptorsinbothtumoralandhealthytissuesderivedfrompatientsdiagnosedwithGCandfoundthatwhereasintumor-derivedtissuetheexpressionofP2Y2issignificantlyincreased,theexpressionofP2X4issignificantlydecreased,ascomparedtohealthytissues.Takentogether,theseresultsdemonstratetheinvolvementofdifferentpurinergicreceptorsandsignalinginGC,andthepatternofexpressionchangesintumoralcells,andthischangeprobablydirectsATPandnucleotidesignalingfromananti-proliferativeeffectinhealthycellstoaproliferativeeffectintumoralcells.

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106)THYROID HORMONE INDUCES LIVER PROTEIN DISULFIDE ISOMERASE AND ENDOPLASMIC RETICULUM OXIDO REDUCTIN-1α BY A REDOX-SENSITIVE MECHANISM.

Cornejo, P1.,Fernández,Virginia2.,Vargas,Romina3.,Carrasco,Juan4.,Fernández,Javier4.,Videla,Luis4.,1EscueladeTecnologíaMédica,FacultaddeSaludyOdontología,UniversidadDiegoPortales.2ICBM,ProgramaFarmacología,Medicina,UniversidadDeChile.3ICBM;Programafarmacología,Medicina,UniversidadDeChile.4ICBM,ProgramadeFarmacología,Medicina,UniversidadDeChile.(SponsoredbyAcknowledgements:ProyectoFONDECYT1150104)

T3triggersoxidativestress(OxS)intheliver,linkedtoitscalorigeniceffectwithconcomitantendoplasmicreticulumstressandPERKactivation,inresponsetoproteinoxidation(UPR).TheeffectofT3andtheantioxidantN-acetylcysteine(NAC)onthePDI/ERO1αsystem,catalyzingdisulfidebondsgenerationinUPR,wasevaluated.FollowingtheadministrationtoSpragueDawleyratsof0.1mgT3/kgand/or0.5gNAC/kgfor3consecutivedays,oxidativestressparametersandtheexpressionofPDIandERO1α(qPCRandWestern-blot)wereassessed.T3inducedsignificantenhancementsintherectaltemperature(p<0.05)andinthelivercontentof8-isoprostanesandoxidizedproteins,withconcomitantlyelevatedmRNAandproteinlevelsofPDIandERO1α.AstheseeffectsweresuppressedbyNAC,itisconcludedthatOxShasacausalroleinPDI/ERO1αinduction,essentialforUPRanditsroleinproteinhomeostasisinliverOxEinjury.

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107)Glycine Receptor β Subunit: A Critical Target for Pain Sensitization

Galaz, Pablo1.,Cespedes,Nicole1.,Jorquera,Manuel4.,Treuer,Adriana2.,Ponce,MaríaJosé4.,Utreras,Jonathan4.,Coronado,Cesar3.,Mariqueo,Trinidad4.,1DepartamentodeBioquímicayBiologíaMolecularUniversidadDeChile.2InstitutodeQuímicadelosrecursosnaturalesUniversidadDeTalca.3DepartamentodeMorfo-función,Escuelademedicina,UniversidadDiegoPortales.4EscuelademedicinaUniversidadDeTalca.(SponsoredbyProgramaDeExcelenciaDeInvestigaciónEnQuímicaYBioorgánicaDeRecursosNaturales(PIEI-QUIM-BIO),UniversidadDeTalca)

Glycinereceptors(GlyRs)arethemaininhibitorychloridechannelsthatcontroltheexcitabilityofspinalcord.Plasticchangesintheexcitabilityofperipheralandcentralnociceptivepathwaysaccountsfordevelopmentofchronicpain.Previousstudieshaveshowntherelevanceofα3homomericGlyRsinpainsensitizationinducedbyspinalPGE2inflammation.AlimitednumberofworkshaveinvestigatedtheallostericmodulationofβsubunitinαβheteropentamericGlyRs.Recently,wereportedthathomomericGlyRsaremoresensitivetotheeffectsofethanolandGprotein;therefore,βsubunitmaybenegativelymodulatingα1βpharmacologicalprofileinresponsetoethanol(Mariqueoetal.,2014).Consideringthepharmacologicpotentialofthismodulation,thepresentstudyexaminestheexpressionofβsubunitinaratneuropathicchronicpainmodel(CCI).WesternblotanalysisofCCIspinalcordsamplesrevealedanincreaseinβsubunitexpressionincomparisonwiththecontrol(Sham),after3daysofpaindevelopment(mean±SEMrelativeabundance18±1and28±1A.U.intheShamandCCIslices,respectively,P˂0.05,n=6).Similarresultswereobservedbyimmunofluorescenceofβsubunitinspinalslices(mean±SEMfluorescenceintensity15.6±1and47.6±2A.U.intheShamandCCIslices,respectively,P˂0.001,n=4).RT-qPCRanalysisshowedincreasedlevelofβgeneexpressionat10daysafterCCIsurgery(2.2-fold,P˂0.001,n=5).Theseresultshighlighttheimportanceofdeterminingtheroleofβsubunitinallostericmodulationofglycinereceptorsasastrategyagainstchronicpainestablishment.

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108)Cx43 hemichannels play a critical role in neuroinflammatory responses promoted by prenatal stress or epilepsy

Maturana, Carola1.,Lagos,Carlos2.,Sáez,Juan1.,1Fisiología,CienciasBiológicas,PontificiaUniversidadCatólicaDeChile.2Endocrinología,Medicina,PontificiaUniversidadCatólicaDeChile.(SponsoredbyAceleradorUCPMIPUC1206AndP09-022-FFromICM-ECONOMIAGrants)

Uponinflammasomeactivationcellsreleaseinflammatorymediators(e.g.,IL-1β)thatmayplayacrucialroleinthedevelopmentofneuroinflammationpresentinneurodegenerativedisorderssuchasepilepsyanddifferentpsychiatricdisorders.Thepossibleroleofconnexin(Cxs)andpannexin1(Panx1)hemichannelaswellasP2X7receptor(P2X7R)intheATP-inducedinflammasomeactivationhasbeensuggested.Now,wehypothesizethatCx43hemichannelsplayarelevantroleincellsensorsoftheCNSmicroenvironment,allowinga strong response to stressoranepileptogenic condition,whilePanx1hemichannelsandP2X7Rmightbe critical formaintainingtheneuroinflammation.Tothisend,weusedoffspring(1dayold)ofcontrolanddexamethasonetreated(last5dayofgestation)mice.Thehemichannelactivitywasdeterminedusingtheethidium(Etd,“snapshot”)uptakeassayinacutehippocampalslices.Thehemichannelactivityincreasedinmicroglia,astrocytesandoligodendrocytesfromoffspringofstressedmothersandwasinhibitedbyhemichannel(10panx1andGap26peptides)andP2X7R(A740003)blockers.Thisresponsepersistedforatleastfourpostnatalweeks.Therefore,theinflammasomeofglialcellsisactivatedbystressviaPanx1/Cx43hemichannelsandP2X7Rsandsince ismaintainedfora longpostnataltime itmightalterperinatalneurogenesisandconnectomic.WealsohypothesizethatincreasedneuronalactivitypromotesactivationofglialcellCxhemichannels,whichmightperpetuateaconditionthatfavorsrecurrenceofneuronalactivityinepilepsy.WeusedD4,aselectiveCxhemichannelblockeridentifiedbyvirtualscreeningoftheNCIdatabasetowardstheCx26crystalstructure.Adultmalemiceweretreatedwithpentylenetetrazol (PTZ),anepileptogenicagentthatactsasnonselectiveagonistofGABAAreceptors.Seizureswereevidentat~7minafterPTZadministrationfollowedbyaperiodofseveralhours,inwhichmicepresentedverylowmotoractivityandsporadiccontractionswith~60%survival.However,D4-pretreatedanimalsshowedonlyabriefconvulsionperiodat~7minpost-PTZadministrationandthen,behavedascontrolanimalswith100%survival.Inaddition,D4inhibitedcompletelythePTZ-inducedglialcellHCactivity.Thus,weproposetheglialcellCxHCsasnovelanticonvulsanttargets.

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109)Effect of supraphysiological aldosterone level on adipogenesis of human liposarcoma cell line SW872

Fuentes-Zuñiga, Cristobal1.,Gonzalez-Gomez,LuisMartin1.,Allende,Fidel2.,Fuentes-Ibacache,Nataly1.,Ortiz-Canales,David1.,Campino,Carmen1,5.,Carvajal,Cristian1,5.,Cifuentes,Mariana3.,Solari,Sandra2.,Owen,Gareth4.,Kalergis,Alexis5.,Lagos,Carlos1,5.,Vecchiola,Andrea1,5.,Fardella,Carlos1,5.,1DepartmentofEndocrinology,SchoolofMedicine,PontificiaUniversidadCatolicadeChile.2DepartmentofClinicalLaboratories,SchoolofMedicine,PontificiaUniversidadCatolicadeChile.3InstituteofNutritionandFoodTechnology(INTA)UniversidaddeChile.4DepartmentofPhysiology,FacultyofBiologicalSciences,PontificiaUniversidadCatolicadeChile.5IMIIMillenniumInstituteonImmunologyandImmunotherapy.(SponsoredbySupportedByProyectoSOCHED13-6,IMIIP09/016-F,CORFO13CTI-21526-P1AndFONDECYT1150437&1130427Grants.)

Background Excessive adipose tissue growth has been correlated with cardiovascular risk factors included in the metabolicsyndrome. The adipose tissue differentiation is characterized by increased expression of gene markers (C/EBPβ, PPARγ andHSD11B1)andtriglycerideaccumulation.Cortisol isaknownpromoterofdifferentiationcommitmentbysignalingthroughtheglucocorticoid andmineralocorticoid receptors (GR&MR) in the adipocyte. Recent reports show that Aldosterone (ALD), thenaturalligandoftheMR,canbesynthesizedinfat.Inprimaryaldosteronism,ALDlevelscanincreaseupto1000-fold(10nM),buttheeffectofsupraphysiological levelsofALDintheproliferationanddifferentiationofhumanpreadipocyteshasnotbeenwellestablished. Aim TostudytheeffectofasupraphysiologicalconcentrationofALD inproliferationanddifferentiationofahumanliposarcomacell lineSW872invitro.Methods TheeffectofALD(0.1and10nM)inproliferationofpreadipocytes,wasstudiedinSW872cellculturesmaintainedingrowingmediumplus1%corticoid-freefetalbovineserumfor12and24hr.DNAwasmarkedwithpropidiumiodideandquantifiedbyflow-cytometry.ThepopulationinproliferationwasdefinedasthesumofS,G2andMcellularcycle(%oftotalcellpopulation).ThedifferentiationprocesswasinducedwithanadipogenicMIXfor48hr.TheeffectofALD(0.1and10nM)addedintheMIXwasevaluatedfor7days.Differentiationmarkersexpressionwasstudiedfor48hrbyqRT-PCR.LipidaccumulationwasmeasuredbyOilRedOstainingandquantifiedbyspectrophotometryondays3and7ofdifferentiation.Results Untreatedpreadipocytespopulationinproliferationatinitialtimewas36%,reducingto23%at12hrandanincreaseof27%at24hrs.At0.1nMofALDshoweda25%proliferationat12hr(2%lessrespecttotimecontrol)anda26%at24hr.10nMALDgeneratesnochangesat12hrrespecttothecontrol,butshowedadecreaseto24%at24hr(3%lessrespecttotimecontrol).ThelevelsofdifferentiationmarkersC/EBPβ,PPARyandHSD11B1mRNAshowedanincreasewheninducedwithMIXrespecttountreated.WhenMIXplusALD0.1nMwasadded,HSD11B1showedashift(24hours)initsmaximumpeak.MIXwith10nMALDdidnotaffectmarkersexpression,andlipidaccumulationexperimentsshowedthatMIXwith10nMALDincreasetriglyceridesaccumulationatday7respecttotheMIXcondition.ConclusionSupraphysiologicalaldosteronetreatmentstendtoreduceproliferationandpromotedifferentiationofpreadipocytes.

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110)Environmental enrichment alters the expression of hypothalamic genes associated with food intake

Hernández, Sergio1.,Guzmán,Luis1.,Kerr,Bredford2.,1LaboratoriodeBiologíaCentrodeEstudiosCientíficos-CECs,UniversidadAustraldeChile.2LaboratoriodeBiologíaCentrodeEstudiosCientíficos-CECs.(SponsoredbyFondecyt1140162,PFB01/2007)

Thepropercontrolofbodyweightrequirestheintegrationofmetabolicsignalsbythearcuatenucleusofthehypothalamus(Arc),whichthroughtheprojectionofneuronalaxonstowardtheparaventricularnucleus(PVN)drivesfeedingbehavior.PomcandAgrpexpressingneuronsarefirstorderneuronslocatedintheArcandthroughthesensingofperipheralsignalreleaseendogenousagonistorantagonistofmelanocortinreceptor4 (MC4R) inthePVN,drivesatietyorappetite,respectively.Previousevidenceexistsdemonstratingthatthesensitivityofhypothalamicneuronstometabolicsignalscanbemodulatedbyenvironmentalfactors.Moreover,thehypothalamusmaintainshighlevelsofplasticityevenduringadulthood,whichstronglysuggestsaroleforneuronalplasticity inbodyweightcontrol.Totestourhypothesis,wehoused129/B6wildtypemalemice,sinceweaningat3weeksofage,inenvironmentalenrichmentconditions(EE),whichisawidelyusedparadigmtoinducesynapticplasticity.TodeterminetheeffectoftheEEonmousebodyweight-relatedphenotype,micewereweighedweeklyandlocomotoractivityandfoodintakewereassessedinmetaboliccagesat7weeksofage.Todeterminechangesintheexpressionoffeedingbehavior-associatedgenes,weevaluatedtheexpressionofhypothalamicgenesfrom7-weekoldmicehousedinEEorstandardconditionsbyqRT-PCR.

OurresultsshowthattheexposuretoEEincreasedmousefoodintake,howeveritdidnotalterbodyweight.WealsoobservedthatEEinducedanincreaseinlocomotoractivity,whichmaycontribute,atleastinpart,tomaintainingbodyweightinspiteoftheincreasedfoodintake.TodeterminethemechanismunderlyingtheincreasedfoodintakeobservedinmiceexposedtoEE,weassessedhypothalamicgeneexpression.WefoundanincreaseintheexpressionofAgrp,whichisapivotalgenethatcontrolsfoodintakeandwedidnotdetectanychangeintheexpressionofeitherPomcorMC4R.Bodyweightmaintenanceinspiteofincreased food intakeobserved inmice exposed to EE, strongly suggests the remodeling of neuronal circuits driving feedingbehaviorandenergyexpenditure.Toassessthishypothesis,wearecurrentlyperformingmorphologicalanalysisinhypothalamifrommiceexposedtoEEorstandardconditionstoevaluatethesynapticplasticity-associatedparameterinArcandPVNneurons.Insummary,ourresultsshowthat EEexposurealtersgeneexpressionandfeedingbehaviorwithoutmodifyingbodyweight,whichstronglysuggestsremodelingofhypothalamicneuronalcircuitsdrivingfeedingbehaviorandenergyexpenditure.

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111)Cold stress decreases serotonin release in rat paraventricular nucleus

Jara, Pablo1.,Lara,Hernán2.,Espinosa,Pedro3.,Sotomayor-Zárate,Ramón3.,1LaboratoriodeFarmacología,FacultaddeQuímicayBiología,UniversidaddeSantiagoDeChile.2LaboratoriodeNeurobioquímica,FacultaddeCienciasQuímicasyFarmacéuticas,UniversidaddeChile.3LaboratoriodeNeuroquímicayNeurofarmacología,FacultaddeCiencias,UniversidaddeValparaíso.

HypothalamicParaventricularNucleus(PVN)isabrainstructurethatsynthesizeandreleaseneuroendocrinehormonescontrollingendocrinefunctionsandisresponsibleforintegratestresssignalsandmodulatetheirresponse.ColdstressisaphysiologicstressormodulatedbyPVN;inthiscontext,ratsexposedtodifferentcoldstressprotocolsshowedincreasedperiphericsympatheticnervousactivityandactivatingcentralthyrotropin-releasinghormone(TRH)neuronsinthePVN;thisactivationismediatedbymultiplebrainregionsrelatedtonoradrenergic,glutamatergicandGABAergictransmission.Thepresentstudyexploretheeffectofcoldstressontheserotonin(5-HT)and5-Hydroxyindoleaceticacid(5-HIAA,itsmajormetabolite)releasebymeasuringthesemoleculesinthePVN,inananimalmodelofcold-stress.Forthispurpose,wemeasured5-HTand5-HIAAlevelsinhypothalamicPVNtissuesection incontrol (roomtemperature)andstressed female rat (4ºC for64hours)usinganHPLCcoupled toaelectrochemicaldetector.Intheseexperiments,weobservedadecreasein5-HTand5-HIAAlevels(50%and65%,respectively)incoldstressedrats.Furthermore,theratioof5-HIAA/5-HTshowedatendencyofincrease,whichcouldindicatesanaugmented5HTmetabolization.Insummaryourresultsshowsthatcoldstressinducesadecreaseinserotoninand5-HIAArelease,probablyduetoanincreaseintheserotoninergicactivity,supportingtheroleofserotoninintheactivationoftheTRH-neuronsbutnotdiscardingotherPVNneurons.Grantsupport:DICYT021443JP,UniversidaddeSantiagodeChiletoP.Jara,FONDECYT1020581,1050765and1090036toH.E.Lara.

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112)Individual susceptibility to obesity and the role of the orexin and dynorphin peptides.

Perez-Leighton, Claudio2,1.,Gac,Lily2.,Mella,Ricardo2.,Ramirez,Beatriz2.,1DepartmentofFoodScienceAndNutritionUniversityofMinnesota.2FacultaddeMedicinaUniversidadAndresBello.(SponsoredbyProyectoCONICYTPAI8230017,ProyectoFONDECYTREGULAR1150274,ProyectoInternoUNABDI-523-14/R)

Understandingtheneuronalandbehavioralmechanismsmediatingenergyexpenditureiskeytodevelopingsuccessfulanti-obesitytherapies.Ourlaboratoryfocusesonhowthehypothalamicorexin/dynorphin(ox/dyn)modulateenergybalanceinanimalmodelsofobesity.Asanobesitymodel,wefedmiceacafeteria(CAF)diet.OurdatashowsthatmicefedCAFdietcanbeclassifiedaseitherobesityprone(OP)orobesityresistant(OR)basedonwhethertheirincreaseinfatmasspercentwashigher(OP)orlower(OR)comparedtomaximumvalueofcontrolfedmice.Ourdatashowsthat increasedspontaneousphysicalactivity(SPA),butnotdifferencesincaloricintake,iskeyforexpressionoftheORphenotypeandthatOP,butnotORmiceshowdecreasedsucrosepreference. Interestingly,ORmicefedwithaCAFdietpersonalizedfor individualsusceptibilitytoobesityshowedhyperphagiacomparedtoOPmice,butfailedtodevelopobesity.Thesedatahasvalidatedamodelofdifferentialsusceptibilitytoobesitytoexplorethefunctionoftheorexinanddynorphinneuropeptides.Thehypothalamicparaventricularnucleus(PVN)isanimportantbrainsiteforregulationoffeedingbehaviorandphysicalactivity.Werecentlydemonstratedthenon-opioidDYNpeptideDYN-A2-17inPVNincreasesfoodintake,SPAandinteractswithorexin-A(OXA)toenhancetheirindividualeffectsinfoodintake.Currently,weareexploringtheeffectsoforexinanddynorphinpeptidesonfoodintake.Intheseexperiments,wemeasuredresponsestoorexinanddynorphinpeptidesinPVNbeforeanafterfeedingwitheithercontrolCAFdiet.Ourcurrentdataindicatethat,priortothedietaryinterventions,DYN-A1-13andOXAbothsignificantlyincreasedshort-termchowintake.InOPandcontroldiet-fedmice,weobservednochangeintheabilityofOXAorDYN-A1-13toincreaseshort-termchowintakerelativetothepre-dietarytreatment.Incontrast,ORmiceincreasetheirfoodintakeafterOXAinjectionandnochangesintheeffectivenessofDYN-A1-13.Wearerepeatingtheseexperiments andextending these results testing if voluntaryphysical activity (wheel running) alters geneexpressionoftheorexinanddynorphingenes inthelateralhypothalamusandbehavioraleffectsofthesepeptidesinjectedintoPVN.Theseexperimentswill improveourunderstandingofthebehavioralandneuronalmechanismsmediatingindividualsusceptibilitytoobesity.

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113)Amyloid-β peptide increases P2X2 receptor levels, modifying the intracellular distribution of Fe65 which affects the amyloidogenic pathway

Godoy, Pamela1.,Barra,Karen1.,Silva-Grecchi,Tiare1.,Fuentealba,Jorge1.,1Physiology,BiologicalSciences,UniversidadDeConcepción.(SponsoredbyThisWorkHasBeenFundedByTheFONDECYTProject1130747.)

Alzheimer’sdisease(AD)isaneurodegenerativedisease,mainlycausedbyanimbalanceintheprocessingoftheAmyloidPrecursorProtein(APP),inwhichtheproductionoftheamyloid-βpeptide(Aβ)isincreased.RecentstudieshavesuggestedthatthepurinergicreceptorsP2XmayhavearoleintheAβtoxicitymechanisms.Ontheotherhand,Fe65isamultidomainadaptorproteinwhichinteractswiththeYEMPTYsequencepresentintheamyloidintracellulardomain(AICD)ofAPP;thissequenceisalsoknownasanendocytosissignalforAPPanditenablestheamyloidogenicpathwaywhichtakesplacemostlyatendocyticcompartments.ItiswidelyknownthatFe65interactswithAICDandTip60-anacetyltransferasehistone-andthiscomplextranslocatetothenucleustoparticipateinthetranscriptionofdifferentgenes.But,thereiscontradictoryevidencefortherolethatFe65hasintheAPPprocessing;nonetheless, the interactionwiththeYEMPTYsequencecouldpreventtheendocytosisofAPPandAβproduction.Besides, ithasbeendescribedthatFe65 interactswiththeP2X2receptor, interactionthatwecorroborated.Furthermore,wehaveseenanincreaseintheP2X2expressionaftertreatmentwithAβ.TheaimofthisworkwastostudyhowtheincreaseontheexpressionlevelsoftheP2X2receptoraffecteditsinteractionwithFe65,theAPPprocessingandAβformation.Toachievethis,westudiedtheP2X2andFe65presenceanddistributioninPC12cellsaftertreatmentwithAβ(0.5µM,24h)byimmunofluorescence,andalsoweoverexpressedP2X2bytransfectiontoquantifypossibleAβchangesinthecellularlysates.WeobservedanincreaseintheP2X2presenceaftertreatmentwithAβ(C:100±3%,Aβ:130±7%),whileFe65didnotshowanyglobalchanges(C:100±3%,Aβ:97±4%);however,adecreaseofitspresenceinthenucleuswasobserved(C:100±5%,Aβ:84±5%).Atlast,weobservedanincreaseinAβlevelsfromcellularlysatesaftertransfectionwithP2X2(C:100%;P2X2:216±48%).Inconclusion,thesedatashowthatAβpeptideinducesanincrementofP2X2expressionlevels,whileFe65expressionremainsstablebutitshowsadiminishmentonitsnuclearlocalization.ThiscansuggestthattheincreaseinthereceptorcouldgenerateasequestrationofFe65,whichwouldexplainthatthisprotein is lessavailableto interactwithAICDandthereforetotranslocatetothenucleus.Theseresultsalongwiththe increasethatweobserved intheAβproductionaftertransfectionwithP2X2,couldmeanthatthe increase inP2X2RsequestratesFe65,reducingavailabilityofthisproteintointeractwithAPP,whichcouldleadtoanincreaseinendocytosisofAPPandthesubsequentproductionofAβpeptide.

191

114)Effect of dichlorvos in spatial learning and memory during the ontogeny of Sprague-Dawley rats

Pancetti,Floria1.,Gámiz, Fernando1.,1CienciasBiomédicas,Medicina,UniversidadCatólicaDelNorte.(SponsoredbyThisResearchWasSupportedByTheFONDECYTPostdoctoralGrantNº3140437AndFONDECYTRegularGrantN°1140856)

Dichlorvos(DDVP)isanorganophosphate(OP)thathasbeenmainlyusedasapesticideandalsoasacognitiveenhancerduetoitsinhibitoryeffectonacetylcholinesterase(AChE)enzyme.Howeverrecentstudieshaveshownthatthisdrugmayactthroughalternativemechanisms toAChE inhibition.Previousstudieshaveshownthatwhentheenzymeacylpeptidehydrolase (APEH)is selectively inhibited by low doses of DDVP positive changes in neural plasticity and cognitive performance are observed.NeverthelesswehavefoundthatthesameDDVPdosagegivenduringtheontogenyofratsproducesbiphasicresponsetriggeringbeneficialpharmacologicaleffect inyoungratsthroughtheenhancementof learningandmemoryandatoxicologicaleffect inoldratsimpairingsuchprocesses.InordertodeterminetheDDVPdosesdisplayingnootrophicortoxiceffectsinlearningandmemorywehavechronicallyinjectedduring28daysSprague-Dawleyratsof1and3month-oldand1year-oldwitharangeofDDVPdoses:0.03,0.1,and2mg/kgperday.Afterthetreatment,ratsweretestedintheMorriswatermazetoassessthespatiallearningduringfivetrainingdaysandmemory24hafterthetraining.Afterthis,ratsweresacrificedandtheAChEandAPEHactivitieswereassessedinhomogenizedhippocampus.Besidesthis,synapticplasticityparameterswerealsomeasuredexvivo.DataobtainedatthismomenthaveshowedthatchronictreatmentofDDVP2mg/kghavenoeffectin1month-oldrats,adeterioratingeffectin3months-oldratsandatoxiceffectin12months-oldratsinlearningandmemory.TreatmentwithDDVP0.1mg/kgproducesanimprovementinlearningandmemoryin1month-oldrats.Howevernoeffectandadeterioratingeffectisobservedin3and12months-oldratsrespectively.FinallythetreatmentwithDDVP0.03mg/kgshowednoeffectin12monthsrats.AtthismomentwearetestingDDVP0.03mg/kgin1and3monthsoldratsandDDVP0.01mg/kgin12monthsoldrats.AtalltheseDDVPdoses,onlyhippocampalAPEHactivitywasspecificallyinhibited,remainingAChEactivityunaffected.Takentogether,theseresultsindicatethatthemagnitudeofAPEHinhibitioncouldbeusedasapredictorofpharmacologicalortoxicologicaleffectswhenlearningandmemoryareusedasendpoints.

192

115)Vulnerability of dopaminergic neurons following recurrent metabolic insults: effects of perinatal asphyxia in organotypic cultures.

Sehrt, M1.,1TecnologíaMédica,Medicina,UniversidaddeChile.

Vulnerabilityofdopaminergicneuronsfollowingrecurrentmetabolicinsults:effectsofperinatalasphyxiainorganotypiccultures.

Sehrt-Uribe, M.,Pérez-LobosR.,Palacios,E.,Bustamante,D.,Morales,P.,Herrera-Marschitz,M.

Millenium Institute BNI-Chile; Programme of Molecular & Molecular Pharmacology, ICBM, Medical Faculty, University of Chile, Santiago, Chile.

Perinatalasphyxia(PA)featuresapoptoticandneuroinflamationmechanisms, leadingtoshort-and long-termneuronaldeath.PAaffectsneuronalnetworksdependendingontheinsulttimingandmetabolicrequirements,beingbasalganglianeurocircuitryparticularlypronetovulnerability,inducingfunctionalimpairmentsofdopaminergicsystems.

WeinvestigatedwhetherPAprimesthevulnerabilityofdopaminergicneuronstorecurrentmetabolicinsults,usingbasalgangliaorganotypicculturesfromasphyxia-exposedneonaterats.

Culturesfromasphyxia-exposedandcontrolanimalsweresubjectedtoH2O2(45mM)andglucosedeprivationfor18hatdayin vitro18(DIV18).Twodaysaftertheinsult,culturesweretreatedforcellviabilityin vitro,orformalinfixedtoanalyseneuronal(MAP-2),neurochemical(tyrosinehydroxylase,TH)phenotype,caspase-3(CASP-3)asapoptoticmarker,andDAPItoanalysecellnumberandmorphologicalnuclearchanges.

Afterasecondmetabolic insult,therewas: (i)adecreasedcellviability inorganotypiccultures, (ii)Snrecurrent insultcauseadecreaseincellnumberandasphyxiaexposedcontroldecreasessignificantlyaboutitsnon-exposedcontrol.Strrecurrentinsultcauseadecreaseincellnumberonlyforitsasphyxiaexposedgroup.Cxdecreaseincellnumberonlyforitscontrolgrouptreatedfora recurrent insult.Further,asphyxiaexposedgroupcellnumberdecreasedabout itscontrol, (iii) increase inapoptoticcelldeath;(iv)increaseinactivationofCASP-3-dependentapoptoticpathwaysinneurons,and(v)increasednumberofTH+/CASP-3+neuronsoforganotypicculturesfromasphyxia-exposed,comparedtonon-asphyctictissue.Furthermore,morphologicalanalysisrevealeda(vi)decreasednumberofapoptoticbodiesintissuesubmittedtoarecurrentmetabolicinsult,suggestingtheactivationofdifferentdeathpathwaysindopaminergicneurons,dependingontheprimingmechanismsinducedbyPA.

Thepresentworkdescribesdifferentialvulnerabilityaffectingneuronsfrombasalganglia,confirmingdopaminergicneuronsasamaintargetfortheimpairingeffectsofrecurrentmetabolicinsultsfollowingPAinCNS.

Sponsor: Herrera-Marschitz,M.

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116)REGULATION OF VOLTAGE SENSING STRUCTURES OF CaV1.2 CALCIUMCHANNEL BY THE AUXILIARY b-SUBUNIT (β3)

De Giorgis, Daniela2.,Contreras,Gustavo2.,Savalli,Nicoletta1.,Navarro-Quezada,Nieves2.,Gonzalez,Carlos2.,Olcese,Riccardo1.,Neely,Alan2.,1DepartmentofAnesthesiology,DivisionofMolecularMedicine,DavidGeffenSchoolofMedicine,UniversityofCalifornia.2CentroInterdisciplinariodeNeurocienciasdeValparaíso,Ciencias,UniversidadDeValparaíso.(SponsoredbyFONDECYT1120802AndACT1104(GC),NIH.R01GM110276(RO).TheCentroInterdisciplinarioDeNeurocienciaDeValparaísoIsAMillenniumInstituteSupportedByTheMillenniumScientificInitiativeOfTheChileanMinistryOfEconomy,DevelopmentAndTourism.)

High Voltage-Activated channels (HVA) translate membrane depolarizations to local increases in intracellular Ca2+. HVAs arecomposedof four similarbutnon-identical repeats, eachonewith itsownvoltage-sensingdomain. The cardiacHVACaV1.2 isco-expressedwithtwoauxiliarysubunits,α2δandβ,thatregulatechannelfunction.Totrackthemovementofindividualvoltagesensorsweintroducedcysteineresiduesatstrategiclocationsinonevoltagesensoratatime,attachedatetramethylrhodaminebasedfluorophoreandmonitoredfluorescencechangesproducedbymembranedepolarizations(Cut-openoocytevoltageclampfluorometry).Usingthisapproachwehaveshownthatindividualvoltage-sensorscontributetochannelopeningandgatingcurrentstoadifferentdegree(Pantazisetal.2014,PNAS.).Hereweshowthatβ-subunit(β3)co-expressionregulatestheequilibriumbetweentherelaxedandactiveconfigurationsofthevoltage-sensor.Voltage-sensorsofallmembersofthevoltage-gatedionchannelfamilyhavethreemainconformations:resting,activeandrelaxed.Normalchanneloperationinvolvestransitfromtherestingtoactiveconformation,andthistransitionpromoteschannelopening.Themorestablerelaxedconfigurationthatispopulatedbyprolongeddepolarizationsdoesnot contribute to channelopening.Once in the relaxed state, stronghyperpolarizations (~ -100mV)arenecessarytorestoretherestingstate.Incontrast,duringnormalfunctiontheresting/activeratioat+40mVapproaches0.5.WhenCaV1.2channelsareco-expressedwithβ3,membranehyperpolarizationismoreeffectiveindrivingthevoltagesensorofthefirstrepeatoutoftherelaxedstate,sothatatvoltagesneartherestingpotentialthemajorityofthevoltagesensorswillbeavailableforactivation.Intheabsenceofβ3,~50%ofthevoltage-sensorsfromthefirstrepeatremainintherelaxedstateattherestingpotential,explaining,atleastinpart,whyCaV1.2channelslackingtheβ-subunitdonotopenatphysiologicaldepolarizations.

194

117)Role of cytoskeleton and RhoA in regulation of Gap Junction Channels and Hemichannels

Jara, Oscar1.,Maripillán,Jaime1.,Momboisse,Fanny1.,García,Isaac1.,Pinto,Bernardo1.,Cárdenas,AnaMaría1.,González,Carlos1.,Martínez,Agustin1.,1CentroInterdisciplinariodeNeurocienciasdeValparaíso,FacultaddeCiencias,UniversidadDeValparaíso.

TheroleoftheactincytoskeletoninthefunctionalregulationofGapJunctionchannel(GJC)hasbeenwellestablished;howeverlessisknownaboutthemolecularmechanismsunderlyingitsactiononHemichannels(HC).Tothisend,wetreatedHeLacellsexpressing connexins (Cx)with the actin-depolarizing agent Cytochalasin B and the effect onHCswere functional addressedperformingdyeuptake,patchclamprecordingsandsingle-cellTIRFmicroscopy.Wefoundthat incellsstablyexpressingCx43,thetreatmentsignificantly reducedthesizeofGJCplaquesaswellas thedyeandelectricalcoupling. Incontrast, therelativeamountandactivityofHCsinnon-appositionalplasmamembranesweresignificantlyincreased.Ontheotherhand,incellsstablyexpressingCx26,actindepolimerizationalsoreducedthesizeofGJplaques,butdidnotaffectthefunctionalstateofGJCsandHCs.SincemembersoftheRhoAGTPasefamilyhavebeenshowntoregulatemanyaspectsofintracellularactindynamics,andtheyhavebeenimplicatedintheregulationofHCsandGJCs,westudiedtothecontributionofRhoAsignalingtothismechanism.WefoundthatcellstransfectedwithadominantnegativeformofRhoAorwithasiRNAagainstRhoAexhibitadecreasedofGJCplaquesincellsexpressingeither,Cx26orCx43.TheseconstructsalsopromotesagainoffunctionofCx43HCsbutnotonCx26HCs.TheseresultssuggestthatGJCsandHCscomposedbyCx26orCx43aredifferentialregulatedbytheactincytoskeleton,andRhoAregulatesthetrafficbutnotthefunctionofCx26HCs.

ThisworksupportedbyMECESUPUVA0603toOJ,FONDECYT1130855andANILLO-ACT1104toADM.TheCentroInterdisciplinariodeNeurocienciasdeValparaísoisaChileanMillenniumInstitute(P09-022-F).

195

118)KCNN4 Attenuates Chronic Allergic Asthma Features in an Ovalbumin Mouse Model

Philp, Amber1,2.,Flores,Carlos2.,1Bioquimica,FacultaddeCiencia,UniversidadAustralDeChile.2BiologíaCentrodeEstudiosCientificos.

Asthmaisacomplexdiseasecharacterizedbychronicinflammation,hyperreactivity,increasedproductionofmucusandairwayremodelling.ManyfeaturesofthisdiseasearethoughttoreflectconsequencesofaTh2-dominatedimmuneresponse,wherethemastcellisoneoftheprincipaleffectors.PreviousreportsdemonstratedthatthepotassiumionchannelKCNN4isimportantforcertainmastcellfunctions,suchasdegranulationandmigration.TheaimofthisworkwastostudythepotentialuseofKCNN4toinhibitthefunctionofthemastcellinthedevelopmentofamousemodelofchronicallergicasthma.

Chronicallergicasthmawasinducedthroughimmunizationwithovalbuminfor10weeksincontrolanimals(WT)andanimalswhichlackexpressionofKCNN4.Agroupofanimalswithdeficiencyofmastcells(KitW-sh/W-sh)wasalsoincluded.TheresultsshowedthatWTmiceimmunizedwithovalbuminproducedthickeningofairwayepithelium,increasedcollagendepositsindistalairwaysandincreasedgobletcellnumbersinthetrachea.Kccn4-/-showedsignificantreductionscomparedwithWTanimalsinthedevelopmentofallasthmacharacteristicsanalysedinthiswork.

Thiswork shows the importanceof KCNN4 in thedevelopmentof chronic allergic asthma, nevertheless theprotective effectdeliveredthroughKCNN4inhibitionisonlypartiallyduetotheinhibitionofmastcellsanditisprobablethattheinhibitionofotherimmunecellsandairwayepitheliumfunctionalsocontributestotheobservedattenuations.

FundedbyFONDECYT1151142

196

119)Molecular determinants involved in cold and menthol sensitivity of the TRPM8 channel

Rivera, Bastián1.,González,Alejandro1.,Tralma,Karina1.,Salas,Jeremy1.,Madrid,Rodolfo1.,Pertusa,María1.,1Biología,QuímicayBiología,UniversidadDeSantiagoDeChile.(SponsoredbySupportedByFONDECYT11130144(MP),1131064(RM),AndCONICYTACT-1113(RM,MP).)

TRPM8isthemainionchannelresponsibleforcolddetectioninthesomatosensorysystem.ThisthermoTRPchannelisactivatedbycold,coolingcompoundssuchasmentholandbyvoltage.AmongthedifferentTRPM8orthologs,chickenTRPM8(cTRPM8)displaysdifferentsensitivitytocoldandmentholcomparedtomouseTRPM8(mTRPM8),suggestingthatnon-conservedregionscouldbeable to tune the chemical and thermal sensitivityof this polymodal ion channel.Nevertheless, themolecularbasesunderlyingthedifferencesamongtheseorthologsremainpoorlyunderstood.InordertoidentifystructuraldomainsinvolvedinTRPM8sensitivitytocoldandmenthol,weperformedsetsofchimerasusingtheorthologsmTRPM8andcTRPM8.Weevaluatedtheresponsestocoldandmentholofthesemutants,usingcalciumimagingandpatchclamptechniquesintransfectedHEK293cells. Weidentifyoneregionof30residues intheproximalN-terminusthat is involvedinthesensitivitytocoldandmentholofthischannel.WefoundthatthetransferenceofthisdomainfromcTRPM8tomTRPM8issufficienttoobtainachimerathatincludeincreasedresponsestocoldandmenthol,a3oCshiftinthetemperaturethresholdtowarmertemperatures,andareducedEC50tomenthol.Electrophysiologicalanalysisrevealedthattheenhancedresponsestoagonistsareduetoashiftinthevoltage-dependenceofactivation,increasingtheprobabilityofchannelopeningsatphysiologicallyrelevantnegativemembranepotentials.Thischangeoccursalongwithan increase intheGmaxvalue. OurresultssuggestakeyroleoftheproximalregionoftheN-terminusinthefinetuneofcoldandmentholsensitivityofthispolymodalthermoTRPchannel.

197

120) Characterization of chaos in a bursting neuronal model and its interaction with noise.

Caviedes, Mauricio1.,Maidana,JeanPaul1.,Quero,Daniel2.,Aguirre,Pablo2.,Orio,Patricio1.,1CentroInterdisciplinariodeNeurocienciadeValparaíso,FacultaddeCiencias,UniversidaddeValparaíso.2DepartamentodeMatemáticaUniversidadTécnicaFedericoSantaMaría.(SponsoredbySupportedByGrantsFondecyt1130862(PO),Fondecyt3130497(PA),ACT-1103(PA),ACT-1104(PO),ACT-1113(PO),FB0008(PO),PFB03(PA).TheCentroInterdisciplinarioDeNeurocienciaDeValparaíso(CINV)IsAMillenniumInstituteSupportedByTheMillenniumScien)

Manyneuronsinthecentralandperipheralnervoussystemexhibitaburstingfiringpattern.Burstingappearsasaconsequenceofionchannelsthatgatewithslowtimeconstants(suchasIMorIh)orthatrespondtoslowintracellularmechanisms(suchascalcium-activatedpotassiumchannels).Weare studying thebehaviorofaneuronalmodelwithparabolicbursting inspired inthermoreceptors.Apersistantsodiumcurrent,acalciumactivatedpotassiumchannelandahyperpolarization-activatedcurrentprovideaslowmembraneoscillationthatelicitsregularfiringofactionpotentials.Dependingonmodelparameters,theperiodicfiringcantaketheformofburstfiring,tonicfiringoranirregularmultimodalfiringofchaoticnature.Generallyspeaking,chaoticbehaviorischaracterizedbyanirregular,non-periodicbehaviorintheabsenceofanyrandomornoisyinfluence.Italsopresentsashort-termpredictabilitythatvanishesexponentiallywithtime,afeaturequantifiedintheLyapunovexponent.Bifurcationanalysisofthemodelrevealsahomoclinicbifurcationinthetransitionfromtonicfiringtosilence,causingachaoticbehaviorknownasShilnikovchaos.Numericalanalysisof inter-spikeinterval(ISI)sequencesshowsapositiveLyapunovexponentinmanyregionsoftheparameterspace.Wealsoshowthatthechaoticbehaviorisdisruptedbynoise,bothinternal(stochasticopeningofionchannels)andexternal(randomcurrentadded),convertingadeterministicchaoticbehaviorintoarandombehavior.Ongoingworkisfocusedoncomplementingthenumericalchaoticcharacterizationwithentropymeasuresandonthefunctionalconsequencesofchaostotheneuralcoding.

198

121)Comparative study in porcine model anesthesia isofluorane/oxygen with and without acute alcohol administration

MONTERO, EDSON1.,ARANGUIZ,MACARENA1.,AVENDAÑO,CINTIA2.,CONTRERAS,ENRIQUE3.,SEPULVEDA,MARIA4.,1FARMACOLOGIA,FACULTADDEMEDICINAVETERINARIA,UniversidadSanSebastián.2LABORATORIOCLINICO,FACULTADDECIENCIASDELASALUD,UniversidadSanSebastián.3FARMACOLOGIA,FACULTADDEMEDICINA,UniversidadCatólicaDeLaSantísimaConcepción.4FARMACOLOGIA,FACULTADDECIENCIASBIOLOGICAS,UniversidadDeConcepción.(SponsoredbyUSS2011-00007-R)

InChile,thehighvehicularaccidentabilitybecausethecombinationethanolanddriving,astudytoevaluatetheeffectsofethanoladministrationinaporcinemodelwithVolatileInductionandMaintenanceAnesthesia(VIMA)isoflurane/oxygen(I/O),wasdesigned.Therearenostudiestorecommendsafeanestheticprotocols.WecontrolledinaporcinemodelinordertodeterminewhetherplasmaelectrolytesandEKGparametersaremodifiedbyactionofethanolclinicalstudy.Ninepigs,Landrace-Largewhite,ASA 1, averageweight of 13 kgwere used. These are pre-medicatedwith Ketamine-Diazepam. The studywas conducted intwosimilaranestheticsperiods(40min.)withspontaneous inhalationanesthesia. InthefirstperiodheunderwentanestheticinductionusingaVIMAmaskI/OwithamaintenancedoseofI/O2%/2L.min-1.Inthesecondperiod,afterawash-outofthreedays,thesameanestheticprotocolofthe1sthalfofthestudywithadministrationofethanol96dilutedto50%and250mg.kg-1dosewasadministered.EKGwasobtained:Heartrate(HR),PRinterval,QTandQTc,QRScomplexandSTsegment,withplasmaelectrolytes:Na+,K+,Cl-andbicarbonateofasampleofarterialblood0,20and40minutes.StatisticalanalysisStudent’sttestwasused.OurresultsindicatedthatduringtheadministrationofVIMAI/OwithethanolslowsHRandplasmaconcentrationsK+andCl-,comparedtocontrol.DecliningHRisaccentuatedbythesynergisticenhancementbetweenethanolandisofluraneatcentrallevel.WhiletransientlowplasmaconcentrationofK+andCl-ispresentedasaneffectrenalplasmaADHdecreasedbytheadministrationofalcoholinduced,therebydecreasingthereabsorptionofK+andCl-withincreaseddiuresis.Duetotheprincipleof electronegativity plasmaNa + and plasmabicarbonate suffer no significant changes. Therewere no significant differencesbetweenthetwoanestheticperiodsfortherestoftheparametersstudied.Inductiontimeandanestheticrecoveryexperiencednosignificantdifferencesbetweentheperiodsanalyzed.WeconcludethatVIMAI/Oproducesastableanestheticinthepresenceofethanol.

199

122)Triphenylphosphonium alkyl derivatives of gallic acid decrease tumor growth in vivo: potentiation with doxycycline.

Peredo-Silva, Liliana1.,Castro-Castillo,Vicente2.,Saavedra-Olavarría,Jorge2.,Pavani,Mario1.,Ferreira-Parker,Jorge1.,1DepartamentodeFarmacología,FacultaddeMedicina,UniversidadDeChile.2DepartamentodeQuímica,FacultaddeCienciasBásicas,UniversidadMetropolitanaDeCienciasDeLaEducación.

Ithasbeenfoundthatn-alkylgallatesareabletoinduceapoptosisinvarioustumorcelllines,primarilybyactivatingthemitochondrialdependentapoptoticpathwayduetoanassociationwithmitochondrialoxidativemetabolism,alteredinthesecells.Ithasbeenreportedthatincreasingconcentrationsofestersofgallicacid,generateanuncouplingeffectofoxidativephosphorylationsystem,followedbyinhibitionofelectronflowthroughthemitochondrialrespiratorychain(athigherconcentrations),mainlyatNADH-CoQoxidoreductase.Theseeffects,preventthesynthesisofATPthatultimatelyledtocelldeath.Besides,itisalsoknownthatthestructureandlipophilicityofthealkylsidechain(alkyllength),isrelevanttotheantitumoractivityofthiscompounds.Mitochondriaplayaroleinregulatingenergymetabolism,cytosoliccalciumconcentration,ROSproductionandapoptosis.Importantly,betweentumorandnon-tumorcells,themitochondriapresentsignificantdifferencesintermsofoxidativephosphorylation.Additionally,tumorcellsexhibitedincreasedmitochondrialmembranepotential(ΔΨm).Theinnermitochondrialmembraneoftumorcells,haveaΔΨmabout150-180mV,morenegativeontheinside.Thispotentialismuchhigherthananyothercellorganelle,andgreaterthanthatofothertissueandnon-tumorcell.Toenhancethecytotoxiceffectofgallicacidesters,wesynthesizedvariousdelocalizedlipophiliccations,wheregallicacidwithdifferentalkylchainlengths,wereconjugatedwithtriphenylphosphoniumgroup(TPP+).These compounds accumulate selectively inmitochondria of tumor cells, guided by their highermembrane potential. In thiswork,weevaluatedtheantitumoractivityandselectivityin vivo ofthisderivatives,inasyngeneicmousemodel.Specifically,thederivativeofgallicacidwith10carbonatomsconjugatedwithTTP+(TPP+C10)inhibitstumorgrowthin vivoafter30daystreatment,anditscombinationwiththeantibioticdoxycycline,achievesaneliminationof80%oftumorsintumor-bearingmice.Moreover,thesurvivalrateofanimalsfollowingtreatmentcomparedtothecontrolgroupis90%withoutexperiencingrelapseafter60daysoftreatmentends.Furthermore,treatmentwithTPP+C10anditscombinationwithdoxycyclineproducesnosystemictoxicity.Inconclusion,thetreatmentwithTPP+C10incombinationwithdoxycycline,issafetoadministeredinanimalsproducingaselectiveeffectinreducetumorsizeversuscontrolgroups,withoutproducingsystemictoxicity.ThisworkwasfundedbyFONDECYTGrantN°1130772andCONICYTscholarshipN°21110084.

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POSTERS II

201

1)A novel competitive antagonist nAChR α4β2, ((S)-1-methylpyrrolidin-2-yl) methyl benzoate, reduces ethanol intake in UChB bibulous rats

Quiroz,Gabriel1.,Sotomayor-Zarate,Ramon2.,Quintanilla,MariaElena3.,Reyes-Parada,Miguel4.,Iturriaga-Vasquez, Patricio5.,1ProgramadeDoctoradoenFarmacología,FacultaddeCienciasQuímicasyFarmacéuticas,UniversidadDeChile.2CentrodeNeurobiologíayPlasticidadCerebral,FacultaddeCiencias,UniversidadDeValparaíso.3InstitutodeCienciasBiomedicas,FacultaddeMedicina,UniversidadDeChile.4EscueladeMedicina,FaculatddeCienciasMedicas,UniversidadDeSantiagoDeChile.5CienciasQuimicasyRecusosNaturales,FacultaddeIngenieriayCiencias,UniversidadDeLaFrontera.(SponsoredbyFONDECYTN°1150615(PIV),1130185(MRP),1130012(MEQ),CONICYTNationalPhDScholarFellowship(GQ))

Alcohol(ethanol)abuseisrelatedasmaincauseofpreventablediseases.Ethanolandtheirmetabolites(suchasacetaldehydeand salsolinol) acts overmany pharmacological targets in CentralNervous System (CNS)modulating the biological effects ofethanol. Similarly to other drugs of abuse, like cocaine, amphetamine, nicotine, etc., ethanol (and their metabolites) couldmodulatesthemesolimbicpathway,inducinganincrementofdopamineinnucleusaccumbens,consideredthemainkeyofdrugabusereinforcementproperties.Electrophysiologicalevidencesshowedthatethanolincreaseacetylcholinecurrentsofnicotinicacetylcholine receptors (nAChR) subtype α4β2, themost abundant nAChR of CNS andmesolimbic pathway. Pharmacologicalevidences indicate that mecamylamine (non selective nAChRs antagonist) and dihydro-β-erythroidine (selective nAChR α4β2competitiveantagonist)reduceethanolintakeinrestrictiveethanolaccessparadigmratmodel.Furthermore,hasbeenreportedthatvareniclineandcytisine(selectivenAChRα4β2parcialagonists)reducedtheethanolintakeinfreechoiceparadigminUChBrats,apotenthigh-drinkingratmodelforethanol intake.Herein,weshowedtheeffectofnovelcompetitiveantagonistnAChRα4β2,((S)-1-methylpyrrolidin-2-yl)methylbenzoate(namedFPy),inethanolintakeconductinfreechoiceparadigminUChBrats.Briefly,UChBratswereexposedtoethanolfreeaccessfor20daystoreachtheplateauingesting.Fromday21,ratswereseparatedinthreegroupsfortheadministrationofsalinesolution,5mg/Kgor10mg/KgofFPyi.p.dailyforseventeendays.Andinthecourseoftwentydaystheethanol,waterconsumptionandbodyweightwasrecordeddaily.OurresultsshowedthatbothFPydoses(5and10mg/Kgi.pdaily)reducedthevoluntaryethanolintakearound50%ofplateauconsumption.Ourresultsindicatethat,selectiveandcompetitivenicotinicantagonistcouldbeausefultoolfortohelpinpharmacotherapyofalcoholabuse.

202

2)Behavioral characterization of the acute effects in rats of 2,4-DMA (2,4-dimethoxyamphetamine) as precursor of atypical psychotropic derivatives

Klagges-Troncoso, Jorge1,2.,Burgos-Villaseca,Jorge1,3.,Benavente-Schonhaut,Sofia1,2.,Malhue-Olmos,Valeska1,2.,Hernández,Alejandro4.,Burgos,Hector5.,Castro-Castillo,Vicente6.,Sáez-Briones,Patricio1,2.,1LaboratoryofNeuropharmacologyandBehavior,FacultyofMedicalSciences,UniversidadDeSantiagoDeChile.2SchoolofMedicine,FacultyofMedicalSciences,UniversidadDeSantiagoDeChile.3DepartmentofBiology,FacultyofBasicSciences,UniversidadMetropolitanaDeCienciasDeLaEducación.4LaboratoryofNeurobiology,FacultyofChemistryandBiology,UniversidadDeSantiagoDeChile.5SchoolofPsychology,FacultyofSocialSciences,UniversidadCentral.6DepartmentofChemistry,FacultyofBasicSciences,UniversidadMetropolitanaDeCienciasDeLaEducación.(SponsoredbyDICYT-USACHGrant021401SBAndDICYT-USACHGrantForUndergraduateStudents2014-JKT)

2,4-dimethoxyamphetamine (2,4-DMA) is a synthetic psychotropic phenylalkylamine structurally related to the hallucinogenmescalinebuthasbeendescribedtopossessweakhallucinogenicproperties.Nevertheless,thesubjectiveeffectsreportedfor2,4-DMAinhumanssuggestamorecomplexpharmacologicalprofile, includingnotonlystandardstimulant-likepropertiesbutalsoentactogenic-likeeffectsaswell.Thispeculiarconditionas“mixed”drugcouldbeconsideredasastartpointtodevelopnovelderivativesexhibitingsinglepsychotropicproperties.Unfortunately,theavailableinvivodatatosupportthelatterassumptionsarescarceandincomplete.Inthepresentwork,theacutebehavioraleffectsof2,4-DMAhavebeenstudiedinmaleSpragueDawleyratsafteri.p.administrationofsingledosesofthedrugasawater-solublesalt(doserange1–20mg/kg).Thepharmacologicalcharacterization includedmeasurementsof spontaneouspsychomotor activity (e.g.motor activity, locomotion, grooming andrearingbehaviors,head-shakesresponses,stereotypy-inducingresponses),theevaluationofanxiolytic/anxiogeniceffectsattheelevatedplusmazeandtheeffectsonacquisitionusingtheactiveavoidanceconditioningparadigm.Theresultsobtainedindicatethat2,4-DMAexhibitacomplexprofilethatdonotfollowastrictdose-responsecorrelation,includingweakanxiolytic-likeeffectsandadecreaseinlocomotionatdifferentdoses(10mg/kgand1mg/kgrespectively),withnosignificantincreaseinthenumberofheadshakes.Incontrast,rearingbehaviorwasconsistentlydecreasedandacquisitionwasalmostabolishedinadoseindependentmanner(p<0.01).Interestingly,adecreaseinthestereotypy-inducingresponsewasalsoobservedat1mg/kgand20mg/kg.Noneoftheeffectselicitedby2,4-DMAwereconsistentwithanentactogenic-likeprofile.Takentogether,theresultsobtainedareinagreementwiththenotionthat2,4-DMAmaypossessmixedpsychotropiceffects,buttheyappeartoincludehallucinogenic-likeandstimulant-likepropertiesonly.

203

3)Rats exposed prenatally to valproate display decreased colonic permeability to macromolecules

Olavarría-Ramírez, Loreto1.,Moyano-Porcile,Valentina1.,González-Arancibia,Camila1.,Díaz-Zepeda,Camilo1.,Valencia,Martina2.,Aliaga,Esteban2.,Bravo,Javier1.,Julio-Pieper,Marcela1.,1GrupodeNeuroGastroBioquímica,LaboratoriodeQuímicaBiológica.InstitutodeQuímica,FacultaddeCiencias,PontificiaUniversidadCatólicaDeValparaíso.2LaboratoriodeNeurociencias,EscueladeKinesiología,FacultaddeCiencias,PontificiaUniversidadCatólicaDeValparaíso.(SponsoredbyFunding:PUCVDI037.302/2013,Conicyt79112017AndFondecyt1130213)

Theexistenceofarichgut-to-braincommunicationhasledtosuggestthatalterationsintheintestinalbarriermaytakepartinthepathophysiologyofmentaldisorders.Particularlyinautismspectrumdisorder(ASD)patients,gastrointestinalalterationsincludingabdominal pain,diarrhea, constipationandbloatingare frequently reported, althoughpartof theexpertmedical communityfeelsthattheprevalenceoftheseconditions isnotyetcompletelyunderstood.The“leakygut”hypothesissuggeststhat foodcompoundswhichareabletocrossthroughahyper-permeableintestinalmucosacouldinducethebehavioralsymptomatologyofautism.However,theevidenceforincreasedintestinalpermeabilityinindividualswithASDiscontroversialandlimited,withmostreportstodateshowingmethodologicalcaveatsincludinginadequatecontrolsandsmallsubjectpopulations.Theaimofthisinvestigationwastoevaluatewhetherintestinalpermeabilityisincreasedinananimalmodelofautism.FortheASDmodel,pregnantSpragueDawleyratsreceivedvalproate(VPA)atgestationalday12.5(450mg/kgintraperitoneal).Thistreatmentisknowntoinducealterationsinbehaviourandsocialinteractionintheoffspring,resemblingthatofASDinhumans.Controlsweretreatedwithsalineatthesamegestationaltime.Samplesofcolonandileumweretakenfrommalepupsbetweenpostnataldays30and33.Permeabilityevaluationwasperformedexvivobymeasuringthetrafficofdifferentsizefluorescentdextrans(FITC-40kDandTRITC-4.4kD)fromthemucosaltotheserosalsideofintestinaltissue.Tissueswereincubatedfor120minutesandmeasurementsweremadeevery30minutes.Alsoasectionofthetissuewasfixedin4%PFAformorphologicalevaluation.TheresultsshowthatinVPAexposedrats,thepermeabilityoftheileumtomacromoleculesisnotaffected.Incontrast,thecolonofVPAratspresentsasignificantdecreaseinpermeabilitycomparedtothecontrolgroup,forbothmacromoleculesafter120minutesofincubation.Asformorphologicalevaluationofcolon,themucosalthicknesswasnotsignificantlydifferentfromcontrol.Althoughthesefindingsareopposedtowhatisproposedbysomeclinicalevidence,adecreaseincolonpermeabilitymayberelatedtoanalteredgutbrainaxiscommunication,havingapotentialeffectonthecentralnervoussystem(CNS).ItisstillnecessarytoinvestigateifsuchintestinalchangesareacauseoraconsequenceofCNSalterationsdescribedinthisanimalmodel.

204

4)Early-life dysbiosis in infant sprague-dawley rats: effect on anxiety-like behaviors and plasma corticosterone levels

Ponce- Guequen, Excequel1.,Barrera-Bugeño,Camila1.,Eyzaguirre-Velasquez,Johana1.,Escobar-Luna,Jorge1.,Olavarría-Ramirez,Loreto1.,Gotteland,Martín2.,Julio-Pieper,Marcela1.,Bravo,Javier1.,1GrupodeNeurogastrobioquímica,laboratoriodeQuímicaBiológica.,InstitutodeQuímica,FacultaddeCiencias.,PontificiaUniversidadCatólicaDeValparaíso.2Departamentodenutrición.,FacultaddeMedicina,UniversidadDeChile.(SponsoredbyFunding:FONDECYT#1140776)

Intestinalcolonizationinneonatesbeginsatbirthbybacterialacquisitionthroughthebirthcanal.Later,gutmicrobiotacompositionvariesthroughoutpostnataldevelopmentbymanyothersfactors,suchastypeoffeedingorexposuretoantibiotics.Ontheotherhand, thesegutsymbiontsarerecognizedtoaffectkeyaspectsofhostphysiology,e.g.embrionarydevelopmentof thebloodbrainbarrierandthecommunicationbetweenthegutandthecentralnervoussystem.Evidenceshowsthatgerm-freemicehavereducedanxiety-likebehaviors,whilehavinganexaggeratedhypothalamic-pituitary-adrenal(HPA)axisresponse.Inaddition,non-absorbablewide-spectrumantibiotics have anxiolytic effects in adultmice. All theseobservationshavebeenmade in adultsubjects.However, very little is known about the effects of acquiring an altered gutmicrobiota early in life on stress-relatedbehaviorandHPAfunctionininfantrats.Therefore,wehypothesizethatinterventioninmaternalgutmicrobiota,likeexposuretowide-spectrumnon-absorbableantibioticsintheperinatalperiodmodifiestheinfantsgutmicrobiota,impactingonstress-relatedbehavior andHPAaxis function. To test thisweadministereda combinationof neomycin (100mg/kg), bacitracin (100mg/kg),pimaricin(5mg/kg)andvancomycin(100mg/kg)orallytopregnantSprague-Dawleydams,startingthreedaysbeforeparturitionandmaintainedituntilpost-natalday(PD)7.OnPD21pupswereweanedandbehavioraltestingbegunonPD22.Openfieldtestshowedthatmalebutnotfemalepupsexposedtoantibiotics,spentmoretimeinthecentralareaoftheapparatusincomparisontocontrolrats.Also,pupsexposedtoantibioticshadreducednumberoffecalboliduringthistest.OnPD23,elevatedplusmazetestrevealedthatbothfemaleandmalepupsexposedtoantibioticshadhighernumberofentriesandspentmoretimeintheopenarmsthancontrolrats.OnPD24ratsweresubjectedtotheforcedswimtest,howevertherewerenodifferencesbetweengroupsindepression-likebehaviors.Then,30minafterthe lastbehavioral testplasmacorticosteroneconcentrationwasdetermined.Animalsexposedtoearly-lifeantibioticshadmorevariability inplasmacorticosteronethanthecontrolgroup.Nodifferenceinbodyweightwasfoundbetweengroups.Togetherthesedatasuggeststhatacquisitionofaconventionalgutmicrobiotainearly-lifeisimportantforanadequatecouplingbetweenHPAaxisfunctionandbehavioralresponsestostress.

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5)Betamethasone treatment effect in patients with spastic paraparesis associated with HTLV-1 retrovirus

Valenzuela, Maria1.,Alberti,Carolina1.,Puente,Javier1.,Quintremil,Sebastián1.,Medina,Fernando1.,Barriga,Andres1.,Ramírez,Eugenio2.,Cartier,Luis3.,1DepartamentodeBioquimicayBiologiaMolecular,CienciasQuímicasyFarmacéuticas,UniversidadDeChile.2Virología,FacultaddeMedicina,UniversidaddeChile.3DepartamentodeCienciasNeurologicas,FacultaddeMedicina,UniversidaddeChile.(SponsoredbyFondecyt1080396)

ProgressiveHTLV-1associatedTropicalSpasticParaparesisorHAM/TSPisconsideredacentralaxonopathycausedbyanaxoplasmictransportdysregulation.Upto3.0%ofinfectedpeopledevelopthisdisease,remainingtherestasasymptomaticcarriers.Inallpatients,T-CD4+cellsarethemaintargetofHTLV-1,in vivo.TheviralproteinTaxhasbeenassociatedtoalterationsobservedinHAM/TSP.Currently,thereisnospecifictreatmentforHAM/TSP.However,therapybasedoncorticosteroidshasbeensuccessfulin decreasing patient symptomatology. Taking into account the above information, the effect of a treatment using systemicbetamethasone (amonthly injection) inHAM/TSPpatientswas investigated,evaluating clinical andmolecular aspects relatedwith immunologicalmarkers.Tounderstandtheeffectsofbetamethasonewedetermined inPBMCs frompatientsmRNAandprotein of FoxP3 and Tax, using real timePCR andflow cytometry. After treatment, an improvement ofmotor disabilitywasobservedtogetherwithadecreaseinT-CD4+Tax+andanincreaseinT-CD4+FoxP3+populationsatbothproteinandmRNAlevels.In order to determine a relationship between decreasedmRNA Tax and CD4+Tax+ populationwith regard to treatmentwithbetamethasone,wesearchedinthebaseTranscriptionElementSearchSystemforthepresenceofresponseelementsinsideandoutsidethepromoterofHTLV-1,findingaresponseelementintheviralpromoteratposition79correspondingtothe5\’LTRU3region.betamethasone increasedtheamplificationof this regionofHTLV-1virus followedbychromatin immunoprecipitation.Thedecreaseinviralpromoteramplificationinthepresenceofglucocorticoidssuggeststhebindingtoarepressoroftheviraltranscriptionalactivity,representedbythedecreaseinmRNAlevelsofTax.TheseresultssuggestarelationshipbetweentheHTLV-1promoterandglucocorticoidreceptor,indicatingthatbetamethasonetreatmentmightcontributetodecreaseviralloadandraiseTregpopulation.

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6)Neonatal programming with Estradiol Valerate does not produce conditioned place preference to amphetamine in adult female rats

Sanguinetti, N1.,Venegas,Francisca1.,Espinosa,Pedro1.,Renard,Georgina1.,Sotomayor-Zárate,Ramón1.,1InstituteofPhysiology,FacultyofSciences,UniversidaddeValparaíso.(SponsoredbyThisWorkWasSupportedByFONDECYTProjectNo.11121205ForRS-Z)

Theprogramming concept is defined as the physiological redirectionof an organor tissue due to an early insult in sensitivedevelopmentalperiods.Inthiscontext,ourlaboratoryhasshownthatneonatalexposuretoestradiolvalerate(EV)increasestheamountofdopamine(DA)inbraincircuitsassociatedwithrewardandlocomotioninadultfemalerats.However,amphetamine-inducedDArelease(systemicandintra-nucleusaccumbens)issignificantlylowerinEVtreated-femalesthancontrolfemalerats.

So,themainobjectiveofthisstudywastoevaluatetheeffectsofrepeatedadministrationofamphetamineinmaleandfemaleratsexposedtoEVatpostnatalday(PND)1ontheexpressionofconditionedplacepreference(CPP)toamphetamineinadulthood.Inthiswork,Sprague-Dawleyratsofbothsexeswereused.AtPND1,theyreceivedadoseofEV(0.1mg/50μLs.c.insesameoil).Controlmaleandfemaleratswereinjectedwith50μLsesameoils.c.atPND1.AtPND60asevendaysCPPprotocolwasperformed.Thisprotocolconsistedinapretestday,fivedaysofconditioningwithadailydoseofamphetamine(1mg/Kgi.p.),andatestday.Timespendinthecompartmentassociatedwithamphetamineisanindexofthereinforcingvalueofthedrug.Ourresultsshowedthatcontrol females, controlmalesandEV treated-malesexpressedCPP toamphetamine (measuredas the increases intimespendinthecompartmentassociatedwiththeamphetamineatthetestday).However,consistentwithourneurochemicalresults,wefoundthatEVtreated-femaleratsdidnotexpressCPPtoamphetamine.TheseresultssuggestthatneonataladministrationofEVdoesnotproduceCPPbehaviorinadultfemalerats,possiblybyalteredexpressionofthedopaminetransporter(amphetaminemoleculartarget).However,thiswillbeevaluatedinsubsequentworkbyQ-RT-PCRandWB.

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7)Amphetamine conditioned place preference and the vasopressinergic system: a study on male and female rats

Bahamondes, Carolina1.,Ahumada,Catalina1.,Silva,Roxana1.,Cruz,Gonzalo1.,Sotomayor-Zárate,Ramón1.,Renard,Georgina1.,1CentrodeNeurobiologíayPlasticidadCerebral-InstitutodeFisiología,FacultaddeCiencias,UniversidadDeValparaíso.(SponsoredbyFinancialSupport:FONDECYTN°11140065ToGMRAndCommitteeForAidAndEducationInNeurochemistry(CAEN),InternationalSocietyForNeurochemistry(ISN).)

ResearchinneurobiologyofdrugaddictionhasbeenfocusedontheeffectsofdrugsofabuseatrewardcircuitandtheroleofLateralSeptum(LS)hasregainedimportanceinthisfield.ThisnucleusisinvolvedinintegrateddifferentareasofthebrainandhasbeenshownthatvasopressinergicneurotransmissionintheLSisinvolvedinregulationofanxious-likebehavior.Howevertheregulationofdrugsofabuseoverthispeptidehasnotbeenwidelystudied.Theaimofthisresearchwastostudytheeffectofamphetamine(AMPH)administrationduringaconditionedplacepreference(CPP)protocoloverextrahypotalamicvasopressin(AVP)systemofadultfemalesandmalesrats.FemaleandmaleSpragueDawleyrats(55-60daysold)wereused.Thestageoftheestruscyclewasdailydeterminedbyvaginalsmearsexamination.WemeasuredtherewardingeffectofAMPHbyCPPprotocol.CPPapparatusconsistinawhitecompartment,corridorandablackcompartment.First,apre-testwasperformedonday1,inwhichanimalwereallowedtofreelyexploreallcompartmentsfor15minutes.Timespentineachcompartmentwasrecorded.Thenaconditioningphasewasperformed,whereanimals receivedadailyAMPHdose (4days)and then theywereconfinedfor60mininthewhitecompartment.Atday6,animalswereagainallowedtofreelyexploreallthecompartments(test).TimespendinthecompartmentassociatedwithAMPHisanindexofthereinforcingvalueofthedrug.Weconsideredthatananimalwasconditionedifitspends>60%ofthetimeintheAMPH-associatedcompartmentthetestdaycomparedtothepre-testday.Besides,wemeasuredAVPcontentinLSbyELISAkitandAVPmRNAexpressioninMeAbyRT-q-PCR.Ourresultsshowedthat50%offemalerats(6conditionedfemalesfrom12AMPH-treatedfemales)and78%ofmalerats(7conditionedmalesfrom9AMPHtreated)expressedCPPtoAMPH.Infemalecasethisbehaviorwasindependentofthestageofestrouscycletheywere.AMPHtreatmentdidnotproducedifferenceinAVPcontentonLS,butinAMPHconditionedanimalstheAVPmRNAexpressiononMeAwaslowerthancontrolanimals.Therefore,AMPHtreatmentsproducesexualdifferencesinacquisitionofCPPtoAMPHandproducealterationinthevasopressinergicsystem.

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8)Long-term effects of prenatal Fluoxetine on memory and motivation in adult male rat offspring

Tamburini, G1.,Rubio,Francisco2.,Moraga-Amaro,Rodrigo1.,Diaz-Galarce,Raul1.,Ampuero,Estibaliz3.,Wyneken,Ursula4.,Stehberg,Jimmy5.,1LaboratoriodeNeurobiologíaUniversidadAndrésBello.2BehavioralNeuroscienceResearchBranch,IntramuralResearchProgramNationalInstituteonDrugAbuse,NationalInstitutesofHealth,U.S..3CenterforBiomedicalResearch,FacultyofBiologicalSciencesandFacultyofMedicineUniversidadAndresBello.4LaboratoriodeNeurociencias,CentrodeInvestigacionesBiomédicasUniversidaddelosAndes.5LaboratoriodeNeurobiología,CentrodeInvestigacionesBiomédicas,FacultaddeCienciasBiológicasandFacultaddeMedicinaUniversidadAndrésBello.

Fluoxetinehas beenprescribed to treat depression in pregnantwomen for over 30 years. Some studies have suggested thatadministrationoffluoxetineduringearlydevelopment in rodentsmay inducepersistentchanges inemotionalbehaviorof theoffspring.However,theeffectofprenatalfluoxetineonmemoryislessstudied,althoughstudiesinadultratstreatedwithclinicallyrelevantdosesoffluoxetine(0.7mg/kg)suggestlong-lastingmemoryimpairments.Theobjectiveofthisstudyistoevaluatetheeffectsofinuteroexposuretofluoxetineonhippocampal-andnon-hippocampal-dependentmemoryoftheadultmaleoffspring,usingMorrisWaterMazeandtheNovelObjectRecognitionmemory,respectively.Anxiety-anddepressive-likesymptomswerealsoevaluated.Fluoxetinetreatedoffspringshowednovelobjectrecognitionmemoryimpairments24hourspost-training,aswellasincreasedanxietyanddepressive-likesymptoms.Interestingly,treatedanimalsdidnotshowsignificantdifferencesinlearningcapacityor retention24hrspost training in comparison to a control group in theMorrisWaterMaze,but showed retentionimpairmentswhentested15daysaftertraining.Ourdatasuggeststhatprenatalexposuretofluoxetinemayinducelong-lasting,detrimentaleffectsonmemoryandemotionalbehaviorintheadultmaleoffspring,andwarrantstheneedforstudiestoassesshumanmemoryinadultsborntomotherswhotookFluoxetineduringpregnancy.

209

9)Effects of the alkaloid gelsemine on recombinant glycine receptors

Marileo, Ana1.,Lara,Cesar1.,Burgos,Carlos1.,Yévenes,Gonzalo1.,1DepartmentofPhysiologyUniversidadDeConcepción.(SponsoredbySupportedByFONDECYT1140515.SponsoredByDr.JorgeFuentealbaA.)

Glycinereceptors(GlyRs)aretransmitter-gatedanionchannelsthatmediatesynapticinhibitioninthecentralnervoussystem.Despite theirpivotal role inmanyphysiologicalandpathophysiologicalprocesses, theGlyRpharmacology is still very limited.Recentstudieshaveshownthatgelsemine,anaturalalkaloid fromGelsemium sempervirens,binds tospinalGlyRsandexertsantihyperalgesicactionsinrodents.However,thefunctionaleffectsofgelsemineonGlyRsarestilllargelyunknown.Inthisstudy,wecharacterizethefunctionaleffectsofgelsemineonGlyRsusingpatch-clamprecordingsofHEK293cellstransientlytransfectedwith plasmids encoding the threemain GlyR alpha subunits (α1, α2 and α3, i.e. homomeric αGlyRs) in the absence or thepresenceofthebetaGlyRsubunit(i.e.heteromericαβGlyRs).Wefirstevaluatedthesensitivityofhomomericα1GlyRstodifferentconcentrationsofgelsemine.Wefoundthat lowmicromolarconcentrations(0.1-50μM)ofgelseminepotentiatedtheglycine-activatedcurrentsofα1GlyRs.Thepotentiationdisplayedabell-shapedprofile,withapeakpotentiationof80±30%using25μMofgelsemine.Wenextinvestigatedwhethertheα2andα3GlyRsweresimilarlymodulatedbygelsemine.Unexpectedly,theglycine-evokedchloridecurrentsthroughthesereceptorsweresignificantlyinhibitedinaconcentration-dependentmannerbygelsemine(1-100μM).WenextanalyzedtheinfluenceoftheβsubunitonthesepharmacologicalprofilesbystudyingthecorrespondingheteromericαβGlyRs.Our results showedthat thepresenceof theβsubunitdidnotaffect thesensitivityα2andα3GlyRs togelsemine.However,thegelsemine-inducedpotentiationofα1GlyRswassignificantlydiminishedbytheexpressionofβsubunits(-8±7%using25μMofgelsemineinα1βGlyRs).Theseresultsindicatethattheactionsofgelseminearesubunit-specificandthatareinfluencedbythepresenceofβsubunits.ThesedatathussuggeststhepresenceofspecificmoleculardeterminantsforthealkaloideffectsonseveralGlyRsubtypes.Ongoingstudiesusingchimericandpoint-mutatedGlyRswilldefinetheresiduesinvolvedinthesepharmacologicaldifferences.Futureeffortsaimingtoidentifythegelseminebindingandmodulatorysitesmayfacilitatethedevelopmentofnewsubunit-specificGlyRmodulatorswiththerapeuticpotential.

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10)Modulation of spinal glycine receptors by the alkaloid gelsemine.

Murath, Pablo1.,Lara,Cesar1.,Yévenes,Gonzalo1.,1DepartmentofPhysiologyUniversidadDeConcepción.(SponsoredbySupportedByFONDECYT1140515.SponsoredByDr.JorgeFuentealbaA.)

Glycinereceptors (GlyRs)aretransmitter-gatedanionchannels thatmediatesynaptic inhibition in thecentralnervoussystem.GlyRinhibitoryfunctionisparticularlycriticalintheprocessingofnociceptiveandsensorysignalsatthelevelofthespinaldorsalhorn.Recentstudieshaveshownthatgelsemine,anaturalalkaloidfromGelsemium sempervirens,exertpotentanalgesicactionsonbehavioralmodelsofpain.Theseevidences suggest thatgelseminemay interactwith spinalGlyRsand thusmodulate thedorsalhorninhibitorysynaptictransmission.HerewestudiedthesensitivityofspinalGlyRstogelsemineusingculturedmousespinalcordneuronsandwhole-cellpatch-clamprecordings.Wefirstanalyzedtheeffectofdifferentconcentrationsofgelsemineontheglycine-evokedcurrents.Wefoundthatgelsemine(0.1-200μM)inhibitedtheglycine-activatedcurrentsinaconcentration-dependentfashion.Theinhibitiondisplayedapeakinhibitionof-79±3%elicitedby200μMofgelseminewithanIC50of48±7μM.Wenext investigated theeffectsof thealkaloidon theGABA-evokedorAMPA-evokedcurrentson theseneurons.Our resultsshowedthattheagonist-evokedGABAergicorAMPAergiccurrentswerenotsignificantlymodifiedby50μMofgelsemine.Wefinallystudiedtheeffectsofgelsemineontheglycinergicsynapticactivitybystudyingminiatureglycinergicpost-synapticcurrents(mIPSCs).Ourresultsshowedthatgelsemine(50μM)dramaticallydecreasedthefrequencyofglycinergicmIPSCs(-89±4%).Thealkaloidhoweverdidnot significantly altered the glycinergicmIPSCamplitude (13±6%). These results indicate that gelseminenegativelymodulatesspinalGlyRs.Furthermore,ourdatashowedthatthealkaloidstronglymodifytheglycinergicsynapticactivityofculturedspinalneurons.Collectively,thesedatasuggestthatthealkaloidmaymodulateinhibitorysynapticnetworksatthelevelofthespinaldorsalhorn.Thesesynapticactionsmaycontributetoexplaintheanalgesicactivityofgelsemineinbehavioralpainmodels.

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11) TOLL-LIKE-RECEPTOR (TLR4) INDUCES AN INCREASE IN PROINFLAMMATORY CYTOKINES AND ADHESION MOLECULES IN CARDIAC FIBROBLAST AND MYOFIBROBLAST

ANFOSSI, Renatto1.,Humeres,Diego1.,Boza,Pía1.,Muñoz,Claudia1.,Vivar,Raúl1.,Díaz-Araya,Guillermo1.,1FARMACOLOGÍAYTOXICOLOGÍA,CIENCIASQUÍMICASYFARMACÉUTICAS,UniversidadDeChile.(SponsoredbyFONDECYTRegular1130300(GDA).BECAGASTOSOPERACIONALES21120406)

Introduction:Toll-like-receptor(TLR4)playsacriticalroleontheonsetandresolutionof inflammatorycardiovasculardiseasessuchascardiacfibrosis.Thisreceptorhasbeenextensivelystudiedinimmunecellspresentonthesiteofcardiacinjury.However,itsroleoncardiacfibroblasts(CF)andmyofibroblasts(CMF);whicharecentralmediatorsofinflammatoryandfibroticmyocardialremodeling,remainsunknown.WehypothesizethatthisreceptorplaysanimportantroleinCF/CMFproinflammatorycytokinesecretionandadhesionmoleculeexpression.Methods:AdultratCFandCMFweretreatedwithLPS(1μg/ml8-48h)inpresence/absenceofTLR4inhibitorTAK-242.9cytokinessecretedtotheextracellularmediumbyCFandCMF(TNF-α,IL-2,IL-4,IL-5,IL-10,IL-12,IL-13,IFN-γ,MCP-1)werequantifiedbyLUMINEXaftertreatment.Proteinlevelsofintercellularadhesionmolecule(ICAM-1)andvascularcellularadhesionmolecule(VCAM-1)weremeasuredbyWesternblot.ToassessifVCAM-1/ICAM-1werefunctional,weculturedmonolayersofCFandCMFinpresence/absenceofLPSandTAK-242during24h,andperformedadhesionassaysofmonocytesoverthosecellmonolayers.Results:TLR4activationinducesanincreaseinthesecretionofTNF-α,IL-10andMCP-1inbothCFandCMFat24/48hofLPSstimulation.TLR4activationalsogeneratedanincreaseofICAM-1/VCAM-1proteinsexpressioninCFandevengreaterlevelswereobservedinCMF.Theseeffectswereobservedfrom8hofLPSstimulationandonwards.BothICAM-1/VCAM-1werenecessaryforadhesionofmonocytestolayersofCFandCMF,becausetheinhibitionoftheseadhesionmoleculesdecreasedthenumberofmonocytesadhered.TheseresultssuggestthatactivationofTLR4inCF/CMFcanactasakeycomponentinthecrosstalkbetweenimmuneandcardiaccellsbycontributingtotheproinflammatorycytokinemilieuandbyrecruitingimmunecellstothesiteofinjurythroughtheup-regulationofICAM-1/VCAM-1.

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12)Characterization of toxicity and antioxidant effects of selenium nanoparticles biosynthesized by Pantoea agglomerans in HUVEC

Haensgen,A.1.,Saavedra,A.1.,González,M.1.,Rojas,S.1.,Carrasco,I.2.,Rodríguez,S.3.,Rojas,C.3.,1PhysiologyUniversidadDeConcepción.2MicrobiologyUniversidadDeConcepción.3CIPAUniversidadDeConcepción.(SponsoredbyFONDEFCA12-I-10374,BasalConicyt-RegionalR08C1002)

Ithasbeenfoundthatselenium(Se)isanessentialmicronutrientthathasanantioxidantandcardioprotectiverole.Seleniumdeficiency causes cardiovasculardiseasesand this condition is associatedwith lower concentrationof Se in soil, affecting thenutritionalpropertiesof grownvegetables forhumanandanimal consumption.Oneof themaindifficultiesabout theuseofSe like a nutritional supplement is that the soluble formsof Se, selenite (Se IV) or selenate (SeVI), are toxic andunable foruseinhumans.TheelementalSe(Se0)haslowertoxicity,butis insoluble inwater.TosolvethisproblemweproposetheuseofPantoea agglomerans, a bacterium capable of coexisting in enrichedmediumwith selenite/selenite, because reduces themetalloid.P. agglomeranssynthetizesandreleasesSe0nanoparticles(SeNPs),theseSeNPscanbeobtained,filtered,stabilizeswithL-cysteineandencapsulateswithchitosanandtripolyphosphateforpotentialapplicationasanutritionalsupplement.OuraimwasdeterminethetoxicityandantioxidantcapacityofbiosynthesizedSeNPsonhumanendothelialcells.Humanumbilicalveinendothelialcells(HUVEC)wereisolated(collagenasedigestion)andmaintainedinmedium199(M199)withsera(20%).HUVECwereincubated(37°C,24h)withdifferentformsofSeandSeNPsandcellviabilityandreactiveoxygenspecies(ROS)synthesisweredeterminedwithVybrant®MTTCellProliferationKitand2′,7′-dichlorofluorescein(DCF)dye,respectively.TheincubationofHUVECwithselenite(1µg/ml)reducedthecellviabilityto20%,meanwhiletheincubationwithbiosynthetizedSeNPs(1µg/ml)didnotreducedthecellviability,comparedtocontrol.InregardswithROSsynthesis,incubationwithselenite(1µg/ml)increasedtheoxidativestress2.9-foldrelatedtocontrol,filteredSeNPsdidnotchangetheROSlevels,meanwhiletheencapsulatedSeNPsinducedasignificantreductionofROSinHUVECexposedtohighconcentrationofD-glucose.Inconclusion,seleniteenhancesROSsynthesisanddiminishedtheviabilityofhumanendothelialcells.ThesealterationsareavoidedwhenweusedbiosynthesizedSeNPs,especiallythestabilizedandencapsulatedform.

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13)Prolonged activation of connexin-formed hemichannels by angiotensin II-induced NADPH oxidase-mediated superoxide production in endothelial cells

Lazo, V1.,Poblete,Inés1.,Figueroa,Xavier1.,1Departamentodefisiología,Facultaddecienciasbiológicas,PontificiaUniversidadCatólicaDeChile.

Increasesinsuperoxide(O2.-)productionbytheNADPHoxidaseareassociatedwiththedevelopmentofendothelialdysfunction

andhypertension.ItisthoughtthatthedetrimentofvascularfunctionobservedinresponsetoangiotensinII(AII)ismediatedbytheincreaseinNADPHoxidase-derivedO2

.-andthefurtherrapidreactionofthisreactiveoxygenspeciewithnitricoxide(NO),whichsubsequentlyattenuatestheendothelium-dependentvasodilatorresponses.However,inadditiontoNO,connexin-formedchannelsalsoplayanimportantroleinthecontrolandcoordinationofvascularsignaling.Connexinproteinscanformgapjunctionchannelsorhemichannels(halfofagapjunctionchannel)andareexpressinendothelialandsmoothmusclecells.Ithasbeenshownthatprolongedactivationofhemichannelsleadstothedevelopmentofcelldysfunction,buttheeffectofO2

.-onhemichannelactivityinthevascularwallofresistancearterieshasnotbeendetermined.Inthiswork,weanalyzedtheinvolvementofNADPHoxidase-mediatedO2

.-productionintheregulationofhemichannelfunctioninmesentericresistancearteriesandprimaryculturesofmesentericendothelialcells.HemichannelopeningwasassessedbymeasuringethidiumuptakeinresponsetoAII(10nM)ordirectlytotheincrementofO2

.-formationevokedbytheapplicationoftheNADPHoxidasehighaffinitysubstrate,NADH(100µM).Asexpected,AIIandNADHapplicationresultedinaprolongedincreaseofO2

.-formationandethidiumuptakeinintactarteriesandculturedendothelial cells.Both the increase inO2

.- productionandethidiumuptakewereblockedbyapocynin, aNADPHoxidaseblocker,orTEMPOL,aO2

.- scavenger. Inaddition,theconnexin-formedchannelblocker,carbenoxolone,alsoabolishedtheincrementinethidiumuptake.TheseresultssuggestthatNADPHoxidase-derivedO2

.-formationleadstoprolongedactivationof connexin hemichannels in endothelial cells of resistance arteries,whichmay explain, at least in part, the development ofendothelialdysfunctiontypicallyobservedinresponsetoAII.ProyectoFONDECYT1150530

214

14)NADPH oxidase regulation by polycystin-1 in cardiomyocytes

Córdova-Casanova, A1.,Olmedo,I1.,Donoso,P1.,Sánchez,G1.,Pedrozo,Z1,2.,1InstitutodeCienciasBiomédicas,FacultaddeMedicina,UniversidadDeChile.2AdvancedCenterforChronicDiseases(ACCDiS),FacultaddeMedicina,UniversidadDeChile.(SponsoredbyFondecyt1150887,1130407,3140449.ProgramaU-IniciaConcursoDeReforzamientoDeInserciónProductivaDeNuevosAcadémicosVID2014UniversidadDeChile,FONDAP15130011)

NADPHoxidase2(Nox2),isoneofthemostimportantsourcesofreactiveoxygenspecies(ROS)inthecardiomyocyteandplaysa crucial role in cardiac physiology and pathology.Mechanical stretch activates Nox2 and increases ROS generation but themechanosensorimplicatedinthisactivationisunknown.Polycystin-1(PC1),atransmembraneproteinthatactsasamechanosensorindifferentcells,isalsoexpressedincardiomyocytesbutitsphysiologicalfunctionisnotfullyunderstood.ToinvestigatetheroleofPC1intheregulationofNox2activitywemeasurednitratedproteins,asanindicationofROSgeneration,inneonatalratventricularcardiomyocytestransfectedwithsiRNAspecifictoPC1orinhearthomogenatesfromPC1knockoutmice.

WeobservedthatthedecreasedPC1expressionintheneonatalcardiomyocytestransfectedwiththePC1siRNAcausedasignificantincrease innitratedproteins.Apocynin, aNox2 inhibitor,prevented this increase.Hearthomogenates fromPC1KOmicealsoshowedan increase innitratedproteinsascomparedtocontrols,confirmingthedataobtained incardiomyocytes.OurresultssuggestthatPC-1isanewregulatorofNox2incardiomyocyteswhichinhibitsNox2,andpreventsthegenerationofROSinbaselineconditions.

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15)Effect of oxHDL on the expression and distribution of endothelial proteins involved in coagulation and fibrinolysis

Pérez, Lorena1.,Simon,Felipe1,2.,1DepartamentodeCienciasBiológicas,FacultaddeCienciasBiológicasyMedicina,UniversidadAndrésBello.2MillenniumInstituteonImmunologyandImmunotherapySantiago,Chile.(SponsoredbyFondecyt1121078AndMIIP09-016-F)

Highdensitylipoprotein(HDL)hasbeenalwaysknownasaprotectiveagentagainstcardiovasculardiseasessuchasatherosclerosisand thrombosis.However, there is controversyabout the roleplayedby theoxidized formofHDL (oxHDL)on thesediseases.Oxidative modification of native HDL can take place as a consequence of high oxidative stress mainly due to inflammatorystimuli.Ontheotherhand,theendotheliumplaysakeyroleonthrombosisregulation,throughexpressionand/orsecretionofproteinsinvolvedincoagulation(thrombusformation)andfibrinolysis(thrombusdegradation).Duringpathologicalcondition,theendotheliumisinpermanentcontactwithseveralmediatorsofinflammationincludingoxHDL.Nevertheless,theeffectproducedbyoxHDLonthrombosisregulationisnotknown.Thus,theaimofthisworkistostudytheeffectofoxHDLontheexpressionanddistributionofendothelialproteinsinvolvedinthrombosisregulation.Inordertoassessthatissue,ratprimaryculturesofmesentericendothelialcells(RMEC)wereexposedto50µg/mLofnativeHDLoroxHDLfor18h,andtheproteinexpressionanddistributionwasassessedbywesternblotand immunofluorescence, respectively.Tissue factor (TF)andtissue factorpathwayinhibitor(TFPI),wereevaluatedasindicatorsofcoagulation.Plasminogenactivator(t-PA),plasminogenactivatorinhibitor(PAI-1) and thrombin-activatable fibrinolysis inhibitor (TAFI) were evaluated as indicators of fibrinolytic activity. Our results showthatRMECexposedtooxHDLchangestheexpressionanddistributionofTF,TFPI, t-PA,PAI-1,andTAFI,suggestingapotentialmodulationonthrombusformation/degradationrate.WeconcludethatoxHDLisinvolvedintheexpressionanddistributionofTF,TFPI,t-PA,PAI-1,andTAFIonRMEC.

216

16)Inhibition of signal transducer and activator of transcription 3 expression induces ALK-5-dependent SMAD4 mobilization in endothelial cells.

Rojas, Macarena1.,Becerra,Alvaro1.,Simon,Felipe1,2.,1Cienciasbiológicas,Cienciasbiológicas,UniversidadAndrésBello.2IMIIMillenniumInstituteonImmunologyandImmunotherapy.(SponsoredbyFondecyt1121078AndMIIP09-016-F)

Introduction:Ithasbeendemonstratedthatduringtheconversionofendothelialcells(ECs)intoactivatedfibroblast,thepro-fibroticcytokinetransforminggrowth factor-β1 (TGF-β1) is increased.UponbindingofTGF-β1to its type I receptoractivingreceptor-likekinase5(ALK-5),SMADproteinsconformanheterologouscomplexwiththecommon-mediatorSMAD4.Subsequentlythisproteincomplextranslocateintothenucleuswhereitregulatestheexpressionofpro-fibroticgenespromotingfibrosis.Ontheotherhand,thesignaltransducerandactivatoroftranscription3(STAT3)isatranscriptionfactorwhichisactivatedinresponsetocytokinesandgrowthfactors.IthasbeendescribedthatinhibitionofSTAT3expressioninducesspontaneousfibrosis,however,theunderlyingmechanismhasnotbeendescribed.ThereforeouraimwastostudywhetherinhibitionofSTAT3expressioninducesthemobilizationofSMAD4intothenucleus,mediatedbyaTGF-β1/ALK-5signalingpathwaymechanism.Methods and Results: Usingprimaryculturesofratmesentericendothelialcells(RMEC),wedemonstratedthatexpressioninhibitionofSTAT3usingsiRNA-STAT3inducedthesynthesisandsecretionofTGF-β1.Moreover,expressioninhibitionofSTAT3increasedSMAD4mobilizationtonucleus.Furthermore,whenweusedthesiRNA-STAT3combinedwithaninhibitorofALK-5(SB431542)thetranslocationofSMAD4intothenucleuswasblocked. Conclusion:WedemonstratethattheinhibitionofSTAT3expressioninducesincreasedtranslocationofSMAD4intothenucleusdependentonALK-5activity.Furthermore,STAT3suppressioninducesTGF-β1secretionsuggestingitsparticipationonALK-5-dependentSMAD4translocationprocess.

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17)The inhibition of endoplasmic reticulum stress do not reverts the fetoplacental endothelial dysfunction in maternal obesity.

Saavedra, Andrea1.,Rojas,Susana2.,Valdivia,Luz1.,Haensgen,Astrid2.,Cid,Marcela3.,Gonzalez,Marcelo1.,Farias,Marcelo4.,1Fisiología,CienciasBiologicas.,UniversidadDeConcepción.2fisiologia,cienciasbiologicas,UniversidadDeConcepción.3Obstetricia,Medicina,UniversidadDeConcepción.4ObstetriciayGinecología,Medicina,PontificiaUniversidadCatólicaDeChile.(SponsoredbyFONDECYT1121145,11100192.)

Thematernalobesity(MO)isagrowingsyndromeinourpopulation,withaprevalenceof30.7%inChileanpregnantinaccordingtoChileanMinistryofHealth(MINSAL).Inanimalmodelsofobesity,havebeendeterminedthatthereisadeleteriouseffectofendoplasmicreticulumstress(ERS)oninsulinsignalingpathway.Inotherhand,iswellknownthatinsulinincreasesthenitricoxide(NO)synthesisinhumanendotheliumandinsulinresistance(IR)isassociatedwithendothelialdysfunction.WehavehypothesizedthatthereisanassociationbetweenMO,IRandERSinhumanendothelialdysfunction.Objectives:TodeterminetheeffectofERSinhibitor(TUDCA,taurourso-deoxycholicacid)and/orinsulinonplacentalvascularreactivity,phosphorylationofendothelialnitricoxidesynthase(eNOS)andlevelsofnitricoxide(NO)andreactiveoxygenspecies(ROS)inHUVEC(humanumbilicalveinendothelialcells)fromnormalandobesitypregnant.Methods:ChorionicveinsandHUVECwereisolated(collagenasedigestion)fromplacentaandumbilicalcord.Sampleswereclassifiedasnormal(N)orobese(ob)inaccordwithpregnantbodymassindexatendofgestation.Isometrictensionwasdeterminedinvesselspre-constrictedwithincreasingconcentrationsofU46619(5x10-8-5x10-4)andexposedtoinsulin(1nM)and/orTUDCA(500μM).HUVECwereincubatedinpresenceorabsenceofinsulin(8h)and/orTUDCA(100μM,24h)tomeasuretotalandphosphorylatedeNOSratio(p-eNOS/eNOS)bywesternblotting,fluorescencemeasureforNOandROSusedDAFandDCFprobes,respectively.Results:Innormalchorionicvein,theconstrictioninducedbyU46619wasdecreased(p<0.05)byinsulin(41%ofmaximalconstriction).WhenvesselswereexposedtoTUDCA,vasoconstrictiondecreased7%and22%inabsenceorpresenceofinsulin,respectively. InHUVEC-Ninsulinincreasethep-eNOS/eNOSratio,withoutchangesincellsco-incubatedwithTUDCA.InHUVEC-obwasdeterminedhigherp-eNOS/eNOSratiocomparewithHUVEC-N.Insulinreducesp-eNOS/eNOSratioinabsence(32%)orpresence(41%)ofTUDCA,untillevelssimilartonormalcontrol.InHUVEC-obthereisanincreaseofNOproduction(66%)andROS(138%),whichisnotreversedbyinsulinand/orTUDCA.Conclusions:TheERSinhibitionwithTUDCAisnotcapableofpotentiatethevasorelaxationinducedbyinsulininnormalpatients,infactthereisareductionofinsulinrelaxationinpresenceoftheinhibitor.Theseresultsarecorrelatedwithp-eNOS.Inobesity, insulinrestoresthenormalabundanceofp-eNOSinHUVEC,buttherearenochangesincellsincubatedwithTUDCA.ROSandNOproductionareincreasedinobesitypatients,butTUDCAisunabletoincreaseordecreaseeffectsofinsulin.

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18)Allosteric regulation of arginase II by inhibitors of NO synthesis. A new mechanism of regulation of NO synthesis in hypertensive processes?

Taborda, María1.,Betancur,Johana1.,Moraga,Fernando1.,López,Vasthi1.,1CienciasBiomédicas,Medicina,UniversidadCatólicaDelNorte.(SponsoredbyThisInvestigationWasMadeWithFundingFromTheRegionalGovernmentAndTheFICR30137774-0Project.)

INTRODUCTION: Themining exploitation in thenorthof Chile has been settled at sites locatedover 4000m, generating theexposureofhumantochronicintermittenthypobarichypoxia(CIHH).Hasbeendescribed,thathypoxiaproducesvasoconstrictionofthecardiovascularsystem,withthepurposeofimprovethedifusionoftheO2.Although,ifthehypoxiaischronic,woulddevelophypertension.Describedinseveraltypesthehypertension,andecreaseofplasmaticofNO,wouldbeassociatedwithincreasedconcentrationsinhibitorsofeNOS,suchasasymmetricdimethylarginine(ADMA)andhomocysteineandaminorbioavailabilityofL-arginineor is theresultsofthecompetitionbetweennitricoxidesynthase(INOS)andarginase.ThemainpurpuseofthisstudywastodeterminetheeffecttheADMAandhomocysteineonthearginasevia,anditspossiblerolinthedevelopmentthehypertensioninducedbyhypoxia.METHODS: Thehypobarichypoxiawassimulatedinahypobaricchamberat428torr(4600m).Subsequently,oncesacrificedandidentifiedratstolerantsandintolerantstoCIHHaccordingtomethodologydescribedonearlierstudies. The arginase activity and inhibitors concentrations were measured. RESULTS AND CONCLUSION: Hypoxia-inducedhypertensionwoulddevelopby increased concentrationsofADMAandhomocysteine, producing an activationof arginase II,generatingaminoravailabilityofL-arginineforNOsynthesis.

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19)Comparative study of the protein expression and activity of inflammasome NLRP3 in cardiac fibroblast and myofibroblast.

Tapia, Felipe1.,Boza,Pia1.,Díaz,Guillermo1.,1DepartamentodeFarmacologíayToxicología,CienciasQuímicasyFarmacéuticas,UniversidadDeChile.(SponsoredbyProjectFONDECYT1130300.SponsorGuillermoDíaz.)

Theinflammasomeisamultiproteincomplexthatincludespattern-recognitionreceptors(PRRs).Thesereceptorscandetectawiderangeofmolecularpatternexclusiveofmicroorganismsandcanalsorecognizechemicalsignalsassociatedto infectionortissuedamage.TheinflammasomeNLRP3hasbeenwidelystudied,alongwiththereceptor,thecomplexalsoincludestheadapterproteinASCandpro-caspase-1.Thepurposeofthiscomplexitistoactivateandregulatetheinnateimmunesystemthroughthesecretionofthepro-inflammatorycytokineIL-1b.

CardiacFibroblasts(CF)equalstoapproximatelytwo-thirdsofthemyocardialtissuevolumeandareinvolvedinthemaintenanceandhomeostasisoftheextracellularmatrix(ECM).Inaddition,theyalsoparticipateintheheartrepairprocessbydifferentiatingintocardiacmyofibroblasts(CMF)whicharecellsinvolvedintheinflammatoryresponsetoinjury.MyofibroblastsarelargecellswithspecificstressfibersthatdistinguishthemfromCFandtheynormallyarepresentfollowingcardiacinjury.

Even though the inflammasome NLRP3 has been studied in different cellular types, until now there are no evidence aboutcomparativestudiesofproteinexpressionandactivityofthisinflammasomeinCFandCMF.

PrimaryculturesofCFobtainedfromneonatalratsinpassage1wererealizedfortheexperiments.CMFwereobtainedbyincubatingCFwithTGF-b1(5ng/ml)for96hours.BothcelltypeswerestimulatedwithLPS(1mg/ml)for8hours.TheproteinlevelsoftheNLRP3,ASC,pro-caspase-1andpro-IL-1bweremeasuredbyWesternBlot.Theactivityofcaspase1wasobtainedbyfluorometricassaysandtheIL-1bsecretionwasmeasuredbyanELISAKit.

Theresultsobtainedshowedhigherlevelsofpro-IL-1binCFthaninCMF.Theproteinlevelsofpro-caspase-1andthesecretionofIL-1bwerehigherinCMFthaninCF.CMFalsoshowedagreateractivityofcaspase1thanCF.ProteinlevelsofASCandNLRP3didnotchangebetweeneithercelltypes.

Finally,wecanconcludeonthebasisoftheresultsobtainedthatCFhaveapro-inflammatoryroleinacutestatesincomparisonwithCMF.Thiscanbeevaluatedthroughthehigherlevelsobtainedofpro-IL-1b.Ontheotherhand,theCMFpresenthigherlevelsofproteinexpressionandactivityofinflammasomeNLRP3inCMF.

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20)Participation of signal transducer and activator of transcription 3 in fibrosis of vascular endothelial cells

Vallejos, Alejandro1.,Rojas,Macarena1.,Becerra,Álvaro1.,Simon,Felipe1,2.,1DepartamentoCienciasBiológicas,FacultaddeCienciasBiológicas,UniversidadAndrésBello.2IMIIMillenniumInstituteonImmunologyandImmunotherapy.(SponsoredbyFondecyt1121078AndMIIP09-016-F)

Introduction:Mostcardiovasculardiseaseshavecommoninflammatoryconditionswhichinducesendothelialdysfunctionthroughimmunesystemactivation.Inflammatoryconditionspromptsendothelialcells(ECs)toadoptfibroblast-likefeaturescharacterizedbylosstheirendothelialspecificmarkersVE-cadherinandCD-31.Inaddition,ECsacquirefibroticmarkersasfibroblast–specificprotein1(FSP-1)andα-smoothmuscleactin(α-SMA).Furthermore,bymeansofendothelialfibrosis,ECsgaintheextracellularmatrix(ECM)proteinsfibronectin(FN)andcollagentypeIII(ColIII).Concordantly,morphologicalchangeshavebeenobserved.NormalECsshowashort-spindlemorphologywithcobblestoneappearance,whereasfibroticendotheliumacquiresafibroblast-likespindleshapedphenotype.Moreover,endothelialcell–celljunctionsareloss.Signaltransducerandactivatoroftranscription3(STAT-3)isatranscriptionalfactorimplicatedinthegrowthoftumors,inprocesseslikeapoptosisandcellularproliferation.Ithasbeendescribedthat inhibitionofSTAT3expression inducesspontaneousfibrosis.However, itsparticipationonendothelialfibrosisisnotknown.Thus,ouraimwastostudywhetherinhibitionofSTAT3expressioninducesendothelialfibrosis.Methods and Results: Usingprimaryculturesofratmesentericendothelialcells(RMEC),wedemonstratedthatexpressioninhibitionofSTAT3usingapharmacologicalinhibitorandsiRNAagainstSTAT3inducedendothelialfibrosis.ExpressioninhibitionofSTAT3decreasedexpression of VE-cadherin and CD-31 and increased expression of the fibroticmarkers, FSP-1 andα-SMA. Furthermore, ECMproteins,FNandColIII,wereseverelyincreased.Conclusion:WedemonstratethattheinhibitionofSTAT3expressioninducesachangeintheendothelialexpressionpatternproducingendothelialfibrosis.

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21)NADPH oxidase blockade reduces Snitrosylation and opening of Cx43 hemichannels improving heart contractility and rhythmicity in mdx mice

Vielma Z, Alejandra1.,BoricP,Mauricio1.,GonzalezR,Daniel2.,1CienciasFisiologicas,CienciasBiologicas,PontificiaUniversidadCatólicaDeChile.2CienciasBasicasBiomedicas,CienciasdelaSalud,UniversidadDeTalca.(SponsoredbyFONDECYT1120595)

Duchennemusculardystrophy,afatalprogressivegeneticdisease,causesdystrophiccardiomyopathyalteringintracellularcalciumandoxidativestress.Oneofthemostimportantsourcesofreactiveoxygenspecies(ROS)inthecardiovascularsystemisNADPHoxidase(NOX).IthasbeenshownthatROSinterfereswithconnexin43(Cx43)locationtotheintercalateddiscs;andhemichannelsformedbyconnexins(Cx)orpannexins(Px)constituteapotentialpathwayfordissipationofionicgradientsandtissuedamage.

Usingmdxmice,amodelofDuchennedisease,wetestedthehypothesisthatincreasedoxidativestressduetoincreasedNOXactivitycausesS-nitrosylationandlateralizationofCxsand/orderegulatingofCxsorPxshemichannelactivity,leadingtodecreasedinotropismandincreasingarrhythmogenicity.

Isolated hearts from 2 and 10months of agemdx mice(Langendorf) showed a reduced contractility, decreased response toβ-adrenergicstimulation,highernumberofarrhythmicepisodes,andincreasedfibrosis(Massontrichromestain)andincreasednumberofapoptoticcells(TUNEL),ascomparedtocontrols.Atbothages,mdxshowedincreasedexpressionofcardiacp22phoxandgp91phoxsubunits,andhigherNOXactivity,associatedwithincreasedlipidperoxidationinserum,skeletalandcardiacmuscle.AlltheseconditionswerereversedtocontrollevelswhenmdxanimalsweretreatedchronicallywithNOXinhibitorapocynin(1monthorally).

WhiletotalcardiacCx43contentwasunchanged,immunofluorescenceandWesternblotanalysisdemonstratedhigherpresenceofCx43atlateralmembranesin2-and10-monthmdxmice,indicatingthatCx43re-localizesfromintercalateddiscstosarcolemma.Inaddition,biotin-switchassaysshowedincreasedS-nitrosylationofCx43andPx1proteins.

Hemichannels opening, evaluatedusing ethidiumpermeabilitywas substantially higher inmdx hearts and this conditionwasnormalizedwhenmiceweretreatedbyapocyninoracutely,usinghemichannelblockerscarbenoxolone(forCx)andprobenecid(forPx).

Theseresultssuggestthat,inDuchennedisease,increasedNOXactivityderegulatesCx43distributionandS-nitrosylation,causinghemichannelsformationand/oractivation,whichmaycontributetoincreasedapoptosisandcardiacdysfunction.

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22)Daily variation of salivary melatonin acute exposure to altitude of 3270 m.

Tapia, Marcelo1.,Wulff,Cristian2.,Silva,Juan2.,DeGregorio,Nicole3.,Behn,Claus3.,1Kinesiologia,CienciasdelaSalud,UniversidadDeAntofagasta.2Biomédico,CienciasdelaSalud,UniversidadDeAntofagasta.3ICBM,FacultaddeMedicina,UniversidadDeChile.

Altitudeisanexternalenvironmentaffectingmultifactorialaslivingorganismsthatareexposeditacutelyorchronically.Themostimmediateresponsestoaltitudearegeneratedrespiratoryandcardiovascularlevelandareintendedtoensuretheavailabilityofoxygentothetissues.Thereisanincreaseinaltitudeinsomniaortroublesleeping,eitherconciliationdeficitinsleeporincreasednumberofawakening,isassociatedwithsubsequentsleepinessduringworkingperiods,affectingtheperformanceofcognitivemotorfunctionsandstatusofpersonsfocusingonproductivityandqualityofworkperformed,andparallelcriticallymanifestinginincreasedriskofaccidents.Inthesleep/wakecycleplaysafundamentalrolemelatonin,knownasthesleephormonewhichalsohasantioxidanteffectandisinvolvedinenergymetabolism.Melatoninsynthesisisinhibitedbyexposuretolight,especiallybluelightspectrum,concomitantlyraisedtheperceptionofbluelightmaybediminishedbytheeffectsofaltitude.Fromthisbackgroundtoassesstheeffectsonmelatoninsecretionduringacuteexposuretoaltitudeappearsasaninterestingproblemthatmayyieldnewinsightforunderstandingthechangesoccurringinthecontrolofthishormoneonphysiologicalfunctionsandthesleep/wakecycleinaltitude.Objective:Theobjectiveofthisprojectwastoevaluatetheeffectofacuteexposuretoaltitudeof3270mintheconcentrationofsalivarymelatonininelevenyoungsubjectsresidentssealevelandrelationshiptophysiologicalparameters.Methodology: Thesamplepopulationischaracterizedbyitsanthropometricsurveysandrightthroughsleephabits,smokingandstresslevel.Profile,respiratoryandcardiovascularparameterswererecordedatrestatsealevelon24hafterexposuretoaltitudeof3270m,usingportableergospirometer.Melatoninmeasurementsforcollectionsalivasampleswasperformedoneachsubjectwithbucalwithbucalswords,daytime(12:00h)andnighttime(0:00h)tosealevelandafter24hofexposureto3270m.Melatoninlevelweredeterminedby inmunoassay for the thestofELISA.Results: Acuteexposure toaltitude (3270m)caused increasedmelatoninconcentrations insalivasamplescomparedtothevaluesofmetabolitecollectedatsea level.Alsostatesthatthereisaninverselinearcorrelationwithchangesinrespiratoryquotient(RQ)withthedifferencesbetweenthelevelofdaytimeandnighttimemelatoninofsealevel.Howevertheaforementionedcorrelationislostwhenindividualsareexposedtoacutehypoxiaconditions.

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23)Low birth weight children associate low 11BHSD2 activity and high lipocaline-2/NGAL

Carvajal, C1.,Tapia-Castillo,Alejandra2.,Lizama-Gonzalez,Jaime1.,Valdivia,Carolina1.,Villarzu,Paula1.,Martinez-Aguayo,Alejandro3.,Fardella,Carlos1.,1Endocrinology,Medicine,PontificiaUniversidadCatólicaDeChile.2Genetica,Medicina,UniversidadDelDesarrollo.3Pediatria,Medicina,PontificiaUniversidadCatólicaDeChile.

Lowbirthweight(LBW)hasbeenassociatedwiththeriskofhypertensioninadults.Recentlyhasbeenproposedthatincreasedinglucocorticoidexposureislikelytobeacriticaldeterminantofgrowthinearlylife.Theamountoffetalexposuretomaternalglucocorticoidsdependsontheexpressionof11beta-hydroxysteroiddehydrogenasetype2enzyme(11beta-HSD2),thatinactivatescortisol(F)tocortisone(E).Thus,impairmentofthe11beta-HSD2wouldallowstheactivationofthemineralocorticoidreceptor(MR)bycortisol,whichaffectthereleaseofcytokinedependentMRasNGAL,thathasbeenlinkedtoprogressionofrenaldisease.

Aim:Tocharacterizetheassociationsbetweenbirthweight(BW)andHSD11B2expression,F/Eratioinserum,andNGALasrenalmarkerinpediatricssubjects.

Methods: Westudied12pediatricpatientsand16controlsubjects(0-18years-old).Wecarriedoutawholeclinicalexamination,andmeasurementof serumaldosterone,plasma reninactivity (PRA), free cortisol (F)(serum), free cortisone (E) (serum), freeNGAL.RNAwasisolatedfromperipheralleukocytes.ThegeneexpressionwasquantifiedbyRT-qPCRandexpressedinrelativeunits(RU).StatisticalanalyseswereperformedbyMannWhitneytesttoevaluatedifferencesbetweengroups.Datawereexpressedasmedian[Q1-Q3]andcomparedwithMannWhitneytest.

Results: WefoundthattheHSD11B2expressionisdecreasedinsubjectswithLBWvsNBW(0.0017[0.00061-0.0036]vs0.01998[0.002912-0.1211]RU;p0.03).Interestingly,F/EinserumisincreasedinsubjectswithLBWvsNBW(5.15[2.72-8.67]vs2.56[2.19–3.73]RU;p0,01)andtheNGALisincreasedinsubjectswhitLBWvsNBW(108.4[91.79-133.1]vs78.32[59.72-83.70]ng/ml;p0.04).Wenotfounddifferenceswiththeothervariablesstudied.

Conclusions: Ourresultssuggestthatchildrenwithlowerbirthweightpresentedreduced11β-hydroxysteroiddehydrogenasetype2activityislikelytobeacriticaldeterminantofgrowthinearlylife,renaldamageandthehypertensiondevelopment.

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24)Aldosterone stimulates immune markers expression related to steroid receptors activation in adipose LS14 but not in SW872 cell line

Gonzalez-Gomez, Luis Martin1.,Fuentes,Cristobal1.,Allende,Fidel2.,Fuentes-Ibacache,Nataly1.,Ortiz-Canales,David1.,Muñoz-Durango,Natalia3.,Campino,Carmen1,3.,Solari,Sandra2.,Cifuentes,Mariana4.,Carvajal,Cristian1,3.,Kalergis,Alexis3.,Lagos,Carlos1,3.,Vecchiola,Andrea1,3.,Fardella,Carlos1,3.,1DepartmentofEndocrinology,SchoolofMedicine,PontificiaUniversidadCatolicadeChile.2DepartmentofClinicalLaboratories,SchoolofMedicine,PontificiaUniversidadCatolicadeChile.3IMIIMillenniumInstituteonImmunologyandImmunotherapy.4InstituteofNutritionandFoodTechnology(INTA),UniversidaddeChile.(SponsoredbySupportedByProyectoSOCHED13-6,CORFO13CTI-21526-P1,FONDECYT1150437&1130427,IMIIP09/016-FGrants.)

Background.Clinicaldatasupportthenotionthataldosteroneisassociatedwithaninflammatorystate,andrecentreportsshowthatthishormoneisnaturallysynthesizedintheadiposetissue.OntheotherhandseveralreportsshowthataldosteronemodulatesT cell polarization, including an inflammatory Th17 phenotype associated with end-organ damage.Thereforealdosterone’sinteractionwiththeadiposetissuemayalterthefunctionoftheimmunesystemandenhanceasystemiclow-gradeinflammation,leading toend-organdamage.Aim. Toassess in vitrowhetheraldosteronemodulates immunogenicactivity,using2differentadiposecell lines.Methods. ForLS14andSW872preadipocytesdifferentiation, cellswereserum-starvedovernight,and thentreatedwithanadipogeniccocktailfor8days.SW872andLS14preadipocytesandadipocytesweretreatedwithincreasingdosesofaldosterone(0.1–100nM),LPS(100ng/mL)orvehiclefor24hrs.RNAwasisolated,andsteroidreceptors(MRandGR)andinflammationmarkersexpression(IL-6,IL-1β,HSP-90andTLR-4)weredeterminedbyqRT-PCR.Results.SW872culturesproliferatemuchfasterthanLS14cultures,butdifferentiationgeneratedmatureadipocytesatsimilartimes,showingamorphologicalshift,fromfibroblast-likeshapestoawiderandroundershape.Upondifferentiation,LS14showasignificantdecreaseinMR(p=0.0204).SW872increasebothMRandGRexpression(p,whicharealsosignificantlyhigherthanLS14(p).100nMaldosteronetreatmenttendstodecreaseMRandincreaseGRexpressioninLS14cells,whereasSW872tendtodecreasebothMRandGRexpressionafterstimulation,whereonlyGRsignificantlydiffersbetweenLS14andSW872preadipocytes(p=0.0153).Inflammationmarkerstudies show that10nMaldosterone treatments significantly increasedHSP-90expression in LS14preadipocytes (p<0.05). InLS14adipocytes,TLR-4expressionpositivelycorrelateswithaldosteronetreatmentconcentrations(p=0.0264)aswellasHSP90(p=0.023),whereasIL-1βexpressionshowsanegativecorrelationwiththesetreatments(p=0.0402).LPStreatmentincreasedtheexpressionofIL-1βinLS14preadipocytesandbothTLR-4andIL-6expressioninLS14adipocytes(p,whereitalsotendstoincreaseIL-1βexpression.SW872celllinedidnotchangeitsimmunemarkersexpressioninresponsetoaldosteroneorLPS.Conclusion.AldosteroneandLPSdifferentiallystimulatesimmunemarkersexpressionassociatedwithsteroidreceptorsinLS14cellcultures,whereasSW872cellculturesdonotrespondeitheraldosteronenortoLPSstimulation.

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25)Expression of free fatty acid receptors 1 and 4 in bovine epithelial endometrial cells

Hidalgo, Maria1.,Larrazabal,Camilo1.,Teuber,Stefanie1.,Loncoman,Carlos1.,Manosalva,Carolina2.,Burgos,Rafael1.,1InstitutodeFarmacologia,FacultaddeCienciasVeterinarias,UniversidadAustralDeChile.2InstitutodeFarmacia,FacultaddeCiencias,UniversidadAustralDeChile.(SponsoredbyFondecyt1151047,FONDEFID14I10050AndDID-UACHS-2014-23)

Freefattyacidsareincreasedinplasmaaroundpartumincows,periodwhichthereisahighincidenceofinfectiousdisease,suchasmetritis.LongchainfattyacidsbindtoG-proteincoupledreceptors,suchasfreefattyacidreceptor1(FFAR1/GPR40)andFFAR4/GPR120,andaroleofthesereceptorsoninnateimmuneresponsehasbeensuggested.Recently,weclonedanddemonstratedthepresenceofthefunctionalFFAR1inbovineneutrophils,cellswithakeyroleinimmuneresponseinendometrium.TheaimofthisstudywastodeterminethepresenceofFFAR1andFFAR4inepithelialendometrialcells.Bovineepithelialendometrial(BEND)cellslinewereculturedandtotalRNAandproteinswereisolated.ByRT-PCRandusingspecificprimerstobovineFFAR1andFFAR4weobtainedaproductofamplificationoftheexpectedsize,254and134bprespectively,whichcorrespondtobothreceptors.UsingantibodiesagainstFFAR1andFFAR4,twoproteinsofapproximately31and42kDa,thepredictedsizeforFFAR1andFFAR4,weredetectedbyimmunoblot.Also, itwaspossibletoobservethepresenceofFFAR1andFFAR4inBENDcellsbyimmunofluorescence.WeshowedthattheagonistsofFFAR1andFFAR4,linoleicanddocosahexaenoicacid,respectively,inducedintracellularcalciummobilizationinFura-2AM-loadedBENDcellsbyspectrofluorometricassay.Inconclusion,ourresultsshowthatBENDcellsexpressFFAR1andFFAR4,andagonistsofbothreceptorsinduceintracellularcalciummobilization,thussuggestingthatBENDcellsfunctioncouldbemodulatedinthepresenceoffattyacids.(SupportedbyFondecyt1151047,FONDEFID14I10050andDID-UAChS-2014-23).

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26)Ugni molinae extracts and its triterpenoids: modulatory effects on β-amyloid aggregation

Jara, D1.,Ana,Riveros1.,Marcelo,Kogan2.,Carla,Delporte2.,1QuímicaFarmacológicayToxicológica,CienciasQuímicasyFarmacéuticas,UniversidadDeChile.2QuímicaFarmacológicayToxicológicaUniversidadDeChile.(SponsoredbyFONDECYT1130155Y1130425,BecaCONICYTN°21130380)

Theβ-amyloidpeptideisa40-42aminoacidmacromoleculeandisconsideredoneofthemajoretiologicalfactorsinAlzheimer’sdisease.Thisproteinisabletoaggregateintomorecomplexstructuressuchasoligomers,protofibrils,fibrilsorotherpossessingvarying degrees of neurotoxicity and directly influence the development of thementioned pathology. Therefore, it is highlyattractivefindingmolecules capable ofmodulating the aggregationprocess of this peptide in order to avoid its toxic effects.Ugni molinae is a native shrub from the south of Chile. The extracts of the leaves of this wild species haveshown anti-inflammatory, analgesic and antioxidant effects that would support its use in folk medicine1,2.Chemical studiesofmurtilla leaveshave shown thepresenceof twomajorgroupsof constituents:phenolic compounds suchas flavonoids, gallic acid, tannins and the derivates compounds of all of them; pentacyclic triterpenoid acids derived fromursane, oleanane and lupane such as asiatic,madecassic, ursolic, oleanolic, corosolic, betulinic, alphitolic andmaslinic acids.Someofthephenoliccompoundsandtriterpenoidspresentintheleavesofmurtillahaveshownmodulatoryeffectsonβ-amyloidaggregationandasaresult,themodulatoryeffectsoftheethanolicextracts(EET),ethylacetate(EAE),thetriterpenoidsursolicandmadecassicacidswerestudiedinthementionedpeptideaggregationprocess.Fortheabove,theTthioflavinassaywasused.Theemissionoffluorescenceofthioflavin(450/480nm)isdirectlyproportionaltotheconcentrationofamyloidaggregates3.1Aguirre,M.C.,Delporte,C.,Backhouse,N.,Erazo,S.,Letelier,M.E.,Cassels,B.K.,Negrete,R.Topicalanti-inflammatoryactivityof2α-hydroxypentacyclictriterpeneacidsfromtheleavesofUgni molinae.Bioorganic & medicinal chemistry,14(16):5673-5677,2006.2Rubilar,M.,Pinelo,M.,Scheuermann,E.,Sinero,J.,Nuñez,M.J.Murtaleaves(Ugni molinae Turcz)asasourceofantioxidantpolyphenols.Journal of Agricultural and Food Chemistry,54(1):59-64,2006.3Levine,H.Protein Science,2:404−410,1993.

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27)Structure-based virtual screening identification of a novel selective connexin hemichannel blocker

Lagos, Carlos F1.,Fernandez,Paola2.,Vargas,Anibal2.,Perez-Acle,Tomás3,4.,Sáez,JuanC2,4.,1DepartmentofEndocrinology,SchoolofMedicine,PontificiaUniversidadCatólicadeChile.2DepartmentofPhysiology,FacultyofBiologicalSciences,PontificiaUniversidadCatólicadeChile.3ComputationalBiologyLab(DLab)FundacionCienciayVida.4CINVInstitutoMilenioCentroInterdisciplinariodeNeurocienciadeValparaíso.(SponsoredbySupportedByFONDECYTProjects1150291&1130652,ProgramaDeFinanciamientoBasalPFB16AndICM09-022-PGrants.)

Connexins are membrane channel proteins, which form hexagonal arrays in the plasma membrane called hemichannels orconnexons.Duringthelastdecade,ithasbeendemonstratedthatunderphysiologicalconditionshemichannelsplayrelevantrolesincell-cellsignalingactingasmembranepathwayforreleasingextracellularsignalingmoleculessuchasATPandNAD+.Thus,theyarecurrentlyconsideredasautocrineandparacrinepathwayforintercellularcommunication.However, indiversepathologicalconditionstheactivityofconnexinhemichannelsisupregulatedandcontributestotheoutcomeofcellulardegeneration.Currently,theavailablehemichannelblockersalsoinhibitgapjunctionchannels,whichplayrelevantrolesincoordinatingnumerouselectricalandmetabolicresponsesofcellularcommunities.Therefore,itisimportanttodiscoverspecifichemichannelblockerwithouteffectongap junctionchannels,whichmightbeuseful todesign rational therapeutic treatmentsofdiversediseases. In thepresentwork,wereportthesuccessfuluseofstructure-basedvirtualscreeningfortheidentificationofnovelchemicalentitiestargetingconnexinhemichannels.Inparticularwehaveidentifiedaconnexinhemichannelblocker(D4)whichblockhemichannelsformedbyconnexins26,32,43and45,butnotgapjunctionchannelsformedbytheseconnexinsorpannexin1relatedchannels.Theidentifiedcompoundsmayserveasstartingpointforthedevelopmentofnovelgenerationofmorepotentandspecificconnexinhemichannelmodulators.

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28)Linoleic acid increases cell migration, MMP-9 activity and MAPK phosphorylation in human keratinocytes

Manosalva, Carolina1.,Mena,Jaqueline2.,Burgos,Rafael3.,Hidalgo,MaríaAngélica3.,1InstituteofPharmacy,FacultyofScience,UniversidadAustralDeChile,Valdivia,Chile.2DepartmentofBiology,FacultyofExactandNaturalSciences,UniversidaddeNariño,Pasto,Colombia.3InstituteofPharmacology,FacultyofVeterinaryScience,UniversidadAustralDeChile,Valdivia,Chile.(SponsoredbyFinancedByDID2014-S13-UACh)

Woundhealingplaysavitalroleinthemaintenanceoftheintegrityoftheskinandmucosalmembranes.Indeed,therearethreemajorskinresponsesafterinjury,includinginflammation,reepithelization(migrationofkeratinocytes)andremodeling(formationofgranulationtissues).Theinflammatoryresponseisakeyeventforacorrectreepithelizationandwoundclosure.Neutrophilsandmacrophagesarepredominantintheinflammationphaseproducingcytokinesandagreatvarietyofgrowthfactorsthatstimulatemigrationandproliferationofkeratinocytes.Thedirectedmigrationofkeratinocytesisinturnessentialforreepithelizationanddefectsinthisfunctionareassociatedwithchronicnon-healingwounds,suchasdiabeticulcers.Ithasbeendemonstratedthatlongchainfreefattyacids,suchasoleicacid(OA)andlinoleicacid(LA),inducesanincreaseinwoundhealingbyinfluencingtheinflammatoryphase,increasingthenumberofneutrophilsinthewound,andbyaffectingthereepithelizationphase.However,theeffectofLAonkeratinocytemigrationhasnotyetbeenexplored.ThisstudydemonstratedthatLAsignificantlyincreasesmigrationofanimmortalkeratinocytecell linefromadulthumanskin,knownasHaCatinascratchwoundhealingassayandatranswellmigrationassay.Matrixmetalloproteinase(MMP)-9iskeyforthemigrationofkeratinocytesduringthewoundhealingprocess.WehavedemonstratedthatLAinducesanincreaseofMMP-9activityandproteinexpression,analyzedthroughzymographyandwesternblot respectively.Additionally,wedemonstrated that LA rapidly (2-5min) stimulatedphosphorylationof ERK1/2andp38MAPK,evaluatedbyimmunoblot.Furthermore,thepresenceofthefreefattyacidreceptor-1(FFAR1),anLAreceptor,wasassessedbyRT-PCR,immunoblottingandfluorescencemicroscopy.Inconclusion,wereporthere,forthefirsttime,thatLAplaysakeyroleincellmigrationandMMP-9activationinkeratinocytes,thereforesupportingitspotentialapplicationinregenerativemedicine;furthermore,theseresponsescouldbeinducedbyFFAR1activation.

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29)Nitric oxide synthase, a target for polyphenols derived from the diet: a molecular approach to the French paradox.

Mateluna, Carlos1.,Calfío,Camila1.,Huidobro-Toro,JPablo1.,Mascayano,Carolina2.,1DepartamentodeBiologia,FacultaddeQuímicayBiología,UniversidaddeSantiagodeChile.2DepartamentodeCienciasdelAmbiente,FacultaddeQuímicayBiología,UniversidaddeSantiagodeChile.(SponsoredbyFundedFONCECYTGrant114-1132,FPB087,CEDENNA)

TheFrenchparadoxisbasedontheobservationthatFrench’s,inspiteofahighlipiddiet,arelesspronetocardiovasculardiseases.SinceFrenchdrinkmorewinethanotherEuropeans; thehypothesiswasrosethatwinemightprotect fromthedevelopmentof vascular diseases.Wines, fresh fruits and vegetables are rich in polyphenols including flavonoids and related compounds.We infer that endothelial nitric oxide synthase (eNOS)might be an interestingmolecular target to examine for the actionofthesechemicals.Insupportofourproposal,NO,thegassignalderivedfromeNOSactivity,isanimportantsignalingmoleculeinbloodvesselsassociatedtoapotentcGMP-dependentvasodilatation.PolyphenolsmayaffectthebioavailabilityofNO.Althoughstudiespostulatedifferentpathwaysforpolyphenolaction,nonehasbeenclarifiedmolecularly.Usingbioinformatics,weassessedwhetherpolyphenolsandflavonoidsmodulatehumaneNOS,actingauniquesitecommontothesechemicals.ThishypothesiswasexaminedusingmoleculardockingbasedinAutoDock4.2.Fortheproteinmodel,weusedtheoxygenasedomainofthedimerofhumaneNOScrystallographicstructure.Weexamined10flavonoidsplustrans/cisresveratrolandpiceatanolasmodelofnaturalstilbenes.Chemicalstructureswereminimized(MMF94xhybridforcefield)onMOEprogram.Resultsareconsistentwithasinglesiteintheextracellularenzymesurfacesuggestingadefinedandrelevant“pocket”intheeNOSoxygenasedomain.Polyphenolsbindingenergiesrangedfrom-8.2to-5.7kcal/mol;whichcorrelatedpositivelywithendothelium-dependentvasodilatoreffectsreportedinliterature(r=0.81,p<0.01).Moreover,eNOSArg107andGlu347werefoundessentialfortheinteractionwith3’-OH,5-OHand7-OHofthesepolyphenols.Furthermore,whenArg107wasartificiallymutatedtoAla107intheeNOStemplate;theaffinitiesfortheproposedpocketdecreasedresultinginrandominteractionsatmultiplesiteswithincreasedbindingenergies.Likewise,whenGlu347wasmutatedforAla107,thebindingenergyincreasedintherangeof0.3-2.2kcal/mol,butthepolyphenolsstillinteractattheproposedsitewithhigherenergies,illustratingtherelevanceofArg107atthedescribedpocket.BindingenergiesofsyntheticpolyphenolseitherlackingthehydroxylgroupsorwithadditionalOHs,orOHsubstitutionsbymethylormethoxylarecompatiblewiththemodel.Weproposeapositiveallostericmodulatorsite intheexternalsurfaceoftheeNOSfordietpolyphenols.Thepolyphenolpocketisclosetothebiopterinbindingsite,allowingustoinferitsrolefavoringNOgenesisbyeNOSactivity.

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30)Effect of Simvastatin upon murine chronic Chagas cardiopathy therapy with benznidazole. Role of simvastatin on endothelial adhesion molecules

Gonzalez-Herrera, Fabiola.,Castillo,Christian1.,Liempi,Ana1.,Kemmerling,Ulrike1.,Maya,Juan2.,1ProgramadeAnatomíayBiologíadelDesarrollo-ICBM,FacultaddeMedicina,UniversidadDeChile.2ProgramadeFarmacologíaMolecularyClínica-ICBM,FacultaddeMedicina,UniversidadDeChile.(SponsoredbyProyectosFONDECYT:1130189(JDM),1120230(UK))

Trypanosoma cruzi causeschronicChagasheartdisease(CCC).ThephysiopathologyofCCCincludesendothelialdysfunctionandmicrovasculardamagethatleadtofocalischemicareasincardiactissue.DuringinfectionwithT. cruzi,vascularendotheliumisactivated,andexpressionofproinflammatorycytokinesincreases.ThisproinflammatorystateismediatedbyactivationofnuclearfactorkappaB(NFκB)thatisinvolvedintheincreasedexpressionofcellularadhesionmolecules(CAMs),amongothereffects,toencouragecellrecruitmentinflammatory.Ontheotherhand,statinsreduceinflammationinthevascularendothelium,NFκBactivation,E-CAMsexpression,andinflammatorycellsrecruitment.Ithasbeendescribedthattheseeffectsaremediatedbytheproductionof15-epi-lipoxinA4,apro-resolutoryinflammationeicosanoid,whichinhibitsNFkBactivation,andCAMsexpression.Benznidazole is theuniquedrugthathasprovenarelativeefficacy in treatingChagasdisease,mainlyduringtheacutephase.However,it ispossiblethatthroughmodulatingkeyphysiopathologicalfactors,theefficacyofbenznidazolecouldbeincreasedduringthechronicphase.Therefore,westudiedtheeffectofsimvastatinonbenznidazoletherapyinanin vivomodelofChagas’sdisease.WeevaluatedtheimpactofthistherapeuticapproachonCAMsexpression,andonthebenznidazoleefficacytotreatthisdisease.Balb/cmiceinfectedwith1000trypomastigotyeofDm28cstrain.weretreatedwithsimvastain4and40mg,benznidazole30and100mg/Kg/day,andthecombinationoforsimvastatin4andbenznidazole30mg/Kg/dayfor20days,startingatthe30thdaypostinfection.Animalswerefollowedduringa90daysperiod,andwereeuthanizedtoobtainbloodandcardiactissueforparasiteload,inflammatoryinfiltrateandCAMsexpressionanalysis.Mortalityratesweresimilaramongallgropusstudied.Asexpected, benznidazole 100 mg/Kg/day decreased parasite load, CAMs expression and inflammation on the cardiac tissue.Similarly,Simvastatin40mg/kg/day,andatalesserextent,4mg/Kg/day,wasabletodecreaseexpressionofVCAM-1,ICAM-1andE-Selectinontheimmunohistochemicalstudies. Interestingly,thisdrugdecreasedsignificantlyparasite loadoncardicatissue.Whenthecombinationofsimvastatinplusbenznidazolewasanalyzed,parasiteloaddecreasedtoalmostzero,andcardiactissueandECAMsexpressionwassimilartocontrol.Inconclussion,Simvastatinaloneprovidesananti-inflammatoryi¡enviromentoncardiactissueinfectedwithT. cruzi,andthecombinationwithbenznidazolecouldimproveChagastreatment.

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31)The inhibition of proteasome prevents Mitofusin 2 and Miro 1 degradation in cardiomyocytes during ischemia -reperfusion.

Olmedo, I1.,Pino,G1.,Anríquez,C1.,Pedrozo,Z1,2.,Donoso,P1.,Sánchez,G1.,1InstitutodeCienciasBiomédicas,FacultaddeMedicina,UniversidadDeChile.2AdvancedCenterforChronicDiseases,FacultaddeMedicina,UniversidadDeChile.(SponsoredbyFONDECYTPostdoctorado3140449,FONDECYT1150887,1130407,FONDAP15130011.)

Duringcardiacischemiareperfusion(I/R)diverseproteinsaredegraded,amongthemmitochondrialproteinssuchasmitofusin2(Mfn2).Thiseventisresponsibleforachangeinmitochondrialdynamicsinducingfragmentedmitochondria(fission)andimpairmentofitsfunction,causingcardiomyocytedeath.OnestrategytoreduceheartdamageduringI/Risproteasomeinhibition,howeverthemechanismbywhichthisinhibitioninducesuchprotectionduringI/Risstillunknown.Miro1isanotherproteinlocatedinmitochondrialmembraneimplicatedintransportanddynamicsofthemitochondria,neverthelesstheconsequencesof I/RonMiro1contentincardiomyocyteshavenotbeenstudied.TheobjectiveofthisworkwastoevaluatewhetherinhibitionoftheproteasomeisabletoprotectthemitochondriafromI/RinjuryandpreservethecontentofMfn2andMiro1.CulturedneonatalratcardiomyocytesweresubjectedtosimulatedI/R(sI/R)intheabsenceorthepresenceoftheproteasomeinhibitorMG132.Celldeathwasevaluatedbylactatedehydrogenaserelease(LDH)andtherelativecontentofmitochondriawasdeterminedbyqPCR.Mitochondrial fusionandfissionwereevaluatedbyconfocalmicroscopyusingmitotrackergreenandtheprotein levelsofMfn2andMiro1wereevaluatedby inmunowesternblot (WB). In theabsenceofproteasome inhibitor, sI/Rdecreasedtherelativecontentofmitochondria,decreasedmitochondrialfusionandmetabolismandproducedcardiomyocytedeath.Also,sI/RdecreasedtheproteincontentofMfn2andMiro1.TheinhibitionoftheproteosomebyMG132avoidedallthemitochondrialchangesinducedbysI/Rmentionedabove,preservedthecontentofMfn2andMiro1andpreventedcardiomyocytedeath.Takentogether,thesedatasuggestthatinhibitionoftheproteasomepreservesmitochondrialfunctionwhichexplainsatleastinparttheprotectiveeffectofproteasomeinhibitioninI/Rinjury.

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32)PARTICIPATION OF TLR4 IN THE ANTIFIBROTIC RESPONSE OF KININS BY AN INCREASE OF PGI2 AND NITRIC OXIDE LEVELS IN CARDIAC FIBROBLASTS

Osorio, José Miguel1.,Muñoz,Claudia1.,Díaz-Araya,Guillermo1.,1QuímicaFarmacológicayToxicológica,FacultaddeCienciasQuímicasyFarmacéuticas,UniversidadDeChile.(SponsoredbyFondecyt1130300-BecaCONICYTDoctoradoNacional21120401)

Tolllikereceptors(TLR)areinvolvedinimmuneinnateresponseandarecapabletosensedangerassociatedmolecularpatterns.TLR4 has been described as a keymediator of cardiac function due to its ability to promote cell survival and inflammatoryresponsebyregulationofcytokinessecretion,adhesionproteins,etc,afterinjury.Ontheotherhand,kininsarepeptides,whicharecapabletobindtheirownreceptors(B1RandB2R)andreducebloodpressureandcollagensecretion,preventingtheprogressofcardiacfibrosisthroughactivationofkininsignalingcascade,includingeNOSandCOXs(byproductionofnitricoxide(NO)andPGI2).Previously,wedemonstratedthatTLR4activationbyLPSincreaseCOX-2andiNOSproteinlevelsincardiacfibroblasts(CF)andmyofibroblasts(CMF),andLPSpretreatmentincreasesthereductionofcollagenIproteinlevelsinducedbyDAKD(B1Ragonist)inCF.Wehypothesize that the increase inPGI2andNO levelsare responsible for theenhanced reductionof collagen I levelsinducedbyDAKDinCFpretreatedwithLPS.ThepurposeofthisworkistodemonstrateifTLR4increasesPGI2andNOsecretion,enhancingkininantifibroticresponse.CFandCMFwereobtainedfrom1-3days-oldSpragueDawleyrats.BothphenotypeswerestimulatedwithLPS1µg/ml,TAK-2422μM,andbothstimuliduring0,24,48and72hours.AftertreatmentCOXsandiNOSlevelsweremeasuredbywesternblot(IWB).PGI2andNOlevelsweremeasuredbyELISA,forthese,CFwerepretreatedwithLPS1µg/mlduring24and48hours,andthenstimulatedwithDAKD100nMduring24and48hours.WedemonstratethatTLR4activationbyLPS inducesan increaseCOX-2and iNOSprotein levels infibroblasts(CF)andmyofibroblasts(CMF)byIWB,and inamajorsecretionofPGI2andNOtotheextracellularmediuminCF,resultinginadecreaseofcollagenIlevels.Finally,wecanconcludethatTLR4hasanantifibroticrolebyincreasingCOX-2andiNOSproteinlevels,andPGI2andNOsecretion,whichcontributestoreducecollagenlevelsinCF.

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33)Effect of recurrent metabolic insults on organotypic cultures from asphyxia-exposed rat pups

Palacios, E1.,Pérez-Lobos,R2.,Lespay-Rebolledo,C2.,Bustamante,D3.,Morales,P4.,Herrera-Marschitz,M5.,1FarmacologíaMolecularyClinica,Medicina,UniversidadDeChile.2ProgrammeofMolecular&ClinicalPharmacology,ICBM,FacultyofMedicine,UniversidadDeChile.3ProgrammeofMolecular&ClinicalPharmacology,ICBM,Medicine,UniversidadDeChile.4MilleniumInstituteBNI-Chile,ProgrammeofMolecular&ClinicalPharmacology,ICBM,,Medicine,UniversidadDeChile.5MilleniumInstituteBNI-Chile;ProgrammeofMolecular&ClinicalPharmacology,ICBM,medicine,UniversidadDeChile.(SponsoredbySupportedByMillenniumInstituteInitiative-Chile(BNIP09-015-F);FONDECYT(1120079,1110263).TheExcellentTechnicalSupportFromMr.JuanSantibáñez,Mr.AlejandroLeivaAndMs.CarmenAlmeydaIsAcknowledged.)

Perinatal asphyxia is a relevant clinical issue associated to long-termdisabilities bymechanisms not yet identified.We havespeculated that the long-term effects are not directly due to deficits produced by the primary insult, but to a permanentvulnerabilitytorecurrentmetabolicorenvironmentaldependinginsultsthatcanorcannotoccuralongthelifeoftheparticularindividual.

Thus,wehavestudiedtheissuebyprofitingoftheorganotypicculturemodel,allowingtoreconstructspecificbrainpathwaysin vitro, tobemonitoredalongdevelopment,evaluatingcelldeath,cellandneurochemicalphenotypeandthere-establishingofproperneurocircuitries.Wesampledbraintissuefrom2-3daysoldratpups,subjectedtoperinatalasphyxia,reconstructingthebasalgangliabyputtingtogetheronacoversliptissuefromsubstantianigra,neostriatumandneocortex,growntogetheronaculturetubecontaining0.75mlofaculturemediumincellincubatorat10%CO2and35

oCinarollerwheelturning1time/minforexposingthesamplestoaqueousandgaseousphases.

Atdayin vitro18(DIV17),theculturesfromasphyxia-exposedandcontrolanimalswereexposedtoametabolicinsultconsistingofahighconcentrationofH2O2for18h,containingornot0.5%glucose.Aftera48hrecoveryperiod,treatedandcontrolcultureswereassayedforcellviabilityusingtheLIVE/DEADviabilitykit(MolecularProbes,Eugene,OR,USA),orformalinfixedforfurtherimmunocytochemistry and confocal microscopy, focusing on cell (glial/neuronal; GFAP/ MAP2) and neurochemical (tyrosinehydroxylase,TH;NOS)phenotype.

Therecurrentmetabolic insult increasedcelldeath incultures frombothasphyxia-exposedandcontrolpups,withaselectiveregionalvulnerability.ThespecificityoftheeffectoncellandneurochemicalphenotypewasstereologicallyquantifiedinpicturestakenbyaFv10iconfocalmicroscope(Olympus,Japan).

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34)Histone deacetylase inhibitors: Synthesis, docking and citotoxicity of thiazolylcoumarins derivatives.

Pardo-Jiménez, Viviana1.,Díaz-Araya,Guillermo2.,Navarrete-Encina,Patricio3.,1QuímicaOrgánicayFisicoquímica,QuímicaFarmacológicayToxicológica,CienciasQuímicasyFarmacéuticas,UniversidadDeChile.2QuímicaFarmacológicayToxicológica,CienciasQuímicasyFarmacéuticas,UniversidadDeChile.3QuímicaOrgánicayFisicoquímica,CienciasQuímicasyFarmacéuticas,UniversidadDeChile.(SponsoredbyBecaDoctoradoNacionalConicytNº21140371)

Histonedeacetylases(HDACs)areafamilyofzinc-dependentenzymes.Theyareinvolvedingeneexpressionthroughregulationoftranscriptionandothercellularprocessesincludingcellcyclearrest,cellproliferation,apoptosisandterminaldifferentiationofvariouscelltypes.HDACinhibitorsareconsideredanewclassofdrugswithmultiplepossibilitiesoftherapeuticaction,amongotherswehaveantineoplastic,anti-inflammatoryandanti-fibroticactivities.Thestructureoftheseinhibitorsfitsapharmacophorestructureincludingthreezones:ahydrophobicheadgroup,a6methyleneorheteroatomshydrophobicspacerchainandaZn+2bindingterminalgroup. Basedonthisgeneralstructure,N-adipoylandN-lipoyl-thiazolylcoumarinsderivativesweredesigned,using the thiazolylcoumarin scaffold asheadgroup, lipoic andadipic acid spacers and terminal Zn+2 chelating carboxilate anddisulfidegroups.Thesynthesiscomprisesthepreparationofthecoumarinnucleus(throughamodifiedPerkinreaction),thiazolylringformationandafurthercondensationreactionwithlipoicandadipicacidtoprovidethefinalcompounds.AdockinganalysisofthepreferredconformationsadoptedbythesecompoundsinthecatalyticsiteofHDACswasdoneusingtheenzymeHDAC8crystalstructureasreference.Thuswecheckthatourinhibitorsdesignmeettheadequateconditionsforenzymeinteractionandaresimilartothepan-inhibitortrichostatinhavinginhibitionconstantsenergiesbetween-10and-15kcal/mole.Todeterminethatthesynthesizedcompoundsarenotcytotoxicinnon-tumorcells,aviabilitytestwasperformedonneonatalratcardiacfibroblastsandwefoundthatthiazolylcoumarinsderivativesintheconcentrationrangeof100nMto10uM,arenotcytotoxic.

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35)New semi-synthetic derivatives of natural catechins: antioxidant-anti-inflammatory activity and effect upon Helicobacter pylori carbonic anhydrase

Pastene, Edgar1.,Parada,Víctor1.,Torres,Eillen2.,Avello,Marcia1.,Alarcón,Julio3.,Felipe,Zuñiga4.,Ormazabal,Valeska5.,Ariel,Saavedra1.,Aranda,Mario6.,García,Apolinaria2.,1Farmacia,Farmacia,UniversidadDeConcepción.2Microbiologia,CienciasBiológicas,UniversidadDeConcepción.3Química,CienciasBásicas,UniversidadDelBío-bío.4BioquímicaClínicaeInmunología,Farmacia,UniversidadDeConcepción.5Farmacología,CienciasBiológicas,UniversidadDeConcepción.6CienciayTecnologiadeAlimentos,Farmacia,UniversidadDeConcepción.

Severalsemi-syntheticderivativesofnaturalcatechinswerepreparedfrompolymericproanthocyanidins(PACs)extractedfromcranberry pomace, Boldus leaves, avocado peels and grape skins. Through acid-catalyzed cleavage and depolymerization ofsuchPACs, formedcarbocationswereattackedwithdifferentphenolicnucleophiles (phloroglucinol, resorcinolandpyrogallol),giving threeadductsofcatechinandepicatechin, respectively.Thenewsemi-syntheticcompoundswerepurifiedand isolatedby preparative techniques such as CPC andHPLC and their formationwas followed and confirmedbyHPLC-DAD-ESI-MS/MS.Introductionof groupsharboringphenolic hydroxyls on the catechins structure increase its antioxidant activity in theDPPH,CUPRAC,ORAC-FLandredbloodcellsprotectionassays.SuchderivativesalsowereabletoreduceIL-8secretioninH.pylori-infectedAGScells.Finally,weevaluatewhethertheintroductionofadditionalphenolicgroupsinnaturalcatechinsactsasmodificationthatfavorsthebindofthenewcompoundstotheactive-sitezincatomofthebacterialenzymecarbonicanhydrase(CA),thereforeincreasingitsinhibitoryproperties.Usinganinsilicoapproach(Docking)themostprobablebindingmodeofthesecompoundsisproposed.Ourpreliminaryresultssuggestthatthesesemi-syntheticphenoliccompoundscouldbeusedaschemicalframeworkforthedesignofnewdrugsthathelptothepreventionandtreatmentofthecolonizationbyH.pyloriandtheinflammatorydamageexertedupongastricmucosa(Financialsupport:Fondecyt1150948;FondequipN°EQM130209).

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36) Salsolinol, an alcohol-derived metabolite, acts as an agonist of the mu-opioid receptor.

Rivera-Meza, Mario1,2.,Berrios-Cárcamo,Pablo1.,Herrera-Marschitz,Mario1,2.,Quintanilla,MaríaElena1.,1ProgramadeFarmacologíaMolecularyClínica,FacultaddeMedicina,UniversidadDeChile.2MilleniumScientificInitiativeBiomedicalNeuroscienceInstitute.(SponsoredbySupportedBy:FONDECYT#11130241:BNIP09-015-F.)

Animal studieshave showed thatacetaldehyde, thefirstmetaboliteofethanol, is amotivationaland reinforcingmolecule. Inthebrain,ethanol-derivedacetaldehydecancondensewithdopaminetogeneratesalsolinol.Thiscompoundisself-administeredintracraniallybyanimals,suggestingthatsalsolinolisthemoleculemediatingtherewardingeffectsofacetaldehydeandethanol.Recentevidenceindirectlysuggeststhatsalsolinolmayexertitsactionthroughanopiatemechanism,whichmayexplaintheefficacyofnaltrexone(anopioid-receptorantagonist)toinhibitethanolintake.However,therearenostudiesshowingthatsalsolinolactsdirectlyonopioid receptors. Toaddress thisquestionwestudied thecapacityofdifferent concentrationsof salsolinol (10-3to10-9M)toactivatethehumanmu-opioidreceptorinacellbasedreceptorassay.Sincethemu-opiodreceptoriscoupledtoaninhibitoryGprotein(Gi),itsactivationbyanagonistresultsinareductionoftheintracellularlevelsofcAMPinducedpreviouslybytheadenylatecyclaseactivatorforskolin.Resultsshowedthatsalsolinoliseffectivetoactivatethemu-opioidreceptorbutpossesalowerpotencycomparedtothefullagonist[Met5]-Enkephalin,measuredasthecapacitytoactivateGiandinhibitthegenerationofcAMP.Theapparenthalfmaximaleffectiveconcentrations(EC50)forsalsolinoland[Met5]-Enkephalinwere7x10-5Mand2x10-8

M,respectively.Thesepharmacodynamicstudiesshowedthatsalsolinolactsasanagonistofthemu-opioidreceptor,showingthesameefficacy,butalowerpotencycomparedtothefullopioidagonist[Met5]-Enkephalin.

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37)Anthocyanins from Aristotelia chilensis inhibit Olanzapine-induced adipogenesis in 3T3-adipocytes

Arena,Pamela1.,Jara,Belen1.,Cubillos-Robles,Karen1.,Tordoya,Isis1.,Gonzalez,Paula1.,Rojo,Leonel1.,1LaboratoriodeFarmacologíayNeurociencias,FACULTADDECIENCIASDELASALUD,UniversidadArturoPrat.(SponsoredbyAcknowledgements:ThisProjectWasFundedByFODECYTProjectN°11140915)

Second-generationantipsychotics(SGAs),clozapine,olanzapineandrisperidone,haveimprovedqualityoflifeofbillionspatientsworldwide and are essential for treatment of schizophrenia (SZ). Off-label applications of SGAs are increasing dramatically,includingmillionsofnon-SZadults, childrenandadolescents.SGAscancausedevastatingcardiometabolic sideeffects leadingtoincreasedprematuremorbi-mortalityinasshortasthreemonthsafterinitiationofthepharmacotherapy.TheSGAs-inducedhyperlipidemiaisassociatedwiththeupregulationofsterolregulatoryelement-bindingprotein1c(SREBP1c),acentralregulatoroflipidbiosynthesisinliverandfattissue.Thereisagrowingbodyofevidencedemonstratingthatanthocyaninsfromfruitsandvegetablesameliorate insulin resistance, inflammationandobesity.Our research is focusedon theevaluationof theeffectofanthocyanins frommaquiberry (Aristotelia chilensis)on theolanzapine-induced lipidaccumulation inadipocytes. IsolationofanthocyaninsfromMaqui(MBA)wasdoneasdescribedbyRojoetal(2012).Briefly,freezedriedmaquiberriesfromcommercialsourceswereblendedwith acidified80%ethanol, lipophilic compoundwere removedwithethyl acetate.AnthocyaninswerethenpurifiedthroughAmberliteandSephadexLH-20columns.Theanthocyaninscontained inthisextractaremainlycyanidinand delphinidin glucosides and the characteristicmarker compound of the extract is delphinidin 3-sambubioside-5-glucoside(D3S5G).Weobservedthatolanzapine,at20µMconcentration,inducedsignificantcelldifferentiationandlipidaccumulationincultured3T3adipocytes.CellulardifferentiationandlipidaccumulationwasanalyzedbydigitalmicroscopyandUVspectrometryafter stainingwithOil Red. Co-incubationof 3T3 cellswithMBA, at 5, 10, 20, 40, 60 and 100µg/mL, produced a significantreductionoftheintracellularlipidaccumulationindifferentiatedadipocytes.ThecytotoxicityoftheMBAwasalsotestedbytheMTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) tetrazolium reductionassay.Cellular toxicitywasobservedwithMBAatconcentrationsaboveat60µg/mL.

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38)Ethyl acetate extracts of different Ugni molinae Turcz. genotypes able to inhibit α-glucosidase

Veas, Rubén1.,Arancibia-Radich,Jorge1.,Peña-Cerda,Marcelo1.,Cortez,Giovanni1.,Seguel,Ivette2.,Delporte,Carla1.,1QuímicaFarmacológicayToxicológica,CienciasQuímicasyFarmacéuticas,UniversidadDeChile.2InstitutodeInvestigaciónAgropecuariaINIA,Carillanca,IXRegión,Chile.(SponsoredbyAcknowledgments:WeAreGratefulToFONDECYTN°1130155,CONICYTGrantsN°21130672AndN°21120377,Chile.ThankYouToINIA(Carillanca,Temuco)ForTheGenotypes.)

Previous studies in our laboratory shown pentacyclic triterpenic acids in different extracts of murtilla leaves (Ugni molinaeTurcz.,Myrtaceae) responsible for inhibitoryactivityagainstvariousenzymes.Between thesesecondarymetabolitescanfind:asiatic,ursolic,madecassicandcorosolicacids,derivativesfromursane;betulinicandalfitolicacidsderivativesfromlupanoandoleanolicandmaslinicacidsderivativesfromoleanane1.Studiescarriedoutinparallelinourlaboratoryindicateahighconcentrationofthesemetabolitesinethylacetateextracts(EAEs)ofleaves.Chileanfolkmedicineattributesmanypropertiestothemurtillaandamongitsusesincludesinfusionsfromitsleavesandbranchestotreatdiabetes2.Thisusecanbeexplainedbywayofinhibitingα-glucosidase,anexo-carbohydrasethatcatalyzesthehydrolysisofcomplexsugarstomonosaccharidesinthebrushborderofenterocytes,andifitisinhibiteddecreasespostprandialglucosepeak3.ThereforetheaimofthisstudywastobenchmarkingofEAEsofleavesfromsevengenotypesofmurtillagrowninequalconditionsofsoilandweather,andsameagronomicmanagement,fromthegenebankoftheInstituteofAgriculturalResearch(IAR)throughthemethodologydescritebyKimetal.(2005)withslightmodifications.StatisticalanalysiswasperformedusingANOVAandTukeymultiplecomparisonsmethod.Theresultsindicatethatthegenotype19-1wasthemostpotentinhibitoragainstα-glucosidasefromSaccharomyces cerevisiae withIC50equalto12.7±0.2μg/mL.Genotype19-1hawasthelesspotentwithIC50of41.0±2.0μg/mL.Howeverallgenotypesshowedbetterpotencythanthereferencedrug,acarbose(IC50of267.2±35μg/mL).Previousstudieswithethanolextractsshowedasimilarbehaviorofgenotypes,wherethe19-1ispartofgenotypeswithacceptablepowerand19-1haaslesspotent.References:1GOITYL.E.,QueupilM.J.,JaraD.,AlegríaS.E.,PeñaM.,BarrigaA.,AguirreM.C.yDelporteC.AnHPLC-UVandHPLC-ESI-MSbasedmethodforidentificationofanti-inflammatorytriterpenoidsfromtheextractsofUgnimolinae.BoletínLatinoamericanoydelCaribedePlantasMedicinalesyAromáticas12(1):108–1163RUBILARM.,PineloM.,IhlM.,ScheuermannE.,SineiroJ.yNúñezM.J.MurtaLeaves(UgnimolinaeTurcz)asaSourceofAntioxidantPolyphenols.JournalofAgriculturalandFoodChemistry.2006,54,59-64.4LORDANS.,SmythT.J.,Soler-VilaA.,StantonC.yRossR.P.Theα-amylaseandα-glucosidaseinhibitoryeffectsofIrishseaweedextracts.FoodChemistry141(2013)2170–2176.

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39)FoxO1 is necessary for the cytoprotective effect induced by TGF-β1 in cardiac fibroblasts

Vivar, R1.,Chiong,M2.,Lavandero,S2.,Díaz-Araya,G2.,1QuímicaToxicológicayFarmacológica,CienciasQuímicasyFarmaceúticas,UniversidaddeChile.2AdvancedCenterforChronicDiseasesUniversidadDeChile.(SponsoredbyBecaPostdoctoral3130657(RV);Fondecyt1130300(GDA);ACCDis(SL,MCh))

Background. Themain function of cardiac fibroblasts (CF) is tomaintain extracellularmatrix (ECM) homeostasis.Whereas incardiacdiseasessuchas,myocardial infarction, theydifferentiate intocardiacmyofibroblasts (CMF) increasing theiractivity topromotetissuehealing.Innormalconditions,CMFdisappearfromtheinjuryzonethroughapoptosis,presumablybytheoxidativeenvironmentthatexistindamagedarea.Howeveritiscurrentlyunknown,whyinpathologicalconditions,CMFdonotdieandtheyevenpersistinthedamagedzonetopromotetheexcessivedepositofECMcomponentsandtissuefibrosis.TGF-β1isacytokinewhich inducesCFdifferentiationand it is crucial incardiacfibrosis.With thisevidence,wesuggest thatTGF-β1 is responsiblefor cytoprotection against oxidative conditions,which favors both CMF survival and cardiac fibrosis. Furthermore, FoxO1 is atranscriptionfactor involved inantioxidantdefense, throughregulationofcatalaseandSOD2expression. Inaddition,wehavedemonstrated that TGF-β1 promotes FoxO1 activation in CF. Therefore, we hypothesized that FoxO1 is important to TGF-β1promotes antioxidant protection in CF, controlling catalase and SOD2 expression.Methodology. Neonatal CF obtained fromSprague-Dawleyratswasused.Toinduceoxidativestress,weusedhydrogenperoxide(H2O2)15µMfor24h.TGF-β1wasusedat10ng/mL.Toevaluateapoptosis,weusedflowcytometryandweevaluatedcaspase3and9fragmentationbywesternblotting.Theproteinlevels(FoxO1,catalase,SOD2,GAPDH)wereassessedbywesternblotting.Celldeathwasdeterminedbycellcount.siRNAwasusedtosilencerFoxO1,catalaseandSOD2.AdenoviruswasusedtoinduceFoxO1overexpression.Results. TGF-β1inhibitedbothcelldeath,apoptosisandcaspases3and9fragmentationinducedbyH2O2inCF.Inaddition,TGF-β1increasedproteinlevelsofantioxidantenzymes,catalaseandSOD2,whereasthesilencingofbothantioxidantsproteinsinhibitedthecytoprotectiveeffectofTGF-β1.FoxO1isacrucialregulatorofcatalaseandSOD2expression.Infact,FoxO1overexpressionincreasedcatalaseandSOD2proteinlevels,bothinthepresenceandabsenceofTGF-β1.FoxO1overexpressionincreasedthecytoprotectiveeffectofTGF-β1inCFsubjectedtooxidativestress.Incontrast,FoxO1silencepreventedproteinlevelsoftheantioxidantenzymescatalaseandSOD2inducedbyTGF-β1,whereasabolishedthecytoprotectiveeffectofTGF-β1inCFstimulatedwithH2O2.Conclusions.Ourresultssuggestthat:i)TGF-β1inhibitsthedeleteriouseffectsofoxidativestressinCFandii)TGF-β1requiresFoxO1toincreasecatalaseandSOD2proteinlevelandtopromoteantioxidanteffectinCF.

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40)Role of EPA/DHA over later stages of liver ischemia-reperfusion: antioxidant and antifibrotic effects

Zuñiga, Jessica1.,Céspedes,Nicole1.,Tamayo,Andrea1.,Arenas,Hector1.,1LaboratoriodeInvestigacionesMédicas,EscueladeMedicina,UniversidadDeTalca.(SponsoredbyThisStudyWasSupportedByTheInternalProjectI002974(DirecciónDeInvestigación,UniversidadDeTalca).)

The chronic administration of omega-3, in particular docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) havepreventive effects related to cardiac injury, brain stroke and hepatic ischemia-reperfusion (IR), action associated to theirprotectiveeffectsagainst inducedhypoxiaand ischemia/reperfusiondamageobserved in these tissues.ThebeneficialofEPAandDHA are associated to their antioxidant and anti-inflammatory capability,mediated by changes inmembrane lipids anddifferential eicosanoidsproduction.Previous studieshavebeendemonstrated that the liver injury inducedby IR ismediatedby the inflammatory response stimulatedby anearly increase (3hpost ischemia) ofNF-κBactivity and late releasesof pro-inflammatorycytokines(IL-1β,IL-6yTNF-α).Ourmainobjectivewastostudytheantioxidantandanti-fibrogenicpotentialinducedbytheoralsupplementationofEPAplusDHAinlaterstagesofliverischemia-reperfusioninjury.MaleSprague-Dawleyratswerepre-treatedbytendayswithdiaryoraldosesofEPA/DHA(375mg/Kg/day),afterthattimetheanimalsweresubjectedto1hofischemiafollowedby3-20-24and48hofreperfusion.ThesupplementedEPA/DHAanimalsshownlessliverdamage(measuredbytransaminaselevelandhistologicalanalysis)thanthenon-supplementedrats,andallthevalueswerenormalizedafterthe24h.Inadditionthenon-supplementedanimalsdisplayasustainedincreasedofcollagendeposit(measurebymasson-goldnertrichromestaining),actionthatwaspreventedinanimalssubjectedtoIRandpre-treatedwithEPA/DHA,withnotdepositofconnectivetissueobservedinthefirst24hoursandonlyaminoraccumulationat48hpost-ischemiawasdetected.Theresultsseeninthisstudycouldberelatedwithapotentialantifibroticcapabilityofomega-3fattyacids,contributingtotheknowledgeofthemechanismofHepatoprotection.

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41)Neurochemical caracterization in cortico-striatal-thalamo-cortical circuit of Eaat3 heterozygous mice: role in neuropsychiatric disoders

Gonzalez, Luis2,3,1.,Espinosa,Pedro1.,Sotomayor-Zárate,Ramon1.,Moya,Pablo2,3.,1LaboratoriodeNeuroquímicayNeurofarmacología,CentrodeNeurobiologíayPlasticidadCerebral,InstitutodeFisiología,Ciencias,UniversidadDeValparaíso.2LaboratoriodeNeurogenética,InstitutodeFisiología,CentrodeNeurobiologíayPlasticidadCerebral,Ciencias,UniversidadDeValparaíso.3MillenniumNucleusinBiologyofNeuropsychiatricDisordersNU-MINDUniversidadDeValparaíso.(SponsoredbyThisWorkWasFundedByMillenniumNucleusNU-MINDNC130011(PRM)AndFONDECYTGrants1141272(PRM)And11121205(RS-Z).)

Obsessive-compulsivedisorder (OCD) isoneof themostprevalentneuropsychiatricdisorders,affectingbetween1-3%of theworldpopulation.AlthoughtheexactcausesofOCDareunknown,manylinesofevidencesuggestthatOCDhasageneticbasis.Recently, ithasgainedstrengththeideaofadysfunctioninglutamatergicneurotransmissioninOCD.SeveralevidencesofthisdysfunctioninOCDincludecertainpolymorphismsintheNMDAreceptorgene,elevatedglutamatelevelsinthebrain-spinalfluidofdrugnaïvepatients,correlationsbetweensymptomsseverityandthe levelsofglutamatemetabolitesandsomedrugsthatmodulateglutamatergicneurotransmissionarecurrentlybeing tested forOCD treatment. Inaddition, linkage, candidategeneaswell asmeta-analysis studies support an associationofSLC1A1 gene andOCD.SLC1A1 encodes the ExcitatoryAmino-AcidTransporters3 (EAAT3) that regulates theclearanceofglutamate insynapticcleft. Atneurocircuitry level, thecortico-striatal-thalamo-cortical(CSTC)circuithasbeeninvolvedinOCDthroughneuroimaging,animalsandpharmacologicalstudies.Inthiscircuit,glutamatergicneurotransmissioniskeyinthecommunicationbetweencortexandstriatummakingsynapseswithmediumspinyneurons(MSNs)thatparticipateinDirectandIndirectpathway.InOCDpatients,anoveractivationofCSTCloop,reflectinglikelyapredominanceofDirectpathwayoverIndirectpathwayhasbeenobserved,suggestingalinktoobsessionsandcompulsions.Inthiswork,wedecidedstudytheneurochemicalregulationinlimbicandCSTCareasinheterozygousmiceforEaat33.Wildtype(WT)andEaat3heterozygous(HET)littermatemiceweresacrificedandtheirbrainwereremovedinice.Cortex,striatum,thalamusandhippocampusweremicrodissectedat4°C,homogenizedinperchloricacidandcentrifugedat12000xg. ThecleanedsupernatantwasinjectedintoaHPLCcoupledelectrochemicaldetectiontodetectdopamine(DA),3,4-dihydroxy-phenylaceticacid(DOPAC),serotonin (5-HT)and5-hydroxy-indoleaceticacid (5-HIAA).The results show increase in striatal andhippocampalDAcontentinEaat3HETmicewithoutaffect5-HTcontentinthesamebrainareas.Ontheotherhand,itwasshownareductionincortical5-HTandDAcontentandonlyareductioninthalamus5-HTcontent.Interestingly,theseresultsshowamonoaminergiccorticalhypofunctionthatcouldbeinvolvedwithalterationsindecision-makingobservedinOCDpatients.

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42)Generation of a novel genetic mouse model for Obsessive-Compulsive Disorder

Moya, Pablo1,2.,Utreras,Elias3.,Martinez,Jonathan1,2.,1LaboratoriodeNeurogenética,InstitutodeFisiología,CentrodeNeurobiologíayPlasticidadCerebral,Ciencias,UniversidadDeValparaíso.2MillenniumNucleusinBiologyofNeuropsychiatricDisordersNU-MINDUniversidadDeValparaíso.3Biología,Ciencias,UniversidadDeChile.(SponsoredbyThisWorkWasFundedByMillenniumNucleusNU-MINDNC130011(PRM)AndFONDECYTGrants1141272(PRM))

SLC1A1 (neuronalglutamatetransportergene,EAAC1,EAAT3)isanattractivecandidategeneimplicatedinObsessive-CompulsiveDisorder(OCD). EAAT3regulates–amongotherglutamatetransporters-extracellularlevelsofglutamateincortico-striato-thalamo-cortical(CSTC)circuitimplicatedinOCD. SLC1A1 isthemostevidentbrain-relatedgeneofinterestlocatedinthechromosomalregion9p24, the region identified in thefirstgenome-wide linkagestudyofmixed largeandsmall familieswithOCD.Also, in thefirstcase-controlstudyofthisgeneinOCDwefoundthatSLC1A1 wasassociatedwithOCD.ThestrongestevidencefromthisstudyindicatedthatacertainhaplotypewasalmosttwotimesmorefrequentinOCDpatientsthancontrols(OR=1.89);twoofthreeSNPsofthishaplotypewerefoundtobeexpressionQuantitative-TraitLoci(eQTLs).Thesefindings,togetherwithfamilylinkagestudiesaswellasbyarecentmeta-analysispointalltowardSLC1A1 asthemostsolidlyestablishedgeneidentifiedinOCD.ThisprojectaimstogeneratetransgenicmousemodelsforCre-dependentconditionalEaat3knockoutandEaat3overexpression,andcharacterizetheirphenotypesusingpharmacological,biochemical,anatomicalandbehavioraltechniques.SuchanimalmodelsareexpectedtoprovideseminalinformationregardingthemechanismofSLC1A1dysfunctionatthegeneregulatory,neurochemical,andanatomicallevelsduringvariousstagesofdevelopment.Inaddition,micewithconditionalSlc1a1expressionalterationsmayofferexcitingpossibilitiesforgeneratingnewanimalmodelsofpsychiatricand/orneurodegenerativedisordersandalsohelpinthedevelopmentofdrugsthattargetglutamateneurotransmittersystemforeffectiveOCDtreatment.Here,wedescribethecurrentstatusofvalidationandcharacterizationofrecentlygeneratedCre-mediatedEaat3alteredexpressionmousemodels.Wepresenttheproof-of-principleforCre-dependentalteredEaat3expression,usingtheCamKII-Credrivermouselineandcomparedtowild-typelittermates,studiedatmRNAandproteinlevelsinfrontalcortex,hippocampus,striatum,thalamus,hypothalamusandpons.Wealsopresentdataofongoingphenotypicalassessmentsevaluatinganxiety-like(openfieldtest,elevated-plusmaze),andOCD-like(groomingindex,marble-burying)behaviors.

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43)Evaluation of the mGluR plasticity processes in the transgenic mice APPswe/PS1δE9 and in the natural model of Alzheimer Disease Octodon degus.

Valdivia, Gonzalo1.,Salazar,Claudia1.,Kirkwood,Alfredo2.,Palacios,Adrián2.,1CentroInterdisciplinariodeneurocienciadeValparaísoUniversidadDeValparaíso.2Mind/BrainInstituteJohnsHopkinsUniversity.(SponsoredbyFinancialSupport:MillenniumInstituteICM-P09-022-F)

AlzheimerDisease(AD)isthemostcommonformofdementiaamongolderpeoplethatcausesaslowdeclineinmemorythinkingandreasoningskills.Thesefailuresarerelated,amongotherthings,totheimpairmentofsynapticplasticitymechanismslikeLTPandLTDthatdependonNMDAreceptorsinmemoryareasofthebrainlikethehippocampus.Here,wereportLTPandLTDformsthatdependonmetabotropicglutamatereceptor(MGLUR)arealsoimpairedinCA1ofhippocampusoftworodentmodelsofAD:thetransgenicADmouseAPPswe/PS1δE9andOctodondegus(degu).Forthiswecombinedelectrophysiologyoffieldpotentialstoevaluateplasticityandwesternblots,toquantifymGluR5content.OurresultsshowthatthemGluRprocessesofLTDandLTParetotallydisruptedintheoldtransgenicmiceandaredifferenttotheoldWildTypeMice.OntheothersideindeguthisprocessesaredisruptedwhentheanimalpresentmemorydeficitsinthebehavioralmemorytestRadialarmmaze(RAM).ThissuggestarelationbetweenthistwoanimalmodelsandthelossofmGluRplasticityinthehippocampusduringtheagingintheAlzheimerdisease,whichpositsasignificanttargetinthestudyofthispathology.

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44)Polymorphisms in ABCB1 and ABCC2 genes in patients with Drug-Resistant Epilepsy at Van Buren Hospital in Valparaíso, Chile

Martinez, Jonathan1,2.,Riquelme,Julio1,2.,Saldias,Cristina1.,Rodriguez,Luciana1.,Moya,Pablo1,2.,1LaboratoriodeNeurogenética,InstitutodeFisiología,CentrodeNeurobiologíayPlasticidadCerebral,Ciencias,UniversidadDeValparaíso.2MillenniumNucleusinBiologyofNeuropsychiatricDisordersNU-MINDUniversidadDeValparaíso.(SponsoredbyFundedByMillenniumNucleusNU-MINDNC130011(PRM))

Epilepsyisadisorderaffecting1-2%ofpopulationworldwide.Despitetreatment,about25%ofpatientsdevelopDrug-ResistantEpilepsy(DRE).PolymorphismsofMultidrugpumpsintheBloodBrainBarrier(ABCFamily)havebeenrelatedtoDRE,inparticularonABCB1andABCC2genes.TheaimofthisstudyistosearchforassociationbetweenABCB1andABCC2polymorphismsandDREinChileanpatients.Thisisanon-experimentalcorrelationaltransversalstudy,approvedbytheScientificandEthicscommitteeofVanBurenHospital,Valparaiso,Chile.Patients(n=140)diagnosedwithEpilepsyaccordingtoILAEwhoattendtheNeurologyClinicwereprospectivelyrecruited.Weclassifiedpatientsintwogroups;thosewhoqualifiedwithinDREdiagnosis(twoormoretrialsofadequatelychosenandtolerateddrugswithoutseizurefreedomwithinoneyear)anddrugresponsivepatients.Allpatientswereinterviewedtorecollectclinicalandepidemiologicaldata.GenomicDNAwasextractedbystandardlysisbufferprocedurefromsalivasamples.DeterminationofABCB1 C3435TandABCC2 c.-24C>TpolymorphismswasperformedbyPCR-RFLP,aspreviouslyreportedinliterature.WeinitiallyreplicatedtheSNPcallingmethodologydescribedusingcommercialhumanDNApanels(Coriell,USA).AllelicdistributionofABCB1andABCC2polymorphismsdonotsignificantlyvaryfromthosereportedinliteratureandUCSCGenomebrowser.Patientrecruitmentisongoing,anddeterminationofSNPfrequencyiscurrentlyunderway.Todate,ourdataindicatethatABCB1 C3435TandABCC2c.-24C>ThavebothsimilarallelicdistributioninChileanEpilepsypatientstothosereportedin literature.Weare currentlydeterminingputativedifferences in allele frequencies inDrug-Resistant vsRespondersEpilepsypatients.

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45)Amyloid-β oligomers induce redistribution of neuronal pSer727-STAT3 in rat primary hippocampal cultured

Muñoz,Yorka1.,Paula-Lima,Andrea2.,Nuñez, Marco Tulio2.,1DepartmentofBiology,FacultyofSciences,UniversidadDeChile.2InstituteforResearchinDentalSciences,FacultyofDentistry,UniversidadDeChile.(SponsoredbyCONICYTPh.D.Fellowship21130445(YM),Fondecyt1150756(APL)AndProjectACT-1114FromPIA,CONICYT(MTN).)

Amyloid-βoligomers(AβOs)inducetheproductionofreactiveoxygenspecies(ROS),oxidativedamageandchangesinmitochondrialdynamicsinculturedneurons,blocklongtermpotentiationinhippocampalslices,AβOsandimpairmemoryinanimalmodels.Signaltransducerandactivatoroftranscription3(Stat3)isacrucialtranscriptionfactorintheCNSthatregulatestheexpressionofsurvival,antioxidantandanti-inflammatorygenes.Stat3isactivatedbyROSviaJak2.Phosphorylationofserine727(pSer727Stat3)modulatesitstranscriptionalactivityinmanydifferentcelltypesinacontext-dependingform.Recently,pSer727Stat3wasfoundassociatedtothemitochondrion,whereitup-regulatesmitochondrialactivityandinhibitstheMPTPformation.Here,weexploredwhetherneuronalpSerStat3expressionanddistribution isaffectedbyAβOs treatment.Primaryhippocampalneuroncultureswereused.ImmunocytochemistryandWesternblotwereemployedtodetectchangesinthedistributionandproteinlevelsofpSerStat3,respectively.Treatmentofmixedneuron-astrocytecultureswith0.5and1µMAβOs(24h) invokedachangeinthedistribution,decreasingthenuclearandmitochondrialcontentofpSer727Stat3andanincreasingitinthecytoplasm.Also,thepresenceofpSer727Stat3decreaseinthemitochondria..However,inastrocyte-depletedneuroncultures,AβOstreatmentdidnotcausepSerStat3redistributionbutincreasedastrocytereactivity,asdeterminedbyGFAPimmunostaining.ThedataisconsistentwiththenotionthatAβOsactivateastrocytestoreleaseanundeterminedsignal(s)thatinducenuclearandmitochondrialdepletionofpSer727Stat3inneurons.

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46)Early handling promotes resilience during childhood in prenatally stressed rats

Pavez-Fox, Melissa1.,Dagnino-Subiabre,Alexies1.,1LaboratoryofBehavioralNeurobiology,CenterforNeurobiologyandBrainPlasticity,InstituteofPhysiology,FacultyofSciences,UniversidadDeValparaíso.(SponsoredbyThisWorkWasFundedByFONDECYTGrantN°1141276ToAD-S.Labsite:Www.stress.cl)

Prenatalstressisariskfactorforseveralpsychiatricdisordersduringchildhoodandadolescence.Conversely,shortstressperiodsonearlylife(i.e.earlyhandling),inducesresiliencetofurtherstressors.So,theaimofthisstudywastodeterminewhetherearlyhandlinginducesresilienceinprenatallystressedrats.Maleprenatallystressedratsweresubjectedtoanearlyhandlingprotocol,whileanimalsofcontrolgroupwerenotsubjectedtoprenatalstress.Afterthatallanimalsreachedtochildhood(postnatalday24),resilientbehaviorswereevaluatedintheopenfieldandelevatedplusmazetests(anxiety-likebehaviors),aswellbysocialinterac-tionandforcedswimtest(depressive-likebehaviors).Ratsthatweresubjectedtoprenatalstressandearlyhandlingshowedlowerlevelsofanxietylike-behaviorscomparedtoprenatallystressedrats.Furthermore,prenatallystressedrats,whichweresubjectedtoearlyhandling,showedhigherescapebehaviorsandsociability.Theseresultssuggestthatearlyhandlinginducesresilienceinprenatallystressedrats.Thus,wehavedevelopedananimalmodelofchildhoodresilience,whichcouldbecomparedwithwell-es-tablishedanimalsmodelofprenatalstress.So,wecancontributetounderstandingoftheneurobiologicalbasisofresilienceandchronicstress.

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47) Is the excitatory amino acid transporter 3 implicated in schizophrenia dysfunction?

Pérez,Miguel1,2.,Morales, Camila1,2.,Arriagada, Jorge1,2.,Moya, Pablo2,3.,Fuenzalida,Marco1,2.,1Laboratorio de PlasticidadNeural,InstitutodeFisiología,FacultaddeCiencias,UniversidadDeValparaíso.2MillenniumNucleus inNeuropsychiatricDisordersNU-MIND,InstitutodeFisiología,FacultaddeCiencias,UniversidadDeValparaíso.3LaboratoriodeNeurogenetica,InstitutodeFisiología,FacultaddeCiencias,UniversidadDeValparaíso.(SponsoredbyThisWorkWasSupportedByGrantsFromMillenniumNucleusNU-MINDNC-130011(M.FAndP.R.M.),FONDECYT1130614(M.F.)And1141272(P.R.M.).)

Although the exact causes of schizophrenia (SZ) remain unknown, emerging evidence over several susceptibility genes isbeginning to shed light on themechanisms underlying the pathophysiology of SZ.One of these genes,SLC1A1, encodes theneuronalexcitatoryamino-acidtransporter-3(EAAT3),amemberofthehighaffinityglutamatetransporterfamilythatco-localizeswithglutamicaciddecarboxylase65(GAD65)ataxonterminalsofGABAergicinterneurons,whichcontributestothesynthesisofGABA;indeed,theblockageofEAAT3resultsinarapidreductionofGABAlevels,whichmayalterstheprecisebalancebetweenexcitationandinhibition(E/I). Inourlab,wehavefoundthatEaat3haploinsufficient(HET)micehavedopaminergicalterationsresemblingsomeanimalmodelsofSZ.However,itisunknownwhetherinEaat3HETmice,theinhibitorysynaptictransmissionisimpaired.Therefore,todetermineifEAAT3contributetoderegulationofGABAergicneurotransmissioninSZ,weevaluatedtheinhibitoryGABAergictransmissionoverpyramidalneuronsinlayerII/IIIofmedialprefrontalcortex(mPFC),inaEaat3HETmiceandconditionalEaat3over-expressing(OE)micecomparedtoapharmacologicalmodelofSZinducedbyketamine(Ket,30mg/kg).Throughelectrophysiologicalassaysweanalyzedthepaired-pulseratio(PPR)ofevokedinhibitorypostsynapticcurrent(eIPSC),the frequency,amplitudeofspontaneous (sIPSC)andminiature IPSCs (mIPSCs).WefoundthatEaat3HETandket-mPFCsliceshad lowerGABAreleaseprobabilitycomparedtoWTmiceorvehicle,whileEaat3OEhad increasedGABAreleaseprobability.Furthermore, conditional Eaat3HETand ket-mPFC slices showeda lowerGABAergic synapticefficacy compared toWT slices,while inEaat3OE the frequencyandamplitudeof sIPSCandmIPSCwere increased.These resultsdemonstratea relationshipbetweenSZ,geneticalterationsonEaat3expressionandchangesonGABAergicsynaptictransmissionandpointtoanewpotentialpresynaptictherapeutictargetattheGABAergicneurotransmissioninSZ.

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48)Dietary n-6 PUFAs induces depressive-like behavior while n-3 has antidepressant effect in a rat model of depression.

Pérez, Catherine1.,Dagnino,Alexies1.,1LaboratoryofBehavioralNeurobiology,CenterforNeurobiologyandBrainPlasticity,InstituteofPhysiology,FacultyofSciences,UniversidadDeValparaíso.(SponsoredbyThisWorkWasFundedByFONDECYTGrantN°1141276ToAD-S.Labsite:Www.stress.cl)

Inhumans,n-3:n-6dietarypolyunsaturatedfattyacid(PUFA)ratioisassociatedwithlongitudinalchangesindepressivesymptoms,with ahigher ratio linked to a slower increase in depressive symptomsovertime. The aimof this studywas to evaluate theeffectsofn-3andn-6PUFAssupplementationonthedevelopmentofdepressive-likebehaviors.MaleSprague-Dawleyratsweresubjectedtochronicunpredictablestress(CUS),ananimalmodelofdepression.Afterward,animalsweresupplementedwithn-3(fishoil),n-6(primroseoil)orwater.Anhedonia(lossofpleasure)andhopelessarethecoresymptomsofmajordepression,bothwereevaluatedintheratsasdepressive-likebehaviorsbysaccharinconsumptionandforcedswimtests(FST),respectively.RatsthatwereexposedtoCUSshowedreducedsaccharinintakeandfloatingbehaviorintheFST,whilesupplementationwithn-3PUFApreventedthesealterations.Conversely,supplementationwithn-6PUFAbyitselfinduceddepressive-likebehaviors,whilen-6hadsynergiceffectswithCUSonsaccharinconsumption.Wespeculatethattheetiologyofdepressive-likebehaviorsinthebrainisrelatedinpartwithalterationsonthePUFAbrainmetabolism.

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49)Large scale integration mechanisms are differentially altered in conscious disorders.

Rivera-Lillo, Gonzalo1,2.,Egaña,Jose3.,Díaz,Violeta4.,Maldonado,Pedro5.,1Kinesiología,Medicina,UniversidadDeChile.2LaboratoriodeNeurosistemas,Medicina,UniversidaddeChile.3Anestesiología,Medicina,UniversidadDeChile.4Neurología,Medicina,UniversidadDeChile.5Fisiología,Medicina,UniversidadDeChile.(SponsoredbySupportedByICMP10-001-F,P09-015-FBiomedicalNeuroscienceInstituteAndCONICYTForGR)

Thevegetativestateisaconsciousdisordercharacterizedbylackofasustained,reproducibleorvoluntarybehavioralresponsestosensorystimulation.Theminimallyconsciousstateisaconditionofseverelyalteredconsciousnesscharacterizedbyminimalbutdefinitebehaviouralevidenceofselforenvironmentalawareness.Previousreportshaveshowedthatsubjectsinthisconditionformaheterogeneousgroup,presentingdifferentevokedandoscillatoryresponsetodifferentcomplexitystimuli.Thisremnantactivitycouldbeassociatedwithdifferentprocessingabilitiesandcanberelatedtotheindemnityofsomebrainregions,ortoremaining connectivity between cortico-cortical and cortico-thalamic structures.Differentmechanismshadbeenproposed toexplaintheconsciousdisorders,themostofthemsharetheideaaboutthelackoflargescaleintegrationsuchasthemainaspectrelatedwithlostofconsciousness.Someofthismechanismsoflargescaleintegrationcouldbeobservedthroughboth,changesinoscillatoryactivityatdifferentfrequencybandsandtemporalcorrelationsbetweentheactivityatdifferentbrainlocations.Weproposethattransitorychanges inoscillatoryactivityamplitudeandphasesynchronyat lowfrequencybands,dependonthedifferentcomplexitystimuliandreflecthowdifferentlargescaleintegrationmechanismsareaffectedinpeoplewithconsciousdisorders.Werecordeddata fromtensubjects invegetativestates, twosubjects inminimallyconsciousstateand tencontrolsubjects.Wemeasuredtheevokedandoscillatoryactivity (24-ChannelEEG)toauditorystimuliusinga threeclassicaloddballparadigmwherethehighcomplexitydeviantstimuluswasthepatient’sownnamepronouncedbyafamilymember(emotionalvalence),middlecomplexitystimuliwasatoneformedbythreefrequenciesandlowcomplexitystimuliwasatoneformedbyonefrequency.Wefoundsignificantchangesinalpha/thetabandpowerspectruminresponsetostimulipresentationinthemostofsubjectsassociatedwithhighcomplexstimuli(activationneuralnetwork),butwithoutaselectiveresponsewhenwecomparebetweenownnamewithotherfirstnames(lackofselectiveresponseofneuralnetwork).Fortheotherside,morespecificlargescaleintegrationmechanismslikephasesynchrony(measuredthroughWeightedPhaseLagIndex)wasaffectedmainlyinthetabandindependentofstimuluscomplexity.Theseresultscontributetothemodelthatproposesalackofcorticalintegrationduetolossoffunctionalconnectivitybetweendifferentcorticalareas,andtoemphasizethatdifferentmechanismsarenotaffectedinasimilarform.

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50)Enhanced tracer coupling between striatal medium spiny neurons in a mouse model of Huntington’s disease

Rozas, Carlos1.,Kadriu,Bashkim2.,Chacon,Marcelo2.,Shlomo,Dellal2.,Faber,Donald2.,1Biology,ChemistryandBiology,UniversityofSantiagodeChile.2NeuroscienceAlbertEinsteinCollegeofMedicine,YeshivaUniv..(SponsoredbyFONDECYT11140430,CHDIFoundationA-5376)

Electrical transmissionbetweenneuronsviagap junctions ispresent throughout thebrain.Gap junctionspermit thediffusionof small signalingmolecules andmetabolites between connected cells. Specifically, theneurotransmitter dopaminehas beendemonstrated tobothweakenandenhance couplingbetween cells in the retina through its actions atD1andD2 receptors,respectively.Huntington’sdisease(HD)isahereditaryneurodegenerativediseasecharacterizedbyseveremotorimpairmentthatinvolvesthelossofstriatalmediumspinyneurons(MSNs)andcorticalprojectionneurons.Dopamine(DA)isacriticalfactorforthenormaloperationofthebasalganglia,andduringtheearlystagesofHDinhumans,DAlevelsareincreasedandDAreceptorexpressionisdecreased.WewereinterestedinwhetherthisdopaminergicdysfunctioninthestriatuminHDleadstoaberranttracer-coupling/electricalconnectivitybetweenMSNs.Tobegintoaddressthisquestion,weperformedwhole-cellpatch-clamprecordingsonMSNswiththediffusiblemorphologicaltracerneurobiotin(NB),whichissmallenoughtocross-gapjunctions.Wethenperformedimmunohistochemistry(IHC)onthesebrainslicesusingafluorophore-taggedstreptavidin,collectedz-stacksviaconfocallaser-scanningmicroscopy,andthencountedthenumberoftracercoupledcells.Wefoundthatonaverage,thenumberofcoupledcellswasthreefoldgreaterforMSNsfromR6/2mice,arapidlyprogressingHDmodel,ascomparedtotheirwildtype(WT)littermates.Thisdifferencewasobservedatboththeearlyandlatetimepointsofthedisease(5and12weeks).Wealsofoundthatwhenbrainslicesweretreatedwiththegapjunctionblockermeclofenamicacid(100μM),therewassignificantlylesstracercouplingandtherewasnolongeradifferenceintracercouplingbetweenWTandHD,indicatingthatthetracercouplingwasdueprimarilytopassageoftheNBfromtherecordedcellacrossgapjunctions.Inagreementwiththisobservation,wefoundthattherewasincreasedexpressionofconnexin36inHDstriatum(R6/2,5weeks),butnotincortex,asassessedbyWesternblotanalysis.TheseresultscomplementpriorfindingsthatHDpathologyischaracterizedbysignificantchangesinbothchemicalandelectricaltransmissioninthestriatum.FutureworkwillaimtodeterminewhethertheincreasesincellcouplinginstriatumresultfromadysregulateddopaminergicsystemandwhetheritcontributestothemotorsymptomsofHD.

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51)Beta-amyloid clearence on Alzheimer’s Disease: Role of APEH on brain and cerebral-vascular function

Sandoval, Rodrigo1.,Moraga,Josefina2.,Valenzuela,Javier1.,Lamas,Guillermo1.,Moraga,Fernando1.,Pancetti,Floria1.,1CienciasBiomédicas,Medicina,UniversidadCatolicadelNorte.2PedagogíaUniversidadMetropolitanaDeCienciasDeLaEducación.

Theaccumulationofamyloidbeta1-42inthebrainisacharacteristicofAlzheimer’sdisease.Anaccumulationofamyloidbeta(AB)1-40inthebrainvasculaturepromotescerebralamyloidangiopathy.Then,themechanismsofdegradationofABwouldhelppreventdiseaseprogression.Themoststudiedenzyme(neprilysinandinsulindegradingenzyme)haveothersubstrates,inthiscontextacetyl-peptidehydrolase(APEH),capableofdegradinginvitro1-40ABbecomesrelevant.Soitisimportanttodeterminewhether inhibitionof thisenzymealters theamountofAB in thebrainandvasculature.Wehipotesized thatAPEH inhibitionresultinincreasedAB1-40,altering1-42/1-40ratio,producingpathophysiologicalchangesincerebralandvascularcharacteristicsofAlzheimer’sdisease.Byusing techniquesof biochemical,electrophysiologicalandbehavioral,usingolder rats injectedwithDDVPAPEHinhibitor,wequantifiedABlevels,ABdegradingenzymesactivityandcontent,synapticplasticityandcerebral-vascularelectrophisiologyandspatiallearning.WefoundthatAPEHinhibitionbyDDVPincreasessynapticlevelsofAB1-40anddecreaseslevels1-42,coincidentwithincreasesintheactivityofneprilysininsynapsesofinjectedrats(P<0.001).Also,APEHinhibitionbyDDVP isassociatedwithadecreaseofcerebralvascularcontractility (p<0.001). Inaddition,AB1-40decreasesNMDAactivityandincreasesalpha7-NAChRactivityinhippocampalsynapses.finally,APEHinhibitionexhibitspatialmemoryimpairment.Theseresultassuggests thatchanges in1-42/1-40ratioduetoalterinABclearencemechanisms inducespathophysiological featurescharacteristicofAlzheimer’sdisease,andpositionsAPEHasaputativetargetforparmacologicaltratmentofthedisease.

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52)ATP-mediated astroglial hyperexcitability in a rat model of chronic epilepsy.

Wellmann, Mario1.,Álvarez-Ferradas,Carla1.,Sáez,JuanCarlos2.,Bonansco,Christian1.,1InstitutodeFisiología,CNPC,FacultaddeCiencias,UniversidadDeValparaíso.2DepartamentodeCienciasFisiológicas,FacultaddeCienciasBiológicas,PontificiaUniversidadCatólicaDeChile.

Itiswidelyacceptedthatastrocytesplayanactiveroleinawiderangeofneuropathologies.Inspiteofthat,howastroglialfunctionchanges inpathologicalconditionsremainsunclear.Previously,weshowedthathippocampalastrocytesfromchronicepilepticratsexhibitanaugmentedincidenceofspontaneousATP-dependentslowCa2+transients(ST),whichupregulatesglutamateCa2+-dependentgliotransmissionandthereforethestrengthofexcitatoryCA3-CA1synapses.BecauseATPisthemaingliotransmitterinvolvedinastrocyte-to-astrocytecommunication,weassessedifitplaysaroleinthisastroglialCa2+-mediatedhyperexcitability.Inorder todetermine theparticipationofnon-vesicularATP releasemediatedbypannexin-1 (Px1-HC)andconnexin-43(Cx43-HC) formedhemichannels,werecordedspontaneousastroglialCa2+ transients inpresenceof thepetidomimetics10-PanxandGap-26 incontrolandkindledhippocampal slices.PX1-HCblockade reduces themeandurationofastroglialCa2+ transients inkindledhippocampalslicesbydiminishingthepercentageandfrequencyofST,withnoeffectinthenumberofoscillationsperarea. Similareffectswereobserved inpresenceof theP2Y1 receptor (P2Y1R) specificantagonistMRS2179.Remarkably,bothantagonistshadnoeffectincontrolcondition,suggestingthatnon-vesicularATPreleaseviaPx1-HCrepresentsapathophysiologicalfeature of the epileptic tissue. Blockade of Cx43-HC doesn’t change any of themeasured parameters in control and kindledhippocampus.Theseevidencessuggestthatastrocyte-to-astrocytesignalingviaPX1-HCandP2Y1R,likelymediatedbyATP,causestheastroglialhyperexcitabilityobservedintheepileptichippocampus.Astroglialdysfunctioncouldrepresentanewkeyinepilepsyphysiopathology, likelycontributingto thereductionof seizure thresholdandepilepsychronicity.Funding:1130491 (CB) fromFONDECYT,CID1/2006 fromDIPUV (CNPC), CONICYT22120213 fellows (MW),UVA08042010 (JM) andUVA0603MECESUPfellows(CAF).

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53)The transcription factor NF-kB is translocated to the nucleus in epilepsy-related excitotoxicity.

Corvalan,Katherine1.,Gómez,Teresa1.,Varas-Godoy,Manuel2.,Wyneken, U3.,1CentroInvestigacionesBiomédicasUniversidadDeLosAndes.2CIBUniversidadDeLosAndes.3CIB,Medicina,UniversidadDeLosAndes.(SponsoredbyFondecyt1140108)

Neurodegenerativedisordersarestronglyassociatedtoexcitotoxicity,i.e.neuronaldeathbypathologicalstimulationofN-methyl-D-aspartate(NMDA)receptorsinsusceptibleareasofthecentralnervoussystem.NeuronaldeathdependsonNMDA-receptormediatedCa2+overloadandactivationofdownstreameffectorssuchasnitricoxidesynthases.Inprimarycellcultures,wehavedescribedanexperimentalconditioninwhichhippocampalneuronsaredifferentiallysusceptibletoanNMDAchallenge(30µMfor1hour)whencomparedtocorticalneurons,thatareresistanttothisinsult.WefoundthatadifferentialsignalingpathwayleadingtonitricoxideproductionandparticularproteinS-nitrosylationpatterns.Specifically,thep65subunitoftheNF-kBtranscriptionfactor is S-nitrosylated in cortical, but not in hippocampal neurons. To testwhether this differential S-nitrosylation pattern isassociatedwithactivationandnucleartranslocationofp65,weobtainednuclearextractsfromcellculturesfollowedbyWesternblots. Immunocytochemical studies are in course, aswell as experimentswith luciferase-NF-kB reporter construct.We foundthatp65translocatestothenucleusafterNMDAstimulationinhippocampal,butnotincorticalneurons.ThisisassociatedtonochangesinthephosphorylationofthecanonicNF-kBactivationpathway.

Anon-canonicpathwayleadingtoNF-kBactivationafteranexcitotoxicchallengemayprovidenoveltargetsforpreventionofcelldeathinneurodegenerativedisorders.

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54)Plasma nanovesicles as potential biomarkers of stress-induced depressive behaviors

Gomez,Cristobal1.,Ramirez,JuanPablo1.,Wyneken, U1.,1CIB,Medicina,UniversidadDeLosAndes.(SponsoredbyFondecyt1140108)

Exosomes,i.e.extracellularnanovesiclesthatoriginateinmultivesicularbodiesofcells,areemergingaspotentialbiomarkersofdiseasesduetotheirmolecularcargoconsistingofproteinsandRNAspecies.Mayordepressivedisorder(MDD)isamultifactorialdiseasewithincreasingevidencefortheexistenceofsub-types.AnimalmodelsofMDDarebasedonexposuretochronicstress.Previousworkinourlaboratorydemonstratedthatchronicstressprotocolsbasedonmovementreduction,eitherbyrestriction(RS)insmallcagesorimmobilization(IS)inplasticbags,for2hduring10days,wereabletoinducedepressive-likebehaviorsthatwereselectivelyrevertedbydifferenttypesofantidepressantdrugs.Moreover,theproteinAldolaseC(AldoC)withinexosomesincreasedintheCSFselectivelyafterstressinducedbyRS,butnotbyIS.

In thiswork,we focusedon thepresenceofputativebrain-derivedbiomarkers inexosomespresent in theplasmaofanimalssubjectedtochronicstressbyRSorIS.Byinuteroelectroporationoftelecephalicastrocytes,weshowthatexosomescontainingAldoCcanbeharvestedfromtheserum.Moreover,endogenousAldoCwasdifferentiallydetectedafterRSorIS.Furthermore,ourresultsshowthatAldoCinexosomesismodifiedbysumoylation.BymassspectrometryandWesternBlotwefoundthatseveralputativebrain-derivedproteinsaredifferentiallypresentinserumexosomesofratssubjectedtobothstressprotocols,includingAldolaseA,GFAP, Synaptofysin andReelin.Additionally,miR26a,highlyexpressed in forebrainastrocytes, is present in serumexosomes.

OurresultsshowthatserumnanovesiclesprovideanovelandusefultooltoidentifyproteinandmiRNAmarkerspresentinstressmodelswithdifferentialpharmacologicalsensitivity,whichcouldhelptodefinemarkersforstressinduceddepressivedisorder.

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55)Metabolic and gene expression changes underlying axonal regeneration during diapause.

CANEO, Mauricio1.,Alkema,Mark2.,Calixto,Andrea1.,1CENTRODEGENOMICA,BIOLOGIA,UniversidadMayor.2NEUROBIOLOGYUniversityofMassachusettsMedicalSchool.

Fooddeprivationcausesprofoundchangesinphysiologyandbehavior.Animalswiththeabilitytoenterdormancyinresponseto lackof foodensure survivalby suppressingoxidativemetabolism tominimizeenergyexpenditure.Hypometabolismduringdormancy is associated with dramatic changes in gene expression, protein repertoire and enzymatic activity. In response tostarvation,thenematodeCaenorhabditis elegans entersdiapauseformingthedauerlarva,astateaccompaniedwithdramaticchangesinmorphologyandmetabolism.WefoundthatdiapauseformationprotectsandregenerateC. eleganssensoryneuronsthathavebeentriggeredtodiebyhyperactivationoftheMEC-4ddegenerin.Themec-4d mutation(A713V)causesconstitutiveopeningofMEC-4channel,sodiumentryandincreasedintracellularCa2+causingneuronaldeathbynecrosis.

One fundamental change that occurs in diapause is the downregulationof the insulin receptorDAF-2,which promotesDAF-16/FOXOactivationand the consequent increase in cellular antioxidant capacity, known tobeneuroprotective (Calixto et al.,2012). However, while daf-2;mec-4d double mutants show extensive neuronal protection they do not regenerate damagedneuronsasdauersdo,suggestingthatothergenenetworksareinvolvedinneuroregenerationduringdiapause.Tounderstandthegeneexpressionchangesthatunderlietheincreasedregenerativecapacityofdauers,weanalyzedtheexpressionlevelsduringdiapauseofgenesknowntobecrucialforregeneration.Akeyregulatorofregenerationisthemitogen-activatedproteinkinaseDLK-1,requiredforaxonregrowthafteraxotomy(Hammarlundetal.,2009),andspontaneousbreakageofaxons(Hammarlundetal.,2007).DLK-1promotesregenerationbysimultaneousactivationofjnk andp38pathways.Wefoundthep38pathwaytobeupregulatedindauersaswellasccpp-6, atargetofDLK-1.Additionally,negativeregulatorsofregenerationsuchasklp-7andefa-6,involvedinmicrotublecatastrophearedownregulated.

TotestwhetherDLK-1hasaroleintheregenerationobservedindauers,wegeneratedadoublemutantofmec-4danddlk-1.mec-4d;dlk-1 mutantsfailtocompletelyregenerate,showingthat,asinaxotomizedaxons,DLK-1isalsoimportantfortheregenerationofdegenerinexpressingaxons.However,weobservedthat50%ofanimalshavewildtypeaxonsattimeofdauerentryincontrastwith8-10%innon-dauers,highlightinganadditionaleffectwhichisdauerdependentbyindependentoftheactivityofDLK-1.Thissuggeststhatthedauerstateconveysalargenumberoffactorsthatareneuroprotectiveandstimulateregeneration.

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56)EFFECTS OF VOLUNTARY EXERCISE ON SPATIAL AND OBJECT RECOGNITION MEMORY OF OCTODON DEGUS DURING AGING.

Salazar, Claudia1.,Palacios,Adrian1.,1CentroInterdisciplinariodeNeurocienciadeValparaíso,Ciencias,UniversidadDeValparaíso.(SponsoredbyMillenniumInstituteICM-P09-022-F)

TheOctodondegus(degu)isarodentthatnaturallydevelopsthemainsignsofAlzheimer’sdisease(deguADlike)duringaging,includingtheincreaseofsolubleAß,andTauhyper-phosphorylationandfailuresassociatedwithlearningandmemory(spatialandobjectrecognition)capacityandalterationsinsynapticplasticity(LTPandLTD).Severalotherstudiessuggestthatexercise(freeaccesstoawheel)canpreventordelaythedeteriorationofcognitionduringneurodegenerativeprocesses.Herewehavestudyindeguofdifferentages,theeffectofvoluntarylong-termphysicalexerciseontheircognitivecapacities.Wehavetestedthere,locomotoractivity,recognitionmemoryandspatialmemorythroughanopenfieldtest,objectrecognitionandradialarmmazeinyoungandoldanimals.Ourresultsshowthatbothyoungandoldexerciseddegushavebettercognitiveperformancecomparedtodeguwithout(wheel)activity.Forexample,after4monthsofwithoutwheelinactivitybothagegroupsshowdeteriorationinbehavioraltest.Thus,voluntaryexercisecouldcorrespondtoaneffectivetherapeuticusage,reducingcognitiveimpairmentcausedbydeguADlike.

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57)Rat olfactory sensory neurons cilia incorporate glucose and may take it from the mucus to use as complementary energy source for odor transduction

BACIGALUPO, JUAN1.,VILLAR,PABLO1.,BLANCHARD,KRIS1.,VILLALOBOS,DANIELA1.,DELGADO,RICARDO1.,VERGARA,CECILIA2.,REYES,JUAN3.,1BIOLOGIA,CIENCIAS,UniversidadDeChile.2BIOLOGIA,CIENCIAS,UniversidadCentralDeChile.3INSTITUTODEQUIMICA,CIENCIAS,PontificiaUniversidadCatólicaDeValparaíso.(SponsoredbyFONDECYT1140520)

Olfactorycilia(~60μmlong,0.2μmdiameter)projectfromtheapicalknobofthesingledendriteofolfactorysensoryneurons(OSNs).OdorantsbindingtoG-proteincoupledreceptorstriggeracAMP-cascadeintheciliathat leadstotheopeningofCa2+-conductive cyclic nucleotide-gated channels (CNGs); Ca2+ itself activates Cl- channels. Both channels generate a depolarizingreceptorpotential.TheciliarequireATPfortheactivityoftheadenylylcyclase,ATPasesandkinases.Themitochondriaintheknobaretheclosesttothecilia,whichlackanyinnermembranes.SlowATPdiffusionfromtheknobandlimitedbasalATPavailabilityintheciliasuggestthepossibleexistenceofadditionalATPsourcestosustaintransductionduringperiodsofintenseodorstimulation.Immunohistochemistrypreviouslyrevealedglucosetransportersintheolfactoryepitheliumciliarylayer,populatedwithOSNsciliaandsupportingcells(SCs)microvilli(Nuñez-Parraetal,ChemSenses,2011).WehypothesizedthatglucoseisreleasedbySCstothemucusandincorporatedbytheciliatoproduceATPbyglycolysis.Wedetectedthepresenceofglucoseinthemucuscoveringtheepitheliawithacolorimetricassay.ByimmunocytochemistryondissociatedcellsweconfirmedthepresenceoftheglucosetransporterGLU-3inOSNbothinOSNciliaandSCmicrovilli.Withafluorescentglucoseanalog(2-NBDG),weobservedthattheciliaincorporatedthissugarfromthemucus.Glycolyticenzymesarepresentinciliarymembranes,asrevealedbyimmunoblotting.Glycolysisaswellasoxidativephosphorylationinhibitorspartlyabolishedodorresponsesinfieldrecordingsfromtheolfactoryepitheliumandsuctionpipetterecordings from isolatedOSNs in thepresenceofexternalglucose (0.2mM).OSNsunderwentfatigueuponglucoseremovalfromtheextracellularsolution.TheseresultssupportthenotionthattheciliatakefromthemucusglucosereleasedbySCsandprocessitbyglycolysistosupplyATPforchemotransduction,inadditiontoATPsuppliedbytheknob.

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58)Chronic phenytoin treatment enhances rat petrosal ganglion responses to acetylcholine

Ortiz, Ignacio1.,Vera,Jorge1.,Alcayaga,Julio1.,1Biología,Ciencias,UniversidadDeChile.(SponsoredbySupportedByGrantFONDECYT1130177)

Thecarotidbody(CB),themainarterialchemoreceptorintherat,transducesarterialgasesandpHandsynapticallydrivestheactivityofafferentneuronswhichsomataarelocatedinthepetrosalganglion(PG).Theseneurons,drivenbytransmittersreleasedbyCBcells,presentapersistentNa+current(INaP),whichacuteblockadebyphenytoinreducesbasalafferentdischarges,andbothincreasedafferentdischargesandventilatoryresponsestoacutehypoxia.However,becausechroniceffectsofphenytoinonthePGneuronshavenotbeenreported,westudiedtheirelectrophysiologicalpropertiesandtransmitter-inducedresponsesinneuronsfrom chronically phenytoin treated rats.Male Sprague-Dawley rats (180-200g), under isoflurane anesthesia, were implantedsubcutaneouslywithanosmoticpump(Alzet2ML4)thatdelivereda10mg/daydose(n=7);controlanimalswereimplantedwithpumpscontainingvehicle(n=3).Bothantibioticandanti-inflammatorywereinjectedattheendofsurgery.After1-4weekstheanimalswereanesthetizedwithsodiumpentobarbitoneandthePGwasobtainedbilaterally.ThetissuewasplacedinachamberunderconstantflowwithHanks´solutionat30°C.Conventionalintracellularrecordingswereperformedwith3MKCl-filledglasselectrodesconnected toamicroelectrodeamplifier (Axoclamp900A); stimuluscontrolanddataacquisitionwascontrolledbysoftware(pClamp10).Acetylcholine(ACh)wasappliednearthePG.Animalsweresacrificedbyananestheticoverdoseattheendoftheexperiment.Phenytointreatmenthadnoeffectonrestingmembranepotential(control:-61.1±7.5mV;Treated:-56.8±0.6mV)orinputresistance(control:23.1±4.3MΩ;Treated:27.1±4.5MΩ).ResponsestoasingledoseofACh(100mM,10µL)producedadepolarizationthatwassignificantlylarger(P<0.05,MannWhitneytest)intreatedrats(ΔV=5.5±1.5mV)thanincontrol(ΔV=2.3±0.5mV).Thus,chronicallyadministeredphenytoindoesnotaffectthemeasuredelectricalpropertiesofPGneurons,whileincreasingtheirresponsestoexogenouslyappliedACh.Itissuggestedthattheseobservedmodificationsmayalterventilatoryresponsesafterchronicphenytointreatment.

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59)Role of TRPM8 channels in the altered sensitivity to cold of corneal mice primary sensory neurons caused by axonal damage.

Piña, R1,2.,Campos,Matías1.,Ugarte,Gonzalo1.,González,Alejandro1.,Bacigalupo,Juan2.,Madrid,Rodolfo1.,1DepartamentodeBiología,FacultaddeQuímicayBiología.LaboratoriodeNeurociencias,UniversidaddeSantiagodeChile.2DepartamentodeBiología,FacultaddeCiencias.LaboratoriodeFisiologíaCelular,UniversidaddeChile.(SponsoredbyGrantsCONICYTAnilloACT-1113(RM,GU),FONDECYT1131064(RM),1140520(JB)AndCONICYTPhDFellowshipAndGrant21110327(RP).)

Injury of corneal sensory fibers is followed by dysesthesias, altered tear production and changes in thermal and chemicalsensitivity. Theneuralandmolecularbasesofthesealterationsarepoorlyunderstood.Both,cold-sensitivityandspontaneousfiringofcornealcold-sensitiveneurons (CCSNs)dependontheexpressionof thecold,voltageandmenthol-activatedchannelTRPM8. In addition of detecting cold, CCSNs expressing TRPM8 are humidity detectors of the eye surface. The role of theseneuronsinthesensoryalterationsinducedbyaxonaldamageremainsunclear.WeusedfocalextracellularrecordingofCCSNs,incombinationwithcalciumimagingandpatchclampingofFM1-43retrogradelabeledcornealneuronsfromtrigeminalganglia,toinvestigatewhethertheirthermal-andchemical-sensitivitywerealteredaftercontrolledmechanicalinjuryofcornealsensoryaxons.WefoundanincreaseinthepercentageofCCSNsandtheirresponsestocoldandmenthol,withnomajordifferencesinthemeantemperaturethresholdcomparedtocontrolanimals.ImmunohistochemistryoftheophthalmicregionoftrigeminalgangliarevealedanincreaseinthepercentageofTRPM8-expressingneuronsafterinjury.UsingtransgenicmiceThy-1YFP,weevaluatedtherecoveryofcornealsensoryfibersafterinjury,andourresultsrevealpartialreinnervationafter21days.Spontaneousfiringandmenthol-sensitivitywerehigherinCCSNsfromdamagedcorneas.Interestingly,theinjuredanimalsexhibitedamorepronouncedbasaltearflow.TheseresultsunveilfunctionalchangesinCCSNsafterinjury,suggestingthatsensoryalterationsinducedbydamageofcornealnervefibersmaybelinkedtoanenhancedTRPM8expression.

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60)Chronic phenytoin treatment reduces rat ventilatory responses to acute hypoxia

Pino, Gabriela1.,Montero,Pablo1.,Alcayaga,Julio1.,1LaboratoriodeFisiologíaCelular,Ciencias,UniversidadDeChile.(SponsoredbySupportedByGrantFONDECYT1130177)

Carotidbody (CB)afferentactivitydrivesventilationfromtheperipherybychanges inarterialgasesandpH.Sensoryneuronsinnervating the CB present a persistentNa+ current (INaP); acute blockade of INaPwith phenytoin reduces normoxic ventilationandresponsestoshorthypoxicchallenges.Becausetheeffectsoflong-termphenytointreatmentonventilationandventilatoryresponsesareunknown,werecordednormoxicventilationandresponsestoawiderangeofoxygeninspiratoryfractions(FIO2)inratschronicallytreatedwithphenytoin.MaleSprague-Dawleyrats(192±3g),underisofluraneanesthesia,weresubcutaneouslyimplantedwithanosmoticpump(Alzet,2ML4)containingvehicle(control;n=9)orphenytoin(10mg/day;n=20);theanimalsreceivedboth an antibiotic and an anti-inflammatory immediately after surgery. From7 to 31days after surgery the animalswereanesthetizedwithsodiumpentobarbitone(60mg/Kg)andplacedinathermoregulatedpad.Thethraqueawascannulatedand connected to a pneunotacograph, connected in turn to a differential pressure transducer to assess ventilatory flow (JV).The left femoral arterywas cannulatedand connected toapressure transducer (StathamP23D) tomeasurearterialpressure(Pa)andassesscardiacfrequency(FC),andarectalprobeusedtomonitorthetemperatureoftheanimal.AnimalsbreathedairspontaneouslyandchangesinFIO2(0–100%range)wereappliedfor30s.Allsignalsweredigitallyacquiredat2KHz/channelandstoredinapersonalcomputer.Tidalvolume(VT)andventilatoryfrequency(FV),respectively,werecalculatedfromJVrecordings,andminutevolume(VE)wascalculatedastheproductVT*FV.Theanimalsweresacrificedwithananestheticoverdoseattheendoftheacuteexperiment.Phenytointreatmenthadnosignificanteffect(P>0.05,ANOVA)onnormoxicventilatory(VT,FV,VE)orcardiovascular(Pa,FC)variables.Phenytoinsignificantlyreduced(P<0.05,Bonferronitestafter2WayANOVA)theincreasesof VT andVE induced by acute hypoxic challenges after 13 and 29 days of treatment,withoutmodifying FV (P > 0.05, 2WayANOVA).Nosignificantchangesincardiovascularresponsestohypoxiawereobserved(P>0.05,2WayANOVA).Ourresultsshowthatphenytointreatmentreducesventilatoryresponsestohypoxia,withoutmodifyingthecardiovascularresponses,suggestingaspecificeffectmediatedbyINaPblockade.

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61)Pannexin 1 modulates the function of the supporting cells of the Organ of Corti.

Prado, Pavel1.,Jara,Oscar1.,Flores,Carolina1.,Maripillán,Jaime1.,Martínez,Agustín1.,1CentroInterdisciplinariodeNeurocienciadeValparaíso,FacultaddeCiencias,UniversidadDeValparaíso.(SponsoredbyWorkSupportedByFONDECYT3150442ToPP,FONDECYT1130855AndANILLO-ACT1104ToADM.TheCentroInterdisciplinarioDeNeurocienciasDeValparaísoIsAChileanMillenniumInstitute(P09-022-F))

Pannexin1(Panx1)isatrans-membraneproteinthatformsnon-selectiveplasmamembranechannelspermeabletoATP.Inthecochlea,thismoleculeisexpressedindifferentcellulargroups,includingthesupportingcellsoftheorganofCorti(OC).LackofPanx1inthecochlearesultsinsensorineuralhearingloss(SNHL).Wehypothesizedthatthistypeofdeafnessmightarisefromthedisruptionofthecochlearpurinergicsignalingpathway,whichfinallyresultsintheabnormalmaturationoftheOC.Togetinsightabout theroleofPanx1 inhearing,cochleaeofwild-typemicewithdifferentpostnatalageswerecollected,andphysiologicalpropertiesofsupportingcellswerecharacterizedasafunctionoftheageandtheeffectofpharmacologicalagentsthatspecificallyblockPanx1channels.Itwasfoundthatwholecellvoltage-dependentioniccurrentsofisolatedsupportingcellsincreasewiththematurationalstageoftheanimal.ThemagnitudeoftheioniccurrentswasimportantlyreducedbyacutetreatmentwithPanx1blockers(probenecidandthemimeticpeptide10Panx1),suggestingacriticalroleofPanx1channelsintheexcitatorypropertiesofcochlearsupportingcell.Furthermore,thebasalreleaseoftheATPbythecochlearsupportingcellswasalsoreducedwhenorganotypicculturesoftheOCwereincubatedwithPanx1blockers.Althoughstillpreliminary,differenceinthesinglechannelactivitywereobservedamonganimalsofdifferentagebracketsTheseresultssupporttheideaofanage-dependentexpressionofPanx1channels in themurinecochlea,whichmightbe important for thecorrect functioningof theOrganofCortiandthedevelopingofhearing.

262

62)Direction seletivity in a network of non-homogeneous Starbust Amacrine Cells (SAC)

Salgado, Simon1.,Castro,Samy1.,Escobar,MaríaJosé2.,Orio,Patricio1.,1CentroInterdisciplinariodeNeurocienciadeValparaiso,FacultaddeCiencias,UniversidadDeValparaíso.2DepartamentodeElectrónicaUniversidadTécnicaFedericoSantaMaría.(SponsoredbySupportedByGrantsFondecyt1130862,1140403,ACT-1104,ACT-1113,FB0008.TheCentroInterdisciplinarioDeNeurocienciaDeValparaíso(CINV)IsAMillenniumInstituteSupportedByTheMillenniumScientificInitiativeOfTheMinisterioDeEconomía(Chile))

Direction-SelectiveRetinalGanglionCells(DSRGC)respondselectivelytostimulimovingtowardsonedirection,whileastimulusmovingintheoppositedirectionproduceslittleornoresponse.ThisactivityisproposedtooriginateintheStarburstAmacrineCells(SACs),whichreleaseGABAwhichinhibitDSRGCswhenstimulusmovescontrarytothepreferreddirection.Intracellularcalciumstudiesinrabbit\\\’sSACshaveshownanasymmetricalresponsetoabarmovingthroughoppositedendrites.Also,biophysicalmodellingsuggeststhatSACdendriticstructuresareintrinsicallyselectivetodirectionbecauseofpassivecableproperties.SACshighlyoverlapwitheachother, and interact throughGABAergic inhibitory synapses,but the roleof this SAC-SAC inhibition isnotwellunderstood.Weareworkinginanovelconductance-basedmodeloftheSACsnetworkconsideringSAC-SACinhibitoryinteractionunder thePyNEURONsimulationenvironment.Weoptimized theparametersof theGABAergic synapses in a SACnetworkstimulatedbyglutamatergicbipolarinputs,andstudiedtheeffectofGABAergicandglutamatergicconductancelevels.Inourmodel,differentSACsshowslightdifferentvelocitytuningcurves,whichcorrespondstothevariabilityfoundinbiologicalexperiments.Asaresult,anon-homogeneouspopulationofSACscanrespondtoawiderrangeofstimulusvelocities.ThereciprocalGABAergicinhibitiongreatlyenhancesthedirectionselectivitymeasured,providingevidencethatanetworkperformsbetterthanisolatedSACs.FutureworkwillbefocusedinintegratingaDSRGCtothemodelaswellasseparateONandOFFbipolar/SAClayers.

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63)Effects of cannabinoid receptor activation in OFF bipolar cells activity

Vielma, Alex1,2.,Schmachtenberg,Oliver2.,Chávez,Andrés2.,Fuenzalida,Marco1.,1CentrodeNeurobiologíayPlasticidadCerebral,FacultaddeCiencias,UniversidadDeValparaíso.2CentroInterdisciplinariodeNeurocienciadeValparaíso,FacultaddeCiencias,UniversidadDeValparaíso.(SponsoredbyPostdoctoralFONDECYT#3140599(AV),RegularFONDECYT#1120513(OS),#1130614(MF),And#1151091(AEC).MillenniumInstituteCINV(OS,AEC)AndMillenniumNucleusNU-MINDNC-130011(AEC,MF).)

Thecannabinoidreceptortype1(CB1R),oneofthemostabundantGprotein-coupledreceptorsinthebrain,hasbeenshowntomediatedanumberofphysiologicalactionssuchas inhibitionofexcitatoryand/or inhibitorysynaptictransmissionthroughinhibitionofvoltage-gatedCa2+channels.Inthemammalianretina,CB1Rsarewidelydistributedintheouterandinnerplexiformlayers (IPL),stronglysuggests thatthis receptormayplayan importantneuromodulatoryrole inregulatingretinal informationprocessing.However, thephysiological significanceofCB1Ractivation in themammalianretinaremainsunclear.Usingwhole-cellrecordingtechniquesinlight-adaptedratretinalslices,weinvestigatedwhetheractivationofCB1RmightdirectlymodulateOFFbipolarcellfunction.ExogenousapplicationofthespecificCB1RagonistWIN55212-2(WIN;1µM)bylocalizedperfusionoftheIPL,generatedmembranehyperpolarizationduetoanoutwardcurrent,recordedat0mVtoisolateinhibitoryCl-currents.Ontheotherhand,thespecificCB1RantagonistAM251(5µM)causedinitialmembranedepolarizationfollowedbyaprolongedhyperpolarization,productofasustainedoutwardcurrentobservedat0mV.NocurrentswereinducedbyWINorAM251at-60mV,theCl-reversalpotentialinourrecordingconditions.TheseresultssuggestthatCB1RmightdirectlymodulatetheintrinsicactivityofOFFbipolarcells.ThepotentialcontributionofeCBsandtheroleofCB1RinregulatinginhibitorysynapticinputsontoOFFbipolarcellsiscurrentlyunderinvestigation.

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64)Sodium Potassium Chloride co-transporter 1 (NKCC1) is responsible of high excitability in chronic epilepsy in adult rats

Lara, Marcelo1.,Lorca,Enrique1.,Rojas,Patricio1.,1Biología,QuímicayBiología,UniversidadDeSantiagoDeChile.

The increase in hippocampal excitability is oneof the cardinal symptomsof temporal lobe epilepsy (TLE), themost commonepilepsytype.InepilepticpatientsaswellasanimalmodelshavebeenobservedanincreaseinDentateGyrusexcitability,whichis thought tobe responsibleofhippocampal foci inTLE.An increase in intracellular chlorideandprotein levelof thechlorideco-transporter1NKCC1,whichdrivestheelectroneutraluptakeofthisanion,havebeenobservedinhumanananimalmodelssuggesting an excitatory GABA (aminobutyric acid) effect. Being similar to what observed in immature neurons during earlydevelopment.InordertoevaluatethecontributionofNKCC1totheelevatedcircuitexcitabilityinchronicepilepsy,weevaluatedDentateGyrusexcitabilitybyextracellularfieldrecordingsinacutebrainsliceofchronicepilepticanimals.Thisallowtonotperturbthe intracellular chloride concentration. Input-output relationships of synaptic and population action potentials needed lessstimulationtoreachsaturatedresponses,consistentwithafacilitationofsynaptictransmission.TheGABAergiccomponentisalsoshiftedtowardlessstimulation.InhibitionofNKCC1byBumetanidedecreases50%oftheamplitudeofGABAergiccomponentinepilepticslices,whileonly10%incontrols.ThiselevatedNKCC1activityresultsinanexcitatoryGABAeffectinepileptictissue.Finally,Bumetanidewasabletodecreasethefrequencyofepileptiform-likefiringinslicesfromepilepticbutnotcontrolanimals.Inoverall,theseresultsshowthatNKCC1isexpressedandactiveindentategyrusofchronicepilepticanimals,anditsblockadeisabletoproduceanti-epilepticeffects.

265

65)The structure of the axon initial segment correlates with basal firing rate in substantia nigra dopaminergic neurons

Meza, R1.,Oñate,Alejandro2.,Henny,Pablo2.,1AnatomiaNormal,Medicina,PontificiaUniversidadCatólicaDeChile.2Anatomia,Medicina,PontificiaUniversidadCatólicaDeChile.(SponsoredbyFundedByFONDECYT1171140AndANILLOACT-1109GrantsToP.H.AndCONICYTGraduateScholarshipToR.M.)

In substantianigracompacta (SNc)dopaminergicneurons theaxonusuallyemerges fromaprimaryorhigherorderdendrite.Actionspotentialsaregeneratedatthemostproximalregionoftheaxon,theaxoninitialsegment(AIS).Thisunmyelinatedregionis enriched in sodiumandother ionic channels, aswell as scaffoldingproteins suchasAnkyrin-G (Ank-G).As shown inothercentralneurons,themolecularcomposition,sizeandlocationoftheAISisknowntoinfluencetheneuron’sactivity.Inordertodescribethemechanismsthat influencefiring inSNcdopaminergicneurons,wecarriedoutaprojecttoexaminewhetherthestructuralcharacteristicsoftheAISrelatetothespontaneoustonicfiringpatternshowedbytheseneurons in vivo.AdultmalemiceSNcneuronswererecordedunderurethaneanaesthesia.Neuronswererecordedduringspontaneousactivityat least15minutesbeforeneurobiotinlabellingusingthejuxtacellularmethod,afterwhichanimalswereperfusedandtheirbrainsremovedandseriallysectioned.Neuronswererevealedusingstreptavidin-Cy3andidentifiedasdopaminergicusingimmunofluorescencefor tyrosine hydroxylase. To determine the shape and localisation of the AIS, entire individual neurons were traced and 3Dreconstructed from labeled fragments acquiredwith a confocalmicroscope. The localisation of the AISwas confirmed usingfurther immunofluorescence staining for Ank-G. Structural analysis show variable dendritic origin and size of the AIS in SNcdopaminergicneurons.ThetwovariablesnonethelessrelateinthatlengthofAISdiminisheswithdistancefromthesoma.Notably,electrophysiologicalanalysesshowthatAISlength/localisationpredictsspontaneousbasalfiringrate,inthatneuronswithlarge/proximalAISfirefasterthanneuronswithsmall/distalAIS.

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66)Serotonin induces inhibitory long-lasting depression by activation of presynaptic 5-HT1 receptors

Morales, Koyam1,2.,Moya,Pablo3,2.,Fuenzalida,Marco1,2.,1Laboratoriodeplasticidadneural,FacultaddeCiencias,UniversidadDeValparaíso.2MillenniumNucleusinNeuropsychiatricDisordersNU-MIND,FacultaddeCiencias,UniversidadDeValparaíso.3LaboratoriodeNeurogenética,FacultaddeCiencias,UniversidadDeValparaíso.(SponsoredbyThisWorkWasSupportedByGrantsFromMillenniumNucleusNU-MINDNC-130011(M.FAndP.R.M.),FONDECYT1130614(M.F.)And1141272(P.R.M.).)

GABAergicinhibitorysynapsesareessentialformaintainingtheexcitationandinhibition(E/I)ratio,whoseimbalanceunderliesvariousbraindiseases.Changesintheefficacyofsynaptictransmissionintheprefrontalcortex(PFC)havebeenproposedastheneural substrateof several cognitiveprocesses such as learning andmemory. Theneurotransmitter serotonin (5-HT) exerts apowerfulcontrolofPFCsynaptictransmission.Giventhewidespreadinnervationofthebrain,itisnotsurprisingthatthe5-HTsystem is the target ofmany drugs used to treat brain diseases. However, the action of serotonergic signalling on inhibitorysynapsesefficacy is stillunknown inPFC.Throughelectrophysiologicalpatch-clamprecordings,westudied thechanges in theinhibitoryplasticitygeneratedby5HT.Ourresultsshowedthatactivationof5-HT1receptorsby5-HT(50μM)orbytheagonist5-carboxamidotryptamine(5-CT, 100nM) induceda long-lastingdepressionof evoked inhibitorypost-synaptic current (eIPSC) inPFCpyramidalneuronsofLayer2/3.Also,weobservedthatactivationof interneuronserotonergic receptorsmight induceanincreaseofpairedpulseratio(PPR)andadecreaseofspontaneousactivity(sIPSC)frequency,suggestingthat5-HTreducesthereleaseprobabilityofGABAergicinterneurons.Thus,ourresultssuggestthat5-HT-dependentchangesintheGABAergicefficacyofPFCcouldbeanimportantfunctionaltargettothetreatmentofdifferentneuropsychiatricdiseasessuchasanxiety,obsessive-compulsivedisorderandschizophrenia.

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67) The effect of a reduced sAHP-conductance on the glutamatergic synaptic plasticity of kindled rats

Morales, Juan1,2.,Fuenzalida,Marco1,2,3.,Bonansco,Christian1,2.,1InstitutodeFisiología,FacultaddeCiencias,UniversidadDeValparaíso.2Centrodeneurobiologíayplasticidadcerebral,FacultaddeCiencias,UniversidadDeValparaíso.3MillenniumNucleusNU-MIND,FacultaddeCiencias,UniversidadDeValparaíso.(SponsoredbyFunding:1130491(CB)And1130614(MF)FromFONDECYT,MillenniumNucleusNU-MINDNC-130011(M.F.),CID1/2006FromDIPUV(CNPC),UVA08042010(JM)MECESUP.)

Theslowafter-hiperpolarization(sAHP)isoneofthethreemainCa2+-dependentK+conductancesactivatedpostburstsofactionpotentials(APs),whichreducesneuronalexcitability,regulatesdendriticintegrationandrestrictsthetemporalcoincidencebetweenpreandpost-synapticactivity requiered for inductionof long termplasticity (LTP) throughshuntingofpost-synapticEPSPs. Inepilepsy,ithasbeenreportedadiminishedsAHP,directlyenhancingneuronalexcitability;howevertheeffectofapathologicallyreducedsAHPonsynapticplasticityisunknown.InthisworkweinvestigateifapathologicallyreducedsAHPcouldfacilitatethepotentiationofglutamatergicsynapses in theepileptichippocampus.Usingwhole-cellpatch-clampconfigurationwerecordedthesAHPinpyramidalneuronsinCA1regionofthehippocampusfromadult(p60)controlandkindled(KD)rats,determiningitseffectonsynapticplasticityinducedbyalowfrequencyspiketimingdependentplasticityprotocol(STDP;pairingbetweenEPSPandabackpropagatedAP).InKDgroup,neuronsshowedaredistributionofthesAHPconductance:lowerthan2.0nS(classifiedasL-sAHP,26.9%oftheneuronsrecorded)andover8.0nS(H-sAHP,23.0%)withoutsignificantchanges inAPfiring,whilethecontrolgroup rarelypresented theL-sAHPphenotype (<8.3%). Inbothgroups, L-sAHPdidnot significantlymodified theEPSPtimecoursenoritsamplitude(noshunting),whichcouldincreasethetemporalwindowforpairingsandthusfacilitatesynapticpotentiation.BothgroupsexhibitedsimilarlevelsofsAHPactivationduringSTDP,butonlyKDneuronswithsAHPlowerthan4.0nSshowedpotentiation,whereasthosewithhighersAHPshoweddepression,similartocontrolsynapses.InKDneurons,STDPprotocolappliedunderconstantsAHPactivationresultedinatransientinitialpotentiation(123.5±8.2%until20minutesafterSTDP)followedbylongtermdepression(LTD,79.0±1.5%after40minutes),neverthelesscontrolneuronsonlyshowedLTD(79.7±7.7%from15minuteson).TheseresultsindicatethatKDL-sAHPneuronsallowtheinductionoftransientinitialsynapticpotentiationfollowedbyLTD.Thisabnormalformofshorttermpotentiationexhibitedbythisneuronalnetworkinepileptichippocampuscouldcontributetothedisruptionofsynapticplasticitythatisrequiredforepilepsyprogression.

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68)The neurovascular coupling-initiated astrocyte Ca2+ signal is mediated by sequential glutamate metabotropic and NMDA receptor activation

Muñoz, Manuel1.,Puebla,Mariela1.,Figueroa,Xavier1.,1DepartamentodeFisiología,FacultaddeCienciasBiológicas,PontificiaUniversidadCatólicaDeChile.

Brain functionsdependonfine regulationof cerebralbloodflowbyamechanismknownasneurovascular coupling.Neuronsand cerebral arterioles are functionally communicated through the astrocytes located between these two cell types and anincreaseinsynapticactivityisrapidlytransducedintoreleaseofvasodilatorsignalsbytheastrocytic-endfeetthatareencasingthecerebralbloodvessels.Theastrocyte-dependentneurovascularcouplingisinitiatedbytheneurotransmittersreleasedduringanincreaseinsynapticactivity,whichactivateanintracellularCa2+signalthatpropagatestotheastrocytic-endfeet.Then,themainneurotransmitterinvolvedinneurovascularcouplingisglutamatethroughtheactivationofglutamatemetabotropicreceptorsonastrocytes.However,theactivationofglutamatemetabotropicreceptorsleadstoD-serineandglutamatereleasebyastrocytes,whichmaycontributetotheCa2+signalingbythestimulationofionotropicNMDAglutamatereceptors(NMDAR).Therefore,weusedprimaryculturesofastrocytes toevaluate theparticipationofNMDAR in the increase in intracellularCa2+ concentration[Ca2+]i initiatedbytheactivationofglutamatemetabotropicreceptorsinastrocytes.ExpressionofNMDAreceptorNR1subunitwasanalyzedbyinmmunofluorescenceandWesternBlotandthechangesin[Ca2+]iwererecordedbyloadingtheastrocyteswiththeCa2+indicator,Fluo-4.Stimulationwithglutamate(30µM)ortheglutamatemetabotropicreceptoragonist,t-ACPD(150µM),evokedanincreasein[Ca2+]ithatshowsanonsetat~20sandapeakat~60s.Inaddition,NMDA(50µM)orNMDA(50µM)plusD-serine(100µM)inducedaCa2+responseofasimilarmagnitude,butinthiscase,thepeakwasobservedat~20s.Interestingly,theincreasein[Ca2+]ielicitedbyglutamateandt-ACPDwasblockedbytheNMDARantagonist,DL-AP5(50µM).NMDARwerefoundtobeexpressedinastrocytes.Then,theseresultsstronglysuggestthatactivationofNMDARisinvolvedintheCa2+signalinitiatedbythestimulationofglutamatemetabotropicreceptorsintheneurovascularcoupling.

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69)Hipercarbic acidosis induce ATP release from brainstem astrocytes in culture

Olivares, María José1.,Donoso,MaríaVeronica1.,Huidobro-Toro,JuanPablo1.,Llona,Isabel1.,Eugenín,Jaime1.,1DepartamentodeBiología,FacultaddeQuímicayBiología,UniversidadDeSantiagoDeChile.(SponsoredbyFondecytRegular#1130874Y#1141132)

Homeostatic regulationofbreathing is achieved through feedback informationprovidedbyperipheral and central respiratorychemoreceptors. Peripheral chemoreceptors senseO2/CO2/H

+ in the arterial blood and central chemoreceptors CO2/H+ in the

cerebrospinalfluid. In thebrainstem, respiratory chemoreceptorsare found,amongother sites, in the retrotrapezoidnucleus(RTN),ventralrespiratorygroup,solitarytractnucleus,ventrallateralmedulla,medullaryraphe,andprebötzingercomplex.Gourineetal,2010,foundthatATPplaysafundamentalrolelikegliotransmitterreleasedbypH/CO2sensitiveastrocytesinresponsetoacidosisattheRTN.TheincreaseinRTNneuronsactivityisattenuatedbythepresenceofP2receptorsantagonistsorapyrase.Inthepresentwork,weevaluatedwhetherastrocytesfromthebrainstemareableofreleasingotherATPderivativestogetherwithATP,likeADP,AMPandAdenosine(ADO).Releaseofpurineswasevaluatedfrombrainstemandbraincortexastrocytesindissociatedcellcultures,whenthesewerestimulatedwithhypercarbicacidosis(10%CO2).TwodaysoldCF1mouseneonateswereanesthetized(3%isofluorane),decapitatedandtheirbrainswereextracted,disaggregated,andculturedinDMEM-F12mediumequilibratedwithaircontaining5%CO2at37°Cfor2weeks.Duringstimulation,DMEM-F12mediumwasreplacedbyartificialcerebrospinalfluid(aCSF)3mMKCl.Astrocytescultureswereexposedatbasalcondition(5%CO2)for45min,orathypercarbicacidosis(10%CO2)for45minat37°C.Sampleswerecollectedat5,15,30,and45minforthetwoconditions.TheconcentrationofATPandderivativesweremeasuredusinghigh-performanceliquidchromatography(HPLC)technique.ATPreleasedfrombrainstemastrocytesincreased3-foldduringhypercarbicacidosiscomparedtothebasalcondition.ADPandAMPconcentrationincreasedduringhypercarbic acidosis to thebasal condition, butADO concentration increased after themaximum releaseofATP frombrainstemastrocytes.Incortexastrocytes,itwasnotobservedanincreaseofATPorADPinducedbyhypercarbia.AMPandADOconcentrationarehigherthanATPandADPconcentrationinbothconditions.AMPandADOmaximumconcentrationnotappeartorespondtohypercarbicacidosis,becauseitbehavesthesamewayasthebasalcondition.Ourresultsindicatethatbrainstemastrocytes,butnotcorticalastrocytesareableofreleaseATPasthemainpurinergicgliotransmitterinresponsetohypercarbia.

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70)The endothelial nitric oxide synthase isoform is present in neuronal synapses and lipid rafts.

Caviedes,Ariel2.,Nualart,Francisco1.,Wyneken, U2.,1Fisiología,Medicina,UniversidadDeConcepción.2CIB,Medicina,UniversidadDeLosAndes.(SponsoredbyFondecyt1140108)

Nitricoxide(NO)modulatesseveralprocesses inthecentralnervoussystemwhile its increase inoxidativestressconditions isassociatedtoneuronaldamage.Itssynthesis inthebrainhasbeenmostcommonlybeenascribedtotheneuronalnitricoxidesynthase (nNOS) isoformalthough the endothelial isoform (eNOS) has been implicated as a retrogrademessenger in cellularplasticity. Inendothelial cells, eNOS is associated toplasmamembrane lipid rafts, specializeddomains rich in cholesterol andsphingolipidsthatcontainspecificproteins likeThy1andcaveolinandprovidesaplatformformultiproteincomplexformationandsignaling.Abettercharacterizationofthepossible localizationofeNOSinneuronswouldhelptoproposefunctionalrolesoftheenzymeinthecentralnervoussystem.Thisshouldbeperformedinacellularsystemdevoidofendothelialcells.WethusstudiedthelocalizationofeNOSbyimmunocytochemistryofprimaryhippocampalandcorticalneuronsandbyWesternBlotsofsubcellularfractions.Finally,cellviabilitywasassessedwiththeTripanbluetest.

Weshowbyconfocalandsuper-resolutionmicroscopythateNOSco-distributeswithpost-synapticmarkers(Shank2andPSD95),butnotwithpre-synapticmarkers,and is localized indendritic spines.Wealso found thateNOSco-distributedwith lipid raftmarkers.Moreover,eNOSisenrichedinsynapticmembranesandinpostsynapticdensitiesisolatedfromneuronalculturesandfromtheratforebrain.eNOSinhibition incorticalcellshasanegative impactoncellsurvivalafterexcitotoxicstimulationwithNMDA. In turn,hippocampalneuronaldeathdependsonnNOS-dependentNOsynthesis andeNOS inhibitiondoesnotaffectneuronalviability.OurresultsshowthateNOSislocatedatexcitatorysynapsesandinlipidraftswhereitcouldrepresentamajorsourceforNOproductionabletomodulatesynapticfunctionandneuronalsurvival.

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71)Transfer of Aldolase C containing exosomes from astrocyte to neurons induces morphological rearrengements in neurons.

Luarte,Alejandro1.,Ramirez,JuanPablo1.,Masalleras,Matias1.,Villalobos,Isabel1.,Varas,Manuel1.,Wyneken, U1.,1CIB,Medicina,UniversidadDeLosAndes.

Astrocytesarethemostabundantglialcellsinthecentralnervoussystemandtheyreleaseseveralsolublefactors(gliotransmitters)thatmodulateneuronalnetworkconnectivity.Furthemore,vesiclessecretedfromastrocytesintotheextracellularspace,suchasexosomes,mayalsoregulatesynapticactivitybytransferringlipids,proteinsandmicroRNAstoneurons.WehavefoundthattheastrocytespecificglicolyticenzymeAldolaseCisreleasedfromastrocyteswithinexosomesthatcanbeinternalizedbyneurons.EndogenousAldolaseCandAldolaseC-GFPcontainingexosomes inducedadose-dependentdecrease in neuriticarborizationselectivelyinhippocampalculturedneurons.Thiseffectscouldbeexplained,inpart,bytheeffectofmicroRNAmiR-26a,thatishighlyenrichedinexosomesandpossiblyassociatedtoAldolaseC,asrevealedbyimmunoprecipitationofAldolaseCfollowedbyRT-qPCR.MorphologicalefectsofexosomesonneuronsarerevertedbyincubationwithmiR-26aantago(inhibitor)andpartiallyreplicatedbymiR-26amimic.Ourresultsshowthatastrocyte-to-neuroncommunicationcanbemediatedbyexosomes,affectingmorphology,whilethemechanismsinvolvedintheregulationofneurotransmissionarecurrentlyunderstudy.

272

72)Change in the position of the action potential initiation site in Granule Cells of the Dentate Gyrus during repetitive firing

Palma-Espinosa, Javier1.,Orio,Patricio2.,Rojas,Patricio1.,1LaboratoriodeNeurociencias,FacultaddeQuímicayBiología,UniversidadDeSantiagoDeChile.2CentroInterdisciplinariodeNeurocienciadeValparaisoUniversidadDeValparaíso.

InGranulecellsofthedentategyrus,actionpotentialsaregeneratedintheAxonInitialSegment(AIS),whichislocated20-40μmfromthesoma,closercomparedtootherneurons.InlayerVpyramidalneurons,whereAISislocated60-100μmfromthesoma,twopeaksappearinthesecondderivativeofVm,wherethefirstpeakcorrespondtoactionpotentialinitiatedattheAIS,andthesecondcorrespondstothesomaticactionpotential.Becauseofacloserinitiationsiteingranulecells,thesecondderivativeofsingleactionpotentialdoesnotshowtheusualtwoseparatepeakscorrespondingtoaxonalandsomaticinitiations.However,inthelatestactionpotentialswithinasuccessivetrain,twopeaksappearsinthesecondderivative,whichismoreevidentas“hump”byinspectingphaseplots.TheapparitionofthehumpinthesecondderivativecouldbeduetoamovementoftheinitiationsiteawayfromthesomaduetoinactivationofvoltagedependentsodiumchannelsintheproximalAIS.Inordertoinvestigatethisbehavior,weusedacomputationalmodelforgranulecellsthatcanreproducefeaturesofsingleactionpotential(Schmidt-HieberC.andBischofbergerJ.JNeurosci.30:10233-42),andmodifyitinordertoreproducefeaturesofspiketrains.Thismodelallowedustoexploreseveralcombinationofvaluesandpositionintheaxonforseveralconductances,inordertoexplainthehumpinthesecondderivativeunderrepetitivefiring.Ourresultssuggestthatactionpotentialinitiationingranulecellsisadynamicprocessthatcanaccountforplasticchangesinfiringpropertiesbymovingpositionandlengthofinitiationsite,ashasbeenproposedforotherbrainstructures.

273

73)Control of neurovascular coupling by S-nitrosylation of astrocytic calcium homeostasis modulator 1 channel.

Puebla, M1.,Muñoz,MF1.,Figueroa,XF1.,1PhysiologyPontificiaUniversidadCatólicaDeChile.(SponsoredbyFondecyt1150530)

Brainfunctionreliesonthecoordinationofneuronalactivitywithcerebralbloodflow,whichisknownasneurovascularcoupling.Thereby,theincreaseinmetabolicdemandassociatedtoanincrementinsynapticactivityissatisfiedbythevasodilationoflocalparenchymal arterioles. The intercellular signaling that functionally connects neuronal activity with arteriolar vasodilation ismediatedbyanincreaseinastrocyteintracellularCa2+concentration,whichpropagatestotheastrocyticend-feetandinducesthereleaseofvasodilatorsignals.Gapjunctionchannelsandhemichannelsformedbyconnexins30(Cx30)and43(Cx43)coordinatethis signalingprocess.As astrocytes express theCa2+-dependentnitric oxide (NO)-synthetizing enzymeseNOSandnNOS, andNO activates hemichannels, we evaluated if NO is involved in the control of neurovascular coupling. Hemichannel activity,changes in intracellular Ca2+ concentration and ATP releasewere evaluated in primary cultures of astrocytes and vasomotoractivityofcortexarterioleswasrecordedinratbrainslices.Stimulationwithglutamateortheglutamatemetabotropicreceptoragonist,1-aminocyclopentane-trans-1,3-dicarboxylicacid(t-ACPD),inducedhemichannelactivation,anincreaseinastrocyticCa2+concentration,ATPreleaseandvasodilationofparenchymalarterioles.Blockadeofhemichannelswiththeconnexinblockingpeptide37,43Gap27inhibitedtheincreaseinCa2+andvasodilation,butnotATPrelease,whichwasabolishedbythecalciumhomeostasismodulator1(CALHM1)channelblocker,rutheniumred.Connexinhemichannelactivitywasalsoblockedbypyridoxalphosphate-6-azophenyl-2’,4’-disulfonicacid(PPADS),apurinergicreceptorantagonist.AstrocytesrelatedtoparenchymalarterioleswerefoundtoexpressbotheNOSandnNOS,andblockadeofNOproductionwithNG-nitro-L-arginine (L-NA)abolishedtheglutamate-ort-ACPD-initiatedastrocyticCa2+signaling,vasodilationandATPrelease.Inaddition,glutamatealsoevokedtheS-nitrosylationofCALHM1channels.TheseresultssuggestthatNOproductionplaysacentralroleinthecontrolofastrocyte-mediatedneurovascularcouplingthroughactivationofCALHM1channelsbyS-nitrosylationandthefurtheropeningofCx43hemichannelviaATPreleaseandpurinergicreceptoractivation.

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74)Pannexin 1 is equally expressed in neurons, microglias and astrocytes of the lamina I-II of the spinal cord in normal and neuropathic rats

Bravo, D1,2.,Maturana,Carola3.,Hernández,Alejandro1.,Juan,Saez3,4.,Constandil,Luis1.,1DepartamentodeBiología,FacultaddeQuímicayBiología,UniversidadDeSantiagoDeChile.2Kinesiología,FacultaddeSalud,DeporteyRecreación,UniversidadBernardoO`higgins.3DepartamentodeFisiologíaPontificiaUniversidadCatólicaDeChile.4InstitutoMilenio,CentroInterdisciplinariodeNeurocienciasdeValparaísoUniversidaddeValparaíso.(SponsoredbyThisWorkWasSupportedByGrantFB0807FromCEDENNA(LCAndDB)AndGrantsFromICM-EconomíaP09-022-F(JCSAndCJM).)

Pannexin1(Panx1)isamembraneglycoprotein,vastlyexpressedinthecentralnervoussystem(CNS)ofmammals.Itformshighconductance,ATPreleasechannelsrelatedwithseveralCNSdiseases,suchasstress,epilepsy,ischemia,neuroinflammationand,recentlydescribed,chronicpain.Althoughtheparticipationofthesechannelsinthesignalingofchronicpaininthespinalcordhavebeendemonstrated,andthepresenceofthePanx1intotalspinalcordhasbeenreported,itremainsunclearwhetherPanx1ispresentinspinalareasrelatedwithpaintransmission.Theseareasincludethedorsalhornofthespinalcord,andspecificallytheLaminaeIandIIofRexed,wherenociceptiveinformationfromtheperipheralnociceptiveneuronsisintegratedtotheCNS.Thissite iswherethemost importantpathologicalneuroplasticityandglialactivationareobservedinchronicpain.Hence,weperformedwesternblotanalysisofPanx1inhomogenatesoftheposteriorquadrantofthe lumbarspinalcord, ipsilateraltoasuralnervelesion(NPrats,apainmodel),andcomparedthemtocontrolrats.Furthermore,weperformedimmunofluorescenceinspinalcordslicestolocatePanx1inneurons,microgliaandastrocytesinLaminaeIandIIofRexedofNPrats.SimilarlevelsofPanx1weredetectedinNPanimals.Panx1waslocalizedinlaminaeIandII,inalltestedcellsofcontrolrats,anditsreactivityinneurons,microgliaandastrocyteswassimilarintissueofNPrats.Hence,Panx1isconstitutivelyexpressedinthedorsalhornofthespinalcordinareasrelatedwithspinalsignalingofpain,probablyinacuteandinchronicstates.ThisconstitutesthefirstevidencethatPanx1isexpressedinthedorsalhornareasrelatedwithpain,openingnewfrontierstopharmacologicalresearchinthistopic.

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75)EXPRESSION OF UNCOUPLING PROTEIN 2 AND 3 DURING RAT SPERMATOGENESIS

Paillamanque,Joaquin3.,Carmona,Emerson3.,Osses,Nelson3.,Santibañez,Cristián3.,Moreno,Ricardo1.,Pino,Jose3.,Bernales,Sebastian2.,Gomez,Francisco2.,Reyes, Juan3.,1DepartamentodeFisiología,FacultaddeCienciasBiológicas,PontificiaUniversidadCatólicaDeChile.2TranslationalResearchGroupFundaciónCienciayVida.3InstitutodeQuímica,FacultaddeCiencias,PontificiaUniversidadCatólicaDeValparaíso.(FinancedByFondecyt1140758)

Oneofthemechanismsassociatedtotheregulationofspermatogenesis isanapoptoticprocessoccurring inthefirstwaveofspermatogenesis,wherepachytenespermatocytesare removedbyapoptosis,apparently tomaintainanadequate relationofgermandSertolicells.Fas,mitochondria,pHi,[Ca2+]iandROSplayimportantrolesinthisprocess.Besidestheclassicalmechanismsassociatedtomitochondria-mediatedapoptoticevents,uncouplingproteins(UCP)andtheiractivitiescanalsolinkmitochondria,ROSandcellapoptosisbothbyuncouplingoxidativephosphorylationandregulatingROSproduction.Inthisresearch,weexploredthepresenceofUCP2andUCP3inrattestisandspermatogeniccellsatdifferentstagesofpostnataldevelopmentusingq-PCRdeterminationsofUCP2and3mRNA,andUCP3proteinexpressionbyWesternblot,immunocytochemistry,andthearachidonicacid-inducedandGDPinhibitionofmitochondrialmembranepotentialdecrease.OurresultsshowdifferentialkineticsoftestisUCP2and3levelsduringpostnataldevelopment.UCP2mRNAincreasesupto25daysofage,decreasingafterwards,whileUCP3onlycanbedetectedat20daysincreasingatleastupto55daysofage.ConsistentwithmRNAexpression,UCP3proteinscanbedetectedin25and60daysoldrats,withsomedifferencesbetweenpuberandadultrats.Inadultratspermatids,arachidonicacidwasabletoinduceaGDP-sensitivedecreaseofmitochondrialmembranepotential,consistentwiththefunctionalexpressionofUCP2and/or3inthesecells.

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76)Role of pannexin hemichannels in calcium mobilization from intracellular stores of mice sperm during ATP induced acrosome reaction.

Torres-Fuentes, Jorge1.,Tomes,Claudia2.,Saez,Juan1.,Darszon,Alberto3.,Treviño,Claudia3.,Moreno,Ricardo1.,1Fisiología,CienciasBiológicas,PontificiaUniversidadCatólicadeChile.2InstitutodeHistologíayEmbriología,FacultaddeCienciasMédicas,UniversidadNacionaldeCuyo.3DepartamentodeGenéticadelDesarrolloyFisiologíaMolecular,InstitutodeBiotecnología,UniversidadNacionalAutónomadeMéxico.(SponsoredbyFONDECYT1150352(RD.M))

Duringtransitthroughthefemalegenitaltract,spermatozoamustundergoseveralbiochemicalandfunctionalchangesbeforetheyarereadyfertilizetheoocyte.Theseeventspreparethespermtoundergothereactionofacrosome(AR),anexocyticeventthatreleaseofenzymes,necessaryforfertilization.TheARcouldbeinducedbyATPorProgesteroneandinvolvesmobilizationofextracellularandinternalstorescalcium.Pannexins(1,2or3)areproteinsthatformhemichannelswiththeabilitytobepermeabletoions,involvedbothextracellularandinternalstorescalciumregulation.Theseproteinsarepresentinmicespermatozoa,buttheirroleinARisunknown.TheobjectiveofthisworkwastocharacterizeinvitrotheparticipationofpannexinhemichannelsinARinducedbyATP.Usingspermatozoaobtainedfromwild-typeandPanx1knock-outmice,presenceandlocalizationwerestudiedbyInmunofluorescenceandWesternBlotting.TheARwasdeterminedbycoomassiebluestaining,hemichannelsfunctionalitybyethidiumuptake,calciummobilizationbysinglecellimagingwithFLUO-3AM,andcalciumstoreparticipationwasstudiedwithSLOtoxinpermeabilizedspermsamplesandcyclopiazonicacid.ResultsshowedthatATPinducestheARalongwitharapidcalciumincreasewhichwaspartiallymodulatedbyPanxinhibitors.Thiscalciumincreaseisrelatedwithcalciummobilizationfrominternalstores.TheseresultsshowthatpannexinsareinvolvedincalciumsignalingfrominternalstoresinARinducedbyATPinmousesperm.

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77)Analysis of pachytene spermatocytes transcriptome treated with arachidonic and cyclopiazonic acid

VASQUEZ, CLAUDIA1.,ACEVEDO,CRISTIAN2.,MORENO,RICARDO3.,OSSES,NELSON3.,REYES,JUAN1.,1INSTITUTODEQUIMICA,CIENCIAS,PontificiaUniversidadCatólicaDeValparaíso.2CENTRODEBIOTECNOLOGIAUniversidadTécnicaFedericoSantaMaría.3CIENCIASFISIOLÓGICAS,CIENCIASBIOLOGICAS,PontificiaUniversidadCatólicaDeChile.(SupportedByFONDECYT1140758AndFSM12041MecesupResearchAssistantshipScholarship.)

Duringmammalianspermatogenesis thepresenceofvariousstagesofgermcells iscorrelatedwithdifferentpatternsofgeneexpression.FactorsderivedfromSertolicellscouldbepartlyresponsibleforthiscoordinatedgeneexpressioninspermatogeniccells.OneofthederivativesoftheSertolicell,arachidonicacid(AA),stimulatesanincreasein[Ca2+]iinratandmousespermatogeniccellsin vitro.TheconsequencesofAAandchangesin[Ca2+]iontranscriptioninthegermcellsisunknown.ThegoalofthisstudywastoanalyzethegeneexpressionofmousespermatocytessubjectedtotheeffectsofAAorcyclopiazonicacid(CPA),anICaSreleaseagent.PachytenespermatocytescellsisolatedandincubatedwithCPAorAA,both4µM,for3h.Subsequently,thecellsampleswereused to synthesize cRNA,whichwashybridized to IlluminaplatformMouseRef-8 containinga total of 25697 sequencesreferencetestsofmousegenome.Atotaloffourindependentcellpreparationsandexperimentswereanalyzed.Thedatawerenormalizedandexpressedasfoldchanges(FC).Weappliedfirst,afilterofthosesequencesthatwerebelowthebackgroundlevelofthemicroplatereader, leaving10532validsequences.Afterward,twoseparatedatafiles,AAtreatmentandCPAtreatment,weregenerated.ThentheLog2ofFCwasappliedtothesesetsofdataandattestwasperformed,selectingthosesequencesthathadap-value<0.05,leavingatotalof650sequencesfortreatmentwithAAand731forCPA.Commonsequencesbetweenthesetwotreatmentswere121.ThelatterwereanalyzedusingthesoftwareMeVMultiExperimentalViewer,formingtwomajorgroupsofsequences.The121genesweresearchedinthedatabaseGeneOntologyforbiologicalprocessesinwhichthesesequenceswouldbeclassified.Thus,AAandchangesin[Ca2+]icanregulategenesthatareimportantformanyprocessesassociatedtocellularcomponentorganizationorbiogenesis,localization,reproductionandmetabolismofrodentspermatogeniccells.

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78)Role of glucose transporters GLUT1 and GLUT8 in proliferation and lactogenesis in murine mammary gland

Villagran, Marcelo1.,Muñoz,Katia1.,Muñoz,Mirna1.,DelPozo,Reginald1.,Mardones,Lorena1.,1CienciasBásicas,Medicina,UniversidadCatólicaDeLaSantísimaConcepción.(SponsoredbyFONDECYT11121367)

Themammaryglandincreasesitsenergeticrequirementsduringpregnancyandlactationtosupportthehighrateofproliferationandthesynthesisofmilknutrients.WestudiedtheroleofglucosetransportersGLUT1andGLUT8inbothprocessesinBalb/Cmice. The temporal changes in the expression of both transporters were analyzed by western-blot, PCR, semi-quantitativeimmunoperoxidaseandcomparedwithtemporalexpressionofproliferativemarkerKi67andlactalbumin(LALB),theregulatorysubunitof lactosesynthetase.GLUT1wasexpressedweakly inover the50%of theglands inall theperiod,although inearlypregnancymoreofthe60%ofthealveolarcellswerestained.SimilarexpressionpatternforGLUT1wasfoundinwholeproteinextracts,reachinganincreaseof4foldinearlypregnancy.ForGLUT8,thepercentageofstainedglandincreasedfrom20to80%duringprogressionfrompregnancytolactationandanincreaseinintensityofGLUT8stainwasalsoobserved.AtmRNAlevelandinwholeproteinextracts,theincreasereached10and3fold,respectively.Ki67increasedtheexpressioninalveolarcellswiththeprogressofthepregnancyandthelactation,from20to100%,accompaniedwithanincreaseintheintensityofthestaining,andLALB,thatwasnotexpressedinvirginmice,showedasimilarpatternthatGLUT8inpregnancyandlactation,increasinginpercentageofstainedgland(20to60%),intensityofthestaining,andexpressionbywestern-blot(3fold).GLUT8,GLUT1andLALBshowedagranularintracellularexpressionandco-localizedbyimmunofluorescence,meanwhileKi67stainedshowedonlynuclearexpression.TheintracellularlocationofGLUT8andthetemporalexpressionofGLUT1discardtheirparticipationinalveolarcellsproliferation.ThetemporalexpressionofGLUT8anditsco-locationwithLALBsuggestthatGLUT8butnotGLUT1,isthetransporterresponsibleofglucoseentranceintoGolgisupportingthelactosesynthesis.Thelactogenesisandtheproliferationofmammaryalveolarcellshareacommonpatterninthismurinemodel.

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79)Pseudogenes and heat shock response for Rnf19a, an E3 ubiquitin ligase in spermatogenesis.

Párraga, Mario1.,Rejas,Carolina1.,SanMartín,Sebastián1.,Villena,Juan1.,DelMazo,Jesus2.,1CentrodeInvestigacionesBiomédicas,Medicina,UniversidadDeValparaíso.2LaboratoriodeBiologíaMoleculardelaGametogénesisCentrodeInvestigacionesBiológicas(CSIC).(SponsoredbyCentroDeInvestiagacionesBiomédicas.CID05/06)

Spermatogenesisisacomplexterminaldifferentiationprocess,whichoccuratlowertemperaturesthanotherprocessesinthebody.Regulationofspermatogenesisreliesonmolecularmechanisms,whichmonitortheprogressionofgermcelldevelopment.Theseareassociatedtodegradationmechanisms,whicheliminatesaberrantspermatogenicandspermcells.Suchmechanismsinvolveevolutionaryconservedproteolyticpathways,asitistheubiquitinationsystem.Alterationsofthesepathwaysoranyoftheircomponentsmayleadtospermatogenicimpairmentandinfertilityasaconsequence.Oneproteinexpressedduringsper-matogenesisandrelatedtotheconservedubiquitinationsystemistheE3ubiquitinligaseRnf19a.TheobjectiveofthisworkistocharacterizeRnf19agenomicstructureincludingthefunctionalityofitspromoter.Toaddressthis,wecarriedoutcomputationalsequence comparisonanalysisusingdifferent genedatabases. Theseanalyses showed thatRnf19a is present inorganismsasdiverseaswormmouseandhuman,accountingforitsevolutionaryconservation.Thisgeneislocatedonchromosome15inthemouse.Rnf19agenomicstructureisdividedinto10exonsand9introns.Furtheranalysisonthemousegenomeshoweddiffer-entRnf19ahomologoussequences inadistinctchromosomethatturnedouttobepseudogenes.OtherspeciesanalyzedalsoshowedpseudogenesforRnf19a.Thesewererat(Rattus norvegicus)andmosquito(Anopheles gambiae).However,noRnf19apseudogeneswerefoundinneitherhumannorC. elegans.Specifically,wefoundthreemousepseudogenesinchromosome9.Thesecontainnointronsandhaveseveralmutationsalongtheirsequencethatdisrupttheopenreadingframe.Accordingtothesecharacteristicsweinferthattheyareprocessedpseudogenes.Analyzingupstreamsequencesfromthegeneandpseudogenesweidentifiedtheirrespectiveputativepromoters.WithinthepromoterwefoundregulatorysequencessuchasTATAbox,GCboxandothers.WealsofoundsomeinterspersedHSEsequencesinthosepromoters.HSEsequencesarecharacteristicofheatshockrespondinggenes.Heatshockproteinsarepartoftheconservedmolecularpathwaysthatinspermatogenesisfunctionwiththeubiquitinationsystemasithasbeendescribed.WeclonedthesepromotersforthegeneandpseudogenesinEGFPcontainingplas-midsandthefunctionofthesepromotersweretestedundernormalandheatstressedconditions.Ourresultsdemonstratethat,in vitro,thesepromotersdriveEGFPoverexpressionrespondingtoheatshock.Furtherexperimentsanalyzingboththeresponsetoheatshockandtheexpressionofthegeneandpseudogenesin vivoshouldbefulfilled.

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80)Development of a lentiviral vector system to study Andes virus entry and neutralization

Varas, Nicolás1.,Starck,MariaFrancisca1.,Beltrán,Camila1.,Fernandez,Yaiza1.,Sánchez,Oliberto1.,1LaboratoriodeBiofármacosrecombinantes,FacultaddeCienciasBiológicas,UniversidadDeConcepción.

Andesvirus(ANDV) isaHantavirusthatcausesanacutediseasecalledHantavirusPulmonarySyndrome(HPS).HPS isahighlypathogenicdiseasewithacase-fatalityrateof40%.ANDVisendemicinArgentinaandChileandatotalof786casesofHPSoccurredduring1995–2012inourcountry.Moreover,ANDVistheonlyHantavirusreportedtospreaddirectlyfromhuman-to-human.Forthestudyandthediagnosticofthisvirusisusuallynecessarytoworkwiththehighlyinfectiousvirusparticles,whatcanonlybedoneinhighbiosafety-levelfacilities.InthepresentstudywedevelopapseudovirionsystembasedonHumanImmunodeficiencyVirus(HIV)vectorpseudotypedwiththeADNV-Gn/Gcenvelopeglycoproteins.ThiswasdonebyreplacingthegenfortheGproteinofVesicularStomatitisVirus(VSVG),presentinacommerciallentiviralexpressionkit,forthegenoftheGlycoproteinprecursorprotein(GPC)thatisposttranslationallycleavedtoformGnandGc.ItiswidelyreportedthatGn/GcareanchoredtotheGolgiapparatusandthatiswhereHantavirussupposedlybud.Ontheotherhandlentivirusesbudinthecellmembrane.HerewepresentanindirectimmunofluorescenceassaythatshowthatGnandGccanalsobefoundinthecellmembraneandthusallowingthepseudotypingoflentiviruses.TheincorporationofGnandGcontoHIV-derivedvectorparticleswasassessedbywesternblot.Inaddition,wetestitinfectivitywithvariouscelllines,includingaHEK293constitutivelyoverexpressingbeta-3integrin(HEK293-ib3),aknownreceptorforHantavirus.Thepseudotyped lentiviruswasableto infectavarietyofcell lines,but in lessamountthatthecommonlentivirusvector.TheuseofHEK293-Ib3causea2-foldincreaseinitsinfectivity.Finally,seraofANDVinfectedhumanswereabletoblockcellentryofthepsedotypedlentivirus.TheGn/GcpseudotypedHIVvectorhasseveraladvantages,hightitervectorproductionandeasyquantificationofcellinfectionbymonitoringGFPreportergeneexpressionbyflowcytometry.Suchpseudotypedlentiviralvectorscanbeusedtodevelopaquantitativeandhigh-throughputpseudovirionassaytostudyearlystepsofANDVcellinfection,neutralizationandscreenforpotentialhantaviruscellentryinhibitors,allofthisinbiosafetylevel2facilities(BSL-2).ThisworkwasfinancedbygrantsFONDECYT1110925andVIUR120015

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81)Study of cellular internalization mechanism of polyamidoamine dendrimers drug nanocarriers

Vidal, F1.,Vásquez,P1.,Soto,R1.,Díaz,C2.,Alderete,J2.,Guzman,L1.,1DepartmentofPhysiology,FacultyofBiologicalSciences,UniversidadDeConcepción.2DepartmentofOrganicChemistry,FacultyofChemicalSciences,UniversidadDeConcepción.(SponsoredbyFONDECYT1131004)

Inmanycasespharmacologicaltargetsareintracellularcomponents,whichmeansthatdrugsmusthavetheabilitytoenterthecell.However,theydonotalwayshavethepropertytocrosstheplasmamembrane,aprocessthatcouldbeimproved.Dendrimericdrugnanocarriersarechemicalstructuresofdiversenaturethatcontain,transportanddeliverthedesireddruginbiologicalsystems.Oneofthedendrimersthathasbeenmostsuccessfullyusedisbasedonpolyamidoamine(PAMAM).Theirversatilearchitectureandeasilymodifiablesurfaceallowtousethemindifferentapplicationsandmakethemthemostpromisingnanocarriersystems.Ingeneral,theirabilitytoenterthecellhasbeendescribedandithasassociatedtoendocyticmechanisms.Endocytosisistheprocessinwhichcellareabletointernalizelargemoleculesbyformingvesiclesfromplasmamembrane.Differentendocytosismechanismhasbeendescribed,suchasclathrin-mediatedendocytosis,caveolae-mediatedendocytosisandpinocytosis.Inthisworkwearefocus in determine the endocytic pathway that PAMAMdendrimers are internalized, understanding that their entry kinetics,intracellulardistribution,associationtoorganellesanddrugreleasedependonthisprocess.Tostudytheircellularinternalization,confocalmicroscopytechnicswereusedandPAMAMdendrimersweremarkedwiththefluorescentdyefluoresceinisothiocyanate(FITC)whichwaslinkedtotheaminogroupsoftheirsurface.Theirinternalizationwasstudiedintwodifferentcelltypes,HeLacellsandmousehippocampalneurons.TodeterminetheinternalizationpathwayofPAMAMdendrimersco-localizationanalysiswasperformedwithanti-clathrin,anti-caveolin-1andanti-Rab-5antibodies.Rab-5isasmallGTPasewhichiscrucialfortheearlyendosomaldynamicsandisusedasendocytosismarker.Theco-localizationobservedwithanti-Rab-5inbothcellulartypessuggestthatPAMAMdendrimersareinternalizedbyaclassicalendocytosisprocess.TheseresultsdemonstratethatPAMAMdendrimersinternalizationinvolvedendocyticpathways,butmorestudiesareneededtodeterminespecificdetails.Also,thestudieswouldbecenteredontheneuronalcellsbecausetherelevanceofthepotentialapplicationsonpharmacologyofthecentralnervoussystem.

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82)The behavioral effects induced by expression of a mutation for PINK and its relationship with dopaminergic neurons in Drosophila melanogaster

Molina-Mateo, Daniela1.,Fuenzalida-Uribe,Nicolás2.,Molina-Fernandez,Claudia2.,Figueroa,Reinaldo3.,Tevy,Florencia3.,Campusano,Jorge2.,1biologiacelularymolecular,cienciasbiológicas,PontificiaUniversidadCatólicaDeChile.2biologíacelularymolecularPontificiaUniversidadCatólicaDeChile.3CentrodeGenómicayBioinformáticaUniversidadMayor.(SponsoredbyFondecyt1141233.)

Parkinsondisease(PD)isadegenerativedisorderassociatedwithseveralmotorsymptomsincludingshaking,rigidity,slownessofmovementanddifficultywithwalking,andisrelatedtothedeathofdopaminergicneurons.Morethan96%ofPDpatientsalsopresentolfactorydysfunction.Althoughthemolecularmechanismsresponsibleforthisdiseasearenotclearithasbeendescribedthatmutationsinspecificgenes,includingthePTEN-inducedputativekinase1(PINK1),wouldberesponsibleforsomecasesoffamilialPD.InthisworkwedescribethebehavioraleffectsinducedbyexpressionofamutationforPINKinDrosophilaanditsrelationshipwithdopaminergicneurons.

WeusedfliesexpressingamutationforPINK1andfliesoftheCanton-Sstrainascontrols.Inbehavioralexperimentssinglemaleflieswereevaluatedatdifferentages (0-3;7-10;14-17;21-24and28-31daysold). Flieswereplaced ina circular arenawithtwocottonsinoppositesides.Behaviorofeachflywasrecordedfor3mininabsenceofanystimulus.Afterwards,cottonsweresoakedwithbenzaldehyde1%ordistilledwaterandflybehaviorwasrecordedforadditional3minutes.UsingtheBuridanTrackersoftwarewetracedthepositionofflies.Olfactorydiscrimination(OD)wasevaluatedasodorPreferenceIndex(PI).Severalmotorparameterswerealsomeasured.Additionally,weusedimmunofluorescenttechniquestovisualizedopaminergicneuronsinyoungandoldmaleflies.

ItispossibletoobservedifferencesinPIbetweenPINKandcontrolsfliesearlyon.Incontrast,changesindifferentmotorparametersareonlyevidentasfliesage,asexpected.WeareevaluatingwhetherthenumberofdopaminergicneuronschangeinmutantsforPINK,althoughpreliminaryresultsshownochangeinyoungversusoldanimals.

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83)Muscarinic Acetylcholine Receptors Contribute to Aversive Olfactory Learning in Drosophila

Molina-Fernández, Claudia1.,Silva,Bryon1.,Ugalde,MaríaBeatriz1.,Campusano,JorgeM1.,1DepartamentodeBiologíacelularymolecular,Facultaddecienciasbiológicas,PontificiaUniversidadCatólicaDeChile.(SponsoredbySupportedByFondecyt1141233)

ThemoststudiedformofassociativelearninginDrosophilaconsistsinpairinganodorant,theconditionedstimulus(CS),withanunconditionedstimulus(US).ThetimelyarrivaloftheCSandUSinformationtoaspecificDrosophilabrainassociationregion,themushroombodies(MB),caninducenewolfactorymemories.Thus,theMBisconsideredacoincidencedetector.IthasbeenshownthatolfactoryinformationisconveyedtotheMBthroughcholinergicinputsthatactivateacetylcholine(ACh)receptors,whiletheUSisencodedbybiogenicamine(BA)systems.Inrecentyears,wehavewitnessedanimportantadvanceinourunderstandingonthespecificneuralBApathwaysandreceptorsinvolvedinolfactorylearningandmemoryinflies.However,littleinformationexistsonthecontributionofcholinergic receptors tothisprocess.Hereweevaluate for thefirsttimethepropositionthat,asinmammals,muscarinicAchreceptors(mAChRs)contributetomemoryformationinDrosophila.ExpressionstudiesshowthatmAChRsareexpressedintheMB.OurbehavioraldatashowthatpharmacologicalandgeneticblockadeofmAChRsinMBdisruptstheformationandretrievalofolfactoryaversivememoryinlarvae.Thiseffectisnotexplainedbyanalterationintheabilityofanimalstorespondtoodorantsortoexecutemotorprograms.TheseresultsshowthatmAChRsexpressedinMBcontributetothegenerationofolfactorymemoriesinDrosophila larvae.

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84)N-3 PUFAs supplementation increases hippocampal neurogenesis and improves memory of stressed rats

Peñaloza Sancho, Valentín1.,Dagnino-Subiabre,Alexies1.,1LaboratoryofBehavioralNeurobiology,CenterforNeurobiologyandBrainPlasticity,InstituteofPhysiology,FacultyofSciences,FacultyofSciences,UniversidaddeValparaíso.(SponsoredbyFONDECYTGrantN°1141276ToAD-SLabsite:Www.stress.cl)

Introduction:Neurogenesisinthehippocampusiskeyforlearningandmemory.Chronicstressreduceshippocampalneurogenesisandimpairsmemory,whileomega-3polyunsaturatedfattyacids(n-3PUFAs)enhanceslearninginrats.

Objective:Theaimofthisstudywastoevaluate,inthesamestressedrats,theeffectsofn-3PUFAssupplementationonhippocampalneurogenesis,learningandmemoryconsolidation.

Methods:MaleSprague–Dawleyratswererandomlyassignedtounstressedandstressed(chronicrestraintstress)experimentalgroups.Afterward,animalsweresupplementedwithn-3PUFAs(DHAandEPAmix)orwater.Neurogenesiswasevaluatedwith5-bromo-2desoxiuridina (BrdU)andquantifiedby immunohistochemistry.LearningandmemorywereanalyzedbytheMorriswatermaze.

Results:Stressedratsthatweresupplementedwithn-3PUFAsshowedhigherlevelsofneurogenesisinthedentategyrusofthehippocampus.Thisresultwascorrelatedwithanimprovementinthememoryconsolidationrespecttonon-supplementedanimals.

Conclusions:Wespeculatethatn-3PUFAssupplementationcouldbeusedinthetreatmentofstress-relatedpsychiatricdisorderswherepatientshaveaffectedthehippocampus,suchasmajordepression.

Support: ThisworkwasfundedbyFONDECYTGrantN°1141276toAD-S.Labsite:www.stress.cl.

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85)Interactions between adrenergic activity and glucocorticoids in the Insular Cortex modulate arousal-induced taste Neophobia

Mendez, Luis1.,JerézBaraona,JuanManuel1.,Rojas,Sebastián1.,Díaz-Galarce,Raul1.,Quintana-Donoso,Daisy1.,Moraga-Amaro,Rodrigo1.,Stehberg,Jimmy1.,1LaboratoriodeNeurobiología,CentrodeInvestigacionesBiomédicas,UniversidadAndrésBello.

Glucocorticoids (GCs) are steroidal stress hormones released from the adrenal glands in response to the activation of thehypothalamic-pituitary-adrenalaxis.PreviousstudiesinhumanshaveshownthatsystemicGCsmayhaveparadoxicaleffects,actingasbothanxiolyticandanxiogenic,butnomechanismorbrainareahasbeensuggestedtomediatesuchparadoxicaleffects.HerewestudiedtheroleofglucocorticoidsattheInsulainthereluctancetonoveltastes(tasteNeophobia),whichcanbeexacerbatedbystressandhigharousalcontexts(HA),andisusedasameasureofanxiety.Thepresentresultssuggestthatglucocorticoidsin the insular cortexmodulate taste neophobia and arousal-induced increases in taste neophobia. Interestingly, intra-insularcorticosteroneinjectionsinducedincreasesinneophobiaatlowdosesanddecreasesinneophobiaathighdoses.Furthermore,intra-insular glucocorticoids restored the behavioral effects of systemic blockage of GC synthesis, and showed anxiogenic oranxiolyticeffectsdependingonconcentrationandonpreviousadrenergicactivation.Theeffectsof intra-insularcorticosteroneshowedtobeparadoxicalnotonlyintasteneophobia,butalsointheelevatedplusmaze,suggestingthattheInsulaisanimportantsitemediatingtheeffectsofGCsinanxiety,andthatGCsmayhaveanxiolyticoranxiogeniceffectswhenactingattheInsula.

FundedbyProyectoFONDECYTN°1130724

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86)Dopamine Receptor type 5 knockout mice (D5RKO) show memory impairments but normal affective behavior.

Moraga, Rodrigo1.,González,Hugo3.,Rojas,Patricio2.,Ugalde,Valentina3.,Pacheco,Rodrigo3.,Stehberg,Jimmy1.,1LaboratoriodeNeurobiología,CentrodeInvestigacionesBiomédicas,UniversidadAndrésBello.2LaboratoriodeNeurociencias.DepartamentodeBiología,FacultaddedeQuímicayBiologíaUniversidadDeSantiagoDeChile.3LaboratorioofNeuroinmunologíaFundaciónCiencia&Vida.

The Dopamine D1 receptor familywhich includes D1 and D5 receptors (D1R and D5R) has been shown to be important forlearningandmemoryinavarietyof learningtasks,brainareasandanimalspecies.FurtheranalysesusinggeneticapproacheshavecorroboratedtherelativecontributionofD1Rinthesecognitivetasks,buttherelativecontributionofD5RinlearningandmemoryremainsunclearduetoalackofstudiesusinggeneticapproachesandtheunavailabilityofdrugsthatcandiscriminatebetweenD1RandD5R.AfewstudieshavereportedlimitedevidencesuggestingthatD1RbutnotD5receptorsmaybeimportantformemory,butstudiestestingD5Rknockoutmice(D5RKO)onmemoryparadigmsarelacking.ThepresentstudywasdesignedtodeterminewhethertheD5RisinvolvedinmemorybytestingD5RKOmiceinabatteryofbehavioraltests.D5RKOmiceshowedunaffectedaffectivebehavior,showingnodepressive-likesymptoms.Theyalsoshowedsignificantimpairmentsinspatialmemoryusing the Morris watermaze, but normal working memory and unaffected object recognition memory. Electrophysiologicalanalysesperformed inD5RKOmicehippocampal slices showed significantdeficits in long-term-potentiation. Further analysesatthemolecularlevelshowedthatgeneticdeficiencyofD5Rresultsinasignificantdown-regulationofthehippocampalNMDAreceptorsubunitNR2B.ThesefindingsdemonstratearoleforD5RinmemoryandsuggestafunctionalinteractionbetweenD5Randhippocampalglutamatergicpathwaysinvolvedinsynapticplasticity.

FundedbyProyectoFONDECYTn°1130724

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87)Adrenergic transmission in the modulation of arousal-induced reluctance to try novel tastes by the insula in the rat

Rojas, Sebastian1.,DiazGalarce,Raul1.,JerezBaraona,JuanManuel1.,QuintanaDonoso,Daisy1.,MoragaAmaro,Rodrigo1.,Stehberg,Jimmy1.,1LaboratoriodeNeurobiologia,CentrodeinvestigacionesBiomédicas,UniversidadAndrésBello.

Reluctancetotrynoveltastes(tasteneophobia)isacommonadaptivebehaviorthatensuresacautiousresponsetoanoveltasteuntilitssafetyhasbeenascertained.However,neophobiaissignificantlyincreasedwhenthenoveltasteispresentedinahigharousalcontext(HA),comparedtowhenthenovelitispresentedinalowarousalcontext(LA).Thisincreasedreluctancetotrynoveltastesinducedbyarousingcontextsisusedtomeasureanxietyinrodentsanddependsontheadrenergicsystem.Todeterminewhetheradrenergicactivityattheinsularegulatesarousal-inducedincreasesinreluctance,acombinationofsystemicandintra-insularmanipulationsofadrenergicactivitywasperformedbeforethepresentationofsaccharin0.1%asachoicetowater,eitherinaHAorLAcontext.Bilateralintra-insularmicroinjectionsofnorepinephrineorthenon-selectivebetablockerpropranololwerefoundtomodulatetheeffectsofarousingcontextsonreluctancetotrynoveltastes.Moreover,systemiceffectsoforalpropranololwere blocked by intra-insular administration of norepinephrine,while intra-insular propranolol blocked epinephrine- inducedreluctancetonoveltastes.Inconclusion,theseresultssuggestacriticalroleforadrenergicactivityattheInsulainregulatingtheeffectsofarousalinthereluctancetotrynoveltastes.FundedbyProyectoFONDECYTN°1130724

288

88)Phenylalanine variability as a determinant factor during neurodevelopment. Outcomes in higher cognitive functions

Santander, Daniela2.,DeLaParra,Alicia1.,Castro,Gabriela1.,Arias,Carolina1.,Cabello,Francisco1.,Cornejo,Veronica1.,Ossandón,Tomás2.,1LaboratoriodeGenéticayEnfermedadesMetabólicas,I.N.T.A.,UniversidadDeChile.2DepartamentodePsiquiatría,FacultaddeMedicinayCentroInterdisciplinariodeNeurociencia,PontificiaUniversidadCatólicaDeChile.

Phenylketonuria (PKU)patientswhohavebeen correctly treateddisplaymilddeficits in cognitive functions likeattentionandworkingmemory,whichemulatethesymptomsobservedinattentiondeficithyperactivitydisorder(ADHD).InbothPKUandADHDtheunderlyingcauseofcognitivesymptomsmayrelatetoadeficitincatecholamines,whichareproductsoftyrosinemetabolism.WecomparedthecognitiveperformancebetweenPKUandADHDchildren,andestablishedaquantitativerelationshipbetweenPhemeansandvariability levelsandhigh-ordercognitive functions inPKUpatients. Clinicaldata from129early-treatedPKUpatientswithnoconcomitant ilnesseswereanalyzed(agerange:6months-15years),sixtyADHDpatientsandsixtycontrols.Asresult, indicatorsofpsychomotor,mentaldevelopment,and IQdataarenegativelycorrelatedwithmeanphenylalaninemialevelsandvariability,especiallystartingfromthe24thmonthsincebirth.AlthoughageandIQareuncorrelated,theIQdropswithage.BothgroupsshowworseverbalIQresultsthanperformanceIQ,andalowscoreinadigitretentionsubtest.WeconcludedthateventhoughphenylalaninemialevelsareuniversallyacceptedasthemostrelevantindicatortoassessthesuccessofPKUtreatment,ourresultshighlighttheimportancetomaintainstablelevelsthroughoutasmuchattheneurodevelopmentstageasattherestofapatient’slife.

289

89)Mechanisms of autonomic regulation during social cognition task

Varas, G1.,Maldonado,Pedro2.,1Fisiología,Medicina,UniversidadeChile.2FisiologiaUniversidadDeChile.

Theperception,interpretationandgenerationofresponsestotheintentionandbehaviorsofothersareknownassocialcognition(3).Therecognitionoffacialexpressionsandtheabilitytoinferthelikelymentalstatesofotherpeopleareanimportantfeatureof social cognition, this ability is called Theory of Mind. The emotions that humans experience while interacting with theirenvironmentareassociatedwith varyingdegreesofphysiological arousal (2).A key system involved in thegenerationof thisphysiological arousal is theautonomicnervous system (ANS).Heart ratevariability (HRV)analysis isemergingasanobjectivemeasureofregulatedemotionalresponding,andfunctionsrelatedtosocialcognitionandTheoryofmind.ThepolyvagaltheoryandtheNeurovisceralintegrationmodelproposesthattheANS,throughvagaltoneactivityandactivityoftheprefrontalcortex,improvestheinteractionsofasubjectwiththeirenvironmentsthroughaninhibitoryeffectonthesino-atrialnode(pacemarker)(1).Thereisevidencethat,atrest,subjectswithspinalcordinjury(SCI)haveapredominanceofsympatheticautonomicactivitywhichcorrelateswithlowHRV(4).OurhypothesisproposesthatthistypeofbasalactivityoftheANSdecreaseautonomicflexibilitythathasbeendescribedasfavorableforsocialcognitiontasks.WemeasuredHRV,asautonomicmarker,in18healthysubjectsand10subjectswithSCI,diagnosedwithparaplegia,whowerepursuingaperiodofadaptationandsocio-laborintegration.A5min.quietsittingperiodatthebeginningoftheassessmentwasusedtocollectbaselineHRV.ThanHRVwasmeasuredduringperformanceoftheTheReadingtheMindintheEyesTest(RMET),whichassessestheaffectivecomponentofthetheoryofmind.BasedonourresultsitwasobservedthatthegroupofsubjectswithSCIhadaworseperformanceinthetest(p=0.001),asignificantlylowerlevelofsecurityonresponsescomparedwiththegroupofhealthypeople(p=0.002),lowerHRVatrest(p=0.005),andasmallerincrease in theHRVduring the task relative to thebaseline condition (0.007). These results suggest that therebealterationsinsocialcognition, insubjectswithSCI,diagnosedwithparaplegia,whowerepursuingaperiodofadaptationandsocio-laborintegration.Ourresultsalsoconfirmapositivecorrelationbetweenlimitationsinautonomicflexibilityandworseperformanceinsocialcognitiontasks.

290

90)Dopamine D4 receptor of the prelimbic cortex is important to the expression of innate fears in rat

Vergara, M1.,Gysling,Katia2.,Fuentealba,José3.,1BiologíaCelularyMolecular,CienciasBiológicas,PontificiaUniversidadCatólicaDeChile.2BiologíaCelularyMolecular,BiologíaCelularyMolecular,PontificiaUniversidadCatólicaDeChile,BiologíaCelularyMolecular,CienciasBiológicas.3QuimicayFarmacia,QuimicayFarmacia,PontificiaUniversidadCatólicaDeChile.

Theaversivememoriesareatypeofmemoryveryimportantinthegenerationofappropriatesbehaviorsandthedecisionmakingofanindividual.Dopamineplaysakeyroleintheregulationofaversivememoriesinthemedialprefrontalcortex(mPFC).Thefacingofinnateaversivestimuli(footshockorpredatorodor)increasesdopaminereleaseinPFC.Thepossiblemediatorinthisresponse is thedopamineD4 receptor (D4R),which ishighlyexpressed in themedialprefrontal cortexandseveralevidencesuggeststhatthisreceptorwouldbethemoleculartargetbywhichdopamineexertsitsactionsrelatedtoaversivememories.InthepresentstudywecharacterizetheroleoftheD4Rintheprelimbiccortex(areaofthemPFCrelatedwhittheexpressionofemotionalmemories).Forthispropose,weperformabilateralinfusionintheprelimbiccortexofL-745,870,aselectiveantagonistoftheD4R,usingthecatodorparadigm.TheinfusionofL-745,870significantlydecreasedtheinnatefearbehaviors(numberandtimethatratsexpressedfreezingbehavior),associatedtothepresenceofcatodor.Also,CamKIIshows,inpreliminarydata,anincreaseinphosphorylationassociatedwiththeinfusionoftheL-745,870.

OurdatashowthatD4RinprelimbicareaofthemPFCplaysaroleintheexpressionofinnatefearbehavior.

291

91)Visual sensory response is differentially affected by the representational format of self-generated thoughts

Villena-González, Mario1.,López,Vladimir1.,Rodríguez,Eugenio1.,1Psicología,CienciasSociales,PontificiaUniversidadCatólicaDeChile.(SponsoredbyThisProjectWasSupportedByAPhDFellowshipFromCONICYT-PCHA/DoctoradoNacional/2014-21140290)

Mindwanderinghasbeenstudiedmostlyasaunitary“offtask”attentionalstate,givinglittleimportancetoitsphenomenologicalcontent.Nevertheless,differencesinrepresentationalformatofthoughts,suchasvisualimageryorinnerspeech,mightaffectthesensoryprocessingofexternalstimuli.Werecordedthebrainactivityof20participants(12women)whiletheywereexposedtoaprobevisualstimulusinthreedifferentconditions:executingataskonthevisualprobe,generatinginnerspeech,andperformingvisualimagery.Event-relatedpotentialsresultsshowedthattheamplitudeofearlyP1/N1,relatedwithsensoryresponse,wassignificantlyattenuatedduringthevisualimagerycondition.Additionally,spectralanalysesshowedthatalpha’spowerinvisualareaswas higherwhen participants engaged in visual imagery than in the other two conditions. Furthermore, an N400-likenegativity,usuallyrelatedwithlanguageprocessing,wasclearlylargerduringtheinnerspeechcondition.Ourresultsshow,forthefirsttimetoourknowledge,thatcorticalresourcesallocationtoexternalstimuliduringself-generatedthoughtsisdifferentiallyaffectedbytherepresentationalformatoftheongoingtrainofthoughts.

292

92)Do I switch tasks better when I feel well?

Vasquez-Rosati,Alejandra1.,López,Vladimir1.,Cosmelli,Diego1.,1EscueladePsicologíaPontificiaUniversidadCatólicaDeChile.(SponsoredbyCONICYT21120514)

Imagineascenariowhereyouarewritingamailandassomeonecomesintotheofficeandstartstalkingtoyou,orthephonebeginstoring.Youwouldhavetostopwhatyouaredoingandstartrespondingtothechangingdemands,switchingbetweendifferentstimuli,operationsandmental sets.Thisability is knownascognitiveflexibility.Now,add to this scenarioa strongemotionalatmosphere that invades you.How youwould behave? Recent findings suggest that emotional states domodulate cognitiveflexibility,butthesefindingsarestillcontroversial.Moreover,thereisnoevidenceoftheunderlyingbrainprocesses.ThepurposeofthepresentstudywasthereforetoexaminesuchinteractionwhilemonitoringchangesinongoingcorticalactivityusingEEG.Wehypothesizedthatpositiveemotionalstatesthatpromoteageneralfeelingofopenness(openstance)wouldfacilitatecognitiveflexibility.Conversely,negativeemotionalstatesthatpromoteageneralfeelingofretreatinginwardly(closedstance)wouldhinderit.Sucheffectsshouldbedetectedasachangeinswitchcostasmeasuredbyreactiontime(RT)anderrorrates,whencomparingrepetitionversusswitchtrials.Inordertoanswerthisquestion,weusedtwomusicalstimulitoinduceemotionalstates(positive/higharousal/openstanceandnegative/higharousal/closedstance).Fourteenparticipantsperformedfirst2blocksoftheMadridCardSortingTask(MCST)inaneutralsilenceconditionandthen4blockswhilelisteningtothecounterbalancedmusicalstimuli.Ourresultsshow,first,atrainingeffectthatisobservablealreadyduringthesilentcondition.Switchcostsofthesecondblockweresmallerthanthoseofthefirstblock.Second,inthefirstblockofemotionalconditions,wefoundthatcomparedtothepositivestimuli,negativestimulidecreaseRTanderrorsforthefirstshiftsignal.OurdatashowsalsothatthevalenceofthefirstemotionalblockisdeterminantintheRTsofthesubsequentblocks.Whenthestimulusvalenceofthefirstblockisnegative,theerrorrateincreases in thesubsequentblocksascomparedto thepositivestimuli.Our resultssuggest that thefirst impressionmadebynegativeemotionshelpsparticipantsfocusonthetaskinitially.However,inthelongterm,itpromotesimpulsivitywhencomparedwithpositiveemotions.Thisfindingsshowthattheinteractionbetweenemotionandcognitiveflexibilityismorecomplexthanpreviouslythoughtandpointsanewwayofunderstandingtheseprocesses.

293

93)Effect of color on long term memory for faces.

Osorio, Marcela1.,Yañez,Rodrigo2.,Toledo,Pedro3.,Rodríguez,Eugenio1.,1EscueladePsicología,FacultaddeCienciasSociales,PontificiaUniversidadCatólicaDeChile.2InstitutodeEconomía,FacultaddeCienciasEconómicasyAdministrativas,PontificiaUniversidadCatólicaDeChile.3DepartamentodeElectrónicaUniversidadTécnicaFedericoSantaMaría.(SponsoredbyAcknowledgementsToVicerrectoríaDeInvestigaciónPontificiaUniversidadCatólica,ConcursoDeInvestigaciónPregradoInvierno2015.)

Memoryisacognitivefunctioninvolvingtheencodingofnewinformation,whichismodulatedbyvariousfactorsinfluencingtheprocessofencodingandsubsequentrecallofinformation.Weareconductinganonlineexperimentoffacialrecognitiontoassesstheeffectofcolorfiltersintheencodingprocess.Usingfacialrecognitionallowsustocontrolformostoftheconfoundingfactorsoftherecognitiontask,suchasown-agebias(OAB),distinctivenessandhometowneffect,lettingustoisolatetheeffectofthecolorintheencodingprocess.Theexperimentisdividedintwoconsecutivedaysessionsof4minutes.Duringthefirstsession,thesubjectsareexposedto36imagesoffacesfor5secondseachone,whichareseparatedbyablacktransitionof1second.Alltheimageshavebeenpreviouslyeditedingrayscale,butonethirdofthemispresentedwitharedfilter,onethirdispresentedwithbluefilter,andtheremainingthirdispresentedwithoutcolorfilter.Thenextday,inasecondsession,wepresent36images,allingrayscale.Onethirdoftheimageswerepresentedthefirstday,meanwhiletheremainingtwothirdsarenewimages.Thetaskconsistsinrecognizethefacesthatwereeffectivelypresentedthefirstsession.Wewillanalyzetheeffectofcolorfiltersintherecognitionoffacesafterasecondpresentationofthepicturesingrayscaleusingthehitandfalsealarmdifferenceinmeanstestforthedifferentfilters,andbymultiplebinomialregressions.Weexpectthattheresultswillrevealanimprovementtendencytotherecognitionoffacesinitiallypresentedwiththeredfilter.

294

94)Chronic stress impairs decision-making and attention in adolescent rats

Ovando, Marcela1.,Dagnino-Subiabre,Alexies1.,1LaboratoryofBehavioralNeurobiology,CenterforNeurobiologyandBrainPlasticity,InstituteofPhysiology,FacultaddeCiencias,UniversidadDeValparaíso.(SponsoredbyThisWorkWasFundedByFONDECYTGrantN°1141276ToAD-S.Labsite:Www.stress.cl)

Medialprefrontalcortex(mPFC)regulatesdecision-makingandattention.Thisbrainareaishighlysensitivetochronicstressduringadolescence. Therefore, the aimof this studywas to determinewhether chronic stress affects decision-making and auditoryattentioninadolescentrats.Bothcognitivefunctionswerequantifiedbytwo-alternativechoicetask(2-ACT),abehavioralparadigmtostudyauditoryattentioninrats.Trainedanimals,thatreachedaperformanceover80%ofcorrecttrials inthe2-ACTduringadelescence,wererandomlyassignedtocontrolandrestraintstressexperimentalgroups.Toanalyzetheeffectsofrestraintstress(7days/3hrs)ondecisión-makingandauditoryattention,trainedratsofbothgroupsweresubjectedto502-ACTtrialsonedaybeforeandonedayafterof thestressperiod.Adifferentscorewasdeterminedbysubstractingtimeof intertrial interval (ITI)andthenumberofcorrecttrials(CT)afterfromthosebeforethestressprotocol.Locomotoractivity(openfield,OF)andanxiety(elevatedplusmaze,EPM)wereevaluatedinallanimals.Chronicstressdidnotaffectlocomotoractivity,whilethenumberofCTinthe2-ACTwaslowerinthestressedratsthanthatofcontrolanimals.Ontheotherhand,ITIandanxietywerehigherinthestressedratscomparedtotheperformanceofcontrolanimals.Ourresultssuggestthatothercognitivefunctionsregulatedbythelimbicsystem,suchascognitiveflexibility,couldbeaffectedbythestress-inducedimpairmentondecision-makingandauditoryattention.

295

95)Changes on growh hormone expression in gills, liver, kidney, intestine and pituitary during smoltification in Salmo salar

Morera, FJ1.,Vargas-Lagos,C2.,Pontigo,JP2.,Oyarzun,R2.,Yañez,A3.,Vargas-Chacoff,L2.,1InstitutodeFarmacologia,CienciasVeterinarias,UniversidadAustralDeChile.2InstitutodeCienciasMarinasyLimnologicas,Ciencias,UniversidadAustralDeChile.3InstitutodeBioquimicayMicrobiologia,Ciencias,UniversidadAustralDeChile.(SponsoredbyFundingByINNOVA-Corfo13IDL2-23565(toLVC,FJMAndAY);Fondap-Incar,No.15110027(toAY),FONDECYTDeINICIACION11130308(toFJM)AndDID-UACH(toFJM))

ThesmoltificationisacriticalpointintheSalmonAquaculture.Duringthisprocess,alsocalledparr-smolttransformation,fishsuffermanychangesunderendocrinecontrol.Growhhormone(GH)isahormoneconsideredofthesamefamilyofProlactin(PRL)andSomatolactin(SL),becauseoftheirstructuralsimilarities.TheaimofthisworkistodescribetheGrowhhormoneexpressionduringtheprocessofsmoltification(inducedbyphotoperiod).Wetooksamplesoftissuesasgills,kidney, liver, intestine(3portions:anterior,mid andposterior) andpituitaryofSalmo salar in 2points of smoltificationprocess: 1) parr, 2) smolt.WeanalysedtheexpressionofGHgenebyRT-Q-PCR.Ingills,kidney,posteriorintestineandpituitarythegeneexpressionofGHsignificantlyincreasedduringsmoltificationprocess,whileanteriorandmidintestinedonotpresentedstatisticalchangesduringthisprocess.WefoundthatthelevelsofGHincreasedduringsmoltificationprocessinseveraltissue,althoughtheintestineintwoportiondonotpresentedchanges,beingtheposteriorportionmoreimportantinthisprocess.ThesedatasuggestthatGHplaysarelevantroleintheosmoregulatorytissuesduringthesmoltificationprocessinS. salar,butfurtherfunctionalstudiesarenecessary.

296

96)Regulation of the expression of the (pro)renin receptor by angiotensin II in renal collecting duct cells.

Reyes-Martínez, Cristian1.,Salinas-Parra,Nicolás1.,Gonzalez,AlexisA1.,1InstitutodeQuímica,FacultaddeCiencias,PontificiaUniversidadCatólicaDeValparaíso.(SponsoredbyFONDECYT11121217)

The(pro)reninreceptor(PRR)isanATPaseH+-transportinglysosomalaccessoryprotein(ATP6AP2)thatalsoactsasareceptorofinactiveproreninandreninactivatingproreninandincreasingreninactivity.Besides,itisinvolvedinaseriesofdifferentprocesses,suchasWnt-signalingandpHhomeostasis,itsactivationhasbeenrelatedtorenalinjuryinhypertensionanddiabetes.Bindingofproreninand renin to thePRRactivatesmitogen-activatedproteinkinases (MAPK)and induces transforminggrowth factor(TGF-β).IthasbeensuggestedthattheexpressionofrenalPRRisupregulatedbycGMP-PKGsignalingpathwayunderlow-sodiumconditions in vivo. Wehave shown that PRR is expressed in collectingduct (CD) cell lineM-1 and that angiotensin II (Ang II)increasestheexpressionofPRRinthiscells.However,themolecularmechanismbywhichAngIImodulatestheexpressionofPRRinCDcellsremainsunknown.WehypothesizedthatAngII-mediatedincreaseinPRRismediatedbytheactivationofproteinkinaseA(PKA)and/orproteinkinaseC(PKC).Inordertoproveit,wetreatedCDcellswithAngIIplusPKAorPKCinhibitionusingH89andcalphostinC,respectively.OurresultsshowedthattheincreaseinPRRproteinlevelsmediatedbyAngIIarepreventedbyPKCandH89independently.Furthermore,forskolin(cAMPenhancer)augmentsPRRhoweverPKCinhibitionpreventedthiseffectinforskolintreatedcells.OurresultssuggestthattheactionofAngIIismodulatedbyPKAandPKC.

297

97)Activation of E-Prostanoid Receptor EP1 and EP4 regulates renin expression in renal collecting duct cells

Salinas-Parra, Nicolás1.,Gonzalez-Vergara,Alex1.,Figueroa,Steffany1.,Gonzalez,AlexisA1.,1InstitutodeQuimica,FacultaddeCiencias,PontificiaUniversidadCatólicaDeValparaíso.(SponsoredbyFONDECYT11121217)

ProstaglandinE2isthemajormetaboliteproducedbycyclooxygenase-2inthekidneyandexertsitseffectsviaGprotein-coupledreceptors EP1 and EP4 in the renal collecting duct (CD) cellswhere it has been demonstrated all components of the renin-angiotensinaxisareexpressed.InthisstudyweevaluatetheeffectoftheactivationofprostaglandinE2receptorEP1andEP4onreninexpressionandmolecularpathwaysinvolvedusingaCDcelllineM-1.OurresultsdemonstratethattheEP1receptorisfoundingreaterextentinM-1cells,whileEP4islessabundant(0.667arbitraryunits(AU)vs.0.304AU).AdoseresponsecurvewithdifferentconcentrationsofprostaglandinE2,showedanincreaseintheexpressionofrenininthenanomolarrange(132±43%overthecontrol).EP1isaproteinkinaseCcoupledreceptorandEP4iscoupledtoGsprotein,bothreceptorsmayactivatePKCand/orPKA.ToestablishtheroleofPKAactivationbycyclicAMP(cAMP)anditspossibledependenceofPKC,weincreasedcAMPlevelswithForskolin(10-6Molar)inthepresenceorabsenceofCalphostinC(10-7Molar),aPKCinhibitor.TheWesternblotanalysisshowedthatforskolinincreasestheexpressionofrenin(135±18%comparetocontrol,P<0,05)andprorenin(136±22%comparetocontrol,P<0.05)proteinlevelswhileforskolinplusPKCinhibitionsuppressesthiseffect.TheseresultssuggestthatincreasedreninandproreninproteinlevelsmediatedbytheactivationofcAMP/PKApathwayisdependentonPKCactivity.

298

98)Altered cortical actin polymerization in dysferlin-deficient skeletal myocytes

Báez, Ximena1.,Gonzalez,Arlek1.,Cea,Luis2.,Bevilacqua,JorgeA3.,Mouly,Vincent4.,Caviedes,Pablo5.,Cárdenas,AnaMaría1.,1CentroInterdisciplinariodeNeurocienciasUniversidadDeValparaíso.2ProgramadeAnatomíayBiologíadelDesarrollo,InstitutodeCienciasBiomédicasUniversidadDeChile.3ProgramadeAnatomíayBiologíadelDesarrollo,InstitutodeCienciasBiomédicas.UniversidadDeChile.4UM76-UPMCUniv.Paris6/U974-Inserm/UMR7247,blddel´Hôpital-G.H.Pitié-SalpétrièreParisUniversidadParis.5ProgramadeFarmacologíaMolecularyClínica,ICBM.UniversidadDeChile.(SponsoredbyThisWorkHasBeenSupportedByGrantsAnilloACT-1121.)

Dysferlinopathiesareagroupofmusculardystrophiescausedbymutationsaffectingtheexpressionofdysferlin(Dysf),aproteinhighlyexpressedinskeletalmuscleandessentialforsarcolemmarepair.Reportedly,animalmodelsofdysferlinopathiesdisplayaderegulatedexpressionofproteinsinvolvedinactincytoskeletondynamics.WethereforehypothesizethatDysfparticipatesintheremodelingoftheactinnetwork.Toaddressthispossibility,weevaluatedwhetherdysferlinopathy-associatedmutationsmodifyactinremodelinginmusclecells.Withthisaim,weusedmyoblastsofcelllinesderivedfromskeletalmusclesofpatientsharboringDysfmutations (ER,AB320,107and379cells).Aspreviously reported,all thesemutations severely reduce theexpressionofdysferlin.Inparallel,weusedRCMHmyoblasts,acelllinederivedfromskeletalmuscleofanormalhumanpatientasacontrol.Actinpolymerizationwasdeterminedbypermeabilizingcellswithdigitonininthepresenceof2mMATP-Mg2+,10mMfreeCa2+

and0.3mMAlexaFluor488G-actinconjugate.Sinceonlyactinpolymersexhibitfluorescence,thefluorescenceintensityreflectstheformationofnewactinfilaments.TheexpressionandoverallorganizationofF-actinwasevaluatedbystainingthecellswithphalloidin rhodamine-B.We found, compared to controlRCMHcells, thatdysferlin-deficientmyoblasts (ER,AB320,107, 379)displayasignificantlyreducedF-actinpolymerizationandexpression.TofurtherevaluatetheroleofDysfinF-actinpolymerization,weexpressedadysferlin-Venusconstructinalldysferlin-deficientcelllines.Wefoundthattheexpressionofthisconstructrestoresactinpolymerizationineitherthemutantcelllines.Altogether,theseresultssuggestthatDysfisinvolvedinF-actinpolymerizationinskeletalmyoblasts,andthatthisprocessisimpairedindysferlinopahies.

299

99)Effect of metformin during gestation in obese rats on reproductive and metabolic parameters in offspring

Olguín, Sofía1.,Alvarez,Daniela1.,Ceballo,Karina1.,Cerda,Tania1.,Cruz,Gonzalo1.,1LaboratoriodeAlteracionesReproductivasyMetabólicas,InstitutodeFisiología,FacultaddeCiencias,UniversidadDeValparaíso.(SponsoredbyFONDECYTINICIACION11130707(GC)AndCentroDeNeurobiologíaYPlasticidadCerebral(CNPC)OfUniversidadDeValparaíso.)

Obesityepidemicisoneofthemajorconcernsintheworld.AmongOECDcountries,Chileisthefourthcountrywithhigherlevelsofobesity.Regardingsexdistribution,obesity ismoreprevalent inwomen(31%)than inmen(19%). In fact,50%ofpregnantwomeninChilehaveeitheroverweightorobesity.Theseconditionsleadtodifferentabnormalitiesinpregnancyanddelivery.Inaddition,recentstudiesshowthattheoffspringofobesemothershasanincreasedprobabilitytosuffercardiovascular,metabolicandreproductivediseases.Ourgroupandothersdemonstratedthatexposuretoahighfatdietisrelatedtoobesity,increasedliverweight,advancedpubertyandincreasedestradiollevelsintheprogeny.Weaimedtodetermineifmetforminpreventsthisdevelopmentalreprogrammingproducedbyahighfatdietexposure.SpragueDawleyratsweredistributedin3groups:ControlDiet(13%Kcalinfat);HighFatDiet(HF)(60%Kcalinfat,ResearchDiet,USA)andHF+Metformin(60%Kcalinfat,ResearchDiet,USA+metformin150-200mg/Kgintapwater).Dietwasadministeredfor1monthprevioustopregnancy,duringpregnancyandnursing.Metforminwasadministeredfrom1weekprevioustopregnancyuntilweaningoftheoffspring.Metformindidnotaffecttheweightgainduringpregnancyandfailinpreventincreasedweightinoffspringofobesemothers.Regardinghepaticweight,maternalobesityincreaseshepaticweightintheoffspring,butmetformindonotpreventthisincrease.Atpostnatalday14thereisanincreaseinestradiolinoffspringofobesemothers,butmetformindoesnotpreventthisincrease.Inconclusion,thedoseofmetforminisinsufficienttopreventalterationstriggeredbymaternalobesityontheoffspring.

300

100)Angiotensin-(1-7) prevents skeletal muscle wasting induced by lipopolysaccharide decreasing proteasomal degradation and autophagy

Rivera, Juan Carlos1,2.,Morales,MaríaGabriela1,2.,Abrigo,Johanna1,2.,Simon,Felipe3,4.,Cabello-Verrugio,Claudio1,2.,1LaboratoriodeBiologiayFisiopatologíaMolecular,Depto.CienciasBiológicas,FacultaddeCienciasbiológicas,UniversidadAndrésBello.2LaboratoriodeBiologiayFisiopatologíaMolecularMillenniumInstituteonImmunologyandImmunotherapy.3LaboratoriodeFisiologíaIntegrativa,Depto.CienciasBiológicas,FacultaddeCienciasbiológicas,UniversidadAndrésBello.4LaboratoriodeFisiologíaIntegrativaMillenniumInstituteonImmunologyandImmunotherapy.(SponsoredbyFunding:Association-FrancaiseContreLesMyopathiesAFM#16670;FONDECYT#1120380,1121078;IMII#P09-016-F;UNABDI-741-15/N.)

Theskeletalmuscleatrophycanbeinducedbylipopolysaccharide(LPS),causingafastandseverelossofmassandmusclestrength.Amongthemechanismsinvolvedistheproteasomaldegradationofmyofibrillarproteinsandautophagy.Angiotensin-(1-7)[Ang-(1-7)],apeptideofthenon-classicalaxisofReninAngiotensinSystem,hasbeneficialeffectsinskeletalmuscleviaitsreceptorMas.WeevaluatedtheeffectofAng-(1-7),andthemechanismsinvolved,onmusclewastinginducedbyLPS.CultureofC2C12myotubesorC57BL/10JmicewereexposedtoLPSinabsenceorpresenceofAng-(1-7).Musclestrength,fiberdiameter,myosinlevelsandmarkersofproteosomalsystem(atrogin-1andMuRF-1)andautophagy(LC3II,Bnip,BnipL,GABARAPandAtg7)weredetermined.OurresultsshownthatAng-(1-7)recoversthedecreasedmusclestrength,fiberdiameterandMHClevels intibialisanteriorofmice. Inaddition,weobservedthatAng-(1-7)preventstheincrementofatrogin-1,MuRF-1,LC3IIandBnipL inducedbyLPS inmice.StudiesinvitrousingC2C12cells,shownsimilareffectsofAng-(1-7)potentiatingtheobservationthatitisapeptidewithanti-atrophicpropertiesinskeletalmuscle.ThemechanismthroughAng-(1-7)producesitanti-atrophiceffectinvolvestheinhibitionofautophagyandproteasomaldegradationofmyofibrillarproteins.

301

101)P2X receptor evolution: sequence and structural comparisons from unicellular green algae to the human receptor subtypes.

Latapiat, Verónica1.,Huidobro-Toro,JPablo1.,1DepartamentodeBiología,FacultaddeQuímicayBiología,UniversidaddeSantiagodeChile.(SponsoredbyFundedFondecyt1141132,PFB0807.)

P2XreceptorsaretrimericATP-activatedionchannelspermeabletoNa+,K+andCa2+;thesereceptorsareinvolvedinmultiplephysiologicalresponses.ThesevenP2Xreceptor(P2XR)cloneshave40–50%identicalaminoacidsequencebetweenthem,withasimilarvariabilitywithinvertebratesubtypes.Eachsubunithastwotransmembranedomainsconnectedbyanextracellularloopof~280aminoacids.TheonlycrystallographicstructureavailableforP2XRisthezebrafishP2X4R(zfP2X4R);theevolutionarytreeofthisfamilyofreceptorsisunkown.TheunicellulargreenalgaOstreococcus tauriisthesmallestfree-livingeukaryotedescribed;aP2XR(otP2XR)wasidentifiedinthisalgawhichisover1,000millionyearsold.Therefore,thequestionarrisesastotheevolutionandputativephylogenyoftheP2XRfamily,whichisnowaddressed.ToexamineanevolutionarypatternofP2XRinthetreeoflife,weusedbioinformaticstoolstodeterminestructuraldifferencesbetweenP2XRinseveralorganismsfromunicelularalgaetomulticelularred,greenandbrownalgaeandhumans.WealsousedmolecularmodelingbasedonthezfP2X4Rtemplate(PDB:4DW1),togeneratetheotP2XRtridimensionalstructure.Moreover,moleculardynamicssimulationtechniqueswereappliedtoestablishanevolutiveanalysisoftheseP2XRs.Blastalignmentprogram(tblastn)wereusedtoidentifypossibleP2XsequencesinChlamydomonas andUlva compressagenomes,aswellasotherrepresentativeredandbrownalgae,usingallP2XRsequencespresentsinNCBIdatabase.ComparativehomologymodellingandmultiplesequencealigmentshowedthattheotP2XRcontains4/5conserveddisulfidebondscharacteristicofvertebrateP2XR; it lacksC217,C227of thedorsalfin.Key functional residues,suchasconservedK68andK309arelikewiseconserved,summinga45%sequenceidentitywithzfP2XR4.SimilaranalysiswereundertakentostudytheconservedhomologyoftheotP2XRwithmulticelularalgaeP2XRs.PreliminarydataisconsistedwithP2X-likeaminoacidicsequencesinthegenomeof Chlamydomonas,aswellasmulticellularalgae;furtheranalysishavebeenundertakentoprecisethevalidityofthesequencehomologiesfound. ThesequenceidentityofOtP2XRandzfP2X4Rallowsproposingconvergenceevolutionarymechanism.TheconservedstructuralandfunctionalcharacteristicsofP2X-likereceptors indifferentgenomeswill illustrateevolutionary relationships amongorganisms through thephylogenetic tree. This studyalsooffersnewinsightsintothemolecularpharmacologyofP2XRs;futurein-silicodockingassayswillassesstheATPorthostericsiteandcomparethepharmacophore.

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102)Mechanisms controlling Nur77 expression and activity: implications in neuronal plasticity

Olivares, Montserrat1.,Parada,Guillermo1.,Andrés,MaríaEstela1.,1BiologíaCelularyMolecular,CienciasBiológicas,PontificiaUniversidadCatólicaDeChile.(SponsoredbyFondecytProjectN°1150200)

Nur77isatranscriptionfactorandorphanmemberofthenuclearreceptorsuperfamilyencodedbyanearlygene.Nur77expressionin brain is regulated by dopamine and glutamate in nuclei of themotivated circuit. In the striatum, nucleusAccumbens andprefrontalcortex,Nur77expressionisinducedbystressandexposuretopsychostimulants.EventhoughNur77hasbeenhighlyimplicated inpathologicalprocessesasaddictionandParkinsondisease, its role in thebrainor themechanismsregulating itsexpressionhavenotbeenelucidated.Here,weusedbioinformatics,chromatinimmunoprecipitation(ChIP)andreporterassaystolearnaboutmechanismscontrollingNur77expression.TheanalysisofavailablebioinformaticsdatashowedthatNur77bindingsitesarepresentinstrongenhancersandactivepromoters.Inpromoters,Nur77bindingisabundantneartoTSS(±500bp)andthemajorityofNur77bindingsiteslackofNBREelements.Interestingly,thedatashowedthatNur77targetsseveralgenesofneuronalplasticity.Recently,itwasshownthatlysine-specifichistonedemethylase1(LSD1,alsoknownasKDM1a)playsasignificantroleinplasticityandneuronalexcitability.Here,weshowevidencesuggestingthatLSD1regulatesNur77expression.Reportergeneassays,surprisinglyandcontrarytowhatwasexpected,showedthatLSD1andtheneuronalvariantLSD1-8aincreaseluciferasereporteractivitydrivenbythehumanNur77promoter. Inaddition,preliminarydatafromChIPassayscarriedoutwithmousestriatumchromatin,showedthatLSD1ispresentinNur77promoter.AresultsupportedbybioinformaticsanalysesofreportedChIPdataassaysshowingthatthehumanNur77promoterbindsHDAC2andCoREST1,bothpartnersofLSD1.Takentogether,theevidencesuggeststhatLSD1maybeinvolvedinmodulatingplasticgenesthroughregulatingtheexpressionofNur77

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103)Unravelling the coordination of zinc in ionotropic receptors: from structural to allosteric modulator sites.

Peralta, Francisco1.,Huidobro-Toro,Juan1.,1DepartamentodeBiología,FacultaddeQuímicayBiología,UniversidadDeSantiagoDeChile.(SponsoredbyFundedByFondecytGrant114-1132,CONICYTBasalGrant087(CEDENNA)AndCONICYT21140407DoctoralFellowship.)

Zincisatransitionmetalselectivelystoredinsynapticvesiclestogetherwithneurotransmitters,fromwhereitisco-released.Theextracellularsynapticspacecontainsvariableamountsofthemetal,whichmayreachmicromolarconcentrations.Zinccoordinateswith structuralproteinmotifsas in zincfingersand/or in thecatalytic siteofenzymes.Themetal is anallosteric regulatorofalmostallionotropicreceptors,eitherasapositiveoranegativemodulator.Moreover,azinc-activatedchannelwasdescribed,anindicationthatthismetalmayalsoplayaroleasaligandactingonareceptorchannelparticularlysensitivetozinc.Wehypothesizedthatthecoordinationofthemetalisdifferentforeachofthesethreefunctions.Toassesshowzincinteractswithligand-gatedreceptors,weexaminedhowzinccoordinatestoproteins,differentiatingbetweenstructuralandcatalyticroles.Tocharacterizethezincproteincoordinationgeometriesandinvestigatethepreferredligandsinthesefunctions,wecomparedthezincligandsfoundinproteinsaccomplishingthethreeidentifiedfunctions.Tothisaim,wesearchedinthedatabankofproteinscrystallizedwithzincandinpapersdescribingzinccoordinationmotifs.Basedonthisdoublescrutiny,wediscoveredthatthezinccoordinationtoionotropicreceptorsissimilartothatfoundinenzymescatalyticmotifsbutdifferenttothezincmotifsofstructuralproteins.Oursearchconsistentlyshowedthatinthecoordinationofstructuralproteins,thefrequencyofCysasametalligandwas75%,His(20%)andAsp/Glu(3%).Incontrast,incatalyticorallostericproteins,themostfrequentligandforzincwasHis(46and66%,respectively),followedbycarboxylicacid(35and18%,respectively)withCysbeingtheleastcommonligand(20and6%).Wealsoshowedthatthezinccoordinationligandsforpositiveandnegativeallostericmodulatorsaredifferent;whereasinthepositivemodulatorsiteswefound11%ofCys,nonewasfoundinthenegativemodulators(0%).Moreover,thepresenceofLysandGlnwasfound4-timesmorefrequentlyinthenegativemodulatorscomparedtothepositivemodulators(19versus5%).Finally,wecomparedthezinccoordinatingspheresinP2X4andP2X2receptors,tworeceptorspositivelymodulatedbyzinc)andconcludethatthesereceptorshavetwodifferentzincallostericmodulationpocketsinrelativecloseregionsoftheextracellulardomain,consistentwith thefindingsatotherpositivemodulatorsites.This studyprovidesnovel structuraldeterminantsof zincmetalcoordinationinallostericregulatorymechanismsofreceptorchannels.

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104)The expression of scFv anti-LDL oxidized is affected negatively for low temperatures in cultures of yeast Pichia pastoris.

Zepeda, A1,2.,Figueroa,C1,2.,Figueroa,E1.,Pessoa,A2.,Farías,J1.,1DepartamentodeIngenieríaQuímica,FacultaddeIngenieríayCiencias,UniversidadDeLaFrontera.2DepartamentodeCienciasBioquímicasyFarmacéuticas,FacultaddeCienciasFarmacéuticas,UniversidadedeSaoPaulo.(SponsoredbyAcknowledgments:ScholarshipsForPhDInChile,CONICYT.CoordinationOfSuperiorLevelStaffImprovement(CAPES,Brazil),NationalCouncilForScientificAndTechnologicalDevelopment(CNPq,Brazil)YSãoPauloResearchFoundation(FAPESP,Brazil).)

Modified forms of low density lipoprotein (LDL) are important to the formation of foam cells andmediators involved in theprogression of atherosclerosis immuno-inflammatory process. Recombinant antibody fragments (scFv) anti-LDL oxidized areapromisingbiotechnologyproduct for theprophylaxis, treatmentanddiagnosisof cardiovasculardiseases. Inmethylotrophicyeasts,whichareabletobeusedassolecarbonsourceasmethanol,Pichia pastorisisanexcellentmodelfortheexpressionofheterologousproteins,largelybecausethisorganismcarriesoutpost-translationalmodifications,suchasglycosylation,verysimilartohuman,whichisreflectedinthelargenumberofproteinsthathavebeenobtainedinthebiologicalmodel.Forthisreason,theaimofthisstudyistodeterminethemostoptimalgrowingconditionforP. pastorisrecombinantallowstoobtainglycosylatedandhighconcentrationofrecombinantprotein.Toconductthestudy,weevaluatedthreecriticalfactorsfortemperature(14ºC,18ºC,22ºC)andmethanolconcentration(1%,1.5%,2%).Thepurificationoftherecombinantproteinwasperformedusingionicchromatography, thequantificationwasusing theBradfordAssayand toanalyze thepurityand formsobtainedwasused thepolyacrylamidegelelectrophoresis.Theresultsshowthatafter72hofinductionwithmethanoltheculturesto18°C+1,5%Mand22°C+2%Mexhibitthemosthighestconcentrationwithrespecttheotherscultureswithamediaof27,25mg/Land23,39mg/L,respectively.Subsequentinthisperiodisobservedthattheonlyonesculturesthatproduce100%ofglycosylatedproteinaretheculturesperformedto22°C,independentlyofthemethanolconcentration.Inconclusion,themostfavorablecultureconditionstoproduceanoptimalantibodyfragmentarethosewithatemperatureof22°C,butthemoreeconomicallyprofitableproductionisa22°C+2%.

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105)Transforming growth factor-β1 increases Cdk5 activity and TRPV1-dependent Ca2+ influx in trigeminal neurons

Lazcano,Pablo2.,Wilson,Carlos1.,Gonzalez-Billault,Christian1.,Utreras, Elias2.,1LaboratoryofCellularandNeuronalDynamic,DepartmentofBiology,FacultyofScience,UniversidaddeChile.2LaboratoryofCellularandMolecularMechanismsofPain,DepartmentofBiology,FacultyofScience,UniversidaddeChile.(SponsoredbySupportedByGrantsACT1114AndFondecyt1140325ToC.G.-BAndFondecyt11110136,1151043AndPAI-79100009ToE.U.)

Inflammationispositivelycorrelatedwithseveralmolecularmechanismsinvolvedinorofacialpain.IncreasedlevelsofTransforminggrowthfactor-β1(TGF-β1)havebeenreportedinfluidsofmigrainepatients,suggestingitsroleduringorofacialpain.WereportedearlierthatCyclin-dependentkinase5(Cdk5)playsacrucialroleinpainsignaling.Cdk5isakinaseactiveinneurons,whereitsactivatorp35ispredominantlyexpressed.WealsoreportedthatperipheralinflammationpromotesCdk5activitybyenhancingtranscriptionofp35.Additionally,wedescribedthatCdk5phosphorylatestheTRPV1channelatthreonine407, increasingCa2+

influxinnociceptiveneurons.Interestingly,wedemonstratedthatTGF-β1increasesCdk5activityinDRGneuronsandodontoblast-likecells.However,thisregulationhasnotbeenstudiedinthetrigeminalganglion(TG).Consideringthisdata,weevaluatedthecontributionofTGF-β1intheregulationofCdk5activitywhichinturnaffectsTRPV1phosphorylationandfunctioninanexvivomodelofTGaswellasculturedTGneuronsfrommice.Totestourhypothesis,weinducedalocalinflammationinthemousevibrissaepadthroughinjectionofl-carrageenanduring3handthenweevaluatedTGF-β1levels,andactivationofcanonicalpathwayandnon-canonicalpathways(phospho-Smad2andphospho-ERK1/2,respectively).Moreover,weevaluatedimmunodetectionofCdk5,p35andEgr1(transcriptionfactorforp35expression),andphospho-T407-TRPV1inmouseTGafterinflammation.Alternatively,culturedTGneuronsweretreatedwithrecombinantTGF-β1for24hinabsenceorpresenceofSB431542(theinhibitorofTGF-β1type1receptor),toevaluatetheexpressionofthealreadymentionedproteins.OurresultsshowedthatorofacialinflammationincreasedTGF-β1 levelsandactivated itssignalingpathways locally inTG. Inaddition, inflammationincreasedCdk5,p35,Egr1inmmunodetection aswell as phospho-T407-TRPV1 in TG compared to controlmice. Remarkably, the treatmentwith TGF-β1increasedbothp35andphospho-T407-TRPV1 inculturedTGneurons,whichwasblockedbySB431542pre-treatment.Finally,TGF-β1treatmentincreasedCa2+influxmediatedbyTRPV1,suggestingafunctionalregulationofthechannelinaninflammatorycontext.Takentogether,ourresultsshownthatTGF-β1increasestheexpressionofp35andCdk5activityduringaninflammatoryprocess,affectingthefunctionofTRPV1channel.TheseresultssuggestamolecularmechanisminvolvingTGF-β1,Cdk5andTRPV1betweeninflammationandtheincreasedpainsensationintheorofacialregion.

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106)Study of protein interaction sites of Gβγ and Glycine Receptor by peptides

Neira, Luis1.,González,Daniela1.,Nova,Daniela1.,Guzmán,José1.,1Physiology,BiologicalSciences,UniversidadDeConcepción.(SponsoredbyThisWorkHasBeenFundedByTheFONDECYTProyect1131004,IDeA-FONDEFProyectCA12I10280AndCMABío-Bío)

Oneofthemostcommonlyuseddrugsworldwideisethanol,anditsabusehasledtosocial,economicandhealthproblems.Ithasbeendeterminedthatoneofitsmainmoleculartargetsofethanolchannelsinthecentralnervoussystemareligand-gatedion,beingglycinereceptor(GlyR)oneoftheirrepresentativemembersofthisfamily.SeveralstudieshaverevealedthatethanolregulatesthefunctionofthisreceptorincreasingCl-conductance,whichcauseshyperpolarizationoftheplasmamembraneincreasingtheinhibitoryactivityofthisionchannel.IthasbeendemonstratedthatethanolcouldbeactingbyanindirectmechanisminvolvingtheβγdimeroftheGprotein,whichwouldinteractwiththeintracellulardomainofGlyRinregionswithbasicaminoacids(aa),suchaslysineandarginine.Inourlaboratory,wehavedesignedpeptidesderivedfromtheN-terminalregion(316-320aa)oftheintracellulardomainoftheGlyRcapableofinhibittheinteractionwithβγdimer.Therefore,continuingwiththislineofresearch,weperformedexperimentsusingnewpeptidesbasedonintermediateandC-terminalregionsofthecytoplasmicdomainofGlyRsuchasP6(HKDDEGG),P9(RFNFSAY),P10(FSAYGMG),P13(CLQAKDG),P24(KLFIQRA),andP28(RAKKIDK).Hence,abioinformaticstudyofprotein-peptidedockingwasdoneusingtoolslikePyMolandSchrödingertomodelandpredicttheinteractionsitesandmostfavorablecomplexesbetweenthepeptidesandGβγproteins.TheonlinesoftwareFastContactwasusedtocalculatetheenergiesofelectrostaticinteractionbetweenthesurfaceaminoacidresiduesofthedimerandthepeptides:Gβγ-P6(-25.5kcal/mol),P9(-10.9kcal/mol),P10(-8.6kcal/mol),P13(-11.8kcal/mol),P24(-19.6kcal/mol)andP28(-33.4kcal/mol).Subsequently,in vitroanalyseswerecarriedoutusingimmunocolocalizationinHEK293cellstoassesstheentranceofthepeptidesintothecellandtheir interactionwithGβγ.Colocalizationwasevaluatedbymeasuring theManders ratio (%).WefoundthatP28peptidewasoneofthemostfavorabletointeractwithGβγ(36.6±1.4%)andtheir locationswereanalyzedbythree-dimensional(3D)reconstructions.Furthermore, throughelectrophysiologicalassayswefoundthat twopeptides intracellularlyapplied (P24andP28)wereabletoreduceethanolpotentiationofGlyRα1.Using100mMethanol,P24andP28(200uM)inhibitedthepotentiationofethanolfrom43.8±5.2%to35.7±9.9%and34.2±3.9%respectively.Finally,thespecificityofthepeptideswasstudiedinthePLCβ2pathwayusingfluorescentmeasurementsandthedatademonstratedthatthesepeptideshavenoeffectinthispathway.

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107)The gasotransmitter hydrogen sulphide (H2S) modified ciliary activity in mouse tracheal epithelial cell cultures.

Ríos, M1.,Droguett,Karla1.,Althaus,Mike2.,Villalon,Manuel1.,1DepartmentofPhysiologicalScience,FacultyofBiologicalScience,PontificiaUniversidadCatólicaDeChile.2InstituteofAnimalPhysiologyJustus-LiebigUniversityofGiessen.(SponsoredbyFondecytPostdoctorado3150652)

Inairwaymucociliaryepithelium,ciliaryactivity isdeterminant for theeffectivenessofmucociliaryclearance (MCC), thebasicdefensemechanismthatcontributetoeliminatenoxiouscontaminantfromtheinhaledair.Severalchemicalandmechanicalsignalsregulateciliarybeatfrequency(CBF)thereforeaffectingMCCvelocity.ATPislocallyreleasebytheairwayepitheliumandincreasesCBFbyamechanismdependentofchangesin[Ca2+]i.Hydrogensulfide(H2S),anendogenousgas,withthecharacteristicodourofrotteneggsisarecentlyknowngasotransmitterthatinfluencesavarietyofcellularandorganfunction.Inrespiratorysystem,H2Shassignificantvasoactiveandanti-inflammatoryeffects,withraisedlevelsobservedinserumandinsputumsupernatantsofasthmaticspatients.H2SdecreasestransepithelialNa

+transport,implicatedintheregulationofmucociliaryepithelialliningfluid,howeveritisunknownwhetherH2Shasaneffectonotherciliaryepithelialfunction.Usingmousetrachealepithelialcellcultures(MTEC)westudiedtheeffectofH2Sonbasalciliaryactivity,measuringCBFbyvideomicroscopy(SissonAmmonsVideoAnalysis),eATPmeasuredbyluminometricassayusingluciferin/luciferase;[Ca2+]imeasuredusingFURA-2AMandthedyeuptakerateusingbromureethidium.Thesameparametersweremeasured incultures stimulatedbyATP in thepresenceofH2S.Weevaluatedculturesexposedtodifferentconcentrationsofsodiumhydrosulphide(NaHS),aH2S-donatingdrug.NaHSinhighconcentrations(100and300μM)increasedtransientlyCBF(20%)during5mineffectthattemporarilycorrelatewiththeincreasein[Ca2+]i.Lowconcentrations (1and10μM) reducedCBFby10-15%butnochanges in [Ca2+]iwereobserved. LowconcentrationsofNaHS(10μM)increasedtherateofdyeuptakeduring2min(0.4±0.1Vhvs0.9±0.3NaHS(n=4and5respectively)),howeverwedidnotobservechangesineATPlevelsfollowingNaHS(10μM)addition.Furthermore,NaHS(10μM)reducedtheCBFincreaseinresponsetoachemicalstimulusofATP(1μM)(62%,p<0.05),andthechangeofthedyeuptakerate(120.6±32.4ATP/Vhvs83.6±18.6ATP/H2S,n=5).TheseresultsshowedthatH2Shasadualeffectonciliaryactivity,athighconcentrations,H2SincreasedCBFand[Ca2+]iandatlowconcentration,H2SdecreasedCBFandtheincrementofCBFanddyeuptakerateinducedbyATP.ThesefindingsdemostratethatexogenousH2Saffectciliaryactivity,andprovideevidencethatthisgasotransmitterhasphysiologicalandpathophysiologicalimplicationsintheregulationofmucociliarytransportintheairways.

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108)Interaction between CRF-BP and CRF2αR increases CRF2αR in the plasma membrane

Slater, Paula1.,Cerda,Cledi1.,Andrés,María1.,Gysling,Katia1.,1DepartamentodeBiologíaCelularyMolecular,FacultaddeCienciasBiológicas,PontificiaUniversidadCatólicaDeChile.(SponsoredbyFundedByFONDECYTGrantNº1110392And1150244,MSIGrantN°P10/063-FAndCONICYTGrantForPhDThesisResearchToS.P.G.)

Thefamilyofcorticotrophinreleasingfactor(CRF)linksstressandaddiction.Intraventraltegmentalarea(VTA)injectionsofCRFaswellasstressfulstimuliinducerelapsetodrugseekingincocaine-experiencedrats,duetothepotentiationofglutamatergicsynapses onto VTA dopaminergic neurons. These potentiation observed in cocaine-experienced rats depends on CRF bindingprotein(CRF-BP)andontype-2CRFreceptor(CRF2αR).However,themolecularmechanismunderlyingthisfunctionalinteractionispresentlyunknown.WehaveobservedthatCRF-BPandCRF2αRareabletophysicallyinteract.Wehypothesizethatthisinteractionaffectsthesub-cellularlocalizationoftheproteins.Thus,westudiedthesubcellularlocalizationofCRF-BPandCRF2αRintransfectedPC12cells.Weanalyzedco-localizationofCRF-BPorCRF2αRwithdifferentorganellemarkers,usingVanSteenselco-localizationanalysis.WealsoanalyzedthepresenceofCRF2αRintheplasmamembraneasfluorescenceintensitiesofCRF2αRmembrane/total.TheresultsshowedthatCRF-BPislocatedprimarilyinsecretorygranulesandCRF2αRinendoplasmicreticulum.Interestingly,whenCRF-BPandCRF2αRareco-expressedahighdegreeofco-localizationintheendoplasmicreticulumisobserved,evidencingthatCRF2αRretainsCRF-BPinendoplasmicreticulum.Furthermore,thepresenceofCRF2αRintheplasmamembraneincreaseswhenisco-expressedwithCRF-BP.Theinteractionandsub-cellularlocalizationchangesbetweenCRF-BPandCRF2αRarespecific,sincetheyarenotobservedwhentheCRF2βRisoformisused.Insummary,ourresultsshowthatCRF-BPandCRF2αRformaproteincomplexallowingmodificationofthesubcellularlocalizationofbothCRF-BPandCRF2αR.

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109) Traffic of dopamine D2L receptor: basic mechanisms and kappa opioid receptor control

Ureta, Roxana1.,Escobar,Angélica1.,Andres,Maria1.,1BiologiaCelularyMolecular,FacultaddeCienciaBiológicas,PontificiaUniversidadCatólicaDeChile.(SponsoredbyFONDECYTProject#1150200)

Dopamine(DA) intheNucleusAccumbens(NAc) isakeyneurotransmittercontrollingmotivatedbehavior.DopaminesignalingiscarriedoutmainlybyactivationofD2andD1receptorsintheNAc.TwosplicedvariantsofD2receptors(D2R)areexpressedintheNAc.D2shortvariantreceptorsarepresynapticallylocatedindopaminergicterminalswheretheiractivationdecreaseDAextracellularlevels,whileD2longvariantreceptors(D2L)arepostsynapticallylocatedinmediumspinyGABAneuronswheretheiractivationfavorsmotivatedbehavior.DAneurotransmissionintheNAcismodulatedbyKappaopioidreceptors(KOR).KORarelocatedmostlyondopaminergicterminalsorincloseappositiontothem.AcuteactivationofKORdecreasesDAextracellularlevelsintheNAcandreducemotivatedbehaviors.OurpreviousworkshowedthatrepeatedactivationofKORdecreasestheinhibitoryfunction of D2R onDA release in theNAc. Interestingly, co-activation of KOR potentiates locomotor sensitization induced byrepeatedadministrationofquinpirole(QNP),aD2Ragonist.TogetherthesedataindicatethatKORandD2Rfunctionallyinteract,bothpreandpostynaptically.WehypothesizedthatKORactivationmodulateslevelsofD2Rinplasmamembrane,controllingtheinternalizationandtrafficofthisreceptor.Toevaluatethishypothesis,wedesignedavectorSepHluorin-D2L-mCherrytofollowD2Ltrafficking,sincegreenfluorescenceofSepHluorinproteinisemittedonlyatpH7.Thus,ifD2Risendocytedgreenfluorescencedecreases.HEK293TcellsweretransientlytransfectedwithSepHluorin-D2L-mCherryandtreatedwithDA(5µM,10µM,50µMand100µM)orwithQNP(5µM,10µM,50µMand100µM)andgreenfluorescenceintensitywasquantifiedonSynergy2-multimodallector.Preliminaryresultsshowedthatboth5µMand10µMDAandQNPdecreasewhilehighDAconcentrationsincreasegreenfluorescenceintensity,suggestingoppositeeffectsdependingonagonistconcentration.Co-transfectionexperimentswithHA-KORandSepHluorin-D2L-mCherryarecurrentlycarryingout.

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110)INFLUENCE OF T3 ADMINISTRATION ON THE HEPATIC PPAR-Α–FGF21 SIGNALING PATHWAY.

Vargas, R1.,Cornejo,Pamela2.,Fernández,Virginia1.,Tapia,Gladys1.,Videla,Luis1.,1Farmacologia,Medicina,UniversidadDeChile.2EscueladeTecnologíaMédica,Medicina,UniversidadDiegoPortales.(SponsoredbyAgradecimientos:ProyectoFONDECYT1150104)

Enhancedmitochondrialactivityintheliver,withincreasedfattyacidoxidation,O2consumptionandATPgeneration,isoneofthemajoreffectsofT3.ThismetabolicpathwaymayoccurthroughPPAR-α/FGF21.TotestthishypothesisasingledoseofT3(0.1mg/kg)oritsvehicle,weregiventoSprague-Dawleyrats,andparametersofT3-treatment,aswellasPPAR-αandtargetgenesforacetyl-CoAoxidase(ACOX),carnitine-palmitoyltransferase-1α(CPT-1α)andhydroximetilglutaril-CoAsynthase-2(HMGCoAS2),aswellasthatofFGF21,wereevaluated(qPCR).SerumlevelsofT3andtherectaltemperaturewereenhanced(p<0,05),inconcomitancewithlivercontentofmRNAforPPAR-αandtargetgenesforACOX,CPT-1αandHMGCoAS2.Inconcomitancewiththeseeffects,FGF21inductionwasobserved,effectthatwassignificantlycorrelatedwiththatofPPAR-α(r=0.98;p<0.0002).ItisconcludedthatT3activatesPPAR-αsignalingpathway,inassociationwiththatofFGF21,hepatokinecontrollingliverenergyproductionneededforpreconditioning.

Acknowledgment:ProyectoFONDECYT1150104

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111)Domains of the human corticotrophin releasing hormone type-2 receptor involved in the interaction with D1 dopamine receptor

Palominos, Tomás1.,Fuenzalida,Javier1.,Slater,Paula1.,Yarur,Hector1.,Gysling,Katia1.,1DepartamentodeBiologíaCelularyMolecular,FacultaddeCienciasBiológicas,PontificiaUniversidadCatólicaDeChile.(SponsoredbyFundedByFONDECYTGrants1110392&1150244,&MSIGrantN°P10/063F)

IthasbeenreportedthatD1dopaminereceptor(D1R)andcorticotrophinreleasinghormonetype-2receptor(CRH2R)areimportantinrelapsetodrugseekingbehaviortriggeredbystressfulstimuli.Reportsfromourgrouphavesuggestedthepresenceofbothreceptorsinpre-synapticinputstotheventraltegmentalarea(VTA).Additionally,byheterologousexpressioninHEK293Tcells,ithasbeenassessedthephysicalandfunctionalinteractionofthesetworeceptors.Theheteromerizationchangesthesubcellularlocalization of both receptors. In thiswork,we have generated several CRH2R deleteriousmutants in order to determine itsinteractingdomainswithD1R.TheinteractionbetweenD1R,CRH2RandCRH2Rmutantswasevaluatedbycheckingtheircapacitytoshifttheirsub-cellulardistributionwhenco-expressed.Alossofthisshiftindicatesalackofinteraction.Ourresultsshowedthattheintracellularloop3andthecarboxyltermimalofCRH2Rarenotresponsibleintheheteromerformation.Interestingly,ourcontrolswithcorticotrophinreleasinghormonetype-1receptor(CRH1R)revealedthatCRH1RandD1Rarecapableheteromerizewhenco-expressedinHEK293Tcells.Thus,D1RiscapableofheteromerizingwithbothCRHreceptors.

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112)

N-Acetylcysteine restores prefrontal cortical-accumbens LTD in Swiss CD1 mice fed high-fat diet.

Morgan, Carlos1.,Hernández,Alejandro2.,Ramírez,Paulina1.,Reyes,Andrea2.,Burgos,Héctor3.,Constandil,Luis2.,Sáez-Briones,Patricio4.,1UnidaddeNutriciónHumana,INTA,UniversidadDeChile.2DepartamentodeBiología,FacultaddeQuímicayBiología,UniversidadDeSantiagoDeChile.3EscueladePsicología,FacultaddeCienciasSociales,UniversidadCentralDeChile.4EscueladeMedicina,FacultaddeCienciasMédicas,UniversidadDeSantiagoDeChile.

Lossofprefrontalcortical(PFC)neuroplasticcontrolonnucleusaccumbensactivityisinvolvedintriggeringdrugseekingbehavioronseveralsubstancesofabuseduetoimpairedglutamatehomeostasisintripartitesynapsesinthenucleusaccumbenscore(Nacc)receivingafferentsfromPFC.WepreviouslyshowedthatPFC-Nacclong-termdepression(LTD),normallyobservedincontrolmiceissuppressedinyoungmicefedhigh-fatdiet(HFD)ad libitumduring2weeksafterweaning,whodevelopedadietarypreferenceforHFD(primedonHFD).WehypothesizedthatthisLTDsuppressionisinvolvedindevelopingandmaintainingdietarypreferenceforHFD.Our interestwas to restore thePFC-Nacc LTDandassess theeffectondietary consumptionofHFD. Then,PFC-NaccLTDwaselicitedinyoungmicefedHFDthatwereinjectedeitherwith100mg/Kgi.p.N-Acetylcysteine(NAC)orPBS2hbefore.N-Acetylcysteine,acompoundabletoinduceastrocytereleaseofglutamate,waseffectiveinrestoringPFC-NaccLTDin vivo.Inaddition,dailyNACadministrationduring5daystomiceprimedonHFDprogressivelydiminisheditsdietaryconsumption.Ourresultsareconsistentwiththeglutamatehomeostasishypothesisofaddictionandextendpreviousfindingsindrugsofabusetoconsumptionofhigh-fatdiet inmice.Theseneuroplasticchangesmayberelevanttoexplainthehighprevalenceofobesity inwesternsocieties.

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113)Putative molecular mechanisms associated to leptin resistance exhibited by Mecp2 null mice.

Valdivia, Sharin1.,Hernández-Galaz,Sergio1.,Guzmán,Luis1.,Ojeda-Provoste,Patricia1.,Gutiérrez,Noemí2.,Kerr,Bredford2.,1LaboratoriodeBiologíaCentrodeEstudiosCientíficos-CECs,UniversidadAustraldeChile.2LaboratoriodeBiologíaCentrodeEstudiosCientíficos-CECs.(SponsoredbyFondecyt1140162.PFB01/2007.)

Leptinisahormoneproducedbyadiposetissuethatmediateslong-termregulationofenergybalance.LeptinactivatesasignalingpathwaytoincreasetheexpressionoftheanorexigenicgenePomcthroughtheJak2/Stat3pathway.Afterbeingphophorylated,Stat3istranslocatedintothenucleustobindtothePomcpromoterandincreaseitstranscriptionalactivity,responsethatrequirethe dissociation of the Pomc-transcriptional repressor FoxO1 and its nuclear exclusion. Several studies have shown that thephosphatidylinositol3-kinase(PI3K)pathwaymediatestheeffectsofleptinonhypothalamicneurons.Leptin-receptorinteractioninducestheproductionofphospha-tidylinositol(3,4,5)trisphosphates(PIP3)byPI3K,allowingthephosphorylationofAktandits upstream activator PDK1, which allows FoxO1 phosphorylation and nuclear exclusion. In addition, the tumor suppressorphosphataseandtensinhomolog(Pten)inhibitsAktactivitybydephosphorylatingPIP3.Ontheotherhand,MethylCpGbindingprotein-2(MECP2)isanepigenetic-associatedchromatin-remodelingfactorwithadualroleongeneexpression.PreviousevidencefrompatientscarryingMECP2mutationsandresultsfromourlabdemonstratedthatthisproteinhasapivotalroleinthecontrolofbodyweight.UsingMecp2-nullmice,wefoundthattheabsenceofMecp2altersthebasalphosphorylationlevelsofAktandits target protein FoxO1 and therefore the activation ofPomc expression in response to leptin does not occur.However, themolecularmechanisminvolvedinthisdisruptedleptinresponsehasnotbeenfullyelucidated.OuraimwastodeterminatethemolecularmechanismunderlyingdisruptedleptinsignalingobservedinthehypothalamusofMecp2-nullmice.Toaccomplishthis,weperformedashort-termleptinchallengeandevaluatedtheexpressionofgenesandproteinsassociatedto leptinsignalingand itspost-translationalmodificationsby immunofluorescenceandwesternblot inhypothalamus fromMecp2-nullmiceandtheirwildtypelittermates.OurresultsshowthatMecp2-nullmiceareunabletoachieveproperleptinsignaling.WeobservedanalteredsubcellularlocationofStat3andFoxO1,adecreasedlevelofAktphosphorylationinbothnuclearandcytosolicproteinfractionsandadiminishedFoxO1nuclearexclusion.Thisalterationinleptinsignalingcomponentsmaybeassociatedwithamiss-regulationofPten,hypothesisthatisstillbeingevaluated.OurresultsallowustogaininsightintothemechanismunderlyingtheleptinresistanceobservedintheabsenceofMecp2,amolecularbridgebetweenepigeneticmodificationsandthecontrolofbodyweight.

314

114)Aldosterone and IL-17 in the genesis of mineralocorticoid arterial hypertension: an ex vivo study.

Vecchiola, A1,2.,Muñoz-Durango,Natalia2.,Fuentes,Cristobal1.,Gonzalez-Gomez,LuisMartin1.,Allende,Fidel4.,Ortiz-Canales,David1.,Tapia-Castillo,Alejandra1.,Valdivia-Pizarro,Carolina1.,Rojas,Maria3.,Carrasco,Carmen1.,Campino,Carmen1,2.,Solari,Sandra4.,Baudrand,Rene1.,Carvajal,Cristian1,2.,Lagos,Carlos1,2.,Kalergis,Alexis2.,Fardella,Carlos1,2.,1DepartmentofEndocrinology,SchoolofMedicine,PontificiaUniversidadCatolicadeChile.2IMIIMillenniumInstituteonImmunologyandImmunotherapy.3DepartmentofFamilyMedicine,SchoolofMedicine,PontificiaUniversidadCatolicadeChile.4DepartmentofClinicalLaboratories,SchoolofMedicine,PontificiaUniversidadCatolicadeChile.(SponsoredbySupportedByProyectoSOCHED13-6,IMIIP09/016-F,CORFO13CTI-21526-P1,FONDECYT1150437&1130427Grants.NothingToDisclose)

Background In vitroand in vivo studiessuggestabroader role foraldosterone,beyondelevatingbloodpressure.Clinicaldatasupportthenotionthataldosteronecandirectlyalterthefunctionoftheimmunesystemandcanparticipateinsystemiclow-grade inflammation,which leads tobloodpressureelevationandendorgandamage.Objective and Hypothesis: Toassess inhumans,whetheraldosteroneandIL-17plasmalevelsmodulateimmunogenicactivitymarkersinperipheralbloodmononuclearcells(PBMCs).Methods:Werecruited178adultsubjects(11-67years,BMI27.09±4.8kg/m2,61%female)andmeasuredinbloodsamples:aldosterone,plasmareninactivity(PRA),hsCRP,andIL-17.WeisolatedmRNAfromPBMCsandevaluatedMR,RAC-1,TLR-4,CD-14,Hsp-90,NGALandIL-17expressionbyq-RT-PCR. Results:AldosteronecirculatinglevelspositivelycorrelatewithPRA(p<0,00001)andCD14expression(p=0,024).IL-17circulatinglevelspositivelycorrelateswithMR(p=0,001),Rac1a(p<0,0001),NGAL(p=0,005)expression. Conclusion:AldosteroneandIL-17plasmalevelsareassociatedtoPBMCinflammatoryactivationmarkers,whichcouldpredisposetoautoimmunedisorderdevelopment.

315

115)Changes of BK potassium channel mRNA abundance in gills during the seawater adaptation in Salmo salar

Morera, FJ1.,Saravia,J1.,Oyarzun,R2.,Pontigo,JP2.,Strobel,P3.,Contreras,C1.,Loncoman,C1.,Gutierrez,L1.,Vargas-Lagos,C2.,Yañez,A.4.,Vargas-Chacoff,L2.,1InstitutodeFarmacologia,CienciasVeterinarias,UniversidadAustralDeChile.2InstitutodeCienciasMarinasyLimnologicas,Ciencias,UniversidadAustralDeChile.3InstitutodeCienciaAnimal,CienciasVeterinarias,UniversidadAustralDeChile.4InstitutodeBioquimicayMicrobiologia,Ciencias,UniversidadAustralDeChile.(SponsoredbyFundingByFONDECYTDeINICIACION11130308(toFJM),INNOVA-Corfo13IDL2-23565(toLVC,FJMAndAY);Fondap-Incar,No.15110027(toAY)AndDID-UACH(toFJM))

Historically,researchonsmoltificationhasbeenlinkedtotheaquacultureindustry,inparticulartothequalityofsmoltsduringtransfertimefromfreshwater(FW)toseawater(SW).Biologically,smoltification,alsocalledparr-smolttransformation,isacomplexdevelopmental process in salmonids that consists of a number of independent but coordinated changes in the biochemistry,physiology,morphology, and behavior of juvenile salmon in its transition from fresh water to seawater life. Instrumental tosmoltificationprocess is theabilityofsmoltgills tograduallybecomecapableofactivesaltsecretionthroughspecializedcellsknownasmitochondria-rich(MR)cells, ionocytesorchloridecells.NaClsecretionbyteleostgills isthereforeaccomplishedviaasecondaryactivetransportofCl-andpassivetransportofNa+.ThedrivingforceforactivetransportisprovidedbytheNa+,K+-ATPase,whichmaintainsalowintracellularNa+concentrationandhighintracellularK+concentrations.However,thismechanismofNaClsecretionnecessitatesathermodynamicprerequisitetoworkunderconditionsimposedbyhighextracellularsalinityinseawater:therecyclingofextracellularK+,mostlikelythroughpotassiumchannels.TheidentitiesofK+channelsrequiredforNaClsecretionfromMRcellsinseawaterarestillunknownforSalmo salar andonlyrecentlyhavebeguntobestudiedinotherteleosts.PreliminarydatafromourlaboratoryshowedtheexpressionofmRNAfromahighconductancevoltage-andCa2+-activatedK+(BK)channelingillsofSalmo salar.Inthisstudy,weperformedatime-coursestudyofthelevelsofBKpotassiumchannelmRNAingillsofS. salarinaseawaterchallengeexperimentandduringsmoltificationinanindustrialsetting.ThiswasanalyzedinconjunctionwiththeenzymaticactivityofNKA,theonly“molecularmarker”ofsmoltificationcurrentlyinusebytheChileansalmonindustry.WefoundthatthelevelsofBKpotassiumchannelmRNAincreaseduringthefirstweekintheseawaterchallengeexperimentandinasimilarwaytoNKAactivityduringsmoltificationinanindustrialsetting.Thesedatacouldbethefirststeptowardconsideringpotassiumchannelsasnewmolecularmarkercandidatesforsmoltification.

316

116)Evaluation of TASK-3 knockdown effect on breast cancer cell proliferation

Veneciano, Jocelyn1.,Zúñiga,Rafael1.,Valenzuela,Claudio1.,Brown,Nelson1.,Zúñiga,Leandro1.,1EscueladeMedicinaUniversidadDeTalca.(SponsoredbyThisWorkWasSupportedByFONDEF-IDEACA13I10223.J.V.ThanksToUniversityOfTalca,BiomedicalScienceMasterProgram.)

TASK-3(alsoknownasK2P9)isapotassium(K+)channelthatbelongstothefamilyoftwo-poredomain(K2P),displayingK+outwardlyrectifyingcurrentsindependentlyofmembranevoltage.TheTASK-3potassiumchannelhasbeenidentifiedasakeycomponentinthemaintainingofrestingpotentialinexcitablecells.Thischannelalsopresentsanoverexpressioninseveraltypesofhumantumors, including thoseoriginating frombreasttissueand lungs. In these tumors,TASK-3seemstopromoteproliferationandsurvivalofcancercells,mostlikelybyaugmentingtheirresistancetobothhypoxiaandserumdeprivation.Here,weevaluatetheoverexpressionofTASK-3potassiumchannelsindifferentbreastcancercelllines(MCF-10F,MCF-10A,MCF-7yMDA-MB-231),usingimmunofluorescenceandrealtimePCR(qPCR)analysistodeterminategeneexpression.OurresultscorroboratethepresenceofTASK-3indifferentbreastcancercelllineswithanexpressionprofileofMCF-10F>>MCF-10A=MCF-7=MDA-MB-231.Also,weassessthecontributionofTASK-3channelactivityoverproliferativeandtumorigenicpropertiesinMCF-10FandMDA-MB-231,throughshRNAapproach.OurresultshowsthattheknockdownofTASK-3,inMCF-10FandMDB-MB-231cancercells,generatedaproteinreductionof~30–50%,evaluatedatbothproteinandmRNAlevels.Thisreductionwasalsowellcorrelatedwithasignificantreductionincellproliferationprocess.OurresultssuggestthatTASK-3hasacriticalroleintumorcellproliferationandcorroboratethetherapeuticpotentialfordesigningofnewtreatmentformammarycancer.

317

117)Characterization of a molecular site for the modulation of the glycine receptor α3 subunit by 2,6-di-tert-butylphenol

Lara, Cesar2.,Burgos,Carlos2.,Muñoz,Braulio2.,Moraga-Cid,Gustavo1.,Corringer,Pierre-Jean1.,Yévenes,Gonzalo2.,1InstitutePasteurInstitutePasteur.2DepartmentofPhysiologyUniversidadDeConcepción.(SponsoredbySupportedByFONDECYT1140515,FondationDeLaRechercheMédicale,InstitutPasteurAndTheAgenceNationaleDeLaRecherche.SponsoredByDr.JorgeFuentealbaA.)

Glycinereceptors(GlyRs)areligand-gatedionchannelsthatareparticularlycriticalinthespinaldorsalhornprocessingofnociceptivesignals.Recentstudieshavedeterminedthatperipheral inflammationdecreasesthefunctionofspinalGlyRscomposedbytheα3subunit, contributing to thegenerationof chronicpain.Molecules thatenhanceα3GlyRsactivity thusmayexertanalgesicactions.However,onlyfewα3GlyRmodulatorsarecurrentlyavailableandthemolecularsitesinvolvedintheseeffectsarenotdefined.Usingelectrophysiologyandmolecularmodeling,herewecharacterizedamolecularsiteassociatedwiththemodulationof α3GlyRs by the non-anesthetic propofol analog 2,6-di-tert-butylphenol (2,6-DTBP). Our results show that 2,6-DTBP (1-300μM)isapositiveallostericmodulatorofα3GlyRs.Singlechannelrecordingsshowedthat2,6-DTBPsignificantlyincreasestheionchannelopenprobabilitywithoutchangesinthemainconductance.StudyingmutantGlyRs,weidentifiedaphenylalanineresidue(F388inα3GlyR)atthebeginningofthetransmembranedomain4(i.e.MA-stretch)whichisnecessaryforthepositiveallostericmodulationelicitedby2,6-DTBP.Whole-cellrecordingsrevealedthatthemutationofthisphenylalanineresiduetoalanine(F388A)significantlyimpairedthesensitivityto2,6-DTBP(164±20%inwild-typevs10±9.0%inF388Amutant).Atthesingle-channellevel,2,6-DTBPdidnotsignificantlyenhancedtheactivityofF388Amutantα3GlyRs,confirmingthelowsensitivityofthemutatedreceptortomodulationby2,6-DTBP.WenextcharacterizedthepotentialroleoftheF388residueforthebindingof2,6-DTBPinastructuralmodelofα3GlyRs.Threeresidues(F388,M384andP381)appearedascriticalforhydrophobicinteractionsof2,6-DTBPwithα3GlyRs.Moleculardockinganalysesrevealedafavorabletheoreticalenergyofinteractionwiththisputativeacceptorsite(ΔGbinding=-42.09kcal/mol,dockingscore=-2.133).Interestingly,theintroductionoftheF388Amutationinthestructurecausedasignificantdecreaseintheseparameters(ΔGbinding=-29.51kcal/mol,dockingscore=-0.507),suggestingadirectrelationshipbetween thedegreeofpotentiationofα3GlyRsby2,6-DTBPand theenergyof interaction.Our resultsdemonstrate that2,6-DTBPisapositivemodulatortheα3GlyR,whichmaydirectlyinteractwithanacceptorsitewithintheMA-stretchofthereceptor.Thedetailedcharacterizationofthismolecularsitecouldcontributetothescreeninganddevelopmentofnewα3GlyRallostericmodulators.

318

118)Exploring the molecular mechanisms underlying the functional inhibition of the glycine receptor α3 subunit by PKA-mediated phosphorylation.

San Martin, Victoria1.,Lara,Cesar1.,Yévenes,Gonzalo1.,1DepartmentofPhysiologyUniversidadDeConcepción.(SponsoredbySupportedByFONDECYT1140515SponsoredByDr.JorgeFuentealbaA.)

Diminished inhibitoryglycinergicneurotransmissionatthe levelofthespinaldorsalhornmakesarelevantcontributiontothegenerationandmaintenanceofchronicpain. InflammationdecreasessynapticglycinergiccurrentsprimarilythroughthePGE2-mediated activation of spinal EP2 receptors, which leads to a PKA-dependent phosphorylation of glycine receptors (GlyRs)composedofα3subunits.Despitetheimportanceoftheseeventsonchronicpain,themolecularandcellularmechanismsinvolvedinthefunctional inhibitionofα3GlyRsbyPKA-inducedphosphorylationhasbeennotyetestablished. Inthiswork,weexplorethenatureofthesemechanismsusing immunocytochemistryandelectrophysiologyofα3GlyRsexpressedinHEK293cells.Wefirstanalyzedwhethertheα3GlyRphosphorylationbyPKAcanmodifythereceptorstabilityattheplasmamembranethroughimmunocytochemistry.TheresultsshowedthatPKAactivationelicitedbyEP2receptoractivation,byincubationwithforskolinorbytheexpressionofconstitutivelyactiveversionsofGαs(GαsQ-L)orPKA(PKA-CQ-R)didnotalteredtheplasmamembranelocalizationofα3GlyRs.WenextstudiedtheeffectsofPKAphosphorylationatthefunctionallevelusingwhole-cellelectrophysiologyofHEK293cellsexpressingα3GlyRsandEP2receptors.ApplicationofPGE2inhibitedtheglycine-activatedcurrentsthroughα3GlyRsinatime-dependentmanner.Themaximalinhibition(-27±9%)wasreachedafter6minofsustainedapplicationofPGE2.Todeterminewhetherthiscurrentinhibitionisrelatedwithchangesontheapparentaffinityorontheefficacyofα3GlyRs,wenextanalyzedconcentration-responsecurvestoglycinebeforeandafterEP2receptoractivation.Ourdatashowedthattheapparentaffinityforglycine(i.e.theEC50fortheagonistglycine)wasnotsignificantlymodifiedbyphosphorylation(ControlEC50=136±19μMvsPGE2EC50=123±16μM).Ontheotherhand,themaximalefficacy(i.e.maximalcurrent)ofα3GlyRswassignificantlydiminishedbyPKAactivation(controlImax=2119±405pAvsPGE2Imax=1560±319pA).Altogether,thesedatasuggestthatthemechanismsinvolvedinthefunctionalinhibitionofα3GlyRsbyPKA-inducedphosphorylationaremorelikelyrelatedwithbiophysicalchangesontheionchannelratherthanmodificationsinthenumberofreceptorsattheplasmamembrane.Ongoingstudiesusingα3GlyRscarryingmutationsmimickingeitherthephosphorylatedorthenon-phosphorylatedstate(S346EorS346A)combinedwithrapidopticalstimulationofthePKAsignalingcascadeandsinglechannelrecordingswillhelptodefinethenatureofthesebiophysicalchanges.

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119)Influence of lipid rafts in the regulation of two-pore domain potassium channels in rat cerebellar granule neurons

Zúñiga, Rafael1.,Valenzuela,Claudio1.,Brown,Nelson1.,Zúñiga,Leandro1.,1EscueladeMedicina,CienciasdelaSalud,UniversidadDeTalca.(SponsoredbyAcknowledgments:ThisWorkWasSupportedByFondecytGrant11110217.R.Z.ThanksTheUniversityOfTalcaForAPhDFellowship.)

Two-poredomainpotassium(K2P)channelsarehighlyexpressedincentralnervoussystem(CNS),wheretheyhavebeenproposedto play important roles inmodulatingneuronal excitability. These channels give rise to leakK+ currents, voltage-independentandprimarilyimportantinthemaintenancesofrestingmembranepotential.Inneurons,thiscurrent,isknownasIKSO(standing-outwardpotassiumcurrent).Severallinesofevidencehavebeenshownthatthecholesteroliscriticalfortheactivityofdifferentplasmamembrane proteins, such as ion channels. Suggesting that these proteins are intimately associated with cholesterolrichmembranemicrodomains(lipidrafts).Andwhereastheimportanceoflipidraftsintegrityisessentialforneuronalactivity.Weevaluatetheeffectcholesteroldepletionbymethyl-β-cyclodextrin(MβCD)onbackgroundK+currentsincerebellargranuleneurons(CGNs)bypatchclampexperiments.CholesteroldepletionshowedareductionofIKSOcurrentsby∼40%,whentheCGNwereexposedto5mMofMβCD.Inaddition,thepresenceofdifferentK2Psubunitswereconfirmedbyimmunocytochemistryand theirassociationwith lipid raftswas identifiedwith immunocolocalizationwithFlotillin-2 (a lipid raftsmarker inneuron).Finally,thoseresultswereconfirmedbyco-immunoprecipitationwithFlotillin-2.TheseresultssuggeststronglythatbackgroundK+channelsareassociatedtolipidsraftenvironmentsandthatthemodificationofthisbehavior,bycholesteroldepletion,affecttheionchannelactivity.

320

120)Properties of the neural circuit associated to the CCAP AN1-AN4 and motoneurons during the ecdysis into pupa of the Drosophila melanogaster

Pineiro, Miguel1.,Mena,Wilson1.,Orio,Patricio1.,John,Ewer1.,1CentroInterdisciplinariodeNeurocienciaValparaiso,FacultaddeCiencias,UniversidadDeValparaíso.

Ecdysisisthemoultingofthecuticleinmanyinvertebrates,whichallowsthemtocontinuegrowing.TheETHhormonesynthesizedintheInkaendocrinecellsisreleasedintothehemolymphandinitiatestheecdysissequenceintheDrosophilamelanogaster.Thissequencecanbedividedinthreesubphases:pre-ecdysis,ecdysisandpost-ecdysis.OfthedifferentneuronsthatexpresstheETHreceptor,theCCAPneuronsfromthesegmentAN1toAN4havebeenidentifiedasthegeneratorsofthemotorpatternduringtheecdysis.ThewaytheseCCAPneuronsmodulatethemotorpatternisnotevident,mostlybecausethecircuits,neuronsandneuropeptidedynamicsarenotcompletelyunderstood.WeareusingcalciumimagingtocapturetheneuronalactivitypatternsofCCAPandmotoneuronsduringtheecdysis.Wedevelopedaclusteringalgorithmtoperformvideoneuralsegmentationusingpixelintensityvariationascueforpixelsbelongingtoneurons,andcoordinatedpixelintensityvariationascueforpixelsbelongingtoasingleneuron.Pixelintensityvariationismeasuredbythedynamicrangeofpixelintensity,whilecoordinatedpixelvariationismeasuredbyitsPearsoncorrelation.Wearemeasuringdifferentpropertiesoftheneuralactivitypatternduringtheecdysistobettercharacterizeitsdynamics.WehavefoundthattheslowCCAPcalciumoscillationsapparently increasetheprobabilityofmotoneuronstoswitchfromanon-oscillatorytoafasteroscillatorystate.InordertoinferthefunctionalconnectivitybetweenCCAPandmotoneurons,wearefittingacontinuoustimeMarkovchainmodeloftwostates(oscillatoryandnon-oscillatory)withtransitionratesfunctionoftheCCAPtimeseriestotheexperimentaldata.ThismodeliscompatiblewiththeapparentstochasticityoftheoftheCCAP–motoneuroncommunication.

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121) EFFECT OF ANGIOTENSIN CONVERTING ENZYME INHIBITION ON THE DEVELOPMENT OF MESONEPHROS IN CHICKEN EMBRYOS.

Moya, Víctor1.,Olivares,Natalia1.,Ansaldi,Valentina1.,Alexis,Gonzalez2.,1InstitutodeBiología,FacultaddeCiencias,PontificiaUniversidadCatólicaDeValparaíso.2InstitutodeQuímica,FacultaddeCiencias,PontificiaUniversidadCatólicaDeValparaíso.

While the roleof the reninangiotensinaldosterone system (RAAS) in the regulationofbloodpressureandvolume is known,recentevidencesuggest theparticipationofRAAS inthedevelopmentofmammaliankidneyandparticularly indevelopingofmetanephros. Inorder toevaluate theeffectsof captopril (angiotensinc convertingenzyme inhibitor)on thedevelopmentofmesonephrosandexpressionofsomecomponentsRAAS,chickenembryosweretreateddailywith5mgcaptoprilperkgfrom48hrsupto14daysduringthedevelopment.ByhistochemicalandimmunohistochemicaltechniquesmesonephrosmorphologywasanalyzedandthepresenceofcomponentsoftheRAASwasevidenced.ProteinexpressionofRAAScomponentswasanalyzedby western blot. Embryos treated with captopril showed increased weight (43.3%), wide (25.4%) and length (23.8%) of themesonephrosaswellasthenumbersofmesonephrictubules(86.7%)comparedtothecontrol.Renin,AT1receptorandaquaporin2proteinlevelswereslightlydecreasedbycaptopriltreatment.TheeffectsofcaptoprilonchickenmesonephrossuggestthatRAASplaysaroleintheregulationofrenaldevelopmentinbirds,butremainstobedeterminedwhetherthemechanismsareequivalenttothosedescribedinmammals. Aknowledegments:FONDECYT11121217

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122) Role of the PON1Q192R polymorphism in the cognitive performance of agricultural workers exposed to organophosphate pesticides in the north of Chile (Coquimbo Region).

LilianaZúñiga*,SebastiánCorral*1andFloriaPancetti*

*LaboratoryofEnvironmentalNeurotoxicology,FacultyofMedicine,UniversidadCatólicadelNorte,Coquimbo,Chile; 1Presentaddress:DepartmentofPsychology,FacultyofSocialSciences,UniversityofChile,Santiago,Chile.

Organophosphate pesticides (OPs) are widely used worldwide with both domestic and industrial purposes. Studies onsituationsofchronicoracuteexposurehaverevealednumeroushealtheffectsattributedmainlytotheinhibitionoftheenzymeacetylcholinesterase (AChE). Besides the peripheral physiological function of this enzyme, AChE is also involved in cognitiveprocesseswithin thebrain. On theother side, ithasbeendescribed that theparaoxonase1 (PON1)enzyme involved in thedetoxification route of OPs, displays polymorphisms that account for human susceptibility to OP exposure. Although thephysiological and toxicological roleofPON1hasbeenwelldescribed, its role in thecognitive impairmentobserved inpeoplechronically exposed toOPshasnotbeenwell established yet. In this studywewanted toevaluate theeffectof the chronicexposuretoOPsoncognitiveperformanceandontheactivitiesoftheenzymesusedtoevaluateacutepoisoning(erythrocyteAChEandplasmacholinesterase,ChE)of93agriculturalworkersand85non-exposedpeopleandcorrelatethesedatawiththePON1Q192Rpolymorphism. Theneuropsychologicalbatteryconsistedof31 tests thatevaluatedgeneralmental state,memory,attention,praxis,executivefunctions,motorcoordination,languageandmood.

TheresultsindicatethattheAChEandChEactivitiesfromtheexposedgroupdidnotdifferfromthosemeasuredintheunexposedgroup.Intheexposedgroupthemeanscoresof21testsweresignificantlowercomparedtothescoresoftheunexposedgroup.Furthermore,theindividualswhoshowedaperformanceunderthenormalscoresinthe90%ofthetestsbelongtotheexposedgroup.Theanalysisofthesampleafterstratifyingthepopulationaccordingto4levelsofimpairment(0=normal;1=borderline;2=mildimpairmentand3=severeimpairment)revealsthatonlytheactivityofAChEdisplaysasignificantinhibitionshowinglesscatalyticactivityintheindividualswithhigherdeterioration.Finally,ourresultsdonotshowaroleofPON1Q192Rpolymorphisminglobalcognitiveimpairment;however,leadustoputspecialinterestinindividualswithQQgenotype(orQallelecarriers)duetotheyshowedahigherpercentofimpairmentinareassuchasattention,executivefunctionandmotorcoordination.Thisworkprovidesinformationofgreatimportanceintermsofoccupationalhealthandenvironmentaltoxicology,asithasbeendescribedthattheQallele(presentinabout60%ofthestudiedpopulation)islessefficientthantheRalleleformetabolizingchlorpiryfos,themainOPpesticideusedinthestudyarea.

ThisresearchwassupportedbyFONDEF(FondodeFomentoalDesarrolloCientíficoyTecnológico,CONICYTChile)GrantnumberD09I1057andFONDECYT(FondoNacionaldeDesarrolloCientíficoyTecnológico,CONICYTChile)Grantnumber3120231.

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HOTEL ENJOY COQUIMBOIVREGIÓN,CHILE

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Indice

AAbarcaJorge78AbdallaDulcineia115AboitizFrancisco155AbrigoJohanna300AcevedoCRISTIAN277AcevedoMaría81AcuñaMario75AdasmeTatiana45AguayoClaudio73,88AguayoLuis74,79,122AguilarMarcelo155Aguilar-riveraM.I.127AguileraC.180AguileraJocelyn99,100AguileraValeria73AguirrePabla48,120,124AguirrePablo197AgurtoAdolfo133AhumadaCatalina80,207AhumadaJuan141AlarconCecilia71AlarconGloria149AlarcónJulio235AlarconPablo101AlbertiCarolina205AlcayagaJulio134,258,260AldereteJ281AlexisGonzalez321AliagaEsteban203AlkemaMark255AllendeFidel154,178,186,224,314AllendeM33AlmendrasSebastian179AlthausMike307AlvarezDaniela150,299ÁlvarezRocío110Álvarez-ferradasCarla252AlvealNatalia175AmbrosettiValery150AmpueroEstibaliz142,208AnaRiveros226AndreaSaavedra172AndrésCouve31

AndrésMaría62,63,308,309AndrésMaríaEstela302AndreuCatherineI.156AnfossiRenatto211AnríquezC231AnsaldiValentina321ArancibiaRadichJorge102Arancibia-radichJorge109,238ArandaMario235AranedaFelipe90,91AranedaOscar171AranguizMACARENA198AraosPatricio167AraunaDiego98AravenaJ170AravenaMarcela181ArayaClaudio90,91,96ArayaJuan28ArenaPamela237ArenasHector240AriasCarolina288ArielSaavedra235ArmijoLorena30ArredondoCristian62ArriagadaJ247ArriagadaJORGE118,247ArriagadaM157ArtigasClaudio139,163AstorgaCésar31AstridHaensgen172AstudilloAland158AvelloMarcia235AvelloZita102AvendaÑoCINTIA198AzócarV.H.180

BBacigalupoJUAN257,259BáezXimena298BahamondesCarolina80,207BarattiniP45BarraKaren121,125,190BarraRafael181BarreraNelson69

325

BarreraNelsonP.175Barrera-bugeñoCamila204Barrera-bugueñoCamila111BarrigaAndres205BarriosGuillermo97BascuñánD122BascuñanDenisse79BastiasCristian148BaudrandRene154,178,314BecerraAlvaro89,216,220BehnClaus222BeltránCamila280BeltranFelipe119Beltran-castilloSebastián146Benavente-schonhautS84,202Benavente-schonhautSofia84,202BenitezAgustin154BeñaldoFelipe90,91,94,96BernalGiuliano182BernalesSebastian275BerríosPablo22Berríos/cárcamoPablo21Berrios-amaroC153Berríos-cárcamoPablo24,106,236BetancurJohana218BevilacquaJorgeA298BezanillaFrancisco9BlanchardKRIS257BlancoElías148BolivarSamir104BonanscoC267BonanscoChristian141,252,267BoncompteGonzalo166BoricPMauricio221BozaP92BozaPia104,211,219BravoAlex106BravoD274BravoE123BravoJAVIER51,111,203,204BravoJessica105BrownNelson316,319BuendiaIzaskun25BugueñoItalo109BurboaPia93BurgosCarlos75,209,317

BurgosFelipe122BurgosH82,83BurgosHéctor82,83,84,202,312BurgosRafael101,107,225,228Burgos-villasecaJ82,83,84,202Burgos-villasecaJorge82,84,202BustamanteD233BustamanteDiego22,40

CCabelloClaudio170CabelloFrancisco288Cabello-verrugioClaudio300CalfaG.57CalfíoCamila229CalixtoAndrea255CamilaNavia149CamilaReyes73CampinoCarmen154,178,186,224,314CamposGerman139,160,163CamposMatías259CampusanoJorge135,137,162,282CampusanoJorgeM283CaneoMauricio255CardenasAna176CárdenasAnaMaría194,298CarlaDelporte226CarlosVeas88CarmonaEmerson275Carmona-rojasE153CarrascoCarlos124CarrascoCarlosM.124CarrascoCarmen314CarrascoI.212CarrascoJuan183CarrascoRodrigo76Carrasco-pozoCatalina53CarrerH.57CarrettaDaniella107CartierLuis205CarvajalC223CarvajalCristian18,154,178,186,224,314Carvalho-de-souzaJoao9CarvalloClaudia85,143CasselsBruce22,84,177CasselsBruceK.48

326

CastilloA82,83CastilloAmparo82,83CastilloC68CastilloCarolina68CastilloChristian230CastilloKaren34CastilloPablo140CastilloR53CastilloRodrigo56CastilloSebastian96Castillo-galánSebastián91,94CastroGabriela288CastroMaite119CastroSamy262CastroVicente100Castro-castilloVicente108,199,202CatalánM108CatalánMabel108CatrimanDanixsa96CaviedesAriel270CaviedesMauricio197CaviedesPablo298CeaLuis298CeballoKarina299CeciM33CelisTeresa121,125CerdaCledi308CerdaFabián74CerdaM35CerdaMauricio35CerdaTania96,150,299CespedesNicole184,240ChaconMarcelo250ChavezA140ChavezAndres140,144,145,263ChenZhuoming91ChiongM239CidMarcela217CifuentesMariana178,186,224ClarkeG52CoddouC41,182CofréChristian82,83ColmenaINES27ColomboAlicia30ConchaMiguel30ConejerosIván101

ConstandilLuis274,312ContrerasC315ContrerasD85ContrerasDarwin143ContrerasENRIQUE198ContrerasGustavo34,193ContrerasMarcela38,145CordovaJohn35Córdova-casanovaA214CornejoP183CornejoPamela310CornejoVeronica288CoronadoCesar184Corral-zavalaSebastian66CorringerPierre-Jean75,317CortésMagdalena110CortésPaulina38CortezGiovanni109,238CorvalanKatherine253CosmelliDiego156,166,292CouveA29,35CouveAndres35CruzGonzalo80,81,150,207,299Cubillos-roblesKaren237CutiñoAndrea168

DDagninoA.157DagninoAlexies248Dagnino-subiabreAlexies50,161,164,174,246,284,294DangBobo9DarszonAlberto10,276DeGiorgisDaniela193DeGregorioCristian31,168DeGregorioNicole222DeKloetRon11DeLaFuenteErwin38DeLaParraAlicia288DelMazoJesus279DelPozoReginald278DelRioRodrigo134DelgadoClaudia130DelgadoRicardo31,85,257DelorenziA58DelporteCarla102,105,109,114,117,238

327

DeviaChrist165DiLioAlessandra75DíazC281DiazGalarceRaul287DíazGuillermo92,219DiazIgnacio36DíazMarcela96DiazPablo168DíazVioleta249Díaz-arayaG239Díaz-arayaGuillermo104,211,232,234Díaz-galarceR59Díaz-galarceRaul159,208,285Díaz-merinoCamila111Díaz-zepedaCamilo203DonosoMVerónica116DonosoM.Veronica95DonosoMariaVeronica103,146,269DonosoP214,231DroguettKarla69,307

EEbenspergerG96EbenspergerGerman54,90,91,94EbenspergerRenato94,96EgañaJose32,249EgeaJavier25EscobarAngélica63,309EscobarMaríaJosé262EscobarMaria-Jose136Escobar-lunaJorge111,204EscorzaT59EscorzaTomás159EscuderoC73,88EscuderoCarlos73,88EsmarDaniela40EspañaRodrigo63EsparzaAndrés120EspejoP.57Espina-marchantPablo40EspinosaNelson155EspinosaPedro188,206,241EspinozaHilda110EugeninJaime146,269Eyzaguirre-velásquezJohana111,204

FFaberDonald250FardellaCarlos154,178,186,223,224,314FaríasJ304FariasJ.G151FaríasJorge53,56,112,115FariasMarcelo217FariasRicardo85,143FelipeZuñiga235FernándezJavier183FernandezPaola227FernándezRicardo100FernandezRoberto99,113FernándezVirginia183,310FernandezYaiza280Fernández-ramiresRicardo113FernandoisDaniela150FerradaJaviera96FerreiraJorge100,108Ferreira-parkerJorge199FiedlerJenny86FierroAngelica177FigueroaC115,304FigueroaC.A151FigueroaCarolina56,112,115FigueroaCatalina114FigueroaE151,304FigueroaElias56,112,115FigueroaReinaldo282FigueroaSteffany297FigueroaXavier93,213,268FigueroaXF273FlorenzanoFernado76FloresCarlos195FloresCarolina261FloresFrancisco179FloresJorge160FrankenIngmarH.A.156FuentealbaJ28FuentealbaJ.A.127,180FuentealbaJorge68,121,125,190FuentealbaJosé63,290FuentealbaPablo155FuentesChristian69FuentesCristobal178,224,314FuentesEugenia167

328

Fuentes-ibacacheNataly178,186,224Fuentes-zuñigaCristobal186FuenzalidaJavier311FuenzalidaM247,266,267FuenzalidaMarco50,118,141,247,263,266,267Fuenzalida-uribeNicolás135,137,162,282

GGacLily189GajardoIvana126GalazPablo184GallardoLuz152GámizFernando191GandiaL27Gárate-pérezMacarena161GarcíaApolinaria235GarcíaCarolina181GarcíaIsaac194Garcia-beltranOlimpo48Garcia-huidobroJ43Garcia-huidobroJPablo116García-huidobroToroJuanPablo175García-rojoGonzalo86GasparPablo32GaticaR.I.127GiacheroM.57GodoyPamela121,125,190GomezCristobal254GomezFrancisco275GómezKarina146GómezMelissa116GómezTeresa253GonzalezAA20GonzálezAlejandro196,259GonzalezAlexisA17,169,296,297GonzalezAlvaro89GonzalezArlek298GonzalezCarlos34,193,194GonzalezDaniel97,98GonzálezDaniela306GonzalezDanilo176GonzálezHugo286GonzalezL241GonzalezLuis130,241GonzálezM.212GonzálezMagdalena168

GonzálezMarcela62GonzalezMarcelo217GonzalezPaula237GonzalezRDaniel221González-arancibiaCamila111,203Gonzalez-billaultChristian124,305Gonzalez-gomezLuisMartin178,186,224,314Gonzalez-herreraFabiola230Gonzalez-vergaraAlex169,297GormazJuanGuillermo76GottelandMartín204GraceAnthony14GuerraMarcelo150GuerreroNestor30GutierrezD33GutierrezL315GutierrezNicolas88GutiérrezNoemí64,313Gutiérrez-hernándezManuel40Gutiérrez-maldonadoSebastián87GuzmánConstanza90,91GuzmánDaniela108GuzmánJosé306GuzmanL281GuzmanLeonardo74GuzmánLuis64,187,313GyslingKatia62,63,78,87,148,290,308,311

HHaegerPaola38,145HaensgenA.212HaensgenAstrid217HardyPaulina129HärtelS35HärtelSteffen35HebelDan179HennyPablo63,265HenriquezFrancisca130HenriquezJuanPablo26,47HenríquezRicardo169HermosillaCarlos101HermosillaTamara129HernándezAlejandro82,83,84,181,202,274,312HernándezSergio187Hernández-galazSergio64,65,313HerreraEmilio53,90,91,94,96

329

HerreraEmilioA54HerreraPachecoGaspar70Herrera-marschitzM233Herrera-marschitzMario21,22,24,40,106,236HerrerosClaudia162HerrerosEduardo99,113HerzogRubén136,138HetzClaudio30HeviaDaniel167HeviaMaríaJosé182HidalgoAngela68HidalgoC35,45HidalgoCecilia35,145HidalgoMaria107,225HidalgoMaríaAngélica228HidalgoSergio137HinostrozaFernando176HugoEstefanía177Huidobro-toroJPablo229,301Huidobro-toroJuan95,303Huidobro-toroJuanPablo103,146,269HumeresClaudio92,104HumeresDiego211

IInestrosaN46IsoardiN.57IsraelYedy22,24Iturra-menaAnn164Iturriaga-vásquezPatricio24,201

JJaraBelen237JaraD226JaraJose99,100,108,113JaraOscar194,261JaraPablo188JaramilloKaren142JaramilloKarina163JerézBaraonaJuanManuel285,287JimenezD55JimenezVerónica74JinChunyang74JohnEwer320JorqueraManuel184JoséEgaña165

JuanSaez274JujiharaGermán174Julio-pieperMarcela111,203,204JuryNur142

KKadriuBashkim250KalergisAlexis186,224,314KarinaJaramillo163KarmelicD147KemmerlingUlrike230KentStephen9KerrBredford64,65,149,187,313KirkwoodAlfredo243KlaggerJorge83KlaggesJorge82Klagges-troncosoJ84,202Klagges-troncosoJorge84,202

LLagosCarlos87,154,178,185,186,224,314LagosCarlosF227LagosCarlosF.178,179LamasGuillermo251LaraCesar75,209,210,317,318LaraHernán188LaraMarcelo132,264LarrazabalCamilo225LatapiatVerónica301LatorreRamon34LavanderoS239LazcanoPablo305LazoV213Leguina-ruzziAlberto71LeivaE44LespayCarolyne40Lespay-rebolledoC233LiShiHua119LiXiao-Jiang119LiempiAna230LinsambarthSergio128Lizama-gonzalezJaime223LlanosAnibal54,90,91,94,96LlonaIsabel269LobosPedro35LoncomanC315

330

LoncomanCarlos225LopezManuela25LópezVasthi218LópezVladimir156,291,292López-gonzálezIgnacio10Lopez-ortegaAura173LorcaEnrique132,264LorenzoYenisleidy34LuarteAlejandro271

MMadariagaF99MadariagaFrancisco113MadridRodolfo70,82,83,196,259Madrid-rojasMatías108MaidanaJeanPaul197MaldonadoN.57MaldonadoP32MaldonadoPedro165,249,289Malhue-olmosV84,202Malhue-olmosValeska84,202ManosalvaCarolina225,228MaraboliVanessa176MarcelaCid172MarceloFarías172MarceloGonzalez172MarceloKogan226MardonesLorena278Mardones-krsulovicC33MarileoAna209MaripillánJaime194,261MariqueoTrinidad184MarquezYsabel173MartijenaI.57MartínezAgustin194,261MartinezJ242,244MartinezJonathan130,242,244Martinez-aguayoAlejandro223MarzoloMaríaPaz142MasallerasMatias271MascayanoCarolina229MatelunaCarlos229MaturanaCarola185,274MauraRafael73MayaJuan230MayfieldJacob37

MayolRocio32MazaFJ58MedinaFernando205MellaJessica26MellaRicardo189Mella-raipanJaime179MenaJaqueline228MenaNatalia120MenaNataliaP.48MenaWilson320MéndezL59MéndezLuis159,285MendezNatalia39MeynardMargarita30MezaR265MezaRodrigo63MezzanoDiego71MicheaL19MicheaLuis167,168MoenneAlejandra116MolinaAlfredo99,100MolinaDaniela137MolinaV57MolinaVA58Molina-berríosAlfredo113Molina-fernandezClaudia282,283Molina-mateoDaniela282MomboisseFanny194MontecinoMartín142Montefusco-siegmundRodrigo165MonteroEDSON198MonteroPablo260MoragaAmaroRodrigo287MoragaFernando90,91,96,218,251MoragaJosefina251MoragaRodrigo286Moraga-amaroR59Moraga-amaroRodrigo128,159,208,285Moraga-cidGustavo75,317MoralesBernardo82,83,85,143MoralesC247MoralesCAMILA118,247MoralesJ267MoralesJuan267MoralesK266MoralesKoyam266

331

MoralesMaríaGabriela300MoralesP40,233MoralesPaola22,40MoralesRicardo139MoranMarisol98MoreJ45MorenoC129MorenoR153MorenoRicardo152,275,276,277MoreraFJ295,315MorganCarlos82,83,181,312MoulyVincent298MoyaP241,242,244,266MoyaPablo130,241,242,244,247,266MoyaVíctor321Moyano-porcileValentina203Munoz-caroTamara101MuñozBraulio75,317MuñozClaudia104,211,232MuñozDaniela38MuñozKatia278MuñozManuel268MuñozMF273MuñozMirna278MuñozValentina40MuñozYorka245Muñoz-durangoNatalia224,314MurathPablo210MurilloMaríaDivina173

NNanclaresCARMEN27NaranjoDavid36NavarreteCamilo69,175Navarrete-encinaPatricio234NavarroElisa25NavarroMyriam105Navarro-quezadaNieves193NeelyAlan34,193NegronIgnacio155NeiraLuis306NochesVerónica63NovaDaniela306NovoaUlises98NualartFrancisco270NunezMarcoT.48

NúñezM120NúñezMarco120,124NuñezMarcoTulio245

OOjeaA58Ojeda-provostePatricia65,313Olavarría-RamírezLoreto111Olavarría-ramírezLoreto203,204OlceseRiccardo193OleaClaudio114OlguínSofía150,299OlivaresErick70OlivaresJesús138OlivaresMaríaJosé146,269OlivaresMontserrat302OlivaresNatalia321OlmedoI214,231OñateAlejandro265OrioPatricio70,138,158,197,262,272,320OrmazábalValeska73,235OrtizIgnacio258OrtizV57Ortiz-canalesDavid178,186,224,314OsorioJoséMiguel232OsorioMarcela293OssandónTomás288OssesNelson275,277OvandoMarcela294OwenGareth186OyarceMaría134OyarzunR295,315

PPachecoR59PachecoRodrigo167,286PaillamanqueJoaquin275PalaciosAdrián126,136,144,243,256PalaciosE233PalaciosEsteban40Palacios-garciaIsmael163Palacios-muñozAngelina144PalmaKarina30PalmaVerónica147Palma-espinosaJavier272PalominosTomás311

332

PancettiFloria191,251PancettiVaccariFloria66PanesJessica125PanesYessica121ParadaEsther25ParadaGuillermo302ParadaVíctor235Pardo-jiménezViviana234PárragaMario279PasteneEdgar235Paula-limaA35,45Paula-limaAndrea35,245PavaniMario108,199Pavez-foxMelissa246PedrozoZ214,231PennaAntonello72Peña-cerdaMarcelo109,117,238PeñalozaEstefania71PeñalozaSanchoValentín174,284Peñaloza-sanchoValentín50PepperbergDavid9PeraltaFrancisco303Peredo-silvaLiliana199PereiraJaime71PérezCatherine248PérezHernán181PérezLorena215PérezM247PérezMiguel50,118,247PerezNelson79,122Perez-acleTomás227Pérez-arancibiaRodrigo117Perez-leightonC189Perez-leightonClaudio189Pérez-lobosR233Pérez-lobosRonald40PertusaMaría196PessoaA304PessoaAdalberto115Pessoa-mahanaCarlosDavid179Pessoa-mahanaHernan79PetersChristian79,122PezoRafael103PhilpA195PhilpAmber195PineiroMiguel320

PinoG231PinoGabriela260PinoJose275PintoBernardo194PintoCristina47PiñaR259PlazaMiguel173PobleteDaniel90PobleteInes93,213PobletePatricio128Ponce-GuequenExcequel204PonceMaríaJosé184PontigoJP295,315PradoCarolina167PradoL131PradoPavel261PrietoM20PueblaM273PueblaMariela268PuenteJavier205PupoA34PupoAmaury34

QQueroDaniel197QuezadaSebastián94QuilodranRene158QuintanaDonosoDaisy287Quintana-donosoD59Quintana-donosoDaisy285QuintanillaM22QuintanillaMaríaElena21,24,106,201,236QuintremilSebastián205QuirozGabriel24,201

RRadojkovicClaudia73RadojkovicClaudia88RalveniusWilliam75RamiresRicardo99RamirezBeatriz189RamírezEugenio205RamirezJuanPablo254,271RamírezLuisa150RamirezM66RamirezOmar35

333

RamírezPaulina312RamírezSebastían182RebolledoSolange108Recabarren-gajardoGonzalo179ReigGerman30RejasCarolina279RenardGeorgina80,206,207ReyesAldo150ReyesAndrea312ReyesJ153ReyesJuan257,275,277ReyesRoberto54,94ReyesVíctor90,91,96Reyes-martínezCristian296Reyes-paradaMiguel24,201RichersonGeorge123RichterHans39RíosM307RíosMariana69RiquelmeJ244RiquelmeJulio244RisopatronJennie112,151RivarolaM49RiveraBastián196RiveraConstanza114RiveraG249RiveraJ300RiveraJuanCarlos300RiveraMario21Rivera-lilloGonzalo249Rivera-mezaM236Rivera-mezaMario21,22,24,106,236RoaJuan71RodrigoRamón76RodriguezEugenio139,160,163,291,293RodriguezLuciana244RodriguezManzanaresP.57RodríguezS.212RojasC.212RojasDiego108RojasMacarena216,220RojasMaria154,314RojasPatricio119,129,132,264,272,286RojasS59RojasS.212RojasSebastián159,285,287

RojasSusana217Rojas-mancillaEdgardo40Rojas-riveraDiego30RojoLeonel237RomeroDiego71RozasCarlos85,143,250RubioFrancisco208RubioMariana40

SSaavedraA.212SaavedraAndrea217Saavedra-olavarríaJorge199SáezClaudia71SaezJ13SáezJuan185,276SáezJuanC227SáezJuanCarlos252Sáez-brionesP84Sáez-brionesPatricio82,83,84,202,312Sáez-brionesS202SalasJeremy196SalazarClaudia243,256SaldiasCristina244SalgadoSimon262Salinas-parraNicolás169,296,297SanMartínLoreto74SanMartínSebastián279SanMartinVictoria318SanabriaMariana173SánchezG214,231SanchezGina145SanchezM33SánchezOliberto280SánchezSussan181Sánchez-carranzaOscar10SandovalRodrigo251Sandoval-guzmanRodrigo66SanguinettiN206SanguinettiNicole81SanhuezaMagdalena147SantanderDaniela288SantibañezCristián275SaraviaJ315SavalliNicoletta193SchmachtenbergOliver133,138,263

334

SeguelIvette102,109,117,238SehrtM192SepulvedaBetsabet105SepúlvedaEvelyn110SepulvedaMARIA198SepúlvedaNestor53SerthMarcos40ShlomoDellal250ShortS.E151ShortStefania56,112SierraltaJimena31SilvaBryon283SilvaHernan32SilvaJaime163SilvaJuan222SilvaPatricio154SilvaRoxana80,207Silva-grecchiTiare28,68,121,190SimonF89,215,216,220SimonFelipe89,170,215,216,220,300SlagterHeleenA.156SlaterPaula87,308,311SolariSandra154,178,186,224,314SotoR281Sotomayor-zarateRamón78,80,81,188,201,206,207,241SpichigerCarlos39StarckMariaFrancisca280StehbergJ58,59StehbergJimmy128,159,208,285,286,287StrobelP315StutzinAndrés72SusanaRojas172

TTabordaMaría218TamayoAndrea240TamburiniG59,208TamburiniGiovanni159TapiaFelipe92,219TapiaGladys310TapiaMarcelo222TapiaPablo89TapiaV120TapiaVictoria48,120Tapia-bustosAndrea40

Tapia-castilloAlejandra223,314TaubertAnja101TeuberStefanie225TevyFlorencia282ToledoJorge68ToledoJorgeRoberto73,88ToledoPedro293TomesClaudia276TopperLauren37TordoyaIsis237TorresEillen235TorresG12TorresPaulina10Torres-farfanC39Torres-fuentesJorge276ToumaJorge105TralmaKarina196TregerJeremy9TreuerAdriana184TrevinoClaudia10,276Troncoso-escuderoPaulina119TuestaM171TuestaMarcelo171

UUgaldeMaríaBeatriz283UgaldeValentina286UgarteGonzalo85,259UretaRoxana309UrraJonathan21UrrutiaP120UrrutiaPamela120UrrutiaPamelaJ.48UrrutiaPatricia181UrzuaBlanca99,113UtrerasElias242,305UtrerasJonathan184

VValdebenitoIvan112,151ValdesJL45ValdiviaCarolina223ValdiviaGonzalo243ValdiviaL33ValdiviaLuz172,217ValdiviaSharin64,313

335

Valdivia-pizarroCarolina154,314ValenciaMartina203ValenzuelaCarlos37ValenzuelaClaudio316,319ValenzuelaJavier251ValenzuelaMaria205Valenzuela-bustamantePaula117Valle-corvalanGissella179VallejosAlejandro220VallejosGabriel110Valverde-porrasNaizmi119VanZundertBrigitte142VarasG289VarasManuel271VarasNicolás280Varas-godoyManuel253VarelaDiego129VargasAnibal227VargasR310VargasRomina183Vargas-chacoffL295,315Vargas-lagosC295,315VasquezCLAUDIA277VásquezP281Vasquez-rosatiAlejandra292VeasRubén238VecchiolaA314VecchiolaAndrea154,178,186,224VelardeVictoria71VenecianoJocelyn316VenegasAlejandro105VenegasFrancisca81,206VeraGonzalo179VeraJorge258VergaraCECILIA257VergaraM290VidalF281VidelaLuis183,310VielmaA263VielmaAlex133,144,263VielmaZAlejandra221VilchesNatalia86VilchesNelson39VillagranMarcelo278VillalobosDANIELA257VillalobosIsabel271

VillalonEsteban128VillalónManuel69,307VillarPABLO257VillarzuPaula223VillenaJuan279Villena-gonzalezMario163,291VinalesLaura158VirgoliniM23VivarR239VivarRaul92,104,211

WWelchJason37WellmannMario252WillisNaomi88WilsonCarlos305WulffCristian222WynekenU60,253,254,270,271WynekenUrsula208

YYañezA295YañezA.315YañezRodrigo293YarurHector311YévenesGonzalo75,209,210,317,318YujaungKim123

ZZangenAbraham128Zapata-torresGerald24ZeilhoferHanns75ZeiseMarc82,83,85,143ZepedaA304ZepedaA.B151ZepedaAndrea56,112,115ZuñigaFelipe88ZuñigaFerlipe73ZuñigaJessica240ZúñigaLeandro316,319ZúñigaRafael316,319Zuñiga-venegasLiliana66