doc.anet.be faculty of medicine and health sciences molecular imaging to quantify neuromodulation of...
TRANSCRIPT
Cover image:Thefourneuromodulationtechniquesthatareappliedinthisdoctoral
thesis: pharmacological injection of a GABAA agonist or antagonist, deep brain
stimulation,highandlowintensitytranscranialmagneticstimulation(clockwisefrom
top left). The left brain image shows the medial prefrontal cortex target region
overlaid with a recording of some motor evoked potentials after high intensity
transcranialmagneticstimulation.Therightbrainimageshowsaclusterofsignificant
hypermetabolism after deep brain stimulation at 60 Hz. The background image
zoomsinonatransversesliceofafused[18F]-FDGPETandMRimageofaratbrain.
Coverdesign:AnitaMuys,NieuweMediaDienst,UniversityofAntwerp
Print:D.ProvoNV,Brulens23B,2275Gierle
©JokeParthoens,Antwerp,2016.Nopartofthisbookmaybereproduced,storedin
a retrieval system or transmitted in any form or by any means, electronical,
mechanical,photocopying, recording,orotherwise,without thepriorpermissionof
theholderofthecopyright.
FacultyofMedicineandHealthSciences
Molecularimagingtoquantifyneuromodulationofthe
medialprefrontalcortexintherat
Moleculairebeeldvormingvoordekwantificatievan
neuromodulatievandemedialeprefrontalecortexinderat
Proefschriftvoorgelegdtothetbehalenvandegraadvan
DoctorindeMedischeWetenschappenaandeUniversiteitvanAntwerpente
verdedigendoor:
JokePARTHOENS
Promotoren: Prof.dr.JeroenVerhaeghe
Prof.dr.StevenStaelens
Antwerpen,2mei2016
MEMBERSOFTHEJURY
INTERNALJURYMEMBERS
Prof.dr.P.VandeHeyning(FacultyofMedicineandHealthSciences;Chair)Prof.dr.A.VanderLinden(FacultyofPharmacyandBiomedicalSciences)
EXTERNALJURYMEMBERS
Prof.dr.C.Baeken(FacultyofMedicine,UGent)Prof.dr.I.Smolders(FacultyofMedicineandPharmacy,VUB)
TableofContents
TableofContents..........................................................................................i
Acknowledgements......................................................................................v
Abbreviations.............................................................................................vii
Chapter1:Introduction..............................................................................11.1 Introduction...............................................................................................21.2 Neurostimulationtechniques.....................................................................3
1.2.1 TranscranialMagneticStimulation...........................................................41.2.1.1 PrinciplesofTMS...........................................................................................41.2.1.2 ClinicalapplicationsofrTMStargetingthedlPFC........................................111.2.1.3 SmallanimalTMS........................................................................................14
1.2.2 Deepbrainstimulation...........................................................................161.2.3 Pharmacologicalinjections.....................................................................18
1.3 PositronEmissionTomography.................................................................181.3.1 PrinciplesofPET......................................................................................181.3.2 [18F]-FDG,glucosemetabolismandlocalbrainfunction........................191.3.3 PETneuroreceptorimaging....................................................................201.3.4 SmallanimalPET.....................................................................................211.3.5 Quantification.........................................................................................22
1.4 CombinedrTMSandPET...........................................................................241.5 RepetitiveTMScombinedwithotherimagingtechniques........................28
1.5.1 Introduction............................................................................................281.5.2 SinglePhotonEmissionComputedTomography....................................281.5.3 FunctionalMagneticResonanceImaging...............................................32
Chapter2:Objectives................................................................................37
Chapter3:PrelimbiccorticalinjectionsofGABAagonistandantagonist:In
vivoquantificationoftheeffectintheratbrainusing[18F]-FDGmicroPET..433.1 Abstract....................................................................................................443.2 Introduction.............................................................................................443.3 MaterialsandMethods............................................................................47
Tablecontents
ii
3.3.1 Animals...................................................................................................473.3.2 Cannulaplacement.................................................................................473.3.3 Habituationperiod..................................................................................483.3.4 Microinjections.......................................................................................483.3.5 MicroPET-CTimaging..............................................................................483.3.6 Histologicalverificationofthecannulaposition....................................503.3.7 Imageanalysis.........................................................................................51
3.4 Results......................................................................................................523.4.1 Histology.................................................................................................523.4.2 VOI-basedanalysis..................................................................................533.4.3 Voxel-basedSPManalysis.......................................................................56
3.5 Discussion.................................................................................................573.6 Conclusion................................................................................................61
Chapter4:Deepbrainstimulationoftheprelimbicmedialprefrontalcortex:
quantificationoftheeffectonglucosemetabolismintheratbrainusing
[18F]-FDGmicroPET....................................................................................634.1 Abstract....................................................................................................644.2 Introduction.............................................................................................644.3 MaterialsandMethods.............................................................................66
4.3.1 Animals...................................................................................................664.3.2 Surgicalprocedure..................................................................................674.3.3 Deepbrainstimulation...........................................................................674.3.4 MicroPET-CTimaging..............................................................................684.3.5 Histologicalverificationoftheelectrodeposition..................................694.3.6 Dataanalysis...........................................................................................69
4.4 Results......................................................................................................714.4.1 Histology.................................................................................................714.4.2 VOI-basedanalysis..................................................................................734.4.3 Voxel-basedSPManalysis.......................................................................74
4.5 Discussion.................................................................................................764.6 Conclusion................................................................................................79
Chapter5:SmallanimalrepetitiveTranscranialMagneticStimulation
combinedwith[18F]-FDGmicroPETtoquantifytheneuromodulationeffect
intheratbrain...........................................................................................815.1 Abstract....................................................................................................82
Tableofcontents
iii
5.2 Introduction.............................................................................................835.3 MaterialsandMethods............................................................................84
5.3.1 Animals...................................................................................................845.3.2 RepetitiveTranscranialMagneticStimulation........................................865.3.3 MicroPET-CTimaging..............................................................................875.3.4 Imageanalysis.........................................................................................88
5.4 Results.....................................................................................................895.5 Discussion................................................................................................915.6 Conclusion................................................................................................95
Chapter6:Performancecharacterizationofanactivelycooledrepetitive
TranscranialMagneticStimulationcoilfortherat......................................976.1 Abstract....................................................................................................986.2 Introduction.............................................................................................996.3 MaterialsandMethods..........................................................................100
6.3.1 RatTMSsetup.......................................................................................1006.3.1.1 Electricfieldcalculations...........................................................................1026.3.1.2 Coolingperformanceevaluation...............................................................103
6.3.2 Animals.................................................................................................1036.3.3 Motorthresholddeterminations..........................................................103
6.3.3.1 MTdeterminationprotocol.......................................................................1036.3.3.2 EffectofcurrentdirectiononMT..............................................................1046.3.3.3 Intra-andinter-animalvariabilityofMTdeterminations..........................1056.3.3.4 Lateralityofstimulation.............................................................................105
6.3.4 PETrTMSstudy.....................................................................................1056.3.4.1 rTMSprotocols..........................................................................................1056.3.4.2 MicroPET-CTimaging.................................................................................1066.3.4.3 Imageanalysis............................................................................................107
6.4 Results...................................................................................................1086.4.1 RatTMSsetup.......................................................................................108
6.4.1.1 Electricfieldcalculation.............................................................................1086.4.1.2 Coolingperformanceevaluation...............................................................109
6.4.2 Motorthresholddeterminations..........................................................1096.4.2.1 EffectofcurrentdirectiononMT..............................................................1106.4.2.2 Intra-andinter-animalvariabilityofMTdeterminations..........................1116.4.2.3 Lateralityofstimulation.............................................................................112
6.4.3 MicroPETrTMS.....................................................................................1136.4.3.1 VOI-basedanalysis.....................................................................................1136.4.3.2 Voxel-basedanalysis..................................................................................114
6.5 Discussion..............................................................................................115
Tablecontents
iv
6.5.1 Intensityoftheelectricfieldandmotorthreshold...............................1156.5.2 Focalityoftheelectricfielddistribution...............................................1166.5.3 [18F]-FDGPETrTMSstudy.....................................................................117
6.6 Conclusion..............................................................................................118
Chapter7:Generaldiscussionandfutureperspectives............................1217.1 MajorFindingsfromthe[18F]-FDG-PETstudies.......................................122
7.1.1 Pharmacologicalstimulation................................................................1227.1.1.1 Mainfindings.............................................................................................1227.1.1.2 Mechanismofaction..................................................................................122
7.1.2 DeepBrainStimulation.........................................................................1237.1.2.1 Mainfindings.............................................................................................1237.1.2.2 Mechanismofaction..................................................................................123
7.1.3 RepetitiveTranscranialMagneticStimulation......................................1247.1.3.1 Mainfindings.............................................................................................1247.1.3.2 Mechanismofaction..................................................................................125
7.1.4 Directionalityanddistributionoftheresponse....................................1267.1.4.1 Directionality..............................................................................................1267.1.4.2 Distribution................................................................................................127
7.1.5 Interpretationof[18F]-FDG-PETdata....................................................1287.2 MajorachievementsandshortcomingsofthenewratrTMScoils...........129
7.2.1 Stimulationfocalityandintensity.........................................................1297.2.2 Positioningandanesthesia...................................................................1307.2.3 Shamstimulation..................................................................................131
7.3 Futureperspectives................................................................................131
Chapter8:Summary...............................................................................135
Chapter9:Samenvatting.........................................................................141
Chapter10:Listofpublications...............................................................147
Chapter11:References...........................................................................151
Acknowledgements
Dit doctoraatsproject was nooit tot stand gekomen zonder de hulp van een hele
hoopmensendieachtermestonden.Graagzouikhieriedereenwillenbedankendie
medeafgelopenjarengeholpenheeft.
EerstenvooralwilikmijnpromotorenProf.dr.JeroenVerhaegheenProf.dr.Steven
StaelensalsookProf.dr.SigridStroobantsoprechtbedankenommedekanstegeven
ditonderzoektoteengoedeindetebrengen.Ikbedankjullieookvoordehulpbijde
data-analyseenhetschrijvenvandeartikels.
Jeroen,bedanktvoorallehulp,voorhetgeduldenomsteedsweertijdvoormijvrij
temakeninjedrukkeagenda!
Ookwilikgraagdr.TineWyckhuysbedankenvoordebegeleiding,hetvertrouwenen
deleukebabbelsinhetlaboendaarbuiten.
Dankgaatookuitnaaralleledenvandedoctoraatscommissie,Prof.dr.PaulVande
Heyning,Prof.dr.AnnemieVanderLinden,Prof.dr. IlseSmoldersenProf.dr.Chris
Baeken.
DitprojectwasnietgeluktzonderdehulpvanStijnServaes,AlanMiranda,Philippe
Joye,dr.StevenDeleyeenonzecollega’svanderadiofarmacie.Bedanktvooraljullie
hulp en expertise! Stijn, bedankt voor de behulpzaamheid tijdens je thesisjaar en
daarna (de slag opmijn neus is je trouwens al lang vergeven). Alan, thanks for all
yourhelpwiththecalculationsandforyoursupport.Philippe,bedanktvoordeleuke
momenten,degrapjesenvoorhetdelenvanjeexpertise.Dr.Stievie,naastaljehulp
metdescanners,PMODenSPMwilikjebedankenvoordeveleleukemomentendie
we de afgelopen jaren hebben beleefd. Leonie, bedankt om me zo vaak (en met
zoveelgeduld)dingenuitteleggendieikandersnooitgesnaptzouhebben.
Acknowledgements
vi
Ookmijn andere collega’s (Sven,Ann-Marie, Tina,Christel, Lauren, Yanina, Saraen
Caroline)enthesisstudenten(Julia,VikienStijn),wilikenormbedankenvoordevele
leuke momenten in en rond het labo! Sven, mijn afwasbuddy, bedankt voor alle
steun.Ann-Marie,I’venevermetsomeonewithsuchagreatsenseofhumor!Thanks
for the hugs and laughs. Tina, ook jouw hulp heb ik enorm gewaardeerd. Bedankt
voordebestellingenenomaltijdallesvoormeuittezoeken.
Henrik, Anders and Bjørn, thank you very much for the nice cooperation and for
answering all my questions! Pieter-Jan, bedankt om de samenwerking met
MagVentureingoedebanenteleiden.
Ook wil ik graag iedereen van TNW en het Bio-Imaging Lab bedanken die me de
afgelopenjarengeholpenheeft,inhetbijzonderProf.dr.StefanieDedeurwaerdere,
Halima,Johan,ElisabethenCaroline.
TenslottebedankikgraagookWannesenTisse.Wannes,bedanktomtelkensweer
voor100%achteralmijnbeslissingentestaanenomeraltijdvoormetezijn.Tisse,
kleinekapoen,ookzonderjouwasditnooitgelukt.
Abbreviations5-CRTT
5-HT
[14C]-DG
[18F]-FDG
[99mTc]-HMPAO
%ID
%MO
µPET
AC
ANOVA
AP
B-field
CCW
Cg
contra
COV
CP
CT
cTBS
CW
DBS
dlPFC
DV
E-field
EC
ECT
EMG
five-choiceserialreactiontimetask
5-hydroxytryptamineorserotonin
[14C]-deoxyglucose
2-deoxy-2-(18F)fluoro-D-glucose99mTc-hexamethylpropyleneamineoxime
Percentageoftheinjecteddose
PercentageofthemaximumMO
SmallanimalPositronEmissionTomography
Activityconcentration
AnalysisOfVariance
Antero-posterior
Magneticfield
Counterclockwise
Cingulatecortex
Contralateral
CoefficientofVariation
CaudatePutamen
ComputedTomography
ContinuousThetaBurstStimulation
Clockwise
DeepBrainStimulation
DorsolateralPrefrontalCortex
Dorso-ventral
Electricfield
EntorhinalCortex
ElectroconvulsiveTherapy
Electromyography
Abbreviations
viii
FDA
fMRI
FOV
FWHD
GABAA
HIPad
Hyp
ID
IL
imTBS
Ipsi
iTBS
LTD
LTP
MAP-TR
MAR
Med
MEP
ML
MO
mPFC
MRI
MT
MThigh
MTlow
NMDA
OCD
OFC
OSEM
FoodandDrugAdministration
functionalMagneticResonanceImaging
Fieldofview
Full-Width-at-Half-Maximum
γ-aminobutyricacidtypeA
Anterodorsalpartofhippocampus
Hypothalamus
InjectedDose
Infralimbic
IntermediateThetaBurstStimulation
Ipsilateral
IntermittentThetaBurstStimulation
Long-termdepression
Long-termpotentiation
MaximumAPosteriori–Transmission
Metalartifactreduction
Medulla
MotorEvokedPotential
Medio-lateral
MachineOutput
MedialPrefrontalCortex
MagneticResonanceImaging
MotorThreshold
UpperMT
LowerMT
N-Methyl-D-aspartate
Obsessive-CompulsiveDisorder
OrbitofrontalCortex
Orderedsubsetexpectationmaximization
Abbreviations
ix
PAC
PET
PFC
PL
QPS
rCMRglc
RsplC
rTMS
SD
Se
SEM
SPECT
SPM
SupC
TBS
tDCS
TES
TMS
ParietalAssociationCortex
PositronEmissionTomography
PrefrontalCortex
Prelimbic
QuadripulseStimulation
RegionalCerebralMetabolicRateofglucose
RetrosplenialCortex
RepetitiveTranscranialMagneticStimulation
StandardDeviation
Septum
StandardErroroftheMean
SinglePhotonEmissionTomography
StatisticalParametricMapping
SuperiorColliculus
ThetaBurstStimulation
TranscranialDirectCurrentStimulation
TranscranialElectricalStimulation
TranscranialMagneticStimulation
VC
VOI
VisualCortex
Volume-Of-Interest
VTA
WB
VentralTegmentalArea
WholeBrain
Abbreviations
x
Chapter1:
Introduction
Chapter1
2
1.1 Introduction
Forthepastcenturyelectricalstimulationofthebrainisbeingusedincreasinglyasa
therapeutic tool to treat neurological and psychiatric disorders. The use of one of
these promising neurostimulation techniques, Transcranial Magnetic Stimulation
(TMS), has shown an exponential growth since its first description in 1985 (Barker
andJalinous1985).Itspopularitystemsprimarilyfromitsnon-invasivenatureandits
relativelyeaseofapplication.Theadministrationofatrainofpulses(repetitiveTMS
orrTMS)hasbeenshowntohavelong-lastingtherapeuticeffectsonneurophysiology
and behaviour. Since 2008, rTMS of the dorsolateral prefrontal cortex (dlPFC) has
been approved as a treatment of refractory depression by the Food and Drug
Administration.
To date, despite the success of rTMS in the clinic and extensive research, no
consensus has been reached on the exact mechanism of action and optimal
stimulation targets and parameters for the various disorders that can or could be
treatedwith rTMS.PositronEmissionTomography (PET)aswellasother functional
imaging techniques allow non-invasive visualization of the direct and long-term
rTMS-inducedneurophysiologicaleffectsthroughoutthebrainanditsnetworks,and
canthusofferrevealinginsightsintotheworkingofneuromodulationtechniques.
InordertolongitudinallytestrTMSparameters,ethicalconsiderationsaswellasthe
need for large homogeneous subject populations render small animal research
indispensable.However,theuseofexistingTMScoilsdesignedforstimulationofthe
human brain in small animal research is hampered by the obvious dimension
differences and this has limited the translation from small animal experimental
results to the clinic. Until recently, no dedicated miniaturized TMS coil suited for
smallanimalreseachwascommerciallyavailable.
In thisdoctoraldissertationweassess theeffectof stimulationof the ratprelimbic
(PL) region of themedial Prefrontal Cortex (mPFC) using several neuromodulation
techiques by visualizing regional changes in rat cerebral glucose metabolism with
small animal PET (microPET or µPET). In particular, we have considered
Introduction
3
pharmacological stimulation using intracranial injections of a GABA agonist or
antagonist(chapter3),DeepBrainStimulation(DBS)(chapter4),andrTMS(chapters
5 and 6). In order to overcome the limitations of large TMS coils in small animal
researchwehavedevellopedtwosmallTMScoilsfordedicatedstimulationoftherat
brain.ThesecoilswereusedinourcombinedrTMSandµPETexperiments.
This introductory chapter gives a short overview of the used neurostimuation
techniques: rTMS, DBS and intracranial pharmacological stimulation; as well as an
introductiontoinvivobrainimagingtechniques,inparticularPETimaging.Finallyan
overviewisgivenofpreviouscombinedclinicaldlPFCrTMSandimagingexperiments.
1.2 Neurostimulationtechniques
Electrical neurostimulation is performed by administering electrical pulses to the
central or peripheral nervous system in order to activate or inhibit the target
structure.Theearliestdocumentationoftherapeuticuseofelectricalstimulationwas
reportedaround47ADby thephysicianScriboniusLargus.Hediscoveredthatpain
causedbygoutcouldberelievedbystandingonaliveelectricrayor“torpedofish”
(ScriboniusLargus.DecompositionemedicamentorumLiber,CLXIIin(Stillings1975)).
Electricstimulationofthebraintotreatpsychiatricdisorderswasfirstintroducedby
dr.Cerlettianddr.Biniin1938aselectroshocktherapyorelectroconvulsivetherapy
(ECT) (Adams 2015). ECT involves inducing brain seizures under controlled
circumstancesbyadministeringshortelectricalpulsesthroughelectrodesattachedto
thescalp.AlthoughthisECThascausedmuchcontroversyduetosevereside-effects,
badpublicity,misusageorevenabuse(Adams2015),thetechniqueisnowadaysfine-
tuned to a safe and efficient treatment for unipolar and bipolarmajor depression
(Dierckx et al 2012), mania (Malhi et al 2012) and catatonia (Kugler et al 2015).
Currentlythereareanumberofalternativeelectricalneurostimulationmethodsthat
are used in clinical practice such as DBS, first used in the 1970’s (Hosobuchi et al
1973) and TMS, presented by Barker and colleagues in 1985 (Barker and Jalinous
1985).
Chapter1
4
Apart from using electrical stimulation, one can also directly activate or inhibit a
targeted brain region with an intracranial injection of a pharmacological agent.
Altough not used in clinical practice, this technique is often used in preclinical
neuroscienceresearchtoinvestigatebrainfunctionanditsnetworks.
1.2.1 TranscranialMagneticStimulation
TMSisanon-invasiveneurostimulationtechniquethatusesanelectricalcurrentina
coiltogeneratearapidelychangingmagneticfieldtoinduceachangingelectricfield
in the brain. Since the description of the first successful TMS stimulation of the
humanmotorcortexin1985(BarkerandJalinous1985),thistechniquehasevolved
fromabasic research tool forneurophysiologists toanon-invaseneuromodulation
toolfortheeffectivetreatmentofavarietyofneurologicalandpsychiatricdisorders.
1.2.1.1 PrinciplesofTMS
TechnicalaspectsofTMS
ThebasicprincipleofTMSisbasedonFaraday’slawonelectromagneticinduction.A
time-varyingcurrentflowinginthecoilgivesrisetoatime-varyingmagneticfield(B-
field),whichwillinturninduceanelectricfield(E-field).Whenthecoilisheldcloseto
thehead, the induced variabelemagnetic field is able topenetrate the scalp, skull
andmeninges,toeventuallyinduceanelectricfieldinthebrain(Figure1.1).
Introduction
5
Figure1.1Transcranialmagneticstimulationwithfigure-of-eightcoil(adaptedfrom(RiddingandRothwell2007)).
The standard TMS apparatus consists of a stimulator, with one or more large
capacitors, that is connectedwith an insulated electric cable to a copper coil in a
plasticcasing (Figure1.2).Whenthecapacitorsdischarge,avery largetimevarying
electriccurrentpulse(usuallyasinewavelastinglessthan1ms)isgeneratedwitha
peakcurrentthatcanreachvaluesofover5000A(Imax).Theelectricalcurrentgives
risetostrongtimevaryingB-fieldsrangingfrom1–4T(Ampère’slaw)whichinturn
inducesanE-field.AccordingtoFaraday’s law,thestrengthoftheinducedE-fieldis
proportionaltotherateofchangeoftheB-field,whichisproportionaltotherateof
change of the current through the coil (dI/dt). TMS requires very strong and brief
currentpulses tomaximize the induedE field. Indeed, themaximumvalueofdI/dt
(and thus of the induced E-field) depends on the period of the pulse,which is the
inverse of the oscillation frequency (f), and on themaximum value of the current
(Imax)accordingtothefollowingequation:
dIdt = 2πfI!"#
This value also depends on the inductance of the coil (L) and the stimulator’s
capacitor’schargingvoltage(V):
Chapter1
6
dIdt =
VL
Withinductancesofabout10-25µHandchargingvoltagesupto2000V,E-fieldsof
more than 100 V/m can be induced in the brain. Another important factor that
determines the strength of the induced E-field is the distance to the coil, with
decreasing magnitude at larger distances away from the coil. However, the exact
spatialdistributionoftheE-fielddependsalsoonbiologicalparameterssuchasthe
structure of the underlying brain tissue (see below Neural interactions) as well as
physicalparameters suchas coilorientationand shape.Althoughmany coil shapes
have been described (for an overview see (Denget al 2013)), themost commonly
used shapes are the circular and the figure-of-eight coil (Kobayashi and Pascual-
Leone 2003, Rossiet al 2009). The first report on TMSof the humanbrain used a
circularcoil((BarkerandJalinous1985),Figure1.2),whichhasthesimplestgeometry
andhenceistheeasiesttoconstruct.Itcanstimulatedeeperbrainregionscompared
to the figure-of-eight coil, but has a rather unfocal E-field maximum (Deng et al
2013),therebystimulatingalargervolumeofbraintissue.
Figure1.2ThefirstTMSsystem,describedbyBarkeretal.in1985(BarkerandJalinous1985).
Introduction
7
Withtheintroductionoffigure-of-eightcoils,thefocalityofTMScouldbeincreased.
Thiscoiltypeconsistofapairofadjacentcircularcoilswithacurrentflowinopposite
directions(Figure1.3),producingamorefocalE-fieldmaximumunderthecenterof
thecoilwherethetwoloopsmeet(Dengetal2013).
Figure1.3TheMagstimfigure-of-eight70mmcoil,oneofthemostfrequentlyusedcoiltypesinscientificliterature(www.magstim.com).Morerecently,Hesed(H)coilsdesignedtostimulatedeeperbrainareas(Rothetal
2002)attheexpenseofreducedfocality(Dengetal2013)havebeenintroduced.
Neuralinteractions
The induced E-field causes changes in ion flow and subsequently changes in
membranepotentials in the cortical tissueunderneath the coil, causingneurons to
depolarize or hyperpolarize (Rossi et al 2009). Even at high intensities, TMS will
primarilyexciteaxonswithinthecortex,becausethestrengthoftheB-fieldinduced
by the coil falls off very rapidly with increasing distance from the coil. However,
distant brain regions might be stimulated directly through axonal projections or
indirectlythroughfunctionallyconnectedbrainnetworks(Siebneretal2009b).
Even though the B-field can pass through the scalp, cranial bone, meninges and
cerebrospinalfluidwithnegligibleattenuation,thedistributionoftheinducedE-field
is greatly determined by the shape, arrangement and network conductivity of the
cervical tissue. Experimental evidence and theoretical calculation indicate that the
threshold for neuronal stimulation is lower when the E-field points along the
directionofanaxonthatterminatesorbendsandthataxonswith largerdiameters
Chapter1
8
areexpectedtobestimulatedatalowerintensity(Rossietal2009).Additionally,the
inducedcurrentdoesnotnecessarilyaffectregionsdirectlybelowthecoilandhence
might preferentially flow through areas with a higher conductance, such as
cerebrospinal fluid (Wassermann and Zimmermann 2012). This greatly hinders our
precise understanding of the impact of TMS on the brain, because it is difficult to
accuratelypinpointwhichanatomicalstructuresarestimulatedbytheE-field.
SinglepulseTMSandMotorEvokedPotentials
When a single TMS pulse above a certain threshold intensity is applied over the
motor cortex a motor evoked potential (MEP) is generated in the contralateral
extremity muscles (Barker and Jalinous 1985). These MEPs can be recorded by
electromyographic (EMG) measurements by placing a surface electrode over the
muscle. Theamplitudeof thisMEP canbe seenas ameasureof excitabilityof the
stimulated motor neurons (Rossi et al 2009). The threshold intensity needed to
provokeaMEPofapredefinedamplitudeisreferredtoasthemotorthreshold(MT).
TheMTisusuallydefinedasthelowestTMSintensitythatisneededtoelicitMEPsof
50 µV peak-to-peak amplitude in a rested or activated musle in at least 50% of
successive TMS pulses (Fitzgerald andDaskalakis 2012). TheMT gives thus also an
indication of themembrane excitability of corticospinal neurons and interneurons
thatprojectontothemotorcortex.TheTMSstimulationintensityandMTareusually
expressed as a percentage of the maximal machine output (MO) of the TMS
stimulator.
Patients with abnormal MEPs might have a dysfunction at any level along the
corticospinal tract or have a centralmotor conduction failure. For example, it has
beenshownthattheMTisraisedinsomecasesofmigraine(MaertensdeNoordhout
etal1992),multiplesclerosis(Bonifaceetal1991)andepilepsy(Reutensetal1993).
Paired-pulseTMS
Withpaired-pulsestimulation,theTMSstimuliaredeliveredinpairswithinavariable
timeinterval.Theresponseelicitedbyasingletestpulse,administeredtothemotor
Introduction
9
cortex or visual cortex (that is, the MEP or phosphene, respectively) can be
modulatedbytheapplicationofaprecedingconditioningpulse in thesameor ina
connected brain region (Hampson and Hoffman 2010). This technique allows
investigators to examine excitatory and inhibitory processes within the cortex
(WassermannandZimmermann2012,Bunseetal2014).
RepetitiveTMS
When lasting inhibitoryor facilitatorytherapeuticeffectsaredesired,rTMS isused.
Thistechniqueinvolvestheadministrationoftrainsofpulseswiththesameintensity,
deliveredtothetargetbrainareaatagivenstimulationfrequency.Accordingtothe
frequency and pattern of the delivered pulses, different types of rTMS can be
distinguished.Stimulationatalowfrequencyof≤1Hzisusuallyadministeredasone
continuous train, whereas high frequency rTMS of > 1 Hz is either applied
continuouslyorintrainsofseveralpulseswithafixedintertraininterval.Apartfrom
the conventional low and high frequency rTMS protocols, rTMS can also be
administeredasrepetitiveapplicationofshortrTMSburstsatahighinnerfrequency
interleavedbyshortpausesofnostimulation.ThispatternedrTMSprotocolismostly
usedasthetaburststimulation(TBS)inwhichshortburstsof50Hzarerepeatedata
rateinthethetarange(5Hz)asacontinuous(cTBS)orintermittent(iTBS)train(Rossi
etal2009)(Figure1.4).
Chapter1
10
Figure1.4ExamplesofrTMStypes.Leftpanel(ConventionalrTMS).Fromthetop:examplesof 10 s of rTMS at 1Hz and at 5Hz; 1 s of rTMS at 10Hz and a typical example of 20Hzapplicationfortherapeuticpurposes(trainsof2sinterleavedbyapauseof28s).Rightpanel(Patterned rTMS). From the top: 20 s of continuous theta burst (cTBS); intermittent thetaburst (iTBS); intermediate theta burst (imTBS) and protocols of quadripulse stimulations(QPS).(Reproducedfrom(Rossietal2009)).
In general, it is believed that the stimulation frequency and pattern are themost
important factors determining the neurophysiological response to rTMS. Lasting
inhibitoryeffectsfollowinglowfrequencyrTMSandcTBSandfacilitatoryeffectsafter
highfrequencyrTMSandiTBSofthemotorcortexhavebeenobservedasmeasured
byEMGinhealthysubjects(Rossietal2009). Importantly,thisdichotomymightbe
anoversimplification,becausetheneteffectofrTMSoncorticalexcitabilitywillalso
beinfluencedbythetypeofneuronsthatarestimulatedandbythebasicexcitability
levelof theseneurons.Forexample,rTMSmightexcite inhibitory interneuronsand
therebyhaveanindirectinhibitoryeffectonremotebrainareas(Pausetal1998).
Other rTMS parameters that could potentially alter the neurophysiological and
behaviouralresponsearethestimulationintensity,thecoilsizeandshape,thepulse
lengthandthewaveformoftheindividualpulses.
Introduction
11
Toensureeffectivenessandsafety,theintensityordosageiscommonlydetermined
individually for each patient by first determining the patient’sMT, which gives an
estimateofthepatient’scorticalexcitabilityaspreviouslyexplained.The intensities
most frequentlyused in clinical practice correspond to90-120%of themearsured
restingMT(Fitzgeraldetal2006).
Apart from these technical parameters the responsemight alsobe affectedby the
durationoftherTMSsession(usually10-40minutes), thenumberofrTMSsessions
(usullydailyfor2toseveralweeks)and,ofcourse,thebrainregionthatistargeted
bythestimulation.ThebraintargetisdeterminedbytheE-fielddistributionandthe
coil position and orientation with respect to the subject’s head. For EMG
measurements, the motor cortex is targeted, whereas for the treatment of
movementdisorderssuchasParkinson’sdisease,thesiteofstimulationisusuallythe
motor cortex or the supplementary motor area (Vadalà et al 2015) and for the
treatmentofepilepsythecoilispositionedabovetheepilepticfocus(Hsuetal2011).
Fortherapeuticuseinpsychiatricdisorders,thedorsolateralprefrontalcortex(dlPFC)
ismostfrequentlythestimulationtargetofchoice.
1.2.1.2 ClinicalapplicationsofrTMStargetingthedlPFC
RepetitiveTMShastheadvantagethatbrainactivityinaspecificcortico-subcortical
networkcanbemodulatednon-invasively,withneurophysiologicalandbehavioural
effects outlasting the period of stimulation. This makes it a potential therapeutic
alternative to treatneurologicalandpsychiatricdisorders thatarecharacterizedby
altered cortical excitability or disregulated interactions between cortical and
subcorticalstructures.
InOctober2008,leftdlPFCrTMSwasapprovedbytheFoodandDrugAdministration
forthetreatmentofmajordepression(GeorgeandPost2011)anditisbeingusedas
apromisingexperimentaltreatmentforawidevarietyofotherpsychiatricdisorders
such as schizophrenia (for review see (Hovington et al 2013)), drug addiction (for
reviewsee(Protasioetal2015))andanxietydisorders(forreviewsee(Machadoetal
2012)).
Chapter1
12
ThedlPFCisinvolvedinthecontrolofexecutivefunctions,whichincludethecontrol
of attention,motorplanning, decisionmaking, goal-directedbehaviour,monitoring
of current internal and external states, episodic memory retrieval and complex
cognitive behaviour (Miller 2000, Miller et al 2002, Jones et al 2011). It controls
subcortical regions of the reward circuit through connections with the ventral
tegmentalareaandthenucleusaccumbens(Ballardetal2011)andisinvolvedinthe
regulationofstriataldopaminerelease (Koetal2008).Dueto its importantrole in
behaviour, a dysfunction in this region and the related circuitry is believed to be
associatedwith a broad rangeof psychiatric disorders. Indeed, a variety of studies
have shown prefrontal abnormalities, predominantly in the left hemisphere, in
patientswithunipolardepression(Drevetsetal2008),schizophrenia(Hovingtonetal
2013) and addiction (Protasio et al 2015). This frontal dysfunction results in a
disturbance in the regulation of stress hormones as wel as the reward system
(Mayberg et al 2005), most likely leading to the apathy, psychomotor slowness
and/or impaired executive functioning and decision making associated with these
diseases(BaekenandDeRaedt2011).
Therefore,inthetreatmentofthesedisorders,highfrequencyrTMS(10Hzor20Hz)
isappliedtothe leftdlPFC inordertoactivatethishypofunctionalbrainregionand
its subcortical connections. These experimental treatments have shown promising
therapeutic results, for example high frequency rTMS applied to the left dlPFC
improvesmood in depression (Baeken et al 2013, Speeret al 2014), reduces drug
craving (Jansenet al 2013) anddiminishes thenegative symptoms accociatedwith
schizophrenia (Hovington et al 2013), possibly through an upregulation of the
serotonin (Baeken et al 2011) and dopamine (Kanno et al 2004) system. These
beneficial effects of rTMS on behaviour have been reported to outlast the
stimulationperiodupto6monthsafterthetreatment(Janicaketal2010),however
larger scale studies are needed to validate these long-lasting effects (George and
Post2011).
Introduction
13
Inaddition,studiesassessingthepotentiallastingnegativeeffectsareneeded.Until
now, no reports of lasting cognitive, neurological or cardiovascular adverse effects
havebeenmade,butresearchonthistopicremainsscarce.Themostcommonside
effect of rTMS is transient headache caused by the stimulation of nerves and
muscles,whichcanbesolvedbymildanalgesics (Rossietal2009).Further,hearing
might be affected for several hours after the treatmentdue to the clicking sounds
the coil makes during the stimulation. Therefore subjects are fitted with earplugs
during stimulation (O’Reardon et al 2007). Themost serious safety concern is the
induction of a seizure during high frequency stimulation. Only a few cases of
accidental seizure induction have been reported so far, most of which occurred
before the introduction of general safety guidelines in rTMS practice and/or in
patientswithaloweredthresholdforseizureinduction(Walletal2014).
The promising therapeutic effects of rTMS in various disorders, its non-invasive
nature as well as the minimal side effects have attracted the interest of many
researchersactively investigatingthistechnique.Yettheexactmechanismofaction
ofrTMSremainslargelyunknown,hamperingthesearchfortheoptimalstimulation
parameters for treatment of the wide range of diseases that can possibly benefit
fromrTMS.Optimalsettingsforparameterssuchasstimulationintensity,frequency,
dosage,dosingschedule,coilshapeandpositioningwilllikelyvarydependingonthe
targetofstimulationandthetypeofpsychiatricorneurologicalapplication(Mozeg
andFlak1999). Therefore, formanyof thesedisorders, large-scale,double-blinded
studies are still needed. However, because these large-scale rTMS experiments in
humansaredifficult to realizedue toethical considerationsand theneed for large
and homogeneous patient groups, preclinical small animal studies can offer a
valuable contribution to the development and standardisation of effective
therapeutic treatments, elucidationof themechanismof action and assessmentof
potentiallastingadverseeffects(Vahabzadeh-Haghetal2012).
Chapter1
14
Finally,beforediscussingdevelopments incoilminiaturizationforsmallanimalTMS
and the accompanying technical difficulties, one important general aspect in rTMS
research, sham stimulation, needs to be introduced. Sham stimulation is used in
blindedstudiesasacontrolconditiontoruleoutpossibleplaceboeffects.Forsham
stimulation a specifically designed sham coil is needed that looks exactly like a
normal TMScoil and thatproduces the sameclicking soundsandevokes the same
headmuscle contractionswithout producing the accompanying E-field in thebrain
thatcancauseneuronstodepolarize.Sofar,mostrTMSstudieslackedthis“perfect”
sham stimulation and instead used no control group or a control group that was
either not stimulated (e.g. comparing rTMS to baseline (Horacek et al 2007)),
stimulated at a very low intensity (Muller et al 2014) or with the coil held
perpendicularytothehead,sothatthemagneticfieldwouldnotpenetratetheskull
(vanderWerfetal2010).
1.2.1.3 SmallanimalTMS
SincethefirstTMSstudyinrodentsin1990(Ravnborgetal1990),therehasbeenan
exponential increase in the number of rat TMS studies (Vahabzadeh-Hagh et al
2012).TranslationofTMS to rodentmodelshasallowedmoremechanistic insights
intoTMS-derivedmeasuresandrTMS-mediatedchangesincorticalfunction(Muller
et al 2014) at the synaptic (Gersner et al 2011, Vahabzadeh-Hagh et al 2011) and
molecular(Wyckhuysetal2013,Löffleretal2012)levels.rTMShasbeensuccesfully
applied in controlleddisease ratmodels of e.g. epilepsy (Yadollahpouret al 2014),
Parkinson’sdisease(Leeetal2013)orstroke(Shinetal2008).
Foreffectivetranslationofthesepreclinicalresultsinrodentstotheclinic,thereare
severalconcernsthatneedtobeaddressed.Firstly, rTMS inpsychiatricdisorders is
mostly applied to the dlPFC, a region that is unique to primates. However, the
functionalandanatomicalpropertiesattributedtothedlPFCcanalsobefoundinthe
ratPLregionofthemPFC(Uylingsetal2003),makingtheratasuitableanimalmodel
toinvestigatedlPFCrTMSthroughstimulationoftheratPLmPFCregion.ThePLand
infralimbic(IL)regionsoftheratmPFCareshowninFigure1.5.
Introduction
15
Figure1.5VolumerenderingofanMRimageoftheratbrainwiththeprelimbic(green)andinfralimbic(blue)regionofthemPFChighlighted;a)sideviewandb)topview.
Secondly, the use of anesthesia, required for immobilization of the rat, during
stimulation in rats might affect the cortical excitability. This difficulty can be
overcome by investigating the effect of each anesthetic on rTMS effects or by
performingrTMSinawakerats,usingproperrestrainingtechniquesforreproducible
coilplacement(Vahabzadeh-Haghetal2011,Wyckhuysetal2013).
Technologically,themostchallengingobstacle inrodentTMSisthesizeoftheTMS
coil inrelationtothesizeoftheanimal’shead.Until recently,nodedicatedratcoil
wascommerciallyavailable.AsaresultTMScoilsforstimulationofthehumanbrain
have been used in rat research. The much larger coil-to-head size ratio in these
experiments(Figure1.6)leadstodifficulttranslationoftheresultsbacktotheclinic,
because thestimulation in the ratbrainwillbemuch less focal, i.e. simultaneously
stimulating many different brain regions, and will be able to stimulate deeper
structuresintheanimal’sbrain(Tischleretal2011).Toovercomethis,smallercoils
need to be developed. However, the greatest challenge in the development of
miniaturizedratcoilsistheexcessiveresistiveheatproductionduringeffective,high-
intensitystimulation insidethewiresofsuchasmallcoil,emphasizingtheneedfor
activedevelopmentofeffectivecoilcoolingsystems(Liebetanzetal2003).
In this work, we developed two miniaturized TMS coils in chapters 5 and 6,
respectively,andevaluatedtheireffectwith[18F]-FDG-µPET.
Chapter1
16
Figure1.6AMagVenturecircularcoilintendedforhumanuseappliedonarat’shead.
1.2.2 Deepbrainstimulation
Deepbrainstimulation involvesthe implantationofmicroelectrodes intothetarget
brainareabystereotacticsurgerytodeliverlow-currentelectricpulses(severalµAto
mA)intothesurroundingtissuewithfrequenciesusuallybetween1Hzand130Hz.
The microelectrode is connected with a subcutaneous wire to a programmable
stimulatorthatistypically implantedinthesubclavicularspace(Schieferetal2011)
(Figure1.7a)).
Figure1.7Deepbrainstimulationsysteminhumana)andratb-c).a)Themicroelectrodeisimplanted deeply within the brain and is connected to a programmable stimulator that istypicallyimplantedinthesubclavicularspace(reproducedfrom(Schieferetal2011)).b)Themicroelectrodeisimplantedintheratbrainandaconnectorisfixedonthetopofthehead.c)During stimulation the awake rat is connected to a programmable stimulator that ispositionedoutsidethecage.
Introduction
17
For the last fewdecades,DBS isbeing successfullyusedasa treatment for several
movementdisorderssuchasParkinson’sdisease,essentialtremoranddystonia(for
reviewsee(PizzolatoandMandat2012))andasapromisingexperimentaltechnique
in the treatment of a wide variety of neurological or psychiatric diseases such as
drug-resistantepilepsy (Bergey2013), chronicpain (Boccardetal 2015),obsessive-
compulsive disorder (OCD) (Greenberg et al 2010), depression (Taghva et al 2013)
andTourette syndrome (Schrocketal 2014).AlthoughDBS is usedworldwideas a
safe and established treatment of severe neurological diseases, its major
disadvantageistherequirementofaninvasivesurgicalprocedure,whichcanleadto
complications such as infection, coma, seizures or intracerebral bleeding. These
serious potential side-effects cause a controversy in the use of DBS for psychiatric
diseases(Clearyetal2015).
Compared to TMS, this technique has the advantage that it can be used to target
brainregionsthatare locateddeeper in thebrainsuchas thesubthalamicnucleus,
amygdala or the hippocampus. Additionally, DBS can be used to stimulate a very
smallareacomparedtothemuchlessfocalstimulationachievedwithTMS.
SimilartorTMS,theapplicationofDBSinthisbroadrangeofneurologicaldisordersis
possibleduetoflexibilityinstimulationparameterslikethetargetregion,stimulation
frequency and intensity. As in rTMS, the main parameter influencing the
directionalityoftheneuronalresponse,i.e.activationorinhibition,seemstobethe
frequencyof thestimulation.LowfrequencyDBS (20-60Hz) isbelievedtoenhance
corticalexcitability(Goddardetal1969)whilehighfrequencystimulation(upto130
Hz) is beliefed to reduce it (Benabid et al 1998, Wyckhuys et al 2010b). In this
doctoral thesis,wehave investigated theeffectsofhighand low frequencyDBSof
theratPLregionofthemPFC(Figure1.7b-c)).onregionalglucosemetabolicrateas
visualizedby[18F]-FDG-µPET(chapter4).
Chapter1
18
1.2.3 Pharmacologicalinjections
Anotherneuromodulation technique, althoughunlike rTMSorDBSnotused in the
clinicfortherapeuticpurposes,isthemicroinjectionofapharmacologicalsubstance
directly intothetargetbrainregion.Thistechnique iscommonlyused in laboratory
animals in order to unravel the functions of a brain region (Gilmartin et al 2012,
Yoshida et al 1997, Yan 1999). Transient neuronal activation achieved by a
microinjection of e.g. bicuculline, a GABAA antagonist that acutely blocks the
inhibitoryactionoftheGABAAreceptor(Jonesetal2011),orneuronalinactivationby
e.g. an injectionofmuscimol, aGABAAagonist (Murphyetal 2011), is traditionally
followedbybehaviouralreadoutsormicrodialysisstudies.
Despiteasubstantialamountofactivationorinhibitionstudiesinvestigatingtherole
oftheratPLmPFC,noinvivofunctionalneuroimagingstudyhavebeenconductedto
visualize the effect of this treatment on the underlying whole brain network
correlations of the PL mPFC. In this dissertation, we will use pharmacological
injections of respectively biccucilline and muscimol in the PL in combination with
[18F]-FDG-µPETtovisualizechangesinregionalbrainglucosemetabolism(chapter3).
1.3 PositronEmissionTomography
1.3.1 PrinciplesofPETPositronemissiontomographyisanimagingtechniquethathasbeenwidelyusedin
clinical nuclear medicine since the 1980s. It allows the visualization of the 3-
dimensional distribution of systemic administered radioactive marked molecules
calledradiotracersor tracers.Theradioactive labelonthemolecules isanunstable
isotope that decays to a stable energy level by emission of a positron (β+), the
antimatter of the electron. Several of these positron-emitting isotopes can be
produced using a cyclotron and often-used examples include 18F and 11C. The
emitted positron will annihilate with an electron and emit a pair of high-energy
photons(511keV).Bothphotonstravelapproximatelyonthesamestraightlinebut
Introduction
19
in different directions (angle between the two directions is 180 ± 0.5 degrees).
Becauseof theirhighenergy thephotons canescape thebody,which can thenbe
detected by the specialized detectors of the PET scanner.When two photons are
detected in coincidence (i.e.within a 6 ns coincidence interval) it is assumed they
correspondtothesamepositron-electronannihilation,fromwhichitcanbeinferred
that this annihilation process took place somewhere along the line connecting the
detectionpositionsofthetwophotons.Whenmanyofthesecoincidenceeventsare
detected and registered a tomographic image representing the distribution of the
tracer inthebodycanbereconstructed. ThegreatversatilityofPETimaginglies in
the fact that different tracers have been developed which allow us to visualize
specific processes in the body of brain. During this doctoral thesis we have
extensivelyusedthemostwidespreadPETtracer,[18F]-FDG,whichisusedtovisualize
glucosemetabolism.
1.3.2 [18F]-FDG,glucosemetabolismandlocalbrainfunction
PET using 2-deoxy-2-(18F)fluoro-D-glucose ([18F]-FDG) iswidely used as a diagnostic
toolinoncologybuthasalsoproventobeveryusefulinneurosciences.[18F]-FDGisa
glucoseanalogwiththehydroxylintheC-2positionreplacedwitharadioactivefluor-
18(18F)isotope.Likeglucose,[18F]-FDGisactivelytransportedintothecellbyglucose
transportproteins,whereitisthenphosphorylatedbyhexokinase,asthefirststepof
the glycolysis. The lack of the 2’hydroxyl group in the [18F]-FDGmolecule prevents
further metabolization by glycolysis, thereby trapping the [18F]-FDG-6-phosphate
moleculeinthecell.Therateatwhich[18F]-FDG-6-phosphateaccumulatesinthecell
is proportional to the rate at which glucose is consumed by the cell as the
phosphorylationistheratelimitingstepofglycolysis.With[18F]-FDG-PETwecanthus
measure the regional cerebral metabolic rate of glucose (rCMRglc). It has been
shown that rCMRglc is coupled to local brain function and activation primarily
throughtheclearingandrecyclingofglutamatebyglialcellsandthemaintenanceof
iongradients(Herholzetal2004).Asaresultareasinthebrainwithhigheractivity
willhaveahigher[18F]-FDGsignal.Thusthe[18F]-FDG-PETmethodcanbeusedasan
Chapter1
20
indirectmanner to study neuronal activity. An example of a normal [18F]-FDG-PET
imageoftheratbrainisshowninFigure1.8.
Fora typical [18F]-FDGbrainPET scan the tracer is injected ina radial veinand the
tracer is thenallowed todistribute throughout thebodyandbrain for30minutes.
After this uptake period a PET scan is performed (approximately 5 to 10minutes).
Thebrainuptakethatismeasuredprimarilyreflectstheintegratedneuronalactivity
overtheuptakeperiod.Itshouldalsobenotedthatthe[18F]-FDG-PETsignalwillalso
beinfluencedbytheglucoseconcentrationintheblood,withalowerPETsignalfor
higherbloodglucoseconcentrationsdue to thecompetitionbetween the [18F]-FDG
andglucose.Forthisreasonsubjectsshouldfastbeforean[18F]-FDG-PETexam.
AnalternativePETtracerthatcanbeusedtomeasureneuronalactivityis[15O]-H2O.
With [15O]-H2Oone canmeasure regional cerebral blood flow (rCBF),which is also
coupled to brain function. In general [18F]-FDG is preferred over [15O]-H2O as the
longer radioactive half-life of [18F]-FDG (110minutes) is logistically favorable over
theveryshorthalf-lifeof[15O]-H2O(2minutes).
1.3.3 PETneuroreceptorimaging
Apartfrom[18F]-FDGand[15O]-H2OmanyotherPETtracershavebeendevelopedfor
brain imaging (Gunn et al 2015). These tracers interact with and bind to specific
molecular targets,many of which are neuroreceptors. The tracer concentration in
differentregionsisthenproportionaltotheavailabletargetsintheseregionsaswell
as to the affinity of the tracer for these targets. A well-known example is [11C]-
raclopride, a dopamineD2/D3 antagonist. This tracer primarily concentrates in the
striatum,aregionthathasmanyD2/D3receptors.Anexampleofa [11C]-raclopride
imageofthehealthyratbrainisshowninFigure1.8.Theamountofavailabletargets
candifferbetweendifferentconditionsduetoanumberofmechanisms(Morrisetal
2014).Forexample thenumberof receptorscanbealteredduetoadiseasestate,
age,orduetotheeffectofaprolongedtreatment.Thenumberofavailabletargets
canalsobealteredduetocompetitionwithanexogenousdrugchallengeordueto
Introduction
21
altered levels of endogenous neurotransmitter under the given experimental
condition(e.g.duetoincreaseddopaminerelease(Strafellaetal2001)).
Figure1.8RatMRI(top), [18F]-FDG(middle),and[11C]-raclopride(RAC,bottom) images.PETimages are obtained from an average of 8 rats and are overlaid on theMRI. The differentdistribution is apparent. [18F]-FDG accumulates primarily in the cortex, striatum, thalamus,hippocampusandthecerebellarwhitematterwhile[11C]-racloprideaccumulatesonlyinthestriatum.
1.3.4 SmallanimalPET
Driven by the success of PET in the clinical setting dedicated miniaturized small
animal(ratsandmice)PETscannershavealsobeendevelopedinthepast15years.
These microPET (or µPET) scanners have been proven to be of great value in
preclinical research and developments from within preclinical PET research have
mademajor contributions to clinical PET and vice versa. At theMolecular Imaging
Center Antwerp (MICA) two state-of-the-art Siemens Inveon (Siemens Preclinical
Solutions,Knoxville,USA)µPET/CTscannersareavailable.Thesescannershavebeen
Chapter1
22
usedforallPETexperimentsdescribedinthiswork.Theyachieveanimageresolution
of 1.4 mm (Bao et al 2009). Two examples of rat brain PET images are shown in
Figure1.8.
AtypicalµPETexperimentisverysimilartoaclinicalPETscan,withinjectionofthe
[18F]-FDG,generally througha tail vein, followedbya30minawakeuptakebefore
scanning.One particular difference between clinical and preclinical scanning is the
useofanesthesia,usually isoflurane,duringthePETscanto immobilizetheanimal.
Isoflurane is known to reduce [18F]-FDG uptake, however, in the described
experimental setup the [18F]-FDGsignalprimarily reflectsneuronalactivityover the
awakeuptakeperiod.Similar towhat isdone inclinicalpracticeanimalsare fasted
foratleast12hoursbeforescanning(Deleyeetal2014).Asaconsequencehowever
thetimeintervalbetweentwoexperimentaldaysshouldbelongenoughinorderto
minimize theeffectof repeated fasting stress (Deleyeetal 2014). Inaddition,only
one single condition per animal can be tested per scan day as the [18F]-FDG signal
decayswithahalf-lifeof110minanditisgenerallyadvisedtoallowfor10half-lives
between two consecutive PET scans. As a consequence, in our combined
neurostimulationandµPETexperiments(chapters3-6)wehavealwaysallowedforat
leasttwodaysbetweendifferentstimulationconditions.
1.3.5 QuantificationWith brain PET imaging the concentration of a radiotracer and its distribution
throughout the brain ismeasured. The image values represent thus (radio)activity
concentrations and are generally expressed as kBq/ml, where one becquerel (Bq)
corresponds to one disintegration per second. In the case of [18F]-FDG this
concentrationisproportionaltotherCMRglcbutalsodependsonotherfactorssuch
astheamountof injecteddoseandthetotalbodyweightofthepatient.Therefore
theimagesareoftennormalizedeitheraspercentofinjecteddose(%ID)
%ID = ACID
orStandardizedUptakeValue(SUV)
Introduction
23
SUV = ACID/BW
withACequalsthemeasuredactivityconcentrationofthePETimage,IDrepresents
the injected dose and BW is the total body weight of the subject. Alternative
formulations includeanextracorrectiontermfortheplasmaglucoseconcentration
(Deleyeetal2014).
To increase the statisticalpower inagroupanalysiswhereonewants to study the
effect of different conditions (e.g. a therapy or disease) it is often necessary to
perform a regional normalization,where the activity concentration in the image is
dividedby themean activity concentration in a specific region. For [18F]-FDGbrain
imagingthisregionisoftenthewholebrain(wholebrainnormalization).Inthisway
onecanremoveunwantedintraorinter-animalvariationofglobalbrainuptakethat
canbecausedbyfactorsthatarenotof interest tothestudy(injecteddose,stress
level,glucoselevels,naturalinteranimalvariation,perfusion).Unfortunatelythisalso
removes anywholebraineffects causedby the conditionof interest. To avoid this
effect one can make use of another reference region that is expected not to be
affected by the disease or therapy that is being studied. Both techniques increase
statisticalpowerbyreducingvariabilityinglobalmetabolism(Welchetal2013).
Aftercountnormalization(SUV,wholebrainnormalization,…)oftheuptakevalues,
statisticalanalysiscanbeperformedonaregionaloravoxel-basedbasis.Inthefirst
methodtheaverageuptakevaluewithinseveralpredefinedvolumesofinterest(VOI)
(usually structural distinct brain regions) is calculated and these average uptake
valuesarethencomparedbetweenthegroups.Thisapproachisquiterobustasthe
signalfromawholebrainregionisconsidered,howeverthismethodiswillalsolose
sensitivityifthePETsignalofthegroupsonlydifferinasubregionofthelargerVOI.
Thereforeanadditionalvoxel-by-voxel comparison isperformed,a techniqueknow
asstatisticalparametricmapping(SPM)(Pennyetal2006).Todoso,the imagesof
thedifferentsubjectsarefirstspatiallynormalizedtoastandardreferencespaceso
that the same pixel in each image corresponds to the same location in the brain.
Differencesbetweenthegroupsarethentestedvoxel-by-voxelwhileaccountingfor
Chapter1
24
the massive multiple testing. SPM is performed to increase detection sensitivity
independentofVOIdefinitions.
In this work we have count normalized the rat brain images using whole brain
normalization (chapters 4-5), or%ID (chapter 3) and performed both regional and
voxel-basestatisticalanalysis.
1.4 CombinedrTMSandPET
Although evidence supporting the beneficial effects of rTMS in a wide range of
disordersisfastlygrowing,theexactmechanismofhowunderlyingneuralnetworks
are modulated to cause lasting beneficial changes in behaviour is still not fully
understood.Infact,somestudieshavereportedconflictingeffectsofthesamerTMS
protocol on the modulation of cortical excitability. By combining rTMS with
functional imaging techniques, both the instant (imaging during rTMS; online
approach)andlong-lasting(imagingafterrTMS;offlineapproach)neurophysiological
effectscanbemonitored(Siebneretal2009a,Sack2010).Moreover,neuroimaging
allowsassessmentoflocalaswellasremoteeffectsofrTMS,therebyalsorevealing
fullnetworkdynamicsandconnectivitymaps(HampsonandHoffman2010,Reithler
etal2011).Further,whenusedbeforerTMStreatment(offlineapproach),functional
imaging has been successfully used to predict treatment response (Martinot et al
2011,Richierietal2011,Hernández-Ribasetal2013)ortoidentifytargetregionsfor
rTMS(Hoffmanetal2007).
Repetitive TMS combined with [18F]-FDG-PET and [15O]-H2O-PET allows for the
construction of activation maps showing rTMS-induced changes in glucose
metabolismandcerebralbloodflow,respectively.Table1.1and1.2giveanoverview
of[18F]-FDG-PETand[15O]-H20-PETstudies,respectively,usingPETcombinedwithleft
(dl)PFCrTMStostudyitsdirectandlastingeffectsonbrainactivity.Ascanbeseenin
bothtables,mostofthestudiesshowincreasedglucosemetabolismorperfusionin
thebrainareasunderneaththecoilduringorafterhighfrequencyrTMS(10Hzor20
Introduction
25
Hz)anddecreasesduringorafterlowfrequencyrTMS(1Hz).Indistantbrainareas,
hyper- and hypometabolism and –perfusion is often seen for both high and low
frequencystimulation.Thevariabilityofthedifferentresultsintermsofdirectionality
anddistributionoftherTMS-inducedeffectsmightbeexplainedbythesmallsample
sizes,thelackofshamstimulationinthemajorityofcombinedrTMSimagingstudies
as well as the high number of varying stimulation parameters (e.g. frequency,
pattern, intensity, number of sessions), the different time durations between
stimulation and scan, different navigation systems to target the same region and
heterogeneous patient populations often taking different medications that might
interfere with rTMS induced effects (Casula et al 2013, Fidalgo et al 2014). These
limitations highlight the need for sham-controlled small animal imaging studies,
which allow systematic, longitudinal investigation of different rTMS parameters
withinonesubjectinahomogeneoussample.
Chapter1
26
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s
Hypom
etabolism-in-right-PFC-and
-bil.-
ACC
,-basal-ganglia-(L>R
),-hypo
thalam
us,-
midbrain,-CB
Hypermetabolism-in-right-post.-insula-
and-bil.-po
st.-tem
poral-cortex,-occipital-
cortex
Baeken
)et)a
l.)2009
Unipo
lar-de
pression
21Offline;-2-days-after-
10-rTM
S-sessions-vs-
baseline
4039
26.1-s
1560
20-m
in110-%-RMT
10-Hz
Hypermetabolism-in-left-ACC
-and
-only-
for-respon
ders-also-in-right-ACC
Hypom
etabolism-of-abn
ormally-
elevated
-metabolism-in-left-m
iddle-
tempo
ral-cortex-and-fusiform
-gyrus
Hypermetabolism-in-m
iddle-cingulum
,-bil.-somatosen
sory-areas,-precune
us
1800
Kimbrell)et)a
l.)20
02Healthy
7-rTMS,-7-
sham
Online;-rTM
S-du
ring-
uptake-vs-sham
-(both-vs-baseline)
1
Li)et)a
l.)20
10Unipo
lar-de
pression
11-
respon
ders
Offline;-3-m
onths-
after-2-weeks-of-
rTMS-vs-baseline
4020-m
in100-%-M
T10-Hz
[1800
30-m
in80%-M
T1-Hz
4026-s
1600
Table1.1Left(d
l)PFCrT
MScombine
dwith
FDG
-PET.FDG
fluo
rode
oxyglucose,P
ETPositron
Emiss
ionTo
mograph
y,RMTrestingMotorThresho
ld,
rTMSrepe
titiveTran
scranialM
agne
ticStim
ulation,PFCprefron
talcortex,ACC
anteriorc
ingu
latecortex,CBcerebe
llum,bil.bilateral,Lleft,R
righ
t,po
st.p
osterio
r.
Introduction
27
[15O]&H
2O)
visualizing)bloo
d)pe
rfusion
State
NAn
alysis
#trains
#pulses/
train
Intertrain)
interval
#pulses/
session
Session)
duratio
nIntensity
Freq
uency
Results
Speer)e
t)al.)2000
Major&dep
ression
10Offline;&72h
&after&10&
rTMS&sessions&vs&
baseline
11600
1600
26&m
in&
40&s
100&%&M
T1&Hz
Hypo
perfusion&in&right&P
FC,&left&m
edial&
tempo
ral&cortex,&left&basal&ganglia,&left&
amygdala
4040
28&s&
1600
20&m
in100&%&M
T20&Hz
Hype
rperfusio
n&in&PFC&(L>R
),&cingulate&
gyrus&(L>R),&left&a
mygdala,&bil.&insula,&
basal&ganglia,&uncus,&hippo
campu
s,&
parahipp
ocam
pus,&th
alam
us,&CB
Paus)et)a
l.)20
01He
althy
815
1010&s
150
3&min
100&%&RMT
10&Hz
Hypo
perfusion&in&left&inf.&parie
tal&cortex
Hype
rperfusio
n&in&bil.&m
idTdlFC,&ACC
,&rostral¶cingingulate&cortex,&bil.&
fron
topo
lar&cortex,&left&post.&cingulate&
cortex,&retrosplenial&cortex
Speer)e
t)al.)2003b
Healthy
101&for&
each&
intensity
75T
7575&s
80&%,&90&%,&
100&%,&110&
%&and
&120&
%&M
T
1&Hz
Negative&correlation&be
tween&pe
rfusion&
and&intensity
&in&bil.&PFC,&left&m
edial&
tempo
ral&lob
e,&bil.¶him
pocampi,&bil.&
post.&m
iddle&tempo
ral&gyri,&bil.&occipital&
cortex
Positive&correlation&be
tween&pe
rfusion&
and&intensity
&in&left&ACC
,&CB,&right&a
nt.&
insula,&right&prim
ary&auditory&cortex,&
somatosen
sory&cortex
Knoch)et)al.)20
06He
althy
161
60T
601&min
110&%&M
T1&Hz
Hypo
perfusion&in&left&ectorhinal&area,&
right&se
cond
ary&visual&cortex,&left&OFC
Hype
rperfusio
n&in&right&F
EF,&right&
supp
lemen
tary&m
otor&area,&left&
fron
topo
lar&cortex,&right&caudate&bod
y,&
left&anterior&cingulum,&right&precentral&
gyrus
650
5&s&
300
1&min
110&%&M
T10&Hz
Hypo
perfusion&in&left&perirh
inal&area,&
right&su
pplemen
tary&m
otor&area
Hype
rperfusio
n&in&left&fron
topo
lar&
cortex,&left&ven
trolateral&PFC
Online;&during&rTMS&
at&each&intensity
,&correlation&with
&intensity
Online;&during&rTMS&
vs&baseline
Offline;&after&rT
MS&
vs&baseline
Table1.2Left(dl)PFCrTM
Scombine
dwith
H2O-PET.P
ETPositron
Emiss
ionTo
mog
raph
y,RMTrestingMotorThresho
ld,rTM
Srepe
titiveTran
scranialM
agne
ticStim
ulation,PFCprefron
talcortex,ACC
anteriorcing
ulatecortex,C
Bcerebe
llum,b
il.bilateral,L
left,R
righ
t,po
st.p
osterio
r,inf.inferio
r,OFCorbito
fron
talcortex,FEFfron
taleyefields.
Chapter1
28
In addition to visualizing thenet neural activity changes throughout thebrain, PET
canbeusedtomeasurerTMS-inducedchangesinspecificneurotransmittersystems.
Using [11C]-raclopride, Strafella et al. found increased dopamine levels in the left
dorsal caudatenucleuswhenhealthy subjectswere scanned5minutesafter10Hz
rTMS of the left dlPFC, compared to rTMS of the left occipital cortex as a control
region (Strafellaetal2001).However,Kurodaetal.didnotobserveanysignificant
changes indopamine levelswhendepressed subjectswere scanned1day after 10
sessions of 10 Hz dlPFC rTMS compared to their baseline scan, suggesting that
increases indopamine levelsmightbetransientorthatdopaminereleasemightbe
attenuatedfollowingchronicrTMS(Kurodaetal2006).Inadditiontodopamine,10
HzrTMShasalsobeenshowntomodulateserotoninsynthesiswhenhealthysubjects
were scannedwith [11C]-αMtrp 8-9minutes following rTMS treatment (Sibon et al
2007).
1.5 Repetitive TMS combined with other imaging
techniques
1.5.1 IntroductionIn addition to PET imaging, rTMS has also been combined with Single Photon
Emission Computed Tomography (SPECT) and functional Magnetic Resonance
Imaging (fMRI). Although these techniques are not used in this doctoral work we
provide here a short description of these techniques and summarize their main
findingswithrespecttoleft(dl)PFCrTMSforcompleteness.Otherfunctionalimaging
techniquessuchasopticalbioluminescence/fluorescenceandultrasoundwillnotbe
described.
1.5.2 SinglePhotonEmissionComputedTomography
SinglePhotonEmissionComputedTomography isan imagingtechniquethat isvery
similarinprincipletoPETimaging.Italsomakesuseofradiolabeledtracersthatare
Introduction
29
administeredtothesubjectbutinsteadofpositronemittingisotopesthetracersare
labeledwithphotonemittingisotopes.Theemittedhigh-energyphotonscanescape
from the body and can then be detected by the SPECT scanner. However, these
photonsarenotemitted inpairs, sounlikewithPETwecannot infer the linealong
whichthephotonemissiontookplacebydetectingtwophotons incoincidence.To
determine the direction of the photons SPECT cameras make use of a lead or
tungsten collimator grid before the detectors. The collimator only allows photons
travelling along a certain direction to reach the detector while blocking all other
photons. For example,with aparallel hole collimatoronlyphotons travelling along
thedirectionperpendiculartothedetectorplanecanreachthedetector.An image
of the activity distribution can then be reconstructed from the detected photons.
Image resolution of clinical SPECT scanners (8-14mm) (Cherryet al 2012) is lower
thanthatofPETscanners.
OneofthemostusedSPECTisotopesis99mTc,ametastableisotopethatdecaystoa
morestablestatewithemissionofhighenergy(140keV)photons.Thephysicalhalf-
lifeof this isotope is6hours.AswithPETmanydifferentmoleculescanbe labeled
withtheisotopeanddependingontheradiotracermanydifferentprocessescanbe
visualized and quantified. As an example, the tracers 99mTc
HexaMethylPropyleneAmineOxime (99mTc-HMPAO) (Neirinckxetal 1987)and 99mTc
ethylcysteinatedimer(99mTc-ECD,or99mTc-bicisate)(Vallabhajosulaetal1989,Kado
etal2001)aretwoSPECTtracersthatdistribute inthebrain inproportiontorCBF.
These two tracers have both been used to study changes in cerebral blood flow
during or following dlPFC rTMS application. Table 1.3 and 1.4 give an overview of
theirmainfindings.Ascanbeseeninbothtables,mostofthestudiesshowincreased
perfusioninthebrainareasunderneaththecoilduringorafterhighfrequencyrTMS
(10Hzor20Hz)anddecreasesduringorafterlowfrequencyrTMS(1Hz).Indistant
brain areas, hyper-and hypoperfusion has been observed after both low and high
frequencyrTMS.TheseresultsarecomparabletotheresultsobtainedwithPET.
Chapter1
30
[99mTc]'H
MPA
O-
visualizing-blood
-pe
rfusion
State
NAn
alysis
#trains
#pulses/
train
Intertrain-
interval
#pulses/
session
Session-
duratio
nIntensity
Freq
uency
Results
Zhen
g-2000
Depressio
n5
Offline;.48.h.po
st.
rTMS.vs.baseline
3050
30.s
1500
?110%
.MT
10.Hz
Hype
rperfusio
n.in.left.AC
C
Catafau-et-al.-2001
Major.dep
ression
7Online;.during.first.
rTMS.vs.baseline
3040
30.s
1200
?90%.RMT
20.Hz
no.significant.changes
Offline;.one
.week.
after.1
0.daily.rT
MS.
sessions.vs.b
aseline
Hype
rperfusio
n.in.left.PFC
Major.dep
ression
525
2060.s
500
?80%.M
T5.Hz
Hype
rperfusio
n.in.ro
stral.A
CC
525
2060.s
500
?80%.M
T10.Hz
Hype
rperfusio
n.in.ro
stral.A
CC
525
2060.s
500
?80%.M
T20.Hz
Hype
rperfusio
n.in.ro
stral.A
CC
Depressio
n9
1360
Q360
3.min
90%.RMT
1.Hz
Hypo
perfusion.in.left.dlPFC
Hype
rperfusio
n.in.right.A
CC,.bil..parietal.
cortex,.insula,.left.CB
945
153.s
675
6.min
90%.RMT
15.Hz
Hypo
perfusion.in.right.o
rbita
l.cortex,.
subcallosal.gyrus,.left.u
ncus
Hype
rperfusio
n.in.left.dlPFC,.inf..fron
tal.
cortices,.right.dorsomed
ial.frontal.
cortex,.post..cingulate,.
parahipp
ocam
pus
Online;.injection.
after.4
.min.rT
MS.vs.
sham
Online;.injection.
after.2
.min.rT
MS.vs.
sham
Offline;.post.rTM
S.vs.baseline
Shajah
an-et-a
l.-2002
Loo-et-al.-20
03
Table1.3Left(dl)PFCrTM
Scombine
dwith
HMPA
O-SPE
CT.S
PECT
Single-Ph
oton
Emiss
ionCo
mpu
tedTo
mog
raph
y,HMPA
O
hexamethylpropylene
amineo
xime,RMTrestingMotorThresho
ld,rTM
Srepe
titiveTran
scranialM
agne
ticStim
ulation,dlPFC
dorsolateralprefron
talcortex,ACC
anteriorcingulatecortex,C
Bcerebe
llum,b
il.bilateral,Lleft,R
right,p
ost.po
sterior,inf.
inferio
r.
Introduction
31
[99mTc]'b
icisate.
visualizing.bloo
d.pe
rfusion
State
NAn
alysis
#trains
#pulses/
train
Intertrain.
interval
#pulses/
session
Session.
duratio
nIntensity
Freq
uency
Results
Nah
as.et.a
l..1998
Depressio
n6
3640
28/s
1440
18/m
in802100%/
MT
20/Hz
6100
10/s/
600
2/min
60%/M
T10/Hz
Hypo
perfusion/in/left/dlPFC,/m
id2
cingulate,/hypothalamus
George.et.a
l..19
99He
althy
8Online;/injection/
durin
g/2/min/10/Hz
/vs/baseline
610
10/s
600
2/min
60%/M
T10/Hz
Hype
rperfusio
n/in/bil./OFC/(L>R
),/hypo
thalam
us
3640
28/s
1440
18/m
in80%/M
T20/Hz/
(after/10/
Hz)
Hype
rperfusio
n/in/bil./OFC/(L>R
),/hypo
thalam
us,/thalamus
Hypo
perfusion/in/right/P
FC,/bil./ACC
,/ant./tempo
ral/cortex
Tene
back.et.a
l..1999
Depressio
n6/13/
respon
ders
Offline;/324/days/
after/1
0/daily/rT
MS/
sessions/vs/b
aseline
4040
28/s
1600
20/m
in100%
/MT
20/Hz/o
r/5/
HzHy
perperfusio
n/in/cingulate/cortex
Mottaghy.et.al..
2002
Depressio
n9
Offline;/2/weeks/
after/1
0/rTMS/
sessions/vs/b
aseline
2080
52/s
1600
20/m
in90%/RMT
10/Hz
Hypo
perfusion/in/right/F
C
Kito.et.a
l..20
08a
Unipo
lar/d
epression
12Offline;/with
in/48h
/after/1
0/rTMS/
sessions/vs/b
aseline
4250
?1000
?100%
/RMT
10/Hz
Hype
rperfusio
n/in/left/dlPFC,/premotor/
area
Online;/injection/
durin
g/2/min/10/Hz
/rTMS/after/1
8/min/
20/Hz/rTM
S/vs/
baseline
Online;/injection/
durin
g/2/min/10/Hz
/rTMS,/after/18/min/
20/Hz/rTM
S/vs/
baseline
Table1.4
Left(dl)PFC
rTM
Scombine
dwith
bicisa
te-SPE
CT.SP
ECTSingle-Pho
tonEm
issionCo
mpu
tedTo
mog
raph
y,R
MTrestingMotor
Threshold,rTM
Srepe
titiveTran
scranialM
agne
ticStim
ulation,dlPFCdorsolateralp
refron
talc
ortex,OFCorbito
fron
talc
ortex,ACC
anterior
cing
ulatecortex,C
Bcerebe
llum,bil.bilateral,Lleft,R
righ
t,po
st.p
osterio
r,inf.Inferio
r.
Chapter1
32
As with PET, SPECT can also be used to visualize changes in neurotransmitter
systems.TheSPECT-tracer[123I]-iodobenzamide,adopamineantagonist,canbeused
toquantifydopamineD2/D3receptoravailability.Usingthistracerimmediatelyafter
3000pulsesof10HzleftdlPFCrTMSindepressedpatients,reductionsinthebilateral
striatalbindingpotentialscompared tobaselineweredemonstrated, thatwerenot
observed after 3weeks of rTMS treatment (Pogarellet al 2006), suggesting acute,
but transient rTMS-induced increases in bilateral striatal dopamine concentrations
that are possibly attenuated after chronic rTMS. These results are in linewith the
observationsmadebyStrafellaetal.(Strafellaetal2001)andKurodaetal.(Kuroda
etal2006)usingraclopride-PET(see1.4CombinedPETandrTMS).Additionally,also
SPECT studies demonstrated rTMS-inducedmodulation of the serotonergic system
using[123I]-5-I-R91150(Baekenetal2012).
1.5.3 FunctionalMagneticResonanceImaging
FunctionalMagneticResonanceImaging(fMRI)isafunctionalimagingtechniquethat
usesmagnetic resonance imaging (MRI) to indirectly detect andmeasure neuronal
activity.Itmeasureschangesinbloodoxygenation,reflectingchangesinenergyuse
of the brain, through the different magnetic properties of oxy- and
deoxyhemoglobin.ThisformofMRIisalsoknownasBlood-Oxygen-LevelDependent
(BOLD)imaging.
Functional MRI achieves good temporal and spatial resolution, allows short scan
timesanddoesnotmakeuseofionizingradiationandthusradiationexposureisnot
a limiting factor when performing repeated scans in the same subject. When
combined with rTMS, fMRI allows for the mapping of corticocortical and
corticosubcortical connectivity in the brain. The first combined fMRI and rTMS
experimentsweredescribedbyBohningetal.in1999(Bohningetal1999).However,
the combination of rTMSwith fMRI is still technically challenging due tomagnetic
interference of TMS stimulator and the MRI scanner, the introduction of imaging
artifacts caused by the presence of metal in the scanner room, and to possible
torqueing of the TMS coil when used in the scanner field (Hampson and Hoffman
Introduction
33
2010).Table1.5givesasummaryofstudiescombiningfMRIwith left(dl)PFCrTMS.
Ascanbeseeninthistable,thesestudiesshowhyperperfusioninthetargetregion
afterlowfrequencyrTMS(1Hz),andnochangesinthisregionafterhighfrequency
rTMS(5Hzand10Hz),contradictingtheresultsfoundwithPETandSPECT.Aswith
PETandSPECT,indistantbrainregions,hyper-andhypoperfusionhasbeenobserved
afterbothhighandlowfrequencyrTMS.
The contrasting results seen in the target region when comparing studies using
differentimagingtechniquesmightbeexplainedbythedifferenceintimeresolution
ofthetechniqesandhighlighttheneedformore,sham-controlled,combinedrTMS
andimagingstudies.
Chapter1
34
fMRI%visua
lizing%
bloo
d%pe
rfusion
State
NAn
alysis
#trains
#pulses/
train
Intertrain%
interval
#pulses/
session
Session%
duratio
nIntensity
Freq
uency
Results
Nah
as%et%a
l.%2001a
Healthy
57
2121,s
147
?80%,M
T1,Hz
Hype
rperfusio
n,in,bil.,aud
itory,cortex,,
right,m
iddle,tempo
ral,gyrus,,right,insula
721
21,s
147
?100%
,MT
1,Hz
Hype
rperfusio
n,in,right,P
FC,,bil.,
auditory,cortex,,bil.,su
p.,te
mpo
ral,
gyrus,,right,insula,,inf.,fron
tal,gyrus
721
21,s
147
?120%
,MT
1,Hz
Hype
rperfusio
n,in,bil.,PFC,,bil.,aud
itory,
cortex,,bil.,su
p.,te
mpo
ral,gyrus,,right,
inf.,fron
tal,gyrus,,right,m
iddle,tempo
ral,
gyrus,,right,visu
al,cortex,,right,insula,,
bil.,precen
tral,gyrus,,left,m
otor,cortex
Li%et%a
l.%20
04a
Depressio
n14
721
21,s
147
7.35,m
in100%
,MT
1,Hz
Hypo
perfusion,in,right,ven
trom
edial,FC
Hype
rperfusio
n,in,bil.,PFC,(L>R
),,rig
ht,
OFC,,left,h
ippo
campu
s,,bil.,th
alam
us,,
bil.,pu
tamen
,,bil.,parietal,lob
es,,bil.,
insula,,left,m
iddle,tempo
ral,cortex
Roun
is%et%a
l.%2006
Healthy
12Offline;,sc
an,6,m
in,
after,rTM
S,vs,sh
am6
300
1,min
1800
11,m
in90%,AMT
5,Hz
Hypo
perfusion,in,left,ven
trolateral,PFC,,
left,intraparietal,sulcus,,bil.,su
p.,parietal,
gyri,,left,su
p.,te
mpo
ral,gyrus,,left,lateral,
occipital,cortex,,right,p
rim.,
sensorim
otor,area,,right,insula,,right,C
B
Fitzgerald%et%a
l.%2007
Major,dep
ression
12Offline;,with
in,48,h,
after,1
5,rTMS,
sessions,vs,b
aseline
3050
25,s
1500
?100%
,RMT
10,Hz
Hype
rperfusio
n,in,left,precune
us
Healthy
10Offline;,rT
MS,vs,sh
am1
1200
Y1200
20,m
in90%,RMT
1,Hz
Hypo
perfusion,in,bil.,te
mpo
ral,lob
es
Hype
rperfusio
n,in,right,caudate,nucleus
Van%de
r%werf%e
t%al%
2010
Online;,during,rTMS,
vs,baseline
Online;,during,rTMS,
vs,baseline
Table1.5Left(d
l)PFCrTM
Scombine
dwith
fMRI.fMRIfu
nctio
nalM
agne
ticReson
anceIm
aging,RMTrestingMotorThresho
ld,A
MTactiv
eMotorThresho
ld,rTM
Srepe
titiveTran
scranialM
agne
ticStim
ulation,dlPFCdorsolateralp
refron
talcortex,,OFCorbito
fron
talcortex,ACC
an
terio
rcingulatecortex,CBcerebe
llum,bil.bilateral,Lleft,R
righ
t,po
st.p
osterio
r,inf.inferio
r.
Introduction
35
Chapter1
36
Chapter2:
Objectives
Chapter2
38
Asoutlinedinchapter1,TMShassinceitsintroductionin1985(BarkerandJalinous
1985) grown from a simple tool to study neuronal functioning and conduction
pathways to a promising therapy for awide varietyof neurological andpsychiatric
disorders. The dlPFC is often the targeted brain region for rTMS treatment of
psychiatric disorders because of its important role in behavior. Despite the vast
amount of research devoted to this relatively new neurostimulation technique, no
consensus has yet been reached on i) the exactmechanism of how rTMS induces
lasting neurophysiological and behavioral changes and ii) the optimal rTMS
stimulation parameters to effectively treat this wide range of disorders. In vivo
functional neuroimaging offers unique opportunities to shed light on these two
importantquestions. Indeed,whencombinedwith rTMS itallowsnon-invasiveand
longitudinal visualization of rTMS-induced direct and lasting neurophysiological
effectsanditsdependenceonvariousstimulationparameters.However,large-scale
trials in humans are difficult to realize due to ethical considerations, the need for
large homogeneous patient populations not medicated for comorbidities and the
associatedhigh costs. Thereforewebelieve thatourunderstandingof rTMSwould
greatlybenefit from thecombinationof rTMSand small animalmolecular imaging.
One major challenge that needs to be overcome for this preclinical paradigm to
become successful is the development of dedicated miniaturized rat TMS coils
enablingtranslationofpreclinicalresultstotheclinic.Therefore,thegeneralaimof
this doctoral thesis was to advance our understanding of rTMS by developing a
dedicated rat TMS coil for non-invasive targeted stimulation of the mPFC, the
rodent analogue of the human dlPFC, and combining rat rTMS with in vivo
molecularimaging.Tofirstsetavalidationbenchmarkforneurostimulationofthe
mPFC we used [18F]-FDG-µPET to visualize and quantify the effects of invasive
neurostimulationusing intracranial injectionsofpharmacologicalsubstancesaswell
aswithDBSimplantations.Thisallowsustodirectlycomparethespatialpatternand
directionalityoftheeffectsofrTMSneurostimulation.
Objectives
39
Hence,inafirststudyweinvestigatedtheeffectsofintracranialinjectionsintothePL
region of the mPFC of a GABAA agonist and antagonist, substances respectively
knowntoinvokeinhibitionandexcitation.Withthisstudywewantedtovalidatethe
abilityof[18F]-FDG-µPETtodetectincreasedordecreasedrCMRglcinducedbydirect
stimulation of this small brain region. Furthermore, our goal was to visualize the
network correlations of the PL mPFC with other brain regions and to assess the
directionalityof themetabolic response in thesedifferent regionscompared to the
expectedresponse.
ThisstudyisdescribedinChapter3,andhaspreviouslybeenpublishedas:
Parthoens, J.; Servaes, S.; Verhaeghe, J.; Stroobants, S.; Staelens, S. Prelimbic
cortical injectionsofGABAagonistandantagonist: In vivoquantificationof the
effect intheratbrainusing[18F]-FDGmicroPET.Molecular ImagingandBiology.
Vol17(6),2015.pp.856-864.
Inasecondstudywecombined[18F]-FDG-µPETwithDBSoftheratPLregion.Ouraim
was to test if the reported frequency-dependent directional response to electrical
stimulationwas reflected in the [18F]-FDG-µPETanalysis. Inadditionwevalidated if
the effects of this very focal electrical neurostimulation technique could spread to
other regions. Thiswill allow comparison of the spatial pattern of the effectswith
thoseobtainedfromnon-invasivebutlessfocalrTMS.
ThisworkisdescribedinChapter4,andhaspreviouslybeenpublishedas:
Parthoens,J.;Verhaeghe,J.;Stroobants,S.;Staelens,S.Deepbrainstimulationof
the prelimbic medial prefrontal cortex: quantification of the effect on glucose
metabolism in the rat brain using [18F]-FDG microPET. Molecular Imaging and
Biology.Vol16(6).2014.pp.838-845.
Inathirdexperiment,theaimwastodevelopaminiaturizedratfigure-of-eightcoil.
ThedevicewasthenusedforratrTMStargetingthemPFC.TherTMSsessionswere
Chapter2
40
similarly combined with [18F]-FDG-µPET to investigate the glucose metabolism
changeselicitedbyhigh(50Hz)andlow(1Hz)rTMS,comparedtoshamstimulation.
Againthegoalofthisexperimentwastovisualizetheeffectandthedirectionalityof
the response in different brain regions to the stimulation at these different
frequencies.
ThisstudyisdescribedinChapter5,andhasbeenpreviouslypublishedas:
Parthoens, J.; Verhaeghe, J.; Wyckhuys, T.; Stroobants, S.; Staelens, S. Small
animal repetitive transcranial magnetic stimulation combined with [18F]-FDG
microPETtoquantifytheneuromodulationeffect intheratbrain.Neuroscience.
Vol275.2014.pp.436-443.
Our in-housebuilt coildescribed inchapter5couldonly stimulateat relatively low
intensitiesandwaspronetooverheating.Therefore theaimof the lastpartof this
doctoral thesis was to develop and validate an improved dedicated rat rTMS coil.
Therefore,wehave,incollaborationwithMagVentureA/S(Farum,Denmark)oneof
themainmanufacturersofTMScoilsandstimulators,developedadedicatedcircular
ratTMScoilequippedwithanactivecoolingsystem.Ouraimsweretovalidatethe
newcoilbycalculatingthegeneratedE-fielddistributionswithcomputersimulations,
MEPmeasurementsandcombinedratrTMSand[18F]-FDG-µPETimaging.Similarlyto
ourpreviousexperimentswewere interested intheeffectsofdifferentstimulation
frequencies(1,10and50Hz)comparedtoshamstimulation.
ThisstudyisdescribedinChapter6,andhaspreviouslybeenpublishedas:
Parthoens,J.;Verhaeghe,J.;Servaes,S.;Miranda,A.;Stroobants,S.;Staelens,S.
Performance characterization of an actively cooled repetitive Transcranial
Magnetic Stimulation coil for the rat. Neuromodulation: Technology at the
NeuralInterface.5Feb2016.doi:10.1111/ner.12387.
Objectives
41
Chapter2
42
Chapter3:
Prelimbic cortical injections of GABA agonist
and antagonist: In vivo quantification of the
effectintheratbrainusing[18F]-FDGmicroPET
Thischapterhasbeenpublishedas:Parthoens,J.;Servaes,S.;Verhaeghe,J.;Stroobants,S.;Staelens,S.PrelimbiccorticalinjectionsofGABAagonistandantagonist:Invivoquantificationoftheeffectintheratbrainusing[18F]-FDGmicroPET.Molecular ImagingandBiology.Vol17(6),2015.pp.856-864.
Chapter3
44
3.1 Abstract
IntroductionWeevaluatedtheglucosemetabolismaftermicroinjectionsofaGABAA
antagonist, bicuculline, and aGABAA agonist,muscimol, in the rat prelimbic cortex
(PL)bysmallanimalPositronEmissionTomography(µPET).
Methods Followingamicroinjectionof0.5µLbicuculline (0.1mg/mL),muscimol (1
mg/mL) or saline in the left PL of the rat mPFC of eleven healthy male Sprague
Dawley rats (250-275 gr), 2-deoxy-2-18F-fluoro-β-D-glucose ([18F]-FDG) PET images
were acquired. Volume-of-interest (VOI)-based analysis and voxel-based statistical
parametricmappingwereperformed(n=9).
ResultsVOI-basedanalysisrevealedsignificantlydifferent[18F]-FDGuptakefollowing
bicucullineversusmuscimolinPL(p<0.001),infralimbiccortex(p<0.01)andcingulate
cortex (p<0.01). Voxel-based analysis showed bicuculline induced widespread
significanthypermetabolismthroughoutthebrainwhilemuscimolinducedsignificant
localizedhypometabolism.
Conclusion Here we visualize functional GABAA mediated correlations of the PL
followingpharmacologicalstimulation.Thiscouldserveasareferenceandshedlight
ontheworkingandfocalityofotherstimulationparadigmstargetingthisregion.
3.2 Introduction
In themammalianbrain, theprefrontal cortex (PFC) takespart in guidingbehavior
towardacquisitionofadaptivegoalsandmodulatingsubcorticalregions(Duncanand
Owen2000,Milleretal2002,Uylingsetal2003).Itcarriesoutthesecomplextasks
by integratingbothexternal informationfromsensoryandmotorsystemstructures
and internal information from limbic and midbrain structures involved in stress
(Koenigs and Grafman 2009a, Jones et al 2011, Chang et al 2011, Qi et al 2012),
memory(DuncanandOwen2000,Eustonetal2012)andreward(Milleretal2002).
A dysfunction in the dorsolateral part of the human PFC (dlPFC) is believed to be
involved inavarietyofneurologicalandpsychiatricpathologiessuchasParkinson’s
Pharmacologicalinjections
45
disease(Pintoetal2004),depression(Changetal2011,Qietal2012,Koenigsand
Grafman 2009b), addiction (Camprodon et al 2007), post-traumatic stress disorder
(KoenigsandGrafman2009a,Parnelletal2012,Gilmartinetal2012),OCD(Okadaet
al2013)andantisocialbehaviour(DohertyandGratton1999,YangandRaine2009),
possiblythroughitskeyroleintherewardcircuit.Furtherrationaleforinvestigating
the functional correlations of the dlPFC in humans comes from the recent
therapeutic interest to use neuromodulation techniques such as repetitive
Transcranial Magnetic Stimulation (rTMS) (Rossi et al 2009) and Deep Brain
Stimulation (DBS) (Taghva et al 2013) targetting this area to treat the
aforementioned disorders. During the past decennium, numerous studies revealed
theefficacyand safetyof rTMS targeting thehuman leftdlPFC in the treatmentof
depression(Fitzgeraldetal2003,Kurodaetal2006,O’Reardonetal2007), leading
toFDAapproval in2008. In thepast fewyears, stimulationof thisbrain targethas
alsobeensuggestedasanewapproachinthetreatmentoffoodcraving(Uheretal
2005)andvariousdrugaddictions,includingcocaine(Camprodonetal2007,Politiet
al2008),alcohol(Mishraetal2010)andnicotine(Amiazetal2009)addiction,with
promisingresults.FurtherexplorationisthereforerequiredofthedlPFCinhumansas
a stimulation target for disorders that imply a prefrontal dysfunction or a
hyposensitiverewardsystem.Abetterunderstandingofthefunctionofthehuman
dlPFC, through study of its rodent analog, and the cortical networks inwhich it is
involved could be a first step towards the development and optimization of new
treatmentsforthesediseases.
In rats, the prelimbic cortex (PL) is a subregion of the medial prefrontal cortex
(mPFC), which shows anatomical and functional similarities with the human dlPFC
(Uylingsetal2003).Tounravelthefunctionsofthisbrainregionfocalactivationor
inhibitionaretypicallyperformedbyamicroinjectionofapharmacologicalsubstance
after which behavioral readout or microdialysis studies are done (Gilmartin et al
2012,Yoshidaetal1997,Yan1999).NeuronalactivationofthePLwithbicuculline,a
GABAA antagonist that acutely blocks the inhibitory action of the GABAA receptor,
Chapter3
46
influencesstressresponses(Jonesetal2011).Additionally,inactivationofthePLby
injection of tetrodotoxin, a sodium channel blocker, revealed that this region is
critical for the expression of learned fears (Corcoran and Quirk 2007), while PL
injectionofmuscimol,aGABAAagonist,elicited increased impulsivity in ratsonthe
five-choiceserialreactiontimetask(5-CSRTT)(Murphyetal2011).Moreover,Corbit
& Balleine (2003) demonstrated that a neurotoxin (NMDA)-induced lesion of this
area impaired the ability of rats to select an action based on previously encoded
outcomeassociations(CorbitandBalleine2003).Preclinicaltherapeuticstudiesusing
rats showed sustained increases in hippocampal 5-HT levels (Juckel et al 1999),
antidepressant-like behavior (Hamani et al 2010a), reduced cocaine self-
administration(Levyetal2007)andincreasedneuronalactivityinthisregionaswell
as in regionsassociatedwithalertness (Parthoensetal2014a)afterDBS in the left
PL. Current research focussing on miniaturizing rTMS coils for preclinical research
targetedtothePLcouldalsoprovidefurtherinsightintothemolecularmechanisms
involved(Parthoensetal2014b).
Despitetheaforementionedamountofactivationor inhibitionstudies investigating
the roleof thePL inbehavior,no invivo functionalneuroimagingstudyhasshown
the effect of a pharmacological treatment on the underlyingwhole brain network
correlationsofthePL.Suchinsights intotheneuronalnetworksrelatedtoacertain
taskor treatmentcanbeachievedbyPositronEmissionTomography (PET)using2-
deoxy-2-18F-fluoro-β-D-glucose ([18F]-FDG) to visualize the neuronal glucose
metabolism,whichindirectlyreflectschangesinneuronalactivity,intheentirebrain
in vivo.Due to the focality of thesepharmacologicalmicroinjections these findings
canalsofunctionasareferencethatwillallowtoshedlightonpossibledifferencesin
workingmechanisms and in focality of other neuromodulation techniques, such as
DBSand rTMS. Inparticular itwill ensureadiscriminationbetweeneffectsof focal
stimulationofthetargetregionversusnon-focalorindirectstimulation.
Pharmacologicalinjections
47
In the current study, small animalPET (µPET) isused tomap the functionalGABAA
mediated correlations of the left PL following focal pharmacological stimulation
(activationorinhibition)byvisualizingglucosemetabolisminthebrain.
3.3 MaterialsandMethods
3.3.1 Animals
MaleSprague-Dawleyrats(n=11,250-275g,Janvier,France)weretreatedaccording
toguidelinesapprovedby theEuropeanEthicsCommittee (86/609/EEC).Thestudy
protocol was approved by the Antwerp University Ethical Committee for Animal
Experiments(ECD2012-50).Theanimalswerekeptunderenvironmentallycontrolled
conditions (12h light/darkcycles,20-23 °Cand50-55%relativehumidity)with food
andwateradlibitum.
3.3.2 Cannulaplacement
Forcannulaplacement,theratswereanesthetizedwithamixtureof isofluraneand
medical O2 (5% induction dose, 2%maintenance) while 0.05mg/kg Temgesic was
injecteds.c.asanalgesic.Asagittalincisionfollowingthesuperiorsagittalsuturewas
madealongtheskull.A26Gguidecannula(Bilaney)wasstereotaxicallyimplantedin
thelefthemisphereabovethePL(AP+3.7mm,ML+2.0mm,DV-4.0mmrelativeto
bregma,atanangleof22° inthecoronalplane) (PaxinosandWatson2007).These
coordinatesforcannulaimplantationweredeterminedfromaprevioussetup,where
itwasverifiedthatthisresultedininjectionsinthePL.Attheendoftheexperimenta
post-mortem analysis to verify the location of the cannula was done. After
placement,thecannulawassealedoffbyinsertionofadummycannula(Bilaney)to
bereplacedbya33Ginternalcannulaforinjection(Bilaney),protruding1mmfrom
theguidecannulaend.Theguidecannulawassecuredtofivesmallscrewsthatwere
insertedintotheskullbydentalcement.
Chapter3
48
3.3.3 HabituationperiodAfter a 1 week recovery-period, rats were habituated to the microinjection
procedure during a ten-day habituation period during which the animals were
handledandexposedtothesoundoftheinjectionpump(QuintessentialStereotaxic
Injector, Stoelting)while the dummy cannulawas removed and reinserted.On the
lastdaya33Ginternalcannula(Bilaney)wasinsertedfor2minutes.
3.3.4 Microinjections
Onthetestdays,eachratwasinjectedwitheither0.5µLmuscimol(1mg/mLsaline)
(Parnelletal2012,Gilmartinetal2012),bicuculline(0.1mg/mLsaline)(Dohertyand
Gratton1999,Enomotoetal2011)orsalineasacontrolatarateof0.5µL/minusing
theinternalcannulaanda2.5µLHamiltonsyringe.Toallowdiffusionofthesolution,
the internal injectioncannulawasheld inplace foroneadditionalminutebefore it
was removed from the guide cannula and replacedwith the dummy (Doherty and
Gratton1999).
3.3.5 MicroPET-CTimaging
A similar protocol as described by Wyckhuys et al. was followed regarding the
imaging procedure and tracer production of [18F]-FDG (Wyckhuys et al 2014). Ten
minutesafteradministrationofthecompound,abolusinjectionof1mCi[18F]-FDG(±
0.5mL) was injected intravenously in the tail while the animal was awake. The
animalswerealloweda30minutetraceruptakeperiod,ofwhich20minutesawake,
after which the rats were anaesthetized by a mixture of isoflurane and medical
oxygen (5% induction, 1.5% maintenance) and placed onto the thermostatically
heatedbedofaµPET-CTscanner.A20minutestaticacquisitionwasthenstarted30
minutes post tracer injection, i.e. 40minutes post-injectionof the drugor control.
(Figure3.1).
Pharmacologicalinjections
49
Figure 3.1 Scan protocol used for the different experiments. Ten minutes after theintracranial (i.c.) injection of either saline, bicuculline ormuscimol in the PL, the ratswereintravenously (i.v.) injected with 1 mCi of [18F]-FDG. Twenty minutes later, the rats wereanesthetizedandpositionedontheµPET-CTscannerafterwhichthePETacquisition(20min)wasstarted,resultinginatotaltimeof30mintraceruptake.
MicroPETimagingwasperformedontwoSiemensInveonPET-CTscanners(Siemens
Preclinical Solution, Knoxville, TN) (Bao et al 2009). The reconstructed spatial
resolutionisaround1.4mmatthecenterofthefieldofview(FOV)andtheaxialand
transaxial FOVs are 10.0 and 12.7 cm, respectively. All rats received all three
conditions inarandomizedorder,whilethethreescanswerealwaysperformedon
the same scanner for a given animal. Two consecutive scanswere separatedby at
least48hourstoallowacompletewashoutofthepharmacologicalagents(Murphy
et al 2011, Fiske et al 2006, Slattery et al 2011) and to accommodate a minimal
fastingdurationofatleasttwelvehoursbeforeeachPETscantoensureoptimal[18F]-
FDGuptake(Deleyeetal2014).Theaverageweightatthemomentofthescanswas
374.4gramswithastandarddeviationof23.91grams.
ForquantitativeanalysistheµPETimageswerereconstructedusing4iterationswith
16 subsets of the 2D ordered subset expectation maximization (OSEM) algorithm
followingFourierrebinning.Alldatacorrections(deadtime,normalization,randoms,
attenuationandscatter)wereapplied.Attenuationandscattercorrectionarebased
on a segmented attenuation map calculated from a modified CT image that was
elaborately corrected for metal artifacts as follows: i) a Maximum A Posteriori –
Transmission (MAP-TR) reconstruction (Deleye et al 2014) was thresholded to
determine the metal parts (screws and canulla) in the reconstruction; ii) metal
Chapter3
50
artifact reduction (MAR) was performed using a sinogram inpainting method
(Lemmensetal2009,Prelletal2009);andiii)asmallportionoftheskullaroundthe
metalscrewsthatwas lost intheMARreconstructionwasreplacedusingtheCTof
the skull of a healthy non-implanted rat. The final CT imagewas thenobtainedby
combining the imagesof i)metalonly, ii) rat imageand iii)partsof skull thatwere
missing in ii) from a donor CT image of normal rat (Sprague-Dawley) skull (Figure
3.2).
Figure 3.2 Correction for metal artifacts on CT-image. A segmented attenuation map wascalculatedfromamodifiedCTimagetocorrectforthemetalartifactsfromtheoriginalCT(A).Theredarrowmarksthemetalartifacts.ThefinalCTimagewasthenobtainedbycombiningtheimagesofthemetalparts,theratimageandpartsoftheskullthatweremissing(B).
3.3.6 HistologicalverificationofthecannulapositionAfter the imaging experiments the exact position of the internal cannula injection
site and an estimate of the spread of the pharmacological agents in the PL were
assessed. The animals were therefore deeply anesthetized with isoflurane and
fountainpenink(0.5µL)wasinjectedatthesamerate.Theanimalsweresacrificed
by an overdose ofNembutal (i.v., 150mg/kg). The brainswere removed and snap
Pharmacologicalinjections
51
frozeninisopentaneusingliquidnitrogenandstoredat-20°C.Coronalsectionsof30
µm were cut on a cryostat (Leica CM 1950) and stained with hematoxyline.
Verificationofthecannulaplacementwasperformedwiththeaidofaratbrainatlas
(PaxinosandWatson2007)andonlyratswithacorrectcannula implantation(n=9)
wereusedintheimageanalysis.
3.3.7 Imageanalysis
EachPETimagewastransformedintothespaceofan[18F]-FDGtemplate(Schifferet
al 2007) using spatial brain normalization in PMOD v3.3 (PMOD Technologies,
Switzerland). Imageswereexpressedaspercent injecteddose (%ID)bynormalizing
thePETactivityconcentrationtotheinjecteddose(ID)atthetimeofthestartofthe
acquisition.AVOI-basedanalysis,usingpre-definedVOIsavailableinPMODv3.3,was
performed to quantitatively investigate the average changes in [18F]-FDG uptake
between the three injection conditions. A one-way repeated measures ANOVA
followedbypost-hoctestingwithBonferronicorrectionwasperformed inSPSSv20
(IBM corporation, NY, USA). Statistical significance was set at p=0.05. Average
changesinoverallVOI-valuescomparedtosalineadministrationarepresentedwith
theirstandarderrorofthemean.Additionally,voxel-basedSPManalysis,beingmore
statisticallysensitive,wasperformedusingSPM8(WelcomeDepartmentofCognitive
Neurology, London, UK) within a one-way repeatedmeasures ANOVA design. The
images, normalized for injecteddoseandmasked to removeextracerebral activity,
were smoothed using a Gaussian filter (isotropic 1.5 mm full-width-at-half-
maximum).An F-contrast, testing for anydifferencebetween the three conditions,
and four T-contrasts, testing for both hyper- and hypometabolism for both
bicuculline andmuscimol versus saline injection,were defined. Voxels that passed
theomnibusF-testatasignificancelevelof0.05(uncorrected)definedamaskforthe
subsequentpost-hocT-contrasts.T-mapswerethresholdedatasignificancelevelof
0.05 (uncorrected) with a cluster extent threshold of 125 voxels (1 mm3). For
visualization,T-mapswereoverlaidona9.4TMRratbrainimage.Theeffectinthese
significant voxels was then calculated for each animal as ((uptake after
Chapter3
52
pharmacologicalinjection)/(uptakeaftersalineinjection)-1)withuptakebeing[18F]-
FDGuptakevalueoftheunsmoothedimageexpressedin%ID.
3.4 Results
Noabnormalbehaviorwasseenafteramicroinjectionofsalineormuscimol in the
leftPL.Inonerat,administrationofbicucullinecausedmildcontractionsoftheright
forepaw,between7-20minutesaftertheinjection.
3.4.1 HistologyInkstainingandhistologicalexaminationrevealedthatthepositioningofthecannula
was successful in nine out of eleven rats. Two rats had an incorrect cannula
placementandwereexcludedfromfurtheranalysis(Figure3.3a).Thusatotalofn=9
rats was used for the image analysis. As an estimation for the extent of the drug
infiltration,Figures3.3b,canddshowthespreadoftheinjectedfountainpeninkin
one rat as an example, showing a diameter of approximately 1 mm around the
cannulatip.
Pharmacologicalinjections
53
Figure 3.3 a) Results of the histological verification of the cannula implantations on twocoronal slices (4.20 and 3.72mm anterior from bregma). Correct placements are depictedwithadot,wrongordisputableplacementswithacross(adaptedfrom(PaxinosandWatson2007));Histologicalestimationofthespreadofthedrugsbyaninkinjectionwith(b)Pictureof the frozencoronalbrain tissueofa ratatapproximately3.72mmanterior tobregma,c)drawingof this slicewith the leftPLdelineated (adapted from (PaxinosandWatson2007))and d) both figures overlaid. PL: prelimbic area, MO: medial orbital cortex, Cg: cingulatecortex.
3.4.2 VOI-basedanalysisAs shown in Figure 3.4, bicuculline injection causes a global increase in [18F]-FDG
uptake whilemuscimol shows a decreased [18F]-FDG uptake in the frontal regions
compared to saline injection. Further quantification using VOI-based analysis
revealed a global on average increased [18F]-FDG uptake in the brain of +20.7% ±
12.1 % (mean ± SEM) after bicuculline injection compared to saline, albeit non-
significant when considering the whole brain (WB) as shown in Figure 3.5. For
muscimol, a whole brain change in [18F]-FDG uptake of +7.5 % ± 10.9 % was
observed;notsignificantasshowninFigure3.5(WB).Regionally,inthetargetedPL,
bicuculline injectioncausedonaveragean increased [18F]-FDGuptakecompared to
saline injection (+22.3%±12.9%,non-significant),whilemuscimol injectioncaused
Chapter3
54
onaverageadecreased[18F]-FDGuptakecomparedtosaline injection(-5.6%±9.8
%,non-significant).
Figure 3.4 Mean PET-images of the three conditions (saline, bicuculline and muscimolinjection; n=9), normalized for the injected dose, overlaid on anMR-template. Values areexpressedaspercentinjecteddose(%ID).
Whencomparingbicucullineversusmuscimolinjections,significantdifferenceswere
found for [18F]-FDG uptake in the targeted PL (25.3 % ± 3.3 %, p<0.001), the
infralimbiccortex(IL;21.7%±5.2%p<0.01)andthecingulatecortex(16.5%±4.0%,
p<0.01)asshowninFigure3.5.
Pharmacologicalinjections
55
Figu
re3.5M
eanup
takevalue
sexpressedaspercentageofthe
injected
dose(%
ID)forthe
saline,bicucullinean
dmuscimol
cond
ition
inth
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lineatedVO
Is.Errorbarsrepresen
tSEM
,*p<0
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,***p<0.001
with
CP:cauda
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en,C
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IPad:a
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Chapter3
56
3.4.3 Voxel-basedSPManalysis
SPM voxel-based analysis, being statistically more sensitive then a VOI-based
approach, showed that a bicuculline injection induced significant increases in [18F]-
FDG uptake compared to saline bilaterally throughout the entire brain (Figure 3.6
and3.7).Thetotalvolumeofvoxelswithsignificantincreaseduptakewas488mm3
(21.8%ofthetotalbrainvolume).Theaverageuptakeincreaseinthesevoxelswas
22.9%±12.1%.Novoxelswithsignificantreduced[18F]-FDGuptakewerefound.
Figure 3.5 T-maps (cluster size > 125 voxels) showing significant hyper-or hypometabolism(p<0.05,notcorrectedformultiplecomparisons)comparedtosalineinjection,overlaidonanMRtemplate.A)Bicucullineversussaline injection.Onlyhypermetabolismwasdetected.B)Muscimolversussalineinjection.Onlyhypometabolismwasdetected.Thedifferentvolumesof interest are delineated and region labels are as in Figure 3.5. The threshold T-value forsignificance(p<0.05)isindicatedbythe*inthecolorbar(T=1.860).Cg:cingulatecortex,EC:entorhinal cortex, HIPad: anterodorsal part of hippocampus, Hyp: hypothalamus, IL:infralimbic cortex PL: prelimbic cortex, Med: medulla, OFC: orbitofrontal cortex, RsplC:retrosplenial cortex, Se: septum, SupC: superior colliculus, VC: visual cortex, VTA: ventraltegmentalarea,WB:wholebrain.
Muscimolontheotherhandelicitednosignificanthypermetabolisminanyvoxel,but
significanthypometabolismwasseenveryfocallyinaclusterofvoxelslocatedinthe
Pharmacologicalinjections
57
PLandcingulate cortex ipsilateral to the injection (Figure3.6). The total volumeof
hypometabolicvoxelswas1.03mm3andtheaverageuptakedecreaseinthesevoxels
was-10.7%±8.0%.
Figure3.6Additionalcoronalsectionsfromtheanteriorparttotheposteriorpartofthebraindisplaying T-maps with significant (p<0.05, not corrected for multiple comparisons)hypermetabolismwhenbicucullineiscomparedtosalineinjection.Thenumberinthetopleftcornerindicatestheslicenumber.
3.5 Discussion
This[18F]-FDGµPETstudyvisualizesregionalmetabolicchangesinanetworkelicited
by activation or inactivation of a brain region by means of a pharmacological
intervention. As expected our findings show that activation of the rat left PL by
administration of bicuculline causes a widespread bilateral increase in glucose
metabolism. Injection of muscimol at the same location causes a decrease in
metabolism, which is almost exclusively ipsilateral to the target region. It has
previously been shown that cannula implantation has a lasting effect on [18F]-FDG
uptake(Schifferetal2006,Frumbergetal2007).Thiseffecthashowevereffectively
beenaccountedforinourstudydesignbyinclusionofacontrolsalineinjectionanda
randomizedinjectionorder.Furthermore,thesefindingsareinaccordancewithanex
vivo autoradiography study using [14C]-deoxyglucose ([14C]-DG) after injection of
bicucullineintotheventrolateralthalamicnuclei,whichalsomainlyshowedtransient
Chapter3
58
increases in cerebral glucose metabolism, which were situated in the ganglia-
thalamo-cortical motor circuit (QingGeLeTu et al 2009). The changes in glucose
metabolism visualized by [18F]-FDG-PET or [14C]-DG autoradiography provide an
indirectmeasureofregionalchangesinneuronalactivation(Chattonetal2003).The
increase in metabolism after GABAA antagonist injection and the decrease after
GABAAagonist injection seen in this studywere largelyexpected sinceGABA is the
main inhibitory neurotransmitter of the central nervous system of mammals. The
GABAA receptor is part of the ligand-gated ion channel complex mediating the
passage of chloride ions across the membrane, thereby hyperpolarizing and thus
inhibitingneurons(Devlin2001).Administrationofbicucullineindirectlyhindersthe
passage of chloride ions by blocking GABAA receptors and thus prevents
hyperpolarization and inhibition. Muscimol, on the other hand, activates GABAA
receptors, thereby enhancing the passage of chloride ions across the membrane,
resulting in a hyperpolarization, thus reducing the excitability of neurons (Devlin
2001). Nevertheless, the widespread bilateral hypermetabolism after left PL
bicuculline injection versus the much more unilateral and focal decrease in
metabolismaftermuscimolinjectionsuggeststhatneuronalexcitationismorelikely
tobetransferredthroughthebrainincontrasttoinhibition.However,theeffectsof
bicuculline and muscimol should be regarded separately and cannot be directly
compared, since each of the two drugs has their own characteristics regarding
diffusionrate,receptorbinding,efficacy,etc.Forbothbicucullineandmuscimol,the
[18F]-FDG injectionwas given tenminutes after themicroinjection, since this is the
timeframe after which both drugs are expected to start affecting the behavior
(Slattery et al 2011) or the glucose metabolism (QingGeLeTu et al 2009). This
timeframe might however differ slightly between both substances. Regarding the
dosageofbothsubstances,aconcentrationwaschosenthat,accordingtoprevious
rodentstudies,hasaneffectonbehaviorwhilebeingsafe(Parnelletal2012)and,in
case of bicuculline, would not elicit epileptic seizures (Doherty and Gratton 1999,
Enomotoetal2011).Oneratdidshowsomemildcontractionsof its forepawfora
Pharmacologicalinjections
59
fewminutesafterthebicuculline injectionandduringthefirstminutesofthe[18F]-
FDGuptake.SinceVOI-basedanalysisrevealednoaberrantactivationpatternsinthis
ratcomparedtotheothers,thedatafromthisratwasmaintainedfortheanalysis.As
afutureperspective,differentdosesofbothsubstanceswillbecompared.
We excluded two rats from the analysis due to a disputable placement of the
implanted cannula as revealed by histological assessment of the spread of the ink
thatwasinjectedinallratsattheendoftheexperiment.Althoughthespreadofan
intracraniallyinjectedproductwilldependonitspropertiessuchastheviscosityand
sizeofthediffusingparticles,thespreadofthefountainpeninkgivesanindicationof
thespreadofthedrug(ChinandHutchison2008).The ink injectionshowedaclear
and focal bluemark in the PL in the nine rats that were included in the analysis.
Therefore we believe that a large part of this region was affected directly by the
administration of the drug and that changes inmetabolism in other brain regions
wereduetotheanatomicalandfunctionalcorrelationswiththetargetregion.
The unilateral injection of bicuculline resulted in widespread bilateral increases in
[18F]-FDGuptake(Figure3.6and3.7),possiblythroughactivationofinterhemispheric
fiber pathways, namely homotopic and heterotopic callosal projection and/or the
hippocampal and anterior commissures, which are known to be more dense in
specieswithasmallerbrainsizesuchasrodents(Milleretal2002).Theseincreases
inregionalcerebralglucosemetabolismafterbicucullineadministrationweremainly
seen inconnectedregions involved inprocessingsensory information,memoryand
the mesolimbic dopaminergic circuit (for reviews on PL connections see (Vertes
2003) and (Euston et al 2012)). The association between the PL, the IL and the
cingulate cortex (Vertes 2003), aswell as the role of the cingulate cortex inmood
disorders(Drevetsetal2008)forwhichthehumandlPFCisoftenatargetstructure,
couldexplainwhysimilarincreasesinglucosemetabolismareseenintheseregions
afteradministrationofbicuculline.
Thesefindingsprovideavisualizationofthefunctionthatwasrecentlyproposedby
Eustonetal.(2012),suggestingthatthePLandILareinvolvedinintegratingsensory
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60
andvisceralinputtolearnassociationsbetweencontext,locationsandeventsandto
deliver correspondingadaptiveemotionalandsocial responses (Eustonetal 2012).
ThisinterestingviewnecessitatesconnectionsofthePLwithalltheabovementioned
brainsystems.Moreover,theensuingactivationofthedopaminergicrewardsystem
afterPL activation confirms the recenthypothesison its role inmood, anxiety and
movementdisorders. Furthermore, a review investigating themainprojection sites
ofthePL(Vertes2003)isremarkablyparalleltotheregionsfoundtobechangedin
glucose metabolism after administration of bicuculline (Table 3.1). This provides
further evidence for the anatomical and functional involvement of the PL in these
regions.
Table3.1OverviewoftheknownprojectionsitesofthePLofthemPFCoftheratcomparedto significant increases in glucose metabolism displayed by T-maps after bicucullineadministration.ProjectionsitesofthePL(Vertes2003) Changeinglucosemetabolism
Medialfrontalpolarcortex XInfralimbiccortex XAnteriorcingulatecortex XMedialorbitalcortex XAgranularinsularcortex XEntorhinalcortex XPiriformcortex Anteriorolfactorynucleus NotinfieldofviewCaudateputamen XNucleusaccumbens XOlfactorytubercle Claustrum Thalamus XVentraltegmentalarea XSubstantianigraparscompacta Periaqueductalgray XRaphenucleus XAmygdala X
Confirmedincreasesinglucosemetabolisminthevolumeofinterestaremarkedwithan‘X’.
Pharmacologicalinjections
61
Due to the intrinsic focality of pharmacologicalmicroinjections this study can also
serve as a reference and improve our understanding of possible differences in
workingmechanismsandfocalityofotherneuromodulationtechniquessuchasDBS
and rTMS in this target regionbyensuring adiscriminationbetween theeffects of
focalstimulationofthePLversustheeffectofnon-focalorindirectstimulation.
3.6 Conclusion
Weevaluatedtheregionalchangesinglucosemetabolisminducedbymicroinjection
ofaGABAAantagonist,bicuculline,andaGABAAagonist,muscimol, intheratPLas
evaluated by µPET. We showed that bicuculline induced widespread significant
hypermetabolism throughout the brain while muscimol induced significant
hypometabolism, mostly restricted to the target region. We have thereby
demonstratedafastapproachtovisualizethefunctionalcorrelationsofthePL,using
pharmacologicalmodulationfollowedbyinvivomolecularimaging.
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62
Chapter4:
Deepbrainstimulationoftheprelimbicmedial
prefrontal cortex: quantification of the effect
on glucosemetabolism in the rat brain using
[18F]-FDGmicroPET
Thischapterhasbeenpublishedas:Parthoens,J.;Verhaeghe,J.;Stroobants,S.;Staelens,S.Deepbrainstimulationoftheprelimbic medial prefrontal cortex: quantification of the effect on glucosemetabolismintheratbrainusing[18F]-FDGmicroPET.MolecularImagingandBiology.Vol16(6).2014.pp.838-845.
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64
4.1 Abstract
IntroductionPrefrontalcortex(PFC)DeepBrainStimulation(DBS)hasbeenproposed
as a therapy for addiction and depression. This study investigates changes in rat
cerebralglucosemetabolisminducedbydifferentDBSfrequenciesusingµPET.
MethodsOnehourDBSoftheprelimbicarea(PL)ofthemedialPFC(mPFC)(60Hz,
130Hzorsham)inrats(n=9)wasfollowedby2-deoxy-2-18F-fluoro-β-D-glucoseµPET.
ResultsSixtyHzDBSelicitedsignificanthypermetabolism in the ipsilateralPL ([18F]-
FDGuptake +5.2 ± 2.3%, p<0.05). At 130Hz, hypometabolismwas induced in the
ipsilateralPL (-2.5±2.6%,non-significant).Statisticalparametricmappingrevealed
hypo-andhypermetabolismclustersforboth60and130Hzversusshamandshowa
certain state of alertness (increased activity in sensory andmotor related regions)
mainlyfor60Hz.
ConclusionThisstudysuggeststhepotentialof60HzPLmPFCDBSforthetreatment
ofdisordersassociatedwithprefrontalhypofunction.
4.2 Introduction
Deep Brain Stimulation (DBS) is a neurostimulation technique that involves the
implantation of one or more electrodes into a specific brain region in order to
interfere with its neural activity. In the past few decennia, this approach has
establisheditsclinicalrelevanceinthetreatmentofmovementdisorders(forreview,
see(PizzolatoandMandat2012)),andhasproventobeapromisingnewtherapyfor
other neurological diseases such as refractory epilepsy (for review, see (Bergey
2013)). More recently, DBS has been introduced into the field of psychiatric
disorders, yielding promising results in the treatment of OCD (Denys et al 2010),
treatment-resistant depression (Anderson et al 2012, Howland et al 2011), eating
disorders (Mantione et al 2010) and drug addiction (Levy et al 2007, Pierce and
Vassoler2013).However,foreachofthesediseases,furtherresearchisrequiredon
theoptimalstimulationtargetandparameters,themechanismofaction,theclinical
DeepBrainStimulation
65
benefitsandpotentialsideeffectsofDBS(HamaniandNobrega2012).Stimulationat
high frequencies (>100Hz) isknownforcausinga lesioning-like inhibitoryeffect in
thetargetregion(LozanoandLipsman2013,PellouxandBaunez2013)and130Hzis
the frequencymostoftenused inboth clinicalpractice (Lozanoand Lipsman2013,
Benabidetal1998,Lipsmanetal2013)andexperimentalresearch(Wyckhuysetal
2010a,2010b,Hamanietal2010a).LowfrequencyDBS(20–70Hz)ofbrainregions
relatedtothelimbicsystem,onthecontrary,hasbeenreportedtoinduceexcitation
of neurons, eventually even causing convulsions, an effect known as kindling
(Goddardetal 1969, Zhangetal 2012). It shouldbementionedhowever, that the
working mechanism of DBS might be different in the various target regions
(Montgomery2010).
The medial prefrontal cortex (mPFC) has been proposed as a target region for
treatment-resistant drug addiction and depression. High frequency DBS of the rat
mPFC influencedcocaine-seekingbehaviorsand themotivation for itsconsumption
intheratcocaineself-administrationaddictionmodel(Levyetal2007)andinduced
an antidepressant-like response in the forced swim test (Hamani et al 2010a). A
dysfunction of the dorsolateral PFC (dlPFC), the primate analogue of the rodent
prelimbic(PL)areaofthemPFC,isinvolvedinbothdiseases(Drevets2000,Changet
al 2011, Qi et al 2012, Willner et al 2013, Hayashi et al 2013) as well as in OCD
(Okada et al 2013) and post-traumatic stress disorder (Simmons and Matthews
2012).Although itsexact role inbehavior remainsunclear, thedlPFC isbelieved to
integratesensoryandvisceral inputto learnassociations(Eustonetal2012)andto
guide behavior toward the acquisition of adaptive goals bymodulating subcortical
regions (Miller et al 2002, Ballard et al 2011) through its key role in the reward
circuit. The beneficial behavioral effects observed in preclinical DBS research
targeting the mPFC might be explained by a DBS-induced upregulation of the
hyposensitive reward-system that is often seen in the abovementioned disorders.
Indeed,mPFCDBShasalreadyshownto inducesustainedincreases inhippocampal
5-HT levels in rats in amicrodialysis study (Hamanietal 2010a) and stimulationof
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66
thehumandlPFCwith repetitive TranscranialMagnetic Stimulation (rTMS) induced
dopamine release in the caudate nucleus, measured by [11C]-raclopride Positron
EmissionTomography(PET)(Strafellaetal2001).
To further investigate themechanismsbehind these therapeutic effects,molecular
imagingtechniquessuchasPETandSinglePhotonEmissionTomography(SPECT)can
be used to provide insight into the stimulated networks. We have previously
demonstrated that SPECT using 99mTc-hexamethylpropyleneamineoxime ([99mTc]-
HMPAO) and PET using 2-deoxy-2-18F-fluoro-β-D-glucose ([18F]-FDG) are
indispensable tools in the evaluation of experimental neurostimulation paradigms
(Wyckhuys et al 2010b, 2013, Parthoens et al 2014b). Both techniques indirectly
visualizestimulation-inducedchangesinregionalneuralactivityandrevealnetworks,
byvisualizing interconnected regional cerebralblood flowandglucosemetabolism,
respectively. In thecurrent study, smallanimalPET (µPET)withVolume-Of-Interest
(VOI)-basedandStatisticalParametricMapping(SPM)analysiswasusedtovisualize
regionalchangesincerebralglucosemetabolisminducedby60Hzand130HzDBSof
theratmPFC.Becauseoftheabovementionedeffectsofthislowandhighfrequency
stimulation paradigms in other targets, we hypothesized that 60 Hz mPFC would
increaseand130Hzdecreasethebrain’sglucosemetabolism.
4.3 MaterialsandMethods
4.3.1 Animals
MaleSprague-Dawleyrats(n=11,250-275g,Janvier,France)weretreatedaccording
toguidelinesapprovedby theEuropeanEthicsCommittee (86/609/EEC).Thestudy
protocol was approved by the Antwerp University Ethical Committee for Animal
Experiments (2012-50). The animals were kept under environmentally controlled
conditions (12h light/darkcycles,20-23 °Cand50-55%relativehumidity)with food
andwateradlibitum.
DeepBrainStimulation
67
4.3.2 SurgicalprocedureTheratswereanesthetizedwithamixtureofisofluraneandmedicalO2(5%induction
dose,2%maintenance)while0.05mg/kgTemgesicwas injectedsubcutaneouslyas
analgesic. A sagittal incision following the superior sagittal suturewasmade along
the skull. A custom-made bipolar DBS electrode (125 µmdiameter, Bilaney, 1mm
betweentheelectrodetips)wasimplantedintheleftPL(AP+3.7mm,ML+2.0mm,
DV-5.0mmrelativetobregma,atanangleof18°inthecoronalplane)(Paxinosand
Watson2007).Additionally,fivesmallstainlesssteelmountingscrews(Bilaney,1.57
mmdiameter, 3.2mm length)were inserted into the skull to secure theelectrode
withdentalcementontotheskull.
4.3.3 Deepbrainstimulation
Theanimalswereallowedtorecoverfromsurgeryforoneweekbeforethestartof
the experiments. During that week animals were also habituated to the DBS cage
(Bioanalytical systems). On the test days, the rat was put in the DBS cage and its
electrode was connected to the output of a stimulator (DS4 Biphasic Stimulus
Isolator,Digitimer)throughacommutator(Bilaney).Thestimulatorreceiveditsinput
through a computer controlled (Labview 7.0) data acquisition card (NI PCI-6251,
National Instruments). Each rat received 1 hour of DBS (60Hz or 130Hz, biphasic
pulses, 200µspulsewidth, 150µA) and sham stimulation,while the animalswere
awake and freely moving. An amplitude of 150 µA was chosen because in a pilot
study,thiswasthesubtresholdintensitytoprovokeabnormalbehavior(i.e.freezing,
wet dog shakes, obsessive grooming or convulsions) when the stimulation was
turnedonateitherfrequency(datanotshown).
Allratsreceivedallthreeconditions(60Hz,130Hzandsham)inarandomizedorder
and two consecutive treatments were separated by at least 48 hours to allow a
washoutoftheeffects.ForshamstimulationtheratwasputintotheDBScagewith
theelectrodeconnectedtothestimulator,thoughnostimulationwasgiven.
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68
4.3.4 MicroPET-CTimaging
Thirty minutes after the start of the stimulation, 1 mCi [18F]-FDG was injected
intravenouslyinthetailvein(animalswerefastedovernightforatleasttwelvehours
(Deleyeetal2014)).After30minutesofawake[18F]-FDGuptake(thus immediately
after terminationof the60minutes stimulation), the ratswere anaesthetizedby a
mixture of isoflurane and medical oxygen (inhalation, 5% induction, 1.5%
maintenance) and placed onto the thermostatically heated bed of a µPET scanner
(Figure4.1).
Figure 4.1 Protocol of one scan session. Thirty minutes after the start of 1h awake DBSapplication or sham, the animals were intravenously injected with 1mCi of [18F]-FDG.Immediatelyaftertheterminationofstimulation,theratswereanesthetizedandpositionedon theµPET-CTscannerafterwhich thePETacquisition (20min)wasstarted, resulting inatotaltimeof40mintraceruptake.
MicroPET imaging was performed on a Siemens Inveon PET-CT scanner (Siemens
Preclinical Solution, Knoxville, TN) (Bao et al 2009). The reconstructed spatial
resolution is 1.4 mm at the center of the field of view (FOV) and the axial and
transaxialFOVsare10.0and12.7cm,respectively.
ForquantitativeanalysistheµPETimageswerereconstructedusing4iterationswith
16 subsets of the 2D ordered subset expectation maximization (OSEM) algorithm
followingFourierrebinning.Theresultingimageswere128x128x159withavoxel
size of 0.78 x 0.78 x 0.80 mm3. All data corrections (dead time, normalization,
randoms,attenuationandscatter)wereapplied.Attenuationandscattercorrection
are based on a segmented attenuationmap calculated from amodified CT image
that was elaborately corrected for metal artifacts as follows: i) a Maximum A
sham,&60&Hz&or&130&Hz&DBS&
1 mCi [18F]-FDG (i.v.)
Anesthesia and positioning on scanner
ANESTHETIZED AWAKE
µPET CT
20 min 10 min 30 min 30 min 10 min
DeepBrainStimulation
69
Posteriori – Transmission (MAP-TR) reconstruction (Lemmens et al. 2009) was
thresholdedtodeterminethemetalparts(screwsandcanulla)inthereconstruction;
ii)metalartifactreduction(MAR)wasperformedusingasinograminpaintingmethod
(Lemmensetal.2009;Prelletal.2009);and iii)asmallportionof theskullaround
themetalscrewsthatwaslostintheMARreconstructionwasreplacedusingtheCT
oftheskullofahealthynon-implantedrat.ThefinalCTimagewasthenobtainedby
combining the imagesof i)metalonly, ii) rat imageand iii)partsof skull thatwere
missinginii)fromadonorCTimage.
4.3.5 HistologicalverificationoftheelectrodepositionTodeterminetheexactpositionoftheelectrodeaftertheimagingexperimentswere
completed, theanimalsweredeeply anesthetizedwith isofluraneand sacrificedby
an overdose of Nembutal (i.v., 150 mg/kg). Then a direct current of 300 µA was
administered for 10 s, causing iron deposition of the electrode tips into the
surrounding tissue. The brains were removed and stored in 98% formaldehyde
solution (4%, Klinipath, The Netherlands) and 2% ferrocyanide (potassium
hexacyanoferrate(II)trihydrate, Sigma-Aldrich, Germany) for at least 48 hours, to
causebluecoloringoftheironparticlesinthebrain,(Figure4.2a,b).Thenthebrains
weresnapfrozen in2-methylbutane(Sigma-Aldrich,Germany)using liquidnitrogen
and stored at -20 °C. Coronal sections of 30 µmwere cut on a cryostat (Leica CM
1950) and stained with Harris hematoxylin solution (Sigma-Aldrich, Germany).
Verification of the electrode placementwas performedwith the aid of a rat brain
atlas(PaxinosandWatson2007)andonlyratswithelectrodeswithintheboundaries
oftheleftPLmPFCwereconsidered(n=9)forfurtheranalysis.
4.3.6 DataanalysisThe ratbrainwas cropped from thePET imagesand imageswere resampled toan
isotropic voxel size of 0.2 mm. The brain images were then transformed into the
Paxinosstereotaxicspace(PaxinosandWatson2007)usingspatialnormalizationtoa
[18F]-FDGratbraintemplate(Schifferetal2007)inPMODv3.3(PMODTechnologies,
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70
Switzerland). First, we verified the absence of significant global changes in whole
brain [18F]-FDG uptake after count normalization for the injected dose, when
comparingshamversus60Hzand130Hz.Allimageswerethennormalizedtohave
anaveragewholebrain[18F]-FDGuptakeof1.
Thevolumesofinterest(VOI)oftheleftandrightmPFCavailableinPMODv3.3were
subdivided into thePLand ILareasbasedonaT2-weighted ratbrainMR template
andthePaxinosstereotaxicatlas(PaxinosandWatson2007).TheVOIsasshownin
Figure 4.4 are 4.8mm3 and 1.5mm3 for the PL and IL areas respectively. Average
normalizeduptakevalueswithineachofthefourVOIswerecalculatedfromthePET
images for statistical analysis. A one-way repeated measures ANOVA followed by
predefined planned contrasts was performed in SPSS v20 (IBM corporation). Two
one-tailed simple contrasts were thereby tested; 60 Hz versus sham and 130 Hz
versus sham to test the hypotheses that, in the stimulated region, these two
paradigms inducedhyper-andhypometabolism,respectively.Statisticalsignificance
wassetatp=0.05.
Additionally, to further explore the imaging results, a voxel-based Statistical
Parametric Mapping (SPM) analysis was performed using SPM8 (Welcome
Department of Cognitive Neurology, London, UK) within a one-way repeated
measures ANOVA design. The normalized brain images were smoothed using a
Gaussian filter (isotropic 1.5 mm full-width-at-half-maximum) and subsequently
masked to remove extracerebral activity. A F-contrast, testing for any difference
between the three stimulation paradigms, and four T-contrasts, testing for both
hyper- andhypometabolism for 60Hz versus shamand130Hz versus sham,were
defined. Voxels that passed the omnibus F-test at a significance level of 0.05
(uncorrected)definedamaskforthesubsequentpost-hocT-contrasts.T-mapswere
thresholded at a significance level of 0.05 (uncorrected) with an extent cluster
thresholdof130voxels (~1mm3).Theeffect ineachof thesignificantclusterswas
then calculated for each animal as ((uptake stimulation)/(uptake sham) -1), with
DeepBrainStimulation
71
uptake being the mean normalized [18F]-FDG uptake in the cluster, and finally
averagedoverallanimals.
4.4 Results
Noabnormal behaviorwasnoticedduringor following applicationofDBSor sham
stimulation.
4.4.1 HistologyAt the end of the experiment, histological examination was performed for
verificationof theelectrodeplacements,whichrevealedthatthepositioningof the
electrodeintothePLmPFCwassuccessfulinnineoutofelevenrats.Tworatshada
disputable electrode placement (i.e. too deep, in the infralimbic cortex) and were
excludedfromtheimageanalysis(Figure4.2c).
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72
Figure4.2Verificationoftheelectrodeplacements:a)coronalpictureofthebrainwiththeintendedelectrodeplacement,b)pictureoffrozenbraintissuewithabluemarkshowingthelocation of the deepest electrode tip, c) location of the deepest electrode tips of all rats(n=11)depictedontwocoronalslices,withthecorrectplacements indicatedbyablackdot(n=9),andwrongordisputableplacementswitharedcross(n=2).Cg=cingulatecortex,PL=prelimbiccortex,IL=infralimbiccortex(adaptedfrom(PaxinosandWatson2007)).
DeepBrainStimulation
73
4.4.2 VOI-basedanalysisVOI-based analysis after whole brain normalization (Figure 4.3) revealed statistical
significantglucosemetabolismdifferencesbetweenthethreestimulationparadigms
inthe ipsilateralPL(ANOVA,p<0.05)butnot inthecontralateralPLandalsonot in
the infralimbic areas (IL). This allowed for subsequent testing of the predefined
planned contrast in the ipsilateral PL, which showed significant hypermetabolism
([18F]-FDGuptake5.2±2.3%,p<0.05)forthe60HzDBSstimulationversussham.At
130Hz,hypo-metabolismwasinducedintheipsilateralPL(-2.5±2.6%,albeitnon-
significant).
Figure 4.3 Regional average changes in glucose metabolism caused by 60 Hz or 130 Hzstimulation, compared to sham stimulation, revealed by volume-of-interest based analysis.mPFC=medialprefrontalcortex,PL=prelimbicareaofmPFC,IL=infralimbicareaofmPFC,ipsi = ipsilateral, contra = contralateral * = regions with significant average change inmetabolism,comparedtosham(ANOVAfollowedbypredefinedcontrasttest,p<0.05).
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74
4.4.3 Voxel-basedSPManalysis
The voxel-based SPM analysis revealed that both stimulation paradigms induced
significantincreasesaswellasdecreasesinglucosemetabolism,whencomparedto
shamstimulation.
Figure 4.4Voxel-based SPM results for the T-contrasts 60HzDBS versus sham. a) Coronalslices through T-maps, showing clusters of significant hyper- or hypometabolism (p<0.05,uncorrected, clustering threshold of 130 voxels (~ 1mm3)), overlaid on aMR template. T-values for hypometabolism are indicated as negative on the color bar. The location of thecoronalslicesareindicatedinthesagittalslice.Slicesare2mmapart. TheVOIsusedintheVOIbasedregionalanalysisaredelineatedwithblackandwhitelines(PL=prelimbicarea,IL=infralimbic area). b) Volume rendering of the significant T-value clusters. Red and blue arehyper-andhypometabolismrespectively.Fromlefttoright:topview,leftsideviewandfrontview.ThenumberingoftheclusterscorrespondstoTable4.1.Arrowsina)andb)pointtothehypermetabolicclusternearthestimulatedregion.
PL
IL
1 2 3
4 5 6
9
1 9 3 5 7 PL
IL 0
5
-5
a
b
3 mm
I
I I
II
II II
III
III
III
IV
IV IV
V
V
V VI
VI
VI
VII
VII
VII
i
i
i ii
ii
ii
iii
iii iii
iv iv
iv
DeepBrainStimulation
75
The thresholded T-maps are shown in Figure 4.4 and 4.5 and the clusters are
summarizedinTable4.1.Fewerclusterswerefoundfor130Hzthanfor60Hz.
For60Hzstimulationasignificantclusterwasfoundatthelocationofthestimulation
forthehypermetabolismT-test(arrowsinFigure4.4andclusterIIinTable4.1).The
clustersizewas1.5mm3,withanaverageincreaseduptakeof7.9±2.9%.Atotalof
56%oftheclusterwaslocatedinthePLVOIusedintheVOIbasedanalysiswhereit
madeup17%ofthetotalPLVOI.For130Hzstimulationnosignificantclusterswere
foundatthelocationofthestimulation.
Figure 4.5Voxel-basedSPMresults for theT-contrasts130HzDBSversussham.a)Coronalslices through T-maps, showing clusters of significant hyper- or hypometabolism. Color barand location of slices is as in Figure 4.4. b) Volume rendering of the significant T-valueclusters.ColoringandviewpointsareasinFigure4.4.For the 60 Hz contrasts the largest hypermetabolic clusters are located in the
ipsilateral piriform cortex, the contralateral auditory / visual cortex / hippocampus
andthe ipsilateralmotorcortex.The largesthypometaboliccluster is located inthe
medulla/cerebellum(ipsilateral).Forthe130Hzcontrastthelargesthypermetabolic
cluster was found in the contralateral auditory / visual cortex. The only
hypometabolic cluster was found in the ipsilateral piriform cortex / caudate
putamen.
2 5 8
a
b
I
I I
II
II II
III III III IV
IV IV
i
i
i
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76
Table4.1OverviewofallSPMclustersofsignificanthyper-andhypometabolismforthe60HzDBS versus sham and 130 Hz DBS versus sham. The effect expresses the percentagewiseincrease or decrease of normalized [18F]-FDG uptake (SEM= standard error of themean).Coordinates of the center of mass of the clusters are given in the Paxinos stereotaxiccoordinates[34].(AP=anteroposterior,ML=mediolateral, DV=dorsoventral).PositiveMLcoordinatescorrespondtoipsilaterallocations.ClusternumberingisasinFigure4.4and4.5.Anatomicalregions:Am=amygdala,Au=auditorycortex,Cb=cerebellum,Cg=cingulate,CN=cohlearnuclei,Cpu=caudateputamen,Ent=entorhinalcortex,Hipp=hippocampus,IC=insularcortex,Ic=internalcapsule,Md=medulla,Mo=motorcortex,Msc=mesencephalon,Pir=piriformcortex,PL=prelimbiccortex,Vi=visualcortex.
Cluster #Voxels Volumemm3
Effect±SEM
CenterofmassAPMLDV Region
60Hzhyper
I 205 1.64 8.9±3.6% 3.45 -0.99 -1.49 Mo,Cg
II 187 1.496 7.9±2.9% 3.14 0.37 -3.78 PL
III 176 1.408 4.6±1.6% 2.34 4.88 -5.97 IC
IV 1189 9.512 5.1±1.8% 0.55 3.86 -8.76 Pir
V 156 1.248 7.6±2.3% -3.12 5.07 -9.94 Pir,Am
VI 376 3.008 3.9±0.8% -5.63 -5.71 -3.64 Au,Vi,Hipp
VII 130 1.04 4.0±1.0% -7.67 5.05 -6.97 Ent
60Hzhypo
i 224 1.792 -4.0±1.1% -3.86 4.15 -4.36 Hipp,Ic
Ii 245 1.96 -4.3±1.1% -6.86 1.18 -5.42 Msc
Iii 519 4.152 -4.8±1.6% -14.01 1.45 -5.7 Cbiv 1352 10.736 -5.1±1.6% -14.8 -2.41 -7.4 Md
130Hzhyper
I 187 1.496 5.9±1.4% -3.1 -6.8 -5.01 Au
II 822 6.576 5.5±0.9% -5.64 -5.93 -3.14 Vi,Au
III 366 2.928 4.5±1.6% -11.49 -3.46 -6.55 Cb,CNIV 211 1.688 6.0±2.2% -14.09 -1.96 -8.27 Md
130Hzhypo i 1697 13.576 -3.9±1.3% 0.39 4.42 -6.78 Cpu,Pir
4.5 Discussion
Theobjectiveofthisstudywastoevaluatetheeffectsof60Hzand130HzDBSofthe
ratPLmPFContheregionalbrainglucosemetabolism.Changes in[18F]-FDGuptake
reflect changes in metabolic demand following neuronal firing and are hence
DeepBrainStimulation
77
believedtoprovideanindirectmeasureofbrainactivity(Drevets2000,Chattonetal
2003). The main research hypotheses of our study were that, in the stimulated
region, i) 60 Hz DBS would cause increased [18F]-FDG uptake (hypermetabolism)
compared to sham and ii) 130 Hz DBSwould cause hypometabolism compared to
sham. The first hypothesis was based on neuronal activation caused by low
frequency DBS. This was first described by Goddard (1969): stimulation of limbic
structuresatlowfrequencies(20–70Hz)resultedinconvulsions,with62.5Hzbeing
themostfavorablefrequencytodevelopthis“kindling”effect(Goddardetal1969).
The second hypothesis was based on the well-documented reversible inhibitory
effects of high frequency DBS stimulation for movement disorders (Benabid et al
1998)andepilepsy(Wyckhuysetal2010b).
Thefirsthypothesiswasconfirmedbyourstudy(60Hzhypermetabolism,+5.2±2.3
%,p<0.05).However,wecouldnotconfirmthesecondhypothesisalthoughasmall
decrease inmetabolismwas foundalbeit not significant. The lowamplitudeof the
regionalmetabolicchangesseeninourstudycaninpartbeexplainedbythepartial
volumeeffect.ThevolumetricresolutionoftheusedµPETscanneris5mm3(Baoet
al2009)withthePLvolumeusedintheVOIbasedanalysisonlybeing4.8mm3.Using
computersimulationswefoundthat,ifthePLVOIwouldhypotheticallybeuniformly
activated,thePETmeasurementwouldresultinanunderestimationoftheactivation
by72%becauseofthispartialvolumeeffect.Therefore,forthesimulatedcase,the
reported5.2%hypermetabolismwouldhypotheticallycorrespondtoanactual18.6
% increase inmetabolism.Other factors thatmight explain the small changes are:
imperfect spatial normalization to the stereotaxic space, the use of a rather low
intensity(150µAasdeterminedinapreliminarytolerancepilotstudy,comparedto
upto400-500µA(Levyetal2007,Hamanietal2010b)),orbytheshorttreatment
durationbeforetracer injection(30minutes,comparedto6-12months(Lipsmanet
al2013,Smith2012)).Giventhesesmallchangesandthegroupsizeused(n=9)the
statistical power of our studywasmoderate. This could explainwhywe could not
confirmthesecondhypothesis(130Hzhypometabolisminthetargetregion).
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Statisticalparametricmappinghoweverconfirmedourregionalanalysisbydetecting
ahypermetabolicclusteratthesiteofstimulationfor60HzDBS.Moreover,asseen
inotherneuroimagingstudies,forbothstimulationfrequencies,thedirectionalityin
neurophysiological responsewasnotalwaysobservedatbrainregionsdistant from
thefrontalcortex (Lipsmanetal2013,Höflichetal2013).AlthoughtheDBStarget
regionmayforinstancebeinhibited,theoutputofthisregionmightbeincreasedas
aconsequenceoftheactivationofefferentfiberpathways,therebyactivatingdistant
neuronalstructures (Hamanietal2010b,Vitek2002).Bothstimulation frequencies
effectivelyresultedinclustersofbothhyper-andhypometabolicvoxels.ThePLDBS
stimulation inducedhypermetabolism (60Hz) andhypometabolism (130Hz) in the
ipsilateral piriform cortex, involved in olfaction. Both stimulation frequencies also
induce hypermetabolism in the contralateral auditory and visual cortex. A
hypermetabolic clusterwas found in themotorcortex for60Hzstimulation.These
results seem to show a certain heightened state of alertness (increased activity in
sensoryandmotor related regions)andhighlights the roleof the frontal regions in
functionssuchasawareness.
Inthecurrentexperiment,theratPLmPFCwaschosenasatargetregionbecauseof
thepromisingpreclinicalresultsinthetreatmentofdepression(Hamanietal2010a)
and addiction. Combining the results from our current study with imaging studies
that suggest ahypofunctionof thePLmPFCas a contributing factor to thealtered
autonomicandneuroendocrinefunctioninthesediseases(Drevets2000,Volkowet
al2003),thisstudysuggestsDBSofthisbrainregionwithastimulationfrequencyof
60Hz for thetreatmentofbothdisorders.Toevaluatethetherapeuticpotentialof
this treatment approach, preclinical imaging studies using animal models of
depressionoraddictionaretopicoffutureworkevaluatingwhether60HzDBSofthe
PLmPFCcanreversefrontalhypometabolism.
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79
4.6 Conclusion
Thisstudysuggeststhepotentialof60HzPLmPFCDBS,or itshumananaloguethe
dlPFC,forthetreatmentofdisordersassociatedwithprefrontalhypofunctionsuchas
depression or addiction. However, further behavioral testing is required for safe
translationtoclinicalstudies.Thissmallanimalmolecularimagingstudysupportsthe
useof[18F]-FDGPETasanaidintherapeuticdecision-making.
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80
Chapter5:
Small animal repetitive TranscranialMagnetic
Stimulationcombinedwith[18F]-FDGmicroPET
toquantifytheneuromodulationeffect inthe
ratbrain
Thischapterhasbeenpublishedas:Parthoens, J.;Verhaeghe, J.;Wyckhuys,T.; Stroobants, S.; Staelens, S. Small animalrepetitive transcranial magnetic stimulation combined with [18F]-FDG microPET toquantify theneuromodulationeffect in the ratbrain.Neuroscience.Vol 275.2014.pp.436-443.
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5.1 Abstract
Introduction Repetitive TranscranialMagnetic Stimulation (rTMS) is a non-invasive
neurostimulationtechniqueforthetreatmentofvariousneurologicalandpsychiatric
disorders. To investigate the working mechanism of this treatment approach, we
designed a small animal coil for dedicated use in rats and we combined this
neurostimulation method with small animal Positron Emission Tomography
(microPET or µPET) to quantify regional 2-deoxy-2-(18F)fluoro-D-glucose ([18F]-FDG)
uptake in the rat brain, elicited by a low (1 Hz) and a high (50 Hz) frequency
paradigm.
MethodsRats(n=6)wereinjectedwith1mCiof[18F]-FDG10minutesafterthestart
of30minutesofstimulation(1Hz,50Hzorsham), followedbya20minutesµPET
imageacquisition.Voxel-basedStatisticalParametricMapping (SPM) imageanalysis
of1Hzand50Hzversusshamstimulationwasperformed.
Results For both the 1 Hz and 50 Hz paradigm we found a large [18F]-FDG
hypermetaboliccluster (2.208mm3and2.616mm3resp.) (ANOVA,p<0.05) located
in the dentate gyrus complemented with an additional [18F]-FDG hypermetabolic
cluster(ANOVA,p<0.05) located intheentorhinalcortex(2.216mm3)forthe50Hz
stimulation.Theeffecton[18F]-FDGmetabolismwas2.9±0.8%at1Hzand2.5±0.8
%at50Hzforthedentategyrusclustersand3.3±0.5%fortheadditionalclusterin
theenthorhinalcortexat50Hz.Themaximal(4.19vs.2.58)andaveraged(2.87vs.
2.21)T-valuesarehigherfor50Hzversus1Hz.
Conclusion This experimental study demonstrates the feasibility to combine µPET
imaging in rats stimulatedwith rTMS using a custom-made small animalmagnetic
stimulationsetuptoquantifychangesinthecerebral[18F]-FDGuptakeasameasure
forneuronalactivity.
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83
5.2 Introduction
RepetitiveTranscranialMagneticStimulation(rTMS)isaneurostimulationtechnique
thatusesarapidlychangingmagneticfieldtoinduceanelectricfieldinthebrain.The
induced electric field elicits depolarization or hyperpolarization of neurons and
lastingchangesincorticalexcitability(Fitzgeraldetal2006,FunkeandBenali2011).
This non-invasive treatment has provided remarkable therapeutic benefits for
variousneuropsychiatricdisorders,suchasdepression(Kecketal2000,Fitzgeraldet
al 2003), addiction (Camprodon et al 2007, Rose et al 2011) and obsessive-
compulsivedisorder(KumarandChadda2011,2011).
Positron Emission Tomography (PET) using 2-deoxy-2-(18F)fluoro-D-glucose ([18F]-
FDG), a glucoseanalog, has shownabnormal low levelsof [18F]-FDGmetabolism in
thedorsolateral Prefrontal Cortex (dlPFC) in bothdepressed (Biveret al 1994) and
addicted(Volkowetal2011)patients.Therefore,inthetreatmentofthesedisorders,
rTMS ispreferentiallyapplied to this region (KobayashiandPascual-Leone2003) in
order to induce increases inneuronalactivity.PETrTMSstudieshaverevealedthat
PFCrTMSinducesneuronalmetabolicchangesinthestimulatedregionaswellasin
remote brain areas, which were highly dependent on the stimulation protocol
(Reithler et al 2011). Other clinical studies showed that daily PFC rTMS sessions
improvemood indepression (Georgeetal 1995)and reducenicotineconsumption
anddependence(Amiazetal2009)andthatcocainecravingreducesforatleastfour
hours after one single PFC rTMS session (Camprodon et al 2007). The exact
mechanismunderlying these rTMS-inducedeffects isnotclearalthough it iswidely
believed to reflect changes in synaptic efficacy akin to long-term
potentiation/depressionofthestimulatednetwork(Houdayeretal2008).
Since human research regarding the elucidation of the mechanism of action is
restricted, laboratory animals are indispensable. PET and Single Photon Emission
Computed Tomography (SPECT) scanners have been successfully miniaturized for
preclinicalstudiesallowingforhighspatialresolutionwithanacceptablesensitivityin
rats andmice (µPET and µSPECT) (Rowland and Cherry 2008).We have previously
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demonstrated that µSPECT using 99mTc-hexamethylpropyleneamineoxime ([99mTc]-
HMPAO) was an indispensable tool in the evaluation of experimental
neurostimulation paradigms (Wyckhuys et al 2010b, 2013) quantifying regional
cerebral blood flow changes also reflecting neural activity (Shibasaki 2008). In our
previous rat rTMS-SPECT experiment (Wyckhuys et al 2013), we used a figure-of-
eight20mmMagstimhumancoil(outerdiameter±26mm),revealingpredominantly
decreasesinregionalcerebralbloodflowduetoapplicationofboth1Hzand10Hz
rTMS,whichwerewidespreadthroughouttheentireratbrainandnotrestrictedto
delineatedbrainstructures.Here,tofurtherexploretheimpactofhigherfrequencies
wenowconsidered1Hzand50HzrTMS.
Recently,wedevelopedasmallerTMScoil(outerdiameter19mm),anexperimental
TMSstimulatorandadedicatedTMS-deliverysetupthatallowedthestimulationof
awakesmall animals. In thecurrent study this speciallydesignedsmall animalTMS
setupwasusedforthefirsttimetoperformaµPETstudyusingStatisticalParametric
Mapping (SPM)analysisof stimulation-ONversus stimulation-OFF (sham).Minor to
moderatebutsignificantchangesin[18F]-FDGuptakeelicitedby1Hzor50HzrTMS
compared to sham are visualized and their regional distribution and intensity are
quantified.
5.3 MaterialsandMethods
5.3.1 Animals
SixmaleSpragueDawleyrats(275-300gbodyweight,Harlan,theNetherlands)were
treated according to guidelines approved by the European Ethics Committee
(86/609/EEC). The study protocol was approved by the Antwerp University Ethical
Committee for Animal Experiments (2011-30). The animals were kept under
environmentally controlled conditions (12 h normal light/dark cycles, 20-23 °C and
50-55%relativehumidity)withfoodandwateradlibitum.
For reproducible positioning of the TMS coil and to reduce stress during the
experimentaldays,ratsweretrainedaprioriduringfiveconsecutivedayspriortothe
Low-intensityrTMS
85
experiment to lie still in a transparent conical restrainer for 30minutes. After this
habituationperiod,eachratreceived30minutesofoneoftworTMSparadigms(1Hz
and 50Hz) or sham stimulation as illustrated by Figure 5.1. Tenminutes after the
startof rTMSorshamstimulationtheratswere injectedwith1mCiof [18F]-FDG in
thetailveinwhileawakeandundercontinuousrTMS.Duringthisradiotraceruptake
period, theanimalswerekept ina separate space, isolated fromroomactivities to
controlforexternalstimuli.
Figure5.1Protocolofascansession.TenminutesafterthestartofrTMSorshamstimulation,theratswereintravenouslyinjectedwith1mCiof[18F]-FDG.Eachratreceived30minutesofi)continuous1Hzii)trainsof50Hz,1.2sdurationand58.8sintertrainintervalsandiii)shamstimulation. The total number of pulses delivered each minute was the same for bothparadigms(60pulsesperminute).Forshamstimulation,continuous1HzrTMSwasdeliveredwith the coil positioned perpendicular and approximately 4 cm away from the head.ImmediatelyafterterminatingtherTMSorshamstimulation,theratswereanesthetizedandpositionedontheµPET-CTscanner(20minutesPET,10minutesCT).
ImmediatelyafterterminatingtherTMSorshamstimulation,thusafter20minutes
ofawake[18F]-FDGuptake,theratswereanesthetizedusingamixtureof isoflurane
and medical oxygen (inhalation, 5% induction and 2% maintenance dose) and
positionedontheµPETscanneruntiltheyreachedatotalof30minutesof[18F]-FDG
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86
uptake, after which a 20 minutes PET acquisition was started followed by a 10
minutesCTscan(Figure5.1).
PET imaging allows longitudinal studies, so all rats were scanned three times,
receiving all three stimulation parameters (in a randomized order). Hence, the
animals functioned as there own control, reducing inter-animal variation and
increasing statistical power. Consecutive experimental days were separated by at
least 48 hours to allow a fasting duration ofminimally twelve hours (Deleye et al
2014).
5.3.2 RepetitiveTranscranialMagneticStimulation
The symmetric figure-of-eight coilwas placedwith its center positioned above the
medial PFC (mPFC) (± 5 mm anterior to bregma, 0 mm mediolateral). The exact
position of the coil in relation to the rat brain was validated by performing a
ComputedTomography(CT)acquisitionofoneratplacedinaconicalrestrainerwith
thecoilfixatedontopoftherestrainer(Figure5.2a).ThisCTimagewascoregistered
with a rat brainMagnetic Resonance (MR) template (Figure 5.2b). Throughout the
experiment,reproduciblepositioningofthecoilinrelationtothebrainwasensured
bythefixationofthecoilontherestrainerincombinationwiththeconicalshapeof
therestrainer,whichpreventedheadmovement.
A custom-made small animal figure-of-eight coil (each wing: 9 insulated 0.8 mm
diameter copper windings, outer diameter 19.0 mm and inner diameter 2.8 mm,
coveredwithpolyimideinsulationtape)(Figure5.2)wasconnectedtotheoutputofa
gradientamplifier(Techron,7700Series)thatreceiveditsinputthroughacomputer
controlled(Labview7.0)dataacquisitioncard(NIPCI-6251,NationalInstruments).
Eachratreceived30minutesof i)continuous1Hzii)trainsof50Hz,1.2sduration
and58.8s intertrain intervalsand iii) shamstimulation.Thetotalnumberofpulses
(450µs,sinewave,peakamplitudeof1782Aeachwing)deliveredeachminute(60
pulsesperminute)wasthesameforboththe1Hzand50Hzparadigms(Figure5.1).
These specifications are comparable to those in our previous study, using the
smallestcommerciallyavailablehumanfigure-of-eightcoil(Wyckhuysetal2013).For
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87
sham stimulation continuous 1 Hz rTMS was delivered with the coil positioned
perpendicular and approximately 4 cm away from the head to have no electrical
stimulationwhilestillmimickingtheexperimentalmanipulationsandclickingnoiseof
thecoil.Topreventoverheatingofthecoil,externalcoolingwasprovidedduring50
HzrTMSwithicepacksonthecoil,whichwereneverincontactwiththerat'shead
andthushaveno influenceontheresultingPET image.Duringstimulationorsham
stimulation visual inspection of the rat’s behavior was performed to record
abnormalities.
Figure5.2Coilpositioning.A)Toensurereproduciblepositioningofthecoilinrelationtotheratbrain,thecoilwasfixatedontoaconicalrestrainer.B)ComputedTomography(CT)imageofaratplacedinaconicalrestrainerwiththecoilfixatedabovethemedialprefrontalcortex(mPFC),coregisteredwitharatbrainMRtemplate(red);note:streakartefactsappearduetoincompatibilityofmetalwithCT.
5.3.3 MicroPET-CTimaging
MicroPET-CT imaging was performed on two Siemens Inveon PET-CT scanners
(Siemens Preclinical Solution, Knoxville, TN) (Bao et al 2009). The energy and
coincidence timingwindowwas set to350–650keVand3.432nsec, respectively.
Thereconstructedspatial resolution isaround1.4mmatthecenterof theFieldOf
View(FOV)andtheaxialandtransaxialFOVsare10.0and12.7cm,respectively.
A20-minutestaticPETacquisitionwasfollowedbyananatomicalCTacquisition(10-
minute scan). For quantitative analysis, µPET images were reconstructed using 4
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iterations with 16 subsets of the 2D Ordered Subset Expectation Maximization
(OSEM) algorithm following Fourier rebinning. Normalization, dead time, randoms,
CT-basedattenuationandsingle-scattersimulationscattercorrectionswereapplied.
5.3.4 Imageanalysis
Each PET image was spatially normalized into the space of an [18F]-FDG template
(Schifferet al 2007) using brain normalization in PMOD v3.3 (PMOD Technologies,
Switzerland). First,weverified theabsenceof significant averageglobal changes in
[18F]-FDG uptake after count normalization for the injected dose,when comparing
shamversus1Hzand50Hz.Wedefinehypo-orhypermetabolismasthemetabolism
of[18F]-FDGto[18F]-FDG-6-phosphateatallinstancesthroughoutthemanuscript.A
voxel-based Statistical Parametric Mapping (SPM) analysis was performed using
SPM8 (WelcomeDepartment of CognitiveNeurology, London, UK)with a one-way
repeated measures ANOVA design. The spatially normalized brain images were
smoothedusingaGaussianfilter(isotropic1.5mmfull-width-at-half-maximum)and
subsequently masked to remove extracerebral activity. The masked images were
then scaled to have an average brain uptake of 1. An F-contrast, testing for any
differencebetweenthethreestimulationparadigms,andfourT-contrasts,testingfor
bothhyper-andhypometabolismfor1Hzversusshamand50Hzversussham,were
defined. Voxels that passed the omnibus F-test at a significance level of 0.05
(uncorrected)definedamaskforthesubsequentpost-hocT-contrasts.T-mapswere
thresholded at a significance level of 0.05 (uncorrected) with an extent cluster
thresholdof130voxels(1mm3)approachingthespatialresolutionofPET.Theeffect
in each of the significant clusters was then calculated for each animal as ((uptake
stimulation)/(uptake sham) -1), with uptake being the mean normalized [18F]-FDG
uptakeinthecluster,andfinallyaveragedoverallanimals.
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5.4 Results
NoabnormalbehaviorwasnoticedduringorfollowingapplicationofrTMSorsham
stimulation.
Voxel-based SPM analysis revealed that both the 1 Hz and the 50 Hz stimulation
paradigm induced significant (p<0.05) regional increases in [18F]-FDG uptake
comparedtoshamasdocumentedinTable5.1andasillustratedbyFigure5.3(1Hz)
andFigure5.4(50Hz).
Table 5.1OverviewofallSPMclusterofsignificant [18F]-FDGhypermetabolismfor the1Hzversus sham and 50 Hz versus shamwhich are larger than 130 voxels (1mm3). The effectexpresses thepercentagewise increaseofnormalized [18F]-FDG uptake.Coordinatesof thecenterofmassof theclustersaregiven inthePaxinosstereotaxiccoordinates (PaxinosandWatson 2007) (AP = antero-posterior, ML = mediolateral, DV = dorsoventral). ClusternumberingreferstoFig.5.3and5.4for1Hzand50Hz,respectively.Anatomicalregions:DG=dentategyrus,EC=Entorhinalcortex.
Cluster #VoxelsVolume
(mm3)Effect
Tmax
(Tmean)
Centerofmass
APMLDVRegion
1Hz I 276 2.208 2.9±0.8% 2.58(2.21) -5.62 -3.59 -4.18 DG
50HzI 327 2.616 2.5±0.8% 3.34(2.90) -5.55 -3.59 -4.23 DG
II 277 2.216 3.3±0.5% 4.19(2.84) -8.16 5.04 -6.30 Ec
For both the 1Hz and 50Hz paradigmwe found a large [18F]-FDGhypermetabolic
cluster(2.208mm3and2.616mm3resp.)inthedentategyrus(clustersinFigure5.3
and 5.4A). For the 50 Hz stimulation there is an additional hypermetabolic cluster
located in theentorhinal cortex (Figure5.4B - 2.216mm3). Theeffect on [18F]-FDG
metabolism is 2.9 ± 0.8% at 1 Hz and 2.5 ± 0.8% at 50Hz for the dentate gyrus
clustersand3.3±0.5%fortheadditionalclusterintheenthorhinalcortexat50Hz.
Themaximal(4.19vs.2.58)andaveraged(2.87vs.2.21)T-valuesarehigherfor50Hz
versus1Hz.
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Figure5.3SPMresultsforthe1Hzstimulation[18F]-FDGhypermetabolism(p<0.05andsize>1mm3) Coronal, sagittal and horizontal sections showing the hypermetabolic cluster in thedentategyrusoverlaidonanMRtemplate.Thecoordinates in thepanel correspond to thePaxinos stereotaxic coordinates (Paxinos and Watson 2007). Red = hypermetabolic. TheclustercorrespondstothenumberinginTable5.1.
Noteworthy, at 1 Hz we found a second significant hypermetabolic cluster also
locatedexactlyintheentorhinalcortexaswellbutthevolume(0.416mm3)didnot
reachour130voxelsthreshold(1mm3).
Figure5.4SPMresultsforthe50Hzstimulation[18F]-FDGhypermetabolism(p<0.05andsize>1mm3),A)sectionsthroughthehypermetabolicclusterinthedentategyrusoverlaidonanMRtemplate.B) idemforthecluster intheentorhinalcortex.Thecoordinates inthepanelcorrespond to the Paxinos stereotaxic coordinates (Paxinos and Watson 2007). Red =hypermetabolic.TheclustercorrespondstothenumberinginTable5.1.
0
4
AP -6.0 mm ML -3.3 mm DV -4.6 mm
1 Hz
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91
Significant[18F]-FDGhypometabolicclusterswerealsofound,albeitonlyforthe1Hz
stimulation.However, the total volume (0.912mm3)ofall theseclusterswasagain
toosmalltoreachour130voxels(1mm3)thresholdwhiletheaverageeffect(-2.67±
1.19%)inthoseregionswassimilarinmagnitudetothehypermetaboliceffects.
5.5 Discussion
WehavecombinedrTMSwith[18F]-FDG-PETtovisualizeinducedchangesincerebral
[18F]-FDGuptake,asemi-quantitativesurrogateforglucosemetabolism(Huang2000,
Schifferetal2007)whichprovidesanindirectmeasureofneuronalactivitychanges
(Sokoloff1977,MagistrettiandPellerin1996,Sokoloff1999).Ourmain findingwas
that both 1 Hz and 50 Hz rTMS delivered at this low intensity induced minor to
moderatebutsignificant increasedregional [18F]-FDGuptake.Forboth1Hzand50
Hz, the largest cluster of pronounced increased [18F]-FDG uptake is located in the
anterodorsal part of the hippocampus (dentate gyrus), a regionwell known for its
role inmemory consolidation with connections to the prelimbic part of themPFC
targetregionunderstimulationandtheentorhinalcortex(perforantpath)(Lavenex
etal2002),aregionalsoshowingsignificanthypermetabolisminourstudyat50Hz
stimulation(andequallyalsoat1Hzhoweveronlyforasubthresholdvolume).These
findingstogethersuggesthoweverthatthehippocampusmightplayakeyroleinthe
induction of long-term neuroplastic changes of this therapy, possibly through long
term potentiation-like mechanisms. The high increase in metabolism in the
hippocampusduringrTMSadministrationto themPFCmightalsobecausedbythe
factthatthehippocampusisoneofthemostexcitableregionsofthebrain(Uvaetal
2005).
We have found unilateral hypermetabolism in both the dentate gyrus and the
enthorhinalcortex.However,reanalysis(notshown)usingtwopairwiset-tests(1Hz
versusshamand50Hzversussham,respectively)insteadofconsideringtheANOVA
framework that was used to obtain the results presented in this paper, revealed
bilateral hypermetabolic clusters in the dentate gyrus and the enthorhinal cortex.
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The reanalysis considered a less stringent statistical test, but considering the
mediolateralcenteredcoilpositioningandthecoilsymmetryweexpectthebilateral
hypermetabolicclustersintheseregionstobetrueeffectsthatweremissedduetoa
lackofstatisticalpower.
Despitethefactthatweusedasmallcoil,theeffectsofrTMSextendedfurtherthan
thetargetedregion(i.e.themPFC).Theabilityofthecoiltostimulateselectedfocal
targets needs to be further established however, as the regions situated adjacent
andespeciallydorsallyfromthemPFCarealsosubjectedtothegeneratedelectrical
potentials.Inaddition,thecoordinatedfiringofagroupofneuronsislikelytochange
the activity in connected brain regions and possibly also the strength of the
connectionsbetweenthesebrainregions(FitzgeraldandDaskalakis2012).Thelatter
hasalsobeenconfirmedinclinical(Speeretal2000,Kimbrelletal2002,Speeretal
2009)andmonkeyPET-rTMSstudies(Hayashietal2004).
The minor differences between the 1 Hz and that 50 Hz stimulation (a large
hypermetabolicregionlocatedintheentorhinalcortexforthe50Hzstimulationand
more significant T-values)might not only be explainedby thedifferent stimulation
frequencybutalsobytheprotocolusedfor50Hzstimulation(1.2strainsof50Hz,
58.8s intertrain interval,30minutes)comparedtothecontinuous1Hzstimulation
(30minutes)aswekepttheamountofpulsesperminuteconstantforbothprotocols
(60pulses/minute).Thelongintertrainintervalsduringthehighfrequencyparadigm
mighthavehadanimpactontheefficacyofthestimulation(Rossietal2009),since
the effect of rTMS is sensitive to the temporal pattern of the stimulation protocol
andhighlydependsonthe inducedexcitabilitychangesby theprecedingactivation
history (i.e. metaplasticity) (Reithler et al 2011). In the current study, the 58.8 s
intertrain intervalswerenecessaryduring50Hzstimulationtopreventoverheating
of the coil. Resistive heat production inside the coil windings is one of the major
constraints in thedevelopmentofnewstimulationprotocols and is anevenbigger
challenge in small coils. As a consequence of the heat generation inside the coil
during rTMS application, higher frequencies cannot be delivered in a continuous
Low-intensityrTMS
93
fashionforlongerperiods(Rossietal2009),incontrasttolowfrequencies(≤1Hz).
Inaddition, inthecurrentstudy, low intensitieswereused(peakamplitude1782A
eachwing), comparable to thepeak amplitudeof the intensity used for peripheral
nervestimulationwiththeMagstim20mmcoilandMagstimRapid2stimulator(at50
% machine output) (Wyckhuys et al 2013). These relatively low intensities yield
smallermagneticfields(about300mT)andthereforesmallerinducedelectricfields
in the brain (approximately 0.55 V/m), which explains the absence of large
quantitativechangesorofvisualeffectssuchasmotortwitching.Togeneratehigher
magnitude potentials, for small animal coil designs, a dedicated cooling system is
indispensable.Wehaverecentlysucceededindesigninganew,activelycooledsmall
animalcoilachievingupto100V/m intherat’sbrainandarecurrentlyperforming
the validationwith electromyographymeasurements and the further evaluation of
thisnewcoilusingmolecularimagingistopicoffuturework.
OtherstudiescombiningrTMSwithfunctionalneuroimagingmethodstovisualizeits
effects on neuronal activation have mainly focused on humans. These studies
showed that rTMSof the leftdlPFChasboth local and remoteeffectsonneuronal
activity.SPECT[99mTc-HMPAO]studiesindepressedpatientsrevealedcerebralblood
flowincreasesinthetargetregionafter10Hzstimulation(Catafauetal2001)andin
the anterior cingulate cortex after 5, 10 or 20 Hz stimulation (Catafau et al 2001,
Shajahan et al 2002), while a SPECT [99mTc]-bicasate study in healthy volunteers
showed decreases (PFC, anterior cingulate cortex and anterior temporal cortex) as
wellasincreases(thalamus,OFCandhypothalamus)inneuronalactivityafter20Hz
and increases (OFC and hypothalamus) after 10 Hz stimulation of the left dlPFC
(George et al 1999). PET [15O]-H2O revealed increases in blood flow after 20 Hz
stimulation (PFC, cingulate gyrus, amygdala, insula, basal ganglia, uncus,
hippocampus,parahippocampus,thalamusandcerebellum)anddecreasesafter1Hz
stimulation (PFC, medial temporal cortex, basal ganglia and amygdala) of the left
dlPFC in depressed patients (Speer et al 2000). An [18F]-FDG PET study in healthy
volunteersusing1HzleftdlPFCrTMSmainlyshoweddecreasesinmetabolism(PFC,
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anterior cingulate cortex, basal ganglia, hypothalamus, midbrain and cerebellum)
(Kimbrelletal2002).Discrepancieswithinthesehumanfindingsand incomparison
withourratdatamightbeexplainedbythevariationinstimulationparameters(e.g.
stimulation intensity, frequency, duration of stimulus trains and total number of
stimuliadministered)andprotocols(e.g.totaldurationoftherTMSsession,number
ofrTMSsessionsaday,imagingtechnique,radioactivetracerandmomentoftracer
injection)beingused,renderingcomparisonofresultsdifficult.
Our previous small animal rTMS [99mTc]-HMPAO µSPECT study using the smallest
commerciallyavailablecoil(MagStim,20mmfigure-of-eight)predominantlyrevealed
decreasesinregionalcerebralbloodflowintheratbraininducedbytheapplication
ofboth1Hzand10Hzstimulation.Increasesinperfusionweremainlyrestrictedto
structuresinvolvedinsensoryinformation,includingtheentorhinalcortex,whichhas
afferentandefferentconnectionstothetargetedmPFCandmanyprojectionstothe
hippocampus.Nohyperperfusionwasseeninthehippocampus,neitherat1Hznor
at 10Hz. Although a comparable intensity andmagnitude of the electric fieldwas
usedinourcurrentstudyasinouraforementionedpreviousSPECTstudy(Wyckhuys
etal 2013), thediscrepancybetween the resultsmight largelybeexplainedby the
useofadifferentneuroimagingtechnique(µSPECTvs.µPET),tracer([99mTc]-HMPAO
vs. [18F]-FDG) and another post-processing SPM normalization (to cerebellum and
whole brain resp.). While both radiotracers are believed to reflect changes in
neuronalactivitybyvisualizing regional cerebralblood flowand [18F]-FDG (glucose)
metabolismrespectively, itshouldbenotedthatthetracershavedifferentkinetics.
[99mTc]-HMPAO distributesmore rapidly (<2minutes) within the brain (Sharp et al
1986),representinga“snapshot”ofbloodperfusionatthetimeofinjection,whereas
[18F]-FDG is accumulated in the brainmore slowly and therefore requires a longer
uptake period of at least 10 minutes (Schiffer et al 2007). In both our current
experimental protocol as in the SPECT study, it was ensured that the brain was
continuouslystimulatedduringthecompleteawaketraceruptakeperiod,whichwas
20minutesforour[18F]-FDGand5minutesforthe[99mTc]-HMPAOstudy.
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Rodent rTMS studies so far have mainly focused on indirect, invasive or terminal
techniques such as behaviorial changes (Fleischmann et al 1995, Tsutsumi et al
2002),microdialysis(Kannoetal2004)orhistology(Gersneretal2011)tostudythe
effect of rTMS. We are strongly convinced that great opportunity lays in the
exploration and validation of new stimulation parameters using non-invasive
neuroimaging techniques. Molecular imaging in combination with a specially
designed small animal rTMS setup allows longitudinal follow-up of the
neurophysiological responses and potential side effects of rTMS, with clinical
relevance.
5.6 Conclusion
This preclinical study describes a protocol for the use of small animal rTMS in
combinationwithµPET.WedemonstratethepotentialofsmallanimalPETtodraw
conclusions on the location, intensity and spatial distribution of [18F]-FDG uptake
changesinducedbydifferentrTMSparadigms.Suchcanenhanceourunderstanding
of the neurophysiological effects of rTMS, ultimately resulting in more effective
clinicaltreatments.
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Chapter6:
Performance characterization of an actively
cooled repetitive Transcranial Magnetic
Stimulationcoilfortherat
Thischapterhasbeenpublishedas:Parthoens, J.; Verhaeghe, J.; Servaes, S.; Miranda, A.; Stroobants, S.; Staelens, S.PerformancecharacterizationofanactivelycooledrepetitiveTranscranialMagneticStimulationcoilfortherat.Neuromodulation:TechnologyattheNeuralInterface.5Feb2016.doi:10.1111/ner.12387.
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6.1 Abstract
Objectives This study characterizes and validates a recently developed dedicated
circularratcoilforsmallanimalrepetitiveTranscranialMagneticStimulation(rTMS).
MethodsTheelectric(E)fielddistributionwascalculatedina3Dsphericalrathead
modelandcoilcoolingperformancewascharacterized.MotorThreshold(MT)inrats
(n=12) was determined using two current directions, MT variability (n=16) and
laterality (n=11) of the stimulation was assessed. Finally, 2-deoxy-2-(18F)fluoro-D-
glucose ([18F]-FDG) small animal Positron Emission Tomography (µPET) after sham
and 1, 10 and 50 Hz rTMS (n=9) with the new Cool-40 Rat Coil (MagVenture,
Denmark)wasperformed.
ResultsThecoilcouldproducehighE-fieldsofmaximum220V/mandover100V/m
atdepthsupto5.3mminaring-shapeddistribution.Nolateralizationofstimulation
wasobserved.Independentofthecurrentdirection,reproducibleMTmeasurements
wereobtainedat lowpercentages (27±6%)of themaximummachineoutput (MO,
MagProX100(MagVenture,Denmark)).Atthisintensity,rTMSwithlongpulsetrains
is feasible (1Hz: continuous stimulation;5Hz:1000pulses;10and50:272pulses).
Whencomparedtosham,rTMSatdifferent frequencies induceddecreases in [18F]-
FDG-uptake bilaterally mainly in dorsal cortical regions (visual, retrosplenial and
somatosensory cortices) and increasesmainly in ventral regions (entorhinal cortex
andamygdala).
ConclusionThecoil issuitableforrTMSinratsandachievesunprecedentedhighE-
fields at high stimulation frequencies and long durations with however a rather
unfocal rat brain stimulation. ReproducibleMEPs aswell as alterations in cerebral
glucosemetabolismfollowingrTMSweredemonstrated.
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6.2 Introduction
Transcranial Magnetic Stimulation (TMS) is a non-invasive neurostimulation
technique based on the principle of electromagnetic induction. An alternating
current in a coil induces a changingmagnetic field, which penetrates through the
skull and in turn induces an electric field in thebrain that candepolarizeneurons.
Repetitive administration of TMS pulses (repetitive TMS or rTMS) increases or
decreases cortical excitability, dependingon the stimulationparameters (Fitzgerald
etal2006),effects thatcan lastbeyondthestimulationsession.TherTMS-induced
behavioraleffectshavetherapeuticpotentialandhaveprovidedpromisingresultsfor
thetreatmentofvariousneurologicalandpsychiatricdisorders,includingdepression
(Baekenetal2011,Hovingtonetal2013),addiction(Roseetal2011,DeRidderetal
2011)andobsessive-compulsivedisorder(KumarandChadda2011).
Despite extensive research, no clear-cut consensus has been reached on the
underlyingneurophysiologicalmechanismandtheeffectofvariousrTMSparameters
ordosingregimens(Vahabzadeh-Haghetal2012).Basicpre-clinicalrTMSinanimals
allowsfurtherevaluationinawellcontrolledlaboratoryenvironment.Fromthefirst
ratTMSstudyin1990(Ravnborgetal1990),therehasbeenanexponentialincrease
in the number of publications on the subject (Vahabzadeh-Hagh et al 2012),
reflectingthegrowinginterest.Yettheshortcomingofcommerciallyavailablesmall
animalTMSsetupsleadtothepredominantuseofhumanTMScoilsinrodents(Rossi
etal2009).WeareconvincedthatpreclinicalTMSresearchwillbenefitgreatlyfrom
advancementsinminiaturizedcoildesignsdedicatedforrats.Toourknowledge,the
smallest figure-of-eight TMS coil that has been described in rat TMS research is a
custom-made coil with inner diameter 2.8 mm and outer diameter 19 mm
(Parthoensetal2014b).Regardingcircularcoils,TMScoilswithanouterdiameterof
up to 166mm have been used to stimulate the rat brain (Vahabzadeh-Hagh et al
2012). The smallest described circular rat coilwas a custom-made, liquid-nitrogen-
cooledcoilwithanouterdiameterof32mm,consistedof5layersof7windingsand
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wasabletogenerateMotorEvokedPotentials(MEPs)anddeliver1Hzstimulationat
115%MT (Liebetanz et al 2003). Unfortunately, no further reports on this device
havebeenpublishedtodate.
Recently,wehavedesignedadedicateddoubleserialswitchedliquid-cooledratcoil
(circular coil; outer diameter 40 mm), the Cool-40 Rat coil (MagVenture A/S,
Denmark)toperformrTMSexperimentswithhighstimulationfrequencies(upto100
Hz),highmaximalelectriccurrent(190A/µs)andmagneticfieldgradients(18kT/s).
This studypresents a characterizationof this rat coil, includinga calculationof the
inducedelectricfieldsinasphericalratheadmodelandanevaluationofthecooling
performance. Secondly, MT experiments were performed assessing the impact of
stimulator current direction, intra- and inter-animal variability of the MT and
lateralization of the stimulation. Traditionally, modulatory effects of rTMS are
assessed byMEP basedmeasures (e.g. (Muller et al 2014)). To also gain a spatial
insight into the working mechanism of rTMS, combined rTMS and neuroimaging
studies are needed (Siebner et al 2009a). In this work we have therefore used 2-
deoxy-2-(18F)fluoro-D-glucose ([18F]-FDG) Positron Emission Tomography (PET)
imaging to visualize the effects of rTMS on regional cerebral glucose metabolism,
whichprimarillyreflectssynapticactivity(JueptnerandWeiller1995).Arandomized
cross-over rTMS-PET study was performed comparing rTMS at 1, 10 and 50 Hz to
shamstimulation.
6.3 MaterialsandMethods
6.3.1 RatTMSsetup
Herewereportontheperformancecharacteristicsofanewlydevelopeddedicated
ratTMScoil(Cool-40Ratcoil,MagVentureA/S,Denmark)specificallydesignedtobe
used for rTMSpreclinical research.Thecoilwasdeveloped ina collaborativeeffort
betweenour researchgroup (Molecular ImagingCenterAntwerp,Belgium)andthe
manufacturer (MagVenture A/S, Denmark). Our contribution was primarily
formulatingthedesiredspecificationsofthecoil forratbrainstimulationaswellas
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theinitialinvivoproofofconceptexperiments.
The Cool-40 Rat coil (Figure 6.1) is an actively cooled bilayer of a 40 mm outer
diametercircularcoilconsistingof twelvecopperwindings ineach layer.Thecoil is
bendedtobeshapedovertheratheadandhasaself-inductanceof9.3µHandan
electrical resistanceof13.7mΩ.ThecoilhousingconsistsofSLSprintedglass filled
polyamide(nylon),containsanon-conductiveliquid-coolingcircuitandissealedwith
non-magneticstainlesssteelscrews.Thetransducerhead(i.e.coil,coolingcircuitand
casing)is50x50x40mm(WxLXH)and,togetherwiththehandle(20cm),hasa
totalweightof0.5kg.Thecoiltemperatureisregulatedbyacontinuouslycirculating
cooling liquidflow(externallycooledto15°Cbyacompressor) inthecasingofthe
coil. The coil is connected to the MagPro X100 stimulator (MagVenture A/S,
Denmark, 1.4 m cable length, 28.5 mm diameter), which has a built-in thermal
protectionalgorithm,predictingtemperature-risetoprotectthesmallwindingsfrom
overheating, even when running at high repetition rates (up to 100 Hz). The
maximum allowed temperature in the coil element is set to 60 °C, with peak
temperaturesupto70°C.Atemperaturesensorispositionedbetweenthewindings
in the coil element with a measuring delay of 5 to 15 seconds. The surface
temperature of the cooled casing that is in contact with the rat’s head is always
maintainedbetweenasafeintervalof15to20°C.Thestimulatorsetupgeneratesa
biphasicsine-wavewitha282µspulsewidthandamaximalpeakcurrentof6960A
at 100 % machine output (%MO) resulting in a maximum current gradient of
approximately190A/µs (withdI/dt=1.918 (%MO)–2.525,obtained fromplotting
dI/dt as a function of %MO in steps of 5 %MO). At 100 %MO, the peak induced
magneticfieldis3.2Tandhasaninitialtimederivativeofapproximately80kT/sata
distanceof5mmfromthecoilsurface.
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Figure 6.1 The MagVenture Rat Coil. A. Picture of the coil, B. coronal, C. sagittal and D.transversalpicturesofaratwithschematicdrawingsofthecoilandcasing.
6.3.1.1 Electricfieldcalculations
The electric field distribution induced by the coil was calculated in a spherical rat
headmodelusingthefiniteelementmethodinSimNIBS2.0(Thielscheretal2015).
Thebentcoilwiththepreviouslydescribedspecifications(see6.3.1RatTMSsetup)
wassimulatedin3Dusingadipolemodelofthecoil(ThielscherandKammer2002)
consistingof1496dipolesdistributedin4layersconsistingof11rings.Therathead
was modeled by a homogeneous sphere with 1.5 cm radius and isotropic
conductivity of 0.33 S/m, with no differentiation of the distinct head tissue layers
(Dengetal2013).Aspacingof2mmbetweenthecoilwindingsandtheheadmodel
accountedforthecoilcasing.Toquantifythedepthpenetration,themaximalE-field
atdifferentdepthswasalsocalculated.
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6.3.1.2 Coolingperformanceevaluation
The number of consecutive pulses that can be generated before the stimulator is
disabled by the thermal protection circuit due to excessive heat production was
determined at 1, 2, 5, 10 and 50 Hz for at least 6 intensities between 1 and 100
%MO.Forthesemeasurementstheinitialcoiltemperaturewasalwayskeptat16°C.
Inaddition, theefficiencyof the liquid-cooling circuitwasmeasuredby logging the
coil temperatureevery5 s for a totaldurationof150 s, starting from themaximal
allowablecoiltemperatureof60°C.Thismeasurementwasrepeatedfourtimesand
theaveragetemperaturecurvewascalculatedandplotted(±standarddeviation,SD)
foreachtimepoint.
6.3.2 Animals
MaleSpragueDawleyrats(Janvier,France,n=12,n=16andn=11forthedifferentMT
experiments and n=10 for the µPET study) were treated according to guidelines
approvedby theEuropeanEthicsCommittee (86/609/EEC).Thestudyprotocolwas
approved by the Antwerp University Ethical Committee for Animal Experiments
(2011-30).Theanimalswerekeptunderenvironmentallycontrolledconditions(12h
normal light/dark cycles, 20-23 °C and 50-55 % relative humidity) with food and
wateradlibitum.
6.3.3 Motorthresholddeterminations
6.3.3.1 MTdeterminationprotocol
To determine the MT, rats were briefly anesthetized with a mixture of medical
oxygenandisoflurane(5%)forcatheterizationofthetailveinforcontinuousinfusion
of propofol (701.9 ± 1.5 µg/kg/min, Diprivan 1 %). Propofol was used as for
maintaining the anesthesia because it has previously been identified as the
anesthetic of choice compared to halothane, pentobarbital and ketamine and it
maintainedstableMEPresponsesoveraperiodof4hourswhengivenattheselow
doses(Luftetal2001).
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Within2minutesafterthestartofpropofoladministration,theisofluranesupplywas
stopped. Meanwhile, a disposable monopolar EMG needle ground electrode
(Technomed, Europe)was inserted in the tail and the left hind limbwas depilated
uponwhichEMGsurfaceelectrodes(AmbuNeuroline700,20x15mm)wereapplied.
Fifteenminutesafterthestartofpropofolinfusion,singlepulseswhereadministered
to the right hemisphere of the propofol-anesthetized rats while the MEPs were
recordedwithaMEPMonitor(2x104samples/s,100Hz–5kHz,MagVentureA/S,
Denmark). The coil wasmoved bothmediolaterally and rostro-occipitally over the
righthemisphereinstepsof±2mmtosearchforthelocationontheheadwherethe
MEP with the highest amplitude could be measured. These high MEP amplitudes
werereachedwhenthecoilwastiltedapproximately10°fromthedorsoventralaxis.
Atthislocation,anapproximationofthethresholdwasobtainedbystimulatingat20
% of themaximumMO and increasing the intensity by 10 % until a positiveMEP
response was measured. A response is defined here as a MEP with peak-to-peak
amplitude ≥ 50 µV. Then themaximum intensity atwhich 5 consecutive pulses all
produced no response (the lower threshold -MTlow) was found by decreasing the
intensityin1%steps.Next,theminimumintensityatwhich5stimuliallproduceda
positive responsewas determined (the upper threshold -MThigh) by increasing the
intensityin1%steps.WedefinedtheMTas(MTlow+MThigh)/2,asproposedbyMills
andNithi(MillsandNithi1997).TomakesurenolowfrequencyrTMSeffectswould
be elicited that may influence cortical excitability, we allowed a minimum of 8 s
betweenthepulses.Thisinterpulseintervalalsoallowedthecoiltocooldownbefore
administrationofthenextpulse.
6.3.3.2 EffectofcurrentdirectiononMT
To investigate which current direction is the most efficient to stimulate cortical
neurons and thus yields the lowestMTs, theMT of 12 ratswas determined using
both the normal (counterclockwise; CCW) and the reverse (clockwise; CW) current
direction during the same session in a randomized order. In the CCW current
direction thecurrent runscounterclockwiseduring the firstphaseof thesinewave
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whenlookingatthecoilfromabove.TheaverageMTsandlatenciesofMEPselicited
bytheCWandCCWcurrentdirectionwerecomparedusingapairedt-testandare
expressedasanaverageoverallanimals±StandardErroroftheMean(SEM).
6.3.3.3 Intra-andinter-animalvariabilityofMTdeterminations
TotestwhethertheMTchangesovertime,theMTof16ratswasdeterminedon3
test days (day 1, day 3 and day 10). The intra- and inter-animal variability is
expressed as the percentage coefficient of variation (%COV = 100 x (standard
deviationσ)/(averageμ)).Theoverallinter-animalCOVisthevariationwithinagroup
ofanimalsatacertaindayaveragedoverthedifferentdayswhiletheoverall intra-
animal COV is the variation within an animal over time averaged over all the
individual animals. Presence of significant differences in MT was evaluated by a
repeated-measuresANOVA(SPSSv20).
6.3.3.4 Lateralityofstimulation
MTwasdetermined in11 rats for5different configurationswith thecenterof the
coil positioned over either the right and left hemisphere or the interhemispheric
fissureandEMGsignalwasrecordedfromEMGsurfaceelectrodeslocatedoneither
the leftor righthind limb. Thepositioningwasperformedasdescribedabove (see
3.1.),wherethecoilwasonlymovedrostro-occipitallyforthecoilcenteredoverthe
interhemispheric fissure. For the stimulation with the coil centered over the
interhemispheric fissure theMT was determined from EMG recordings in the left
hind limb.Statisticalanalysiswasperformedbya repeated-measuresANOVA(SPSS
v20).
6.3.4 PETrTMSstudy
6.3.4.1 rTMSprotocols
Forreproduciblepositioningofthecoilandtoreducestressduringtheexperimental
procedure,tennaïveratswerehandleddailyandtrainedforaperiodof9daysprior
to the start of the experiment to lie still for 30 minutes in a custom-made semi-
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flexible restrainer (silicone-mold, Belosil, Equator, Belgium) (Wyckhuys et al 2013),
equippedwithatooth-bar.Attheendofthishabituationperiod,theMTofeachrat
was determined according to the protocol described above (see 6.3.3 Motor
thresholddeterminations).ForrTMSadministration(110%oftheindividualMTs)the
conscious rats were positioned inside the silicone-mold with the coil kept in a
constant position by a coil holder fixated onto the restrainer. The restrainer was
designedandpositionedsothatthecenterofthecoilwas locatedoverthemidline
and 14 mm anterior from the interaural line. The coil was oriented with the coil
bendings locatedovertheleftandrighthemisphereas illustratedinFigure6.1B-D.
Eachratreceived30minutesof(i)continuous1Hz,(ii)trainsof10Hz,6-sduration
and54-sintertrainintervals,(iii)trainsof50Hz,1.2-sdurationand58.8-sintertrain
intervalsand(iv)shamstimulation.Forshamstimulationcontinuous1HzrTMSwas
deliveredwiththecoilpositionedperpendicularandapproximately4cmawayfrom
thehead.DuringrTMSandshamstimulationvisual inspectionof therat’sbehavior
wasperformedtorecordabnormalities.
6.3.4.2 MicroPET-CTimaging
TenminutesafterthestartofrTMSorshamstimulation,abolusinjectionof1mCiof
[18F]-FDG (±0.5 mL) was injected intravenously in the tail vein while the rat was
awakeandundercontinuousstimulation.Duringthisradiotraceruptakeperiodand
while being stimulated, the animals were kept isolated in a separate space.
Immediately after terminating the 30min rTMSor sham stimulation, thus after 20
min of awake [18F]-FDG-uptake, the rats were anesthetized using a mixture of
isofluraneandmedicaloxygen(inhalation,5%inductionand2%maintenancedose)
andpositionedontothethermostaticallyheatedbedofaSiemensInveonmicroPET-
CT scanner (Siemens Preclinical Solution, Knoxville, TN) (Bao et al 2009) until they
reached a total of 30 min of [18F]-FDG-uptake, after which a 20-min static PET
acquisitionwasstartedfollowedbya10-minComputedTomography(CT)scan.One
animalhaddiedbeforthestartofthePETexperiments.Allremainingnineratswere
scannedfourtimes,receivingall fourstimulationparadigms inarandomizedorder.
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Consecutive experimental days were separated by at least 48 h to allow a fasting
durationofminimally12h(Deleyeetal2014).MicroPET-CTimagingwasperformed
asdescribedpreviously(Parthoensetal2014b,2014a).
6.3.4.3 Imageanalysis
The ratbrainwas cropped from thePET imagesand imageswere resampled toan
isotropic voxel size of 0.2 mm. Using PMOD v3.3 (PMOD Technologies, Zurich,
Switzerland), thebrain imageswere then spatially normalized into the spaceof an
[18F]-FDGtemplate (Schifferetal2007), smoothedusingaGaussian filter (isotropic
1.5mm full-width-at-half-maximum),masked to remove extracerebral activity and
normalizedtohaveanaveragewholebrain[18F]-FDG-uptakeof1.
AVolumeOf Interest(VOI)-basedanalysis,usingpre-definedbrainVOIsavailable in
PMODv3.3,wasperformedtoquantitativelyinvestigatetheaveragechangesin[18F]-
FDG-uptake between the three active rTMS conditions and sham stimulation.
Statisticalanalysisconsideredaone-wayrepeatedmeasuresANOVA,withinsubjects
followedby3plannedsimplecontrastwithBonferronicorrectionandwasperformed
inSPSSv20(IBMcorporation,NY,USA).Eachpredefinedcontrasttestedforchanges
introduced by one of the three active rTMS paradigms versus sham stimulation.
Statistical significance was set at p<0.05. Average changes in overall VOI-values
comparedtoshamstimulationarepresentedwithSEM.
Additionally a voxel-based Statistical Parametric Mapping (SPM) analysis was
performedusingSPM8(WelcomeDepartmentofCognitiveNeurology,London,UK)
within aone-way repeatedmeasuresANOVAdesign.An F-contrast, testing for any
difference between the four conditions, and six T-contrasts, testing for both
increasesanddecreasesforallthreeactiverTMSparadigmsversusshamstimulation,
weredefined.Voxels thatpassed theomnibus F-test at a significance level of 0.05
(uncorrected)definedamaskforthesubsequentpost-hocT-contrasts.T-mapswere
thresholded at a significance level of 0.05 (uncorrected) with an extent cluster
thresholdof125voxels (1mm3).Forvisualization,T-mapswereoverlaidona9.4T
MRratbrainimage.
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6.4 Results
6.4.1 RatTMSsetup
6.4.1.1 Electricfieldcalculation
Thering-shapedelectricfielddistributioninthesphericalratheadmodelisshownin
Figure6.2AandC.Theresultingfieldshowssomeassymetrywiththemaximalvalue
located underneath sides of the coil that are bended downwards. The maximum
electricfieldcalculatedatthesurfaceofthespherewas220V/m,withahalfpower
region(|E|≥|E|max/ 2) (SalvadorandMiranda2009)on23%of thesurface.The
electricfielddecayswithdepthasdepictedinFigure6.2BandC,withamaximumof
over100V/matdepthsupto5.3mmfromthesurface(Figure6.2B).
Figure6.2Electricfielddistribution.A.Coilorientationandinducedelectricfielddistributionon the surface of the rat brain model by the 2-layered bended circular rat coil. B. Themaximumelectricalfieldinthesphereasafunctionofthethedistancetothebrainsurface(depth).C.Theelectricfielddistributiononthebrainsurface(topview)andatdifferentslicesthroughthesphere(horizontalsliceat4mmdepthandtwocentralverticalslices(sagittalandcoronal)).
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6.4.1.2 Coolingperformanceevaluation
The number of continuous stimuli that could be generated for three stimulation
intensitiesateachfrequencyisplottedinFigure6.3A.Atastimulationintensityof27
%MO,theoverallaverageMTasdeterminedinthemotorthresholdexperiment(see
below: 6.4.2Motor threshold determinations), the TMS setup is able to stimulate
continuously at 1 Hz and can generate 1000 pulses consecutively at 5 Hz and 272
pulsesat10and50Hzbefore thestimulator is shutoffby the thermalprotection.
These272pulsescanalsobegeneratedathigherintensitiesof36%MO(or133%of
averageMT),53%MO(196%ofaverageMT)and75%MO(278%ofaverageMT)for
5,2an1HzrespectivelyascanbeseeninFigure6.3Bshowingthenumberofstimuli
for 5 different frequencies (1, 2, 5, 10 and 50 Hz) as a function of stimulation
intensity(in%MO).Thecurvefor50Hziscomparabletothe10Hzcurve.Figure6.3C
showsthecoolingofthecoilwhenthestimulationisdiscontinued,startingfromthe
maximum temperature of 60 °C. After 60 s, the coil temperature has already
returnedbacktoroomtemperature(21°C)andafter110sthecoilhascooleddown
to16°C.
6.4.2 Motorthresholddeterminations
DuringallMTdeterminationexperiments,theanimalswereonlyslightlysedatedand
still reacted to sensory stimuli such as pinching the paws. At the end of the
experiment, within 10 minutes after termination of the propofol infusion, their
behavior returned back to normal in their home cage. The application of the TMS
pulsesdidnotcauseanynoticeablediscomfortduringoraftertheprocedure.
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Figure 6.3RatCoilperformance incontinuousrTMSprotocols.A.Themaximumnumberofpulsesasafunctionofthefrequencyfor22,27and35%MO,i.e.80%,100%and130%ofthe averagemotor threshold determined in the previous experiments, respectively, B. Themaximumnumberofpulsesthatcanbedeliveredbeforeheatingupofthecoilasafunctionofthepercentageofthemaximummachineoutput(%MO)for1Hz,2Hz,5Hz,10Hzand50HzandC.Thedecrease incoil temperatureover time,starting from60°C,averagedover4measurements(±standarddeviation).
6.4.2.1 EffectofcurrentdirectiononMT
Thedeterminationofthethresholdsatthetwocurrentdirectionstookonaverage23
± 2min per animal. ExampleMEPs atMT for one animal are shown in 6.4A. The
averageMTwas28.4±2.1%MOforthenormalcurrentmode(CCW)and26.9±1.8
%MOforthereversecurrentmode(CW),nosignificanteffectofcurrentdirectionon
MTwasfound(pairedt-test,p-value0.079,Figure6.4B).The latenciesoftheMEPs
averaged 8.23 ± 0.14 ms and 8.38 ± 0.16 ms for the normal and reverse current
mode, respectively. The difference between these latencies was not significant
(paired t-test, p-value 0.407). We did not notice differences in optimal coil
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positioning for the two current directions. Since no significant differences were
observed between the normal and reverse current modes, the default normal
currentmodewasusedintheremainderofourTMSexperiments.
Figure 6.4 EMG results. A. Individual and averaged MEPs at MT in one animal. B. MTsdeterminedatnormal (counterclockwise,CCW)orreverse(clockwise,CW)currentdirectionandC.MTsdeterminedatday1,3and10.Boxplotsrepresentsthe75thpercentiles,withthemedian indicated, whiskers indicate the 10th and 90th percentile and the dots are theminimumandmaximumMTvalues.
6.4.2.2 Intra-andinter-animalvariabilityofMTdeterminations
TheMTs for thedifferentdaysareshown inFigure6.4CandTable6.1.Onaverage
the MT determination in this experiment took 9.6 ± 3.7 minutes per animal,
measured between the first administered pulse until the last pulse. There was no
significantchangeinMTovertime(repeated-measuresANOVA,p-value=0.188)and
theoverallaverageMTwasfoundtobe27±6%MO.TheCOVsfortheinter-animal
variability foreach timepointwere26.2%,17.8%and21.4% forday1,3and10,
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respectively, giving an overall averaged inter-animal COV of 21.8 %. The average
intra-animalCOVovertimewas12.7%.
Table 6.1Motor threshold (MT) determination on three test days (day 1, 3 and 10). Theoverallinter-animalcoefficientofvariation(COV)isthevariationwithinagroupofanimalsata certain day averaged over the different days while the overall intra-animal COV is thevariation within an animal over time averaged over all the individual animals.MTmotorthreshold,%MOpercentageofmachineoutput,SDstandarddeviation.
Rat MT(%MO) AverageMT(%MO)±SD
IndividualCOV
Day1 Day3 Day10
1 25% 23% 18% 22%±4% 16.4%2 26% 26% 23% 25%±2% 6.9%3 41% 38% 44% 41%±3% 7.3%4 38% 28% 32% 33%±5% 15.4%5 30% 22% 25% 26%±4% 15.7%6 18% 21% 25% 21%±4% 16.5%7 23% 22% 23% 23%±1% 2.5%8 17% 23% 23% 21%±3% 16.5%9 30% 31% 32% 31%±1% 3.2%10 19% 27% 24% 23%±4% 17.3%11 21% 28% 27% 25%±4% 14.9%12 28% 26% 32% 29%±3% 10.7%13 21% 30% 27% 26%±5% 17.6%14 22% 25% 30% 26%±4% 15.7%15 27% 31% 30% 29%±2% 7.1%16 24% 35% 31% 30%±6% 18.6%
COVforeachday 26.2% 17.8% 21.4%
Inter-animalCOV:
Intra-animalCOV:
21.8% 12.7%
6.4.2.3 Lateralityofstimulation
TheMTs for thedifferentconfigurations (stimulationandEMGrecordingpositions)
areshown inFigure6.4D.Repeated-measuresANOVArevealedsignificantdifferent
MTsforthedifferentconfigurations(p=0.0312).PosthoctestsshowedthatMTwas
significantlylowerwhenthecoilwascentredontheinterhemisphericfissure(28.3±
6.6 %MO versus 32 ± 7.4 %MO averaged over the 4 other configurations). No
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unilateral stimulation was obtained as no differences were found between MT
thresholds determined from the ipsilateral versus contralateral hind leg EMG
readings.
6.4.3 MicroPETrTMS
6.4.3.1 VOI-basedanalysis
As shown in Figure 6.5, VOI-based analysis revealed statistically significant
differences in regional glucose metabolism when comparing the three stimulation
paradigmsandshamstimulation in thevisual,entorhinal, retrosplenialandparietal
associationcorticesaswellasintheanterodorsalhippocampus.Subsequenttesting
of the predefined planned contrasts in these brain regions revealed significant
increasedglucosemetabolismintheentorhinalcortexfor1Hzstimulationcompared
tosham(+3.46±0.94%).Significantdecreasescomparedtoshamwereobservedin
thevisualcortexforallthreestimulationfrequencies(-3.73±1.02%,-3.67±0.74%
and-4.23±1.17%for1,10and50Hzrespectively)andfor10Hzand50Hzinthe
retrosplenial (-2.91 ± 0.84 % and -3.72 ± 0.9 %) and parietal association cortices
(-3.26±1.05%and-3.82±0.78%).
Figure6.5Regionalaveragechanges inglucosemetabolismcausedby1,10or50HzrTMS,comparedtoshamstimulation,revealedbyvolume-of-interest-basedanalysis. ECentorhinalcortex, PAC parietal association cortex, RSC retrosplenial cortex, VC visual cortex. Asteriskindicatesregionswithsignificantaveragechange inmetabolismcomparedtosham(ANOVAfollowedbypredefinedcontrasttest,p<0.05).
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6.4.3.2 Voxel-basedanalysis
As demonstrated in Figure 6.6, voxel-based analysis revealed that the three
stimulationparadigms induced significant increases aswell as decreases in glucose
metabolismwhen compared to shamstimulation. For all frequencies, clusterswith
increased[18F]-FDG-uptakewere locatedbilaterally intheentorhinalcortexandthe
amygdala and decreased [18F]-FDG-uptake was observed in dorsal cortical regions,
situated bilaterally underneath the coil windings (i.e. the visual, retrosplenial and
somatosensorycorticesandtheanterodorsalhippocampus).Thesignificanceofthe
responsewashighestfor1Hzandlowestfor10HzrTMS(maximumandminimumT-
value5.32and-6.06for1Hzcomparedto4.04and-4.94for10Hz).
Figure6.6Voxel-basedSPMresultsfortheT-contrasts1,10and50Hzversussham.T-mapsshowing clusters of significant hyper- or hypometabolism (p<0.05, uncorrected, clusteringthresholdof125voxels(≈1mm3)),overlaidonaMRtemplate.Tvaluesforhypometabolismare indicated as negative on the color bar. Regionswith prominent clusters are delineatedwithwhitelines(RSCretrosplenialcortex,VCvisualcortex,HipADanterodorsalhippocampus,ECentorhinalcortex,Amamygdala,SSCsomatosensorycortex).
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6.5 Discussion
6.5.1 Intensityoftheelectricfieldandmotorthreshold
Thecalculatedelectricfielddistributionshowedthatthiscoilcaninducehighelectric
fieldsintheratbrainofmorethan100V/matdepthsupto5.3mm.Thesevaluesare
comparabletotheelectricfieldstrengthsusedinhumanTMSapplications(Salvador
and Miranda 2009), and strong enough to hyperpolarize or depolarize neurons.
Positive MEP responses could be evoked using current gradients well below the
maximal current gradient. Indeed, the average MTs were low compared to the
maximumMO (averageMTof 27%MO, corresponding to a current gradient of 49
A/µs).Furthermore,theMTdeterminationwasreproduciblewithamoderateinter-
animalvariability(MaedaandPascual-Leone2003)(averageCOV=21.8%compared
to44.6%describedbyLuftetal.(Luftetal2001))andalowintra-animalvariability
(COV=12.7%).
ThepossibilitytoestablishareproducibleMTatlowpercentagesoftheMOisagreat
advantageofthecoilforTMSandrTMSexperiments,whereintensitiesofupto130
%MT(e.g.35%MOforaMTof27%MO)areoftenused(Hovingtonetal2013,Rossi
etal 2009). Inaddition, for rTMS it is required that thecoil canoperate for longer
times at these stimulation intensities without overheating. Especially for patients
withhighMTs,overheatingofthecoilduringrTMSposeslimitationsoneffectiveand
safeoperation(Rossietal2009).ForsmallTMScoils,thisisevenalargerchallenge
toovercome(Vahabzadeh-Haghetal2012).Duetoitsactivecoolingmechanism,the
TMSsetupdescribedinthecurrentstudypermitsaveryhighnumberofstimulitobe
given at high frequencies at the typical rTMS intensities before overheating of the
coilwindings(i.e.maximally60°Cinsidethecoil,withpeaktemperaturesupto70°C
whilekeepingtheexternalcasingat21°C).Inaddition,thenewcoilcoolsdownfrom
themaximumallowedtemperaturetoroomtemperaturewithinasingleminuteand
to16°Cwithin2minutes(Figure6.3C).ThisfeatureallowsabroaderrangeofrTMS
protocols (i.e. longer stimulation trains, shorter intertrain intervals, higher
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intensities,higherfrequenciesandlongerstimulationsessions)tobeinvestigatedin
ratscomparedtomostavailablesmallhumancoils thatarecurrentlybeingused in
rodents.
6.5.2 FocalityoftheelectricfielddistributionTheinducedE-fieldinthesphericalheadmodelhasaring-shapeddistributionaswas
expectedforacircularTMScoil(Dengetal2013)andtogetherwiththe40mmouter
coil diameter suggests a rather unfocal stimulation of the rat cerebral cortex. This
was confirmed by the lack of laterality in the MT determination. The average
latenciesofthehindlimbMEPsinthepresentstudy(8.23±0.14msand8.38±0.16
ms)werecomparable to thosepreviouslydescribedaftermore focal stimulationof
the rat’s motor cortex using a figure-of-eight coil (8.76 ± 0.29 ms) (Kamida et al
1998). However, using our described coil we found comparable latencies when
placingthecoildirectlyontothespinalcordofapropofol-anesthetizedrat(datanot
shown). This lack of focality and lack of differences in the latency times raises the
questionwhethertheevokedpotentialsmeasuredintherat’slimbsinstudiesusinga
circularcoilmightbe,solelyorinpart,causedbystimulationofregionsfurtherdown
thecorticospinaltract.Thestimulationmightalsoexplainwhynosignificanteffectof
current direction was observed. This lack of focality of circular coils in rat TMS
researchlimitstranslationtoclinicalrTMS.Inarecentreviewofratstudiesutilizinga
TMSand/orrTMSprotocolitwasdescribedthatcircularcoilshavebeenmoreoften
usedthanfigure-of-eightcoilsforratTMSresearch(51%vs.42%ofalltheusedcoil
shapes) (Vahabzadeh-Hagh et al 2012). However, figure-of-eight coils achieve a
betterfocalityofthepeakelectricfieldcomparedtocircularcoils(Dengetal2013).
Therefore, our futurework is the design of aminiaturized figure-of-eight coilwith
comparabledimensionsaspreviouslydescribedbyourresearchgroup(Parthoenset
al 2014b), but including the high-performance cooling system described in the
present study to achieve high E-fields, which may allow lateralized MT
determinationsandhighrepetitionratesatleastat120%MT.
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As a final note, the field calculations described in this study used a simplified
spherical head. More accurate simulations including realistic Magnetic Resonance
Imaging (MRI) and CT based rat brainmodels (Wyckhuys et al 2013, Salvador and
Miranda 2009, Windhoff et al 2013, Wyckhuys et al 2013, Salvador and Miranda
2009)couldprovideamoreaccurateviewof theactual fielddistribution in the rat
brain.Ithaspreviouslybeendemonstratedinamorerealisticheadmodelthat,due
to charge build up at tissue interfaces, the focality of the total electric field
distributionisactuallyimprovedcomparedtothefocalityoftheprimaryelectricthat
is calculated in simple sphericalheadmodels thatdonot take thebrain shapeand
tissuelayersintoaccount(SalvadorandMiranda2009).
6.5.3 [18F]-FDGPETrTMSstudy
DedicatedratTMSstudiesareideallysuitedforcross-overPETimagingexperiments.
We found that rTMS induced widespread bilateral changes in cerebral [18F]-FDG-
uptake in dorsal cortical regions situated directly underneath the coil windings
(bilateral retrosplenial, visual and somatosensory cortices) as well as changes in
ventral regions (bilateralentorhinal cortexandamygdala).All frequencies causeda
decrease in [18F]-FDG-uptakeunderneaththecoilandboth increasesanddecreases
at ventral regions. Although themajority of clinical rTMS studies suggest that the
stimulationfrequencymightbethemostimportantstimulationparameterregarding
the directionality of the physiological response, with low frequency rTMS (≤ 1 Hz)
causing decreases and high frequency rTMS (> 1 Hz) causing increases in brain
activity underneath the coil, this dichotomy seems to be an oversimplification.
Indeed, both high and low frequency rTMS have been shown to induce mixed
excitatory and inhibitory effects (Siebner et al 2009a, Mantione et al 2010,
Lefaucheur et al 2014), a finding confirmed by our study. Other stimulation
parameters that can influence the response include target region, stimulation
duration,patternandintensityandshouldbecarefullyconsideredwhencomparing
different studies. In addition, a general limitation encountered inmany preclinical
rTMS studies concerns a sham stimulation protocol that exactly replicates the
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sensoryeffectsofthestimulation(skullvibration,perceivedsound)highlightingthe
need for dedicated sham coils (Borckardt et al 2008). As a consequence, in our
current study,we cannot completely ruleout possible sensory effects that arenot
replicatedintheshamcondition.
Finally,onecouldalsoverifythecorticalactivity(andexcitability)afterrTMStrainsby
a MEP based measure akin to human rTMS metrics in order to increase the
translationalrelevance.However,itshouldbenotedthatingeneralonecannotdraw
simple parallels between electrophysiological excitability outcomes and regional
neuronalactivity(Siebneretal2009a).
6.6 Conclusion
A new circular liquid-cooled rat coil specifically designed for rTMS, enabled
stimulationathighintensities(MTatonly27%MO)andfrequencies(50Hz)forlong
durations (272 pulses) and achieves unprecedented field strenghts (100 V/m in
tissue). Positive MEP responses could be evoked and significant alterations in
cerebralglucosemetabolismcouldbe induced in ratbrain.Thesimulatedelectrical
fieldsandthedisabilitytoachievelateralizationhoweverwarrantfurtherresearchas
thefocalityislimited.
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Chapter7:
Generaldiscussionandfutureperspectives
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7.1 MajorFindingsfromthe[18F]-FDG-PETstudies
7.1.1 Pharmacologicalstimulation
In chapter 3, we used [18F]-FDG-PET to evaluate the glucose metabolism changes
causedbymicroinjections of aGABAA antagonist, bicuculline and aGABAA agonist,
muscimolintheratPLmPFCcomparedtoasalineinjection.
7.1.1.1 Mainfindings
Asexpected,bicucullineinjectioncausedincreasesandmuscimolinjectiondecreases
inbrainglucosemetabolism.Bicuculline injection inthe leftPL inducedwidespread
significant hypermetabolism bilaterally throughout the brain, mainly in the target
region and in regions with known connections to the target region, such as the
contralateral PL, thebilateral cingulate cortex and theentorhinal cortex.Muscimol
on the other hand, caused a focal hypometabolism, restricted to the ipsilateral PL
andcingulatecortex.Thedifferencesindistributionoftheeffectmightbeexplained
bythefactthatthedifferentdosesofbothsubstancescannotbecompareddirectly,
orthatneuronalexcitationspreadseasierthroughoutthebrainthaninhibition.
7.1.1.2 Mechanismofaction
GABA is the main inhibitory neurotransmitter of the central nervous system of
mammals.TheGABAAreceptortransducesGABAsignaling intoacascadeofevents,
usually initiatedbyCl- influx,whichmediateshyperpolarizationof thepostsynaptic
neuronalmembrane,therebyhinderingthespreadofexcitability(Costa1998,Devlin
2001).
BicucullineisacompetitiveantagonistforGABAAreceptorsthathindersthepassage
of chloride ions and thus prevents hyperpolarization and inhibition (Devlin 2001).
Muscimolontheotherhandisaselectiveagonistforthisreceptorandbindstothe
same site on the GABAA receptor as GABA itself. It thereby enhances Cl- influx,
resultinginhyperpolarisationandthusreducingexcitabilityofneurons(Devlin2001,
Frølundetal2002).
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7.1.2 DeepBrainStimulation
Inchapter4,weused[18F]-FDG-µPETincombinationwithhigh(130Hz)andlow(60
Hz)frequencyDBSoftheratPLmPFC.DBSstimulationofthehumanPFChasbeen
proposedasatherapyforaddictionanddepression.Theprimaryaimwastovisualize
theeffectsofthis focalneurostimulationapproachoncerebralglucosebothonthe
siteofstimulationandatmoredistalbrainregions.Inadditionwewantedtoverifyif
the predicted directionality of the response to both stimulation frequencies, i.e.
inhibitionfor130HzDBSandactivationfor60Hzstimulation,couldbeobserved.
7.1.2.1 Mainfindings
In the left PL, 60 Hz DBS induced significant hypermetabolism and 130 Hz elicited
hypometabolism, albeit non significant. Additionally, for both stimulation
frequencies,voxel-basedanalysisrevealedbothhypo-andhypermetabolismclusters
whencompared to shamstimulation.At60Hz the responsewasmorewidespread
compared to 130 Hz and mainly showed increased activity, most pronounced in
sensoryandmotorrelatedregionsandinthehippocampus.Theneuronalactivation
causedbylowfrequencyDBSobservedinthetargetregionsuggeststhepotentialof
60 Hz PFC DBS for the treatment of disorders associated with prefrontal
hypofunction,suchasdepressionandaddiction.
7.1.2.2 Mechanismofaction
In the treatmentofmovementdisordersaswellas inepilepsy,high frequencyDBS
(usually at 130 Hz) is usually used because of its well-documented reversible
inhibitory or lesion-like effects (Benabid et al 1998, Wyckhuys et al 2010b). Low
frequency stimulation (20 Hz – 70 Hz) on the other hand, is known to cause
convulsionsintheratwhenrepeatedlyappliedtolimbicstructures(mosttypicallyin
the amygdala or hippocampus), a process called “kindling” (Goddard et al 1969,
Wyckhuysetal2010a,Zhangetal2012).
Howevertheexactneurobiologicalmechanismofthesefrequency-dependenteffects
of DBS on neuronal activity is still not fully understood. To explain the inhibitory
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effectsofhigh frequencyDBS,a fewhypotheseshavebeenbrought forward (fora
review see (Breit et al 2004)), including (1) depolarization blocking of neuronal
transmissionthroughinactivationofvoltagedependention-channels,(2)jammingof
information by imposing an efferent stimulation-driven high frequency pattern, (3)
synaptic inhibition by stimulation of inhibitory afferents to the target nucleus, (4)
synapticfailurebystimulation-inducedneurotransmitterdepletionand(5)induction
ofhomeostaticmechanismstocompensate for therepeatedactivations (vanWelie
et al 2004). Less extensive research has been performed on the underlying
mechanismof kindling causedby low frequencyDBS,but it hasbeen suggested to
involvemechanismsrelatedtolong-termpotentiation(LTP)(Matsuuraetal1993).
However, it should be noted that the specificmechanism by which DBS exerts its
effectsarelikelytovaryamongstimulatedtargetregionsanddiseases.
7.1.3 RepetitiveTranscranialMagneticStimulation
Repetitive TMS is a promising neurostimulation tool for the treatment of a wide
variety of neurological and psychiatric diseases. Particularly for the treatment of
depression,leftDLPFCrTMShasshowngreatpotentialasanalternativeoradditional
therapy(forreviewssee(Hovingtonetal2013,Lefaucheuretal2014)).However,no
consensus has been reached yet on its mechanism of action and its optimal
stimulation parameters, highlighting the need for preclinical research. In chapter 5
and6,wedescribedourtwocustom-mademiniaturizedratTMScoilsandtestedthe
effectsoflowandhighfrequencystimulation,targetedatthemPFC,onbrainglucose
metabolismusing[18F]-FDG-µPET.
7.1.3.1 Mainfindings
Usingthefirstratcoilprototype(chapter5),afigure-of-eightcoilthatwasonlyable
tostimulatesub-MT,voxel-basedanalysisrevealedalargehypermetabolicclusterin
theanterodorsalhippocampusforboththe1Hzand50Hzparadigm.Anadditional
hypermetabolicclusterwasfoundintheentorhinalcortexfor50HzrTMS.
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Withthesecondratcoil(chapter6),acircularcoil,rTMSatanintensityof110%MT
at1Hz,10Hzand50Hz inducedwidespreadbilateralchanges in[18F]-FDGuptake.
VOI-based analysis showed that all frequencies caused hypometbalism in regions
underneaththecoil(visualcortexfor1Hz,10Hzand50Hz;retrosplenialcortexand
parietal association cortex for 10 Hz and 50 Hz) and hypermetabolism in the
entorhinalcortexfor1HzrTMS.Forall frequencies,voxel-basedanalysisconfirmed
hypometabolism in dorsal cortical regions underneath the coil windings (i.e. the
visual,retrosplenialandsomatosensorycorticesandtheanterodorsalhippocampus)
andhypermetabolisminthebilateralentorhinalcortexandamygdala.
The results of both studies taken together clearly show that a high stimulation
intensityisrequiredtoevokewidespreadsignificantchangesinglucosemetabolism
in the targetedbrain regions. Indeed,using relatively focal low intensity rTMSwith
the small figure-of-eight coil, changes in [18F]-FDGuptake couldonlybe induced in
thehippocampus.Thehippocampus isknowtobe themostexcitable region in the
brain(Uvaetal2005).Thisregionsisalsoconnectedtotheentorhinalcortex(viathe
perforant path), which can explain the second hypermetabolic cluster in the
entorhinalcortex.The less focal,high intensityrTMSadministeredwiththecircular
rat coil on the other hand, was able to induce metabolism changes in the areas
underneath the coil (i.e. hypometabolism for all frequencies) as well as in distant
brainregions(includinghypermetabolisminthebilateralentorhinalcortex).
7.1.3.2 Mechanismofaction
The exact mechanism of action of rTMS remains largely unknown. An appealing
hypothesisimplicatesthatrTMSinduceschangesinsynapticplasticity.Applicationof
continuous rTMS at a low frequency (≤ 1Hz) is hypothesized to cause neuronal
inhibition throughmechanisms akin to synaptic depression (LTD), possibly because
the incoming pulse coincides with the inhibitory phase produced by the previous
pulse.HighfrequencyrTMS(>1Hz)ontheotherhand,isbelievedtoleadtosynaptic
potentiation(LTP)becausetheincomingpulsearrivesduringthedepolarizingphase
of the previous pulse (Rossi et al 2009, Reithler et al 2011). Although these
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hypotheses seem to be in line with the findings from most electrophysiological
studies,thissimpledichotomyisnotalwaysobserved.Indeed,neuroimagingstudies
haverevealedthatbothhighandlowfrequencyrTMScanresultinmixedexcitatory
and inhibitory effects (Siebner et al 2009a). These contrasting results might be
explained by the fact that TMS activated a huge number of axons, presynaptic
terminalsandpostsynapticsitessimultaneously, leadingtoamassivestimulationof
excitatoryandinhibitorycells(FunkeandBenali2011).
7.1.4 Directionalityanddistributionoftheresponse
7.1.4.1 Directionality
Changes in [18F]-FDGuptakeor regional glucosemetabolismarebelieved to reflect
changes in neuronal activity (Sokoloff 1999, Magistretti and Pellerin 1996). The
observed increased [18F]-FDG uptake after injection of a GABA antagonist
(bicuculline)anddecreasesafterGABAagonistinjections(muscimol)weretherefore
asexpected,especiallyinthetargetregion.
For electrical stimulation (DBS or rTMS), the simple dichotomy between activation
and inhibition followingstimulationatdiffering frequencies is lesswellestablished.
For 60 Hz DBS, we could confirm the hypothesis that this stimulation frequency
wouldcause increases inglucosemetabolisminthetargetregion.Additionally,130
Hz inducedtheexpectedhypometabolism,albeitnon-significantly.Asseen inother
neuroimagingstudies,thisresponsewasnotalwaysobservedinbrainregionsdistant
from the frontal cortex (Lipsman et al 2013, Höflich et al 2013). This might be
attributed to the fact that [18F]-FDG-PET canonly visualize thenet activity changes
integrated over thewhole uptake period of the tracer, whichmight be caused by
excitationaswellasinhibition(JueptnerandWeiller1995).Forexample,itmightbe
possiblethatactivationof inhibitorymechanismsresults inareducedtraceruptake
(Pausetal1998,SackandLinden2003).
Both rTMS experiments were unable to demonstrate frequency-dependent
responses in directionality. This might be explained by (1) the larger amount of
Generaldiscussion
127
different cell types that are stimulated concurrently compared to focal DBS,
activatingbothexcitatoryand inhibitoryneurons (Looetal2003,FunkeandBenali
2011)and/orby(2)thelackofagoodshamstimulationforrTMScomparedtoDBS.
During DBS administration, no sensory effects are normally observed when the
stimulationisswitchedon.ActiverTMSontheotherhand,producesa loudclicking
sound, tickling sensations on the skin as well as contractions of the skin muscles.
Theseeffects cannotall bemimickedbyour shamstimulation (i.e.holding the coil
perpendicularly).Thereforeaspecificallydesignedshamcoilisrequired.
7.1.4.2 Distribution
Regarding thedistributionof the inducedchanges in [18F]-FDGuptake,we found in
chapter 3 that the neuronal activation caused by intracranial bicuculline injections
spread from the targeted region to the contralateral hemisphere, and to a broad
range of interconnected regions involved in memory, processing of sensory
information, and the dopaminergic circuit. Inhibition caused by muscimol on the
otherhand,wasonlyseen inthetargeted leftPLandtheadjacentcingulatecortex
suggestingthatneuronalinhibitionislesslikelytobetransferredthroughthebrainin
contrast to excitation.However, thesedifferences in spatial distributionmight also
beattributedtodifferentdosagesandpotenciesofbothcompounds,whichmakea
directcomparisondifficult.
ForelectricalorelectromagneticstimulationwithDBSorrTMS,thedosageisusually
specifiedby the intensityofstimulation (SiebnerandRothwell2003,Fitzgeraldand
Daskalakis 2012) and the total number of pulses. For the DBS experiment, the
intensityofstimulationwasthesameforeachanimalandforbothfrequencies,but
the totalnumberofpulseswashigher for130Hzcompared to60HzDBS (i.e.130
pulsesx60x60=468000pulsesfor130Hzand60pulsesx60x60=216000pulses
for60Hz).However,as inthepharmacologicalexperiment,theresponsewasmore
widespread at 60Hz DBS,which activated the target region, compared to 130Hz,
whichinducedhypometabolisminthetargetregionalbeitnon-significantly.
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For both rTMSexperiments,we administered the samenumber of total pulses for
eachfrequency(60pulsesperminuteinbothexperiments),thestimulationintensity
wasdeterminedforeachanimalindividuallybasedontheMTandthesameintensity
wasusedforeachfrequency.ForbothrTMSexperiments,comparableresultswere
seen regarding the distribution of the response. However, the high-intensity non-
focalstimulationwiththecircularcoilelicitedmuchlargerhyper-andhypometabolic
clusterscomparedtothelow-intensityfocalstimulation.
Interestingly,inallfourPETstudies,prefrontalcortexstimulationinducedsignificant
metabolicchanges(i.e.increases)intheentorhinalcortexatleastbyoneparadigm:
bybicuculline,bylowfrequencyDBS,byhighfrequencyrTMSatlow-intensityandby
all frequencies at high-intensity rTMS. These results reflect the high excitability of
thisbrainregionanditsconnectionstotheprefrontalcortex.
7.1.5 Interpretationof[18F]-FDG-PETdataAn importantaspect thatneedstobeconsideredwhen interpretingthedata is the
countnormalization.Inchapter4,5and6wehaveusedWBnormalizationinorder
to reduce inter- and intra-animal variability. The whole brain was used for
normalizationbecauseof the lackofagoodreferenceregionthatwasnotaffected
bythedifferentstimulationprotocols.However,onemusttakeintoaccountthatthis
method removes the possibility to detect changes in absolute uptake and that
activation in any volumeof interest (VOI) shouldbe considered in termsofoverall
activityinthewholebrain(Welchetal2013).Therefore,beforeapplyingthewhole
brainnormalizationweverifiedtheabsenceofsignificantdifferencesinwholebrain
uptakebetweenthedifferentconditions.Ontheotherhand,forthepharmacological
challenge experiment (chapter 3) we did find such significant differences, with an
increasein[18F]-FDG-uptakeofmorethan20%afterbicucullineinjection.Therefore,
we used %ID normalization instead of WB normalization in chapter 3. We have
chosentouse%IDoverSUVsincethebodyweightoftheanimalsvariedconsiderably
inthislongitudinalexperimentandithasbeenshownthatinratquantificationusing
SUV overcompensates the effect of body weights on [18F]-FDG brain uptake as a
Generaldiscussion
129
result of lower uptake in white fat tissue (Deleye et al 2014). It should be noted
however thatallexperimentsconsidered randomization toavoidsystematiceffects
duetoweightvariation.
7.2 Major achievements and shortcomings of the new
ratrTMScoils
7.2.1 Stimulationfocalityandintensity
Theuseofhumancoils inpreclinicalresearchishamperedbythelackoffocalityof
thestimulation(Rossietal2009,Vahabzadeh-Haghetal2012)andthelargecoilto
headsizeratiowhichreducestheefficiencyofthemagneticstimulation(Weissman
et al 1992). These two issues render the translation of the results obtained with
these coils in a preclinical setting to the clinic difficult. Indeed the resulting
stimulation that these coils achieve in the rat brain is very different from the
stimulationofhumanbraininclinic.Untilrecently,nodedicatedratTMScoilswere
commercially available. There is thus an obvious need tominiaturize TMS coils for
preclinicalresearch,howevertheminiaturizationisnotatrivialtask.Therefore,one
ofthemaintechnicalcontributionsofthisworkisthedevelopmentoftwodedicated
rat rTMS coils. In particular, the circular rat coil that we have developed in
collaborationwithMagventureA/G(Farum,Denmark) isnowbeingcommercialized
by Magventure as the Cool-40 rat coil. This allows other research groups to
contributetothepreclinicalevaluationofrTMSwithourdevelopedcoil.Inthisway
our contribution to theunderstandingof rTMSwill go beyond the combined rTMS
and[18F]-FDG-PETstudiesdescribedinchapter5and6.
ProbablythebiggestchallengetoovercomeinTMScoilminiaturizationistheheating
ofthecoilwires,duetotheverystrongelectricalcurrents(uptoseveralthousands
amperes) that are needed to induce the high E-fields (above 100 V/m) for
stimulation. This poses problems especially when repeated administration of TMS
pulsesathighfrequenciesandatintensitiesabovetherat’sMTisrequired,asisthe
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case in rTMS. Therefore, the circular rat TMS coil developed in chapter 6 was
equippedwithanactivecoolingsystem.Thecoilcoolingallowedstimulationathigh
frequencies (upto100Hz)at intensitieswellabovetheaverageMT(stimulationof
averageMTcorrespondstoapproximatelyto27%MO)resultinginelectricalfieldsof
morethan100V/mforprolongeddurationswithoutoverheatingthecoil.Inthisway
wecouldmimictypicalrTMSprotocolsthatareusedintheclinic(Rossietal2009).
However, as our EMG measurements, E-field calculations and [18F]-FDG-PET
experiments have indicated, the circular coil still lacked sufficient focality for
effective translation to clinical results. From our findings, in particular our E-field
calculationsithasbecomeclearthatitwillbedifficulttorealizeacircularratcoilthat
willbefocalenoughforratbrainstimulation.Figure-of-eightcoilsenablemorefocal
stimulation (Deng et al 2013). The figure-of-eight coil that we have developed in
chapter5wassmallerandmorefocalthanthecircularcoil,howeverthedesigndid
notallow theuseof thevery strongelectrical currents thatare required to induce
high electrical field strengths. As a result we could not demonstrate MEPs when
stimulating the motor cortex. Moreover the design did not include any cooling
mechanismsothecoilwaspronetooverheating.Therefore,forfutureexperiments
wewouldconsideranimproveddesignusingthebestcoilfeaturesofChapter5and
6resultinginafigure-of-eightcoil includingactivecooling,eventhoughthistypeof
coilismoredifficulttodevelopthanacircularcoil.
7.2.2 PositioningandanesthesiaAnother technical difficulty in rodent TMS is the accurate and reproducible
positioning of the coil. As explained in the methods in chapter 5 and 6, we
approachedthisproblembybuildingarestrainerfortheratwiththecoilfixatedon
top.Doing so, thebrain couldbe targeted ina reproduciblewaywhile the ratwas
awake and fully conscious. This solution avoids the use of anesthesia, which can
influencerTMSresponses,butmightincreasestresslevels.Todecreasestressonthe
test days, a habituation period of several days was consideredwith daily sessions
duringwhichtheratwaspositionedintherestrainer.
Generaldiscussion
131
7.2.3 Shamstimulation
As in many human TMS studies, we held the coil perpendicularly to the head to
administershamstimulation.Asmentionedpreviously,withthisapproachwedonot
perfectlymimictheTMS-inducedsensationsduringrealstimulation,highlightingthe
need for additional developments in the design of a rat sham coil. This sham coil
shouldproducetheclickingsoundsandmusclecontractions,withoutinducinganE-
fieldinthebrain.
7.3 Futureperspectives
Since the success of dlPFC rTMS treatment for depression, new neurological
indicationsforthistherapyareemerging.Weareconvincedthatadditionalresearch
tounraveltheworkingmechanismofrTMSandtooptimizestimulationprotocolswill
greatlybenefitfromtranslationalstudiesinrodentsusingsmallanimalimaging.
OurdedicatedratTMScoils, inparticular thecircularcoilwithactivecooling,area
first step towards truly translational preclinical rTMS research. However, given the
rather unfocal stimulation of the circular coil, additional research efforts into the
design of new rat TMS coils are required. A cooled figure-of-eight coil design,
potentially with eccentric windings and magnetic shielding seems to be the way
forward. New designs should initially be validated through electromagnetic field
calculationsinarealisticratheadmodel(SalvadorandMiranda2009)toassessthe
focality of the E-field and thus of the stimulation. In addition, more research is
neededintothedevelopmentofaratshamcoil.
New figure-of-eight TMS coil prototypes can then be further evaluated in vivo,
initiallyusingEMGmeasurements. Furthermolecular imaging studies that visualize
the direct and lasting neuromodulatory effects of stimulation will provide new
insightsintotheworkingmechanismofvariousrTMSprotocolsinnormalratsaswell
as in disease models, e.g. in a rat model for addiction or obsessive compulsive
disorder. Besides [18F]-FDG-µPET that was used in this doctoral thesis, other PET-
tracers (e.g. glutamatergic tracers) and other imagingmodalities such as fMRI will
Chapter7
132
makeagreatcontributiontopreclinicalTMSresearch. Inadditiontothese imaging
experiments,complementarybehavioraltestscouldbeperformedtoelucidateTMS-
inducedlong-termeffectsoncognitivefunctioning.
Generaldiscussion
133
Chapter7
134
Chapter8:
Summary
Chapter8
136
Transcranial Magnetic Stimulation (TMS) is a non-invasive neurostimulation
technique thatuses rapidly changingmagnetic fields to induceelectric fields in the
brain that can depolarize or hyperpolarize neurons. When applied repetitively
(repetitive TMS or rTMS), lasting changes in the brain’s physiology can be elicited,
withtherapeuticbenefitsforabroadrangeofneurologicalandpsychiatricdisorders.
Forexample, rTMSof thedorsolateralPrefrontalCortex (dlPFC)hasbeenapproved
for the treatment of depression by the Food and Drug Administration in 2008.
However, the exact working mechanism of rTMS is still poorly understood, and
currently no consensus has been reached on the optimal stimulation parameters.
PositronEmissionTomography(PET)with2-deoxy-2-(18F)fluoro-D-glucose([18F]-FDG)
is amolecular imaging technique that allows visualization of the regional Cerebral
Metabolic Rate of glucose (rCMRglc), reflecting neuronal activity. In combination
with rTMS, direct and lasting rTMS-induced changes in neuronal activity can be
visualized in the target region aswell as in remote brain regions. Since large-scale
clinical research is restricted due to high costs, the need for large homogeneous
patient populations and ethical considerations, small animal molecular imaging
combinedwithrTMSoffersgreatopportunitiesinthisresearchfield.Therefore,the
general aim of this doctoral thesis was to investigate rTMS by developing a
miniaturizedratTMScoiltostimulatetheratmedialPrefrontalCortex(mPFC),the
analogue of the human dlPFC, and combining rat rTMS with small animal PET
(µPET). The ability of [18F]-FDG-µPET to visualize and quantify the effects of
neurostimulationof this smallbrain regionwas firstdemonstrated in two focalbut
invasive neurostimulation techniques: (1) intracranial microinjections of
pharmacological substances (chapter 3) and (2) Deep Brain Stimulation (DBS)
(chapter 4). This allowed us to compare the distribution and directionality of their
effectsonneuronalactivitywithnoninvasive,thoughlessfocal,rTMS(chapter5and
6).
Summary
137
Therefore in chapter 3, we investigated the effects of microinjections into the
prelimbic region of the mPFC (PL) of a GABAA agonist (muscimol) or antagonist
(bicuculline), substances that are known to evoke inhibition and excitation,
respectively. As expected, bicuculline caused increased while muscimol caused
decreased brain glucose metabolism. Bicuculline induced widespread significant
hypermetabolismbilaterallythroughoutthebrain,mainlyinthetargetregionandin
regions with known connections to the target region. Muscimol caused focal
hypometabolism,restrictedtotheipsilateralPLandcingulatecortex.Thedifferences
indistributionof theeffectmightbeexplainedby the fact thatneuronalexcitation
spreads easier throughout the brain than inhibition or that the different doses of
bothsubstancescannotbecompareddirectly.
In chapter 4,we combined [18F]-FDG-µPETwithPLmPFCDBSat a low (60Hz) and
high (130 Hz) frequency. We found that in the target region this focal electrical
stimulation induced significant hypermetabolism at 60 Hz and hypometabolism at
130 Hz, albeit non significant. For both stimulation frequencies both hypo- and
hypermetabolism clusters were observed. At 60 Hz the response was more
widespreadcomparedto130Hzandmainlyshowedincreasedactivity.Theneuronal
activationcausedby low frequencyDBSobserved in the target regionsuggests the
potentialof60HzPFCDBSforthetreatmentofdisordersassociatedwithprefrontal
hypofunction,suchasdepressionandaddiction.
Inchapter5,wedevelopedaminiaturizedratfigure-of-eightcoil,whichwasusedto
stimulate the rat mPFC at subthreshold intensities. Repetitive TMS was combined
with[18F]-FDG-µPETtovisualizethedistributionanddirectionalityofthechanges in
rCMRglcinducedbyhigh(50Hz)andlow(1Hz)frequencyrTMScomparedtosham
stimulation.
Both frequencies induced a large hypermetabolic cluster in the anterodorsal
hippocampusandanadditionalhypermetabolicclusterwas found in theentorhinal
Chapter8
138
cortex for 50 Hz rTMS. These results might be explained by the fact that the
hippocampus is the most excitable region in the brain, and thus has the lowest
thresholdforstimulation.
Theaforementionedcoilwasonlyable to stimulateat lowsubthreshold intensities
andwaspronetooverheating.ThereforeanewdedicatedcircularratrTMScoilwith
an active cooling system was developed in collaboration with MagVenture A/S
(Farum,Denmark)andisdescribedinChapter6.Computersimulationswereusedto
calculatetheinducedE-fieldanditwasshownthatthiscoilwasabletostimulatethe
brain at intensities above 100 V/m, with a rather unfocal ring-shaped E-field
maximum underneath the coil windings. Motor Evoked Potential (MEP)
measurementsconfirmedtheseresultsandshowedthatthecoilwasabletoevoke
MEPsforstimulationsaboveanaverageintensity(theMotorThreshold–MT)ofonly
27 % of themaximumMachine Output (MO). However, theseMEPs could not be
elicited unilaterally because of the lack of sufficient focality. At an intensity of 27
%MO long rTMS pulse trains could be administered without excessive heat
production.Inparticularthecoilenabledcontinuousstimulationat1Hzandat5Hz
1000pulsesat10and50Hz272pulsescouldbegeneratedwithoutoverheatingthe
coilandgeneratingelectricalfieldswellabove100V/m.RepetitiveTMSatdifferent
stimulationfrequencies(1Hz,10Hzand50Hz)werecombinedwith[18F]-FDG-µPET
and revealed widespread bilateral changes in [18F]-FDG uptake compared to sham
stimulation.Allfrequenciesinducedbilateralhypometabolismindorsalcorticalbrain
regions underneath the coil windings and hypermetabolism in the bilateral
entorhinalcortex.Theseresultsdemonstratethatthenewratcoilisabletostimulate
athighintensities,butthatmoreresearchisneededintonewcoildesignstoincrease
thefocalityofthestimulation.
Summary
139
Chapter8
140
Chapter9:
Samenvatting
Chapter9
142
Transcraniële Magnetische Stimulatie (TMS) is een niet-invasieve
neurostimulatietechniek die gebruik maakt van snel veranderende magnetische
velden om elektrische velden in de hersenen te induceren waardoor neuronen
gedepolarizeerd of gehyperpolarizeerd kunnen worden. Herhaaldelijke toediening
vanTMS-pulsen(repetitieveTMSofrTMS)kanblijvendeveranderingenveroorzaken
in de fysiologie van de hersenen, met therapeutische voordelen voor een brede
waaier vanneurologischeenpsychiatrischeaandoeningen. Zowerdbijvoorbeeld in
2008 rTMS van de dorsolaterale Prefrontale Cortex (dlPFC) erkend voor de
behandeling van depressie door de Food and Drug Administration. Het exacte
werkingsmechanisme van rTMS is echter nog niet volledig opgehelderd en
momenteelisernoggeenconsensusbereiktoverdeoptimalestimulatieparameters.
PositronEmissionTomography(PET)met2-deoxy-2-(18F)fluoro-D-glucose([18F]-FDG)
is eenmoleculaire beeldvormingstechniek die hetmogelijkmaakt omde regionale
cerebralemetabole snelheidvanglucoseverbruik (regionalCerebralMetabolicRate
of glucose (rCMRglc)) te visualiseren. De rCMRglc is een indirecte maat van de
neuronale activiteit. In combinatie met rTMS kunnen acute en blijvende rTMS-
geïnduceerde veranderingen in neuronale activiteit gevisualiseerdworden in zowel
dedoelregioalsinverdergelegenregio’s.Aangeziengrootschaligklinischonderzoek
beperkt is wegens de hoge kosten, de nood aan grote homogene
patiëntenpopulaties en ethische overwegingen, kan moleculaire beeldvorming in
kleine proefdieren gecombineerd met rTMS een meerwaarde betekenen voor het
rTMSonderzoek.DaaromwashethoofddoelvanditdoctoraatsonderzoekomrTMS
teonderzoekendooreengeminiaturizeerdeTMS-spoelvoorrattenteontwikkelen
voorstimulatievandemedialePrefrontaleCortex(mPFC),dehersenregioinderat
analoogaandehumanedlPFC.VervolgenswerdrTMSbijderatgecombineerdmet
PET beeldvorming van kleine proefdieren (µPET). De waarde van [18F]-FDG-µPET
voorhetvisualiserenenkwantificerenvandeeffectenvanneurostimulatievandeze
kleine hersenregio werd eerst gedemonstreerd bij twee focale maar invasieve
neurostimulatietechnieken, namelijk (1) intracraniële microinjecties van
Samenvatting
143
farmacologische bestanddelen (hoofdstuk 3) en (2) diepe hersenstimulatie (DBS)
(hoofdstuk 4).Hierdoor kondenwede regionale verspreiding endedirectionaliteit
vanhuneffectenopneuronaleactiviteitvergelijkenmetniet-invasieve,maarminder
focalerTMS(hoofdstuk5en6).
In hoofdstuk 3 onderzochten we de effecten van microinjecties met een GABAA
agonist (muscimol) en een GABAA antagonist (bicuculline) in de prelimbische regio
van de mPFC (PL). Van deze stoffen is geweten dat ze respectievelijk inhibitie en
excitatie veroorzaken. Zoals verwacht, induceerde bicuculline verhogingen en
muscimol verlagingen in het glucosemetabolisme in de hersenen. Bicuculline
veroorzaakte bilateraal en wijdverspreide statistisch significante hypermetabole
clusters, voornamelijk in de PL doelregio en in geconnecteerde regio’s. Muscimol
induceerdeeenfocaalhypometabolisme,datbeperktbleeftotde ipsilateralePLen
gyruscingulatus.Dezeverschillenindeverspreidingvanheteffectkunnenverklaard
worden doordat de verschillende dosissen van beide stoffen niet eenvoudig met
elkaarkunnenvergelekenworden,ofdoordatneuronaleexcitatiezichgemakkelijker
doorheendehersenenverspreidtdaninhibitie.
In hoofdstuk 4 combineerden we [18F]-FDG-µPET met laagfrequente (60 Hz) en
hoogfrequente (130 Hz) DBS van de PL. We vonden dat deze focale elektrische
stimulatie in de doelregio significant hypermetabolisme veroorzaakte bij 60 Hz en
hypometabolismebij130Hz(niet-significant).Bijbeidestimulatiefrequentieswerden
hypo- en hypermetabole clusters geobserveerd. Bij 60 Hz was de respons meer
verspreid dan bij 130 Hz en vertoonde vooral verhoogde activiteit. De neuronale
activatie in de doelregio veroorzaakt door laagfrequente DBS suggereert het
potentieel van 60 Hz PFC DBS voor de behandeling van aandoeningen die
geassocieerdzijnmetprefrontalehypofunctie,zoalsdepressieenverslaving.
Chapter9
144
Inhoofdstuk5ontwikkeldenweeengeminiaturiseerdeachtvormigeTMS-spoelvoor
ratten.DezewerdgebruiktomdemPFCvanderattestimulerenmeteenintensiteit
lager dan de drempelwaarde voor motorische stimulatie. Repetitieve TMS werd
gecombineerd met [18F]-FDG-µPET voor het visualiseren van de regionale
verspreidingendedirectionaliteitvandeveranderingeninrCMRglcveroorzaaktdoor
hoogfrequente (50 Hz) en laagfrequente (1 Hz) rTMS ten opzichte van sham
stimulatie. Beide frequenties induceerden een grote hypermetabole cluster in de
anterodorsalehippocampuseneenextrahypermetabole clusterwerd gevonden in
de entorhinale cortex bij 50 Hz rTMS. Deze resultaten zouden kunnen verklaard
worden doordat de hippocampus demeest exciteerbare hersenregio is en dus de
lageredrempelwaardeheeftvoorstimulatie.
Despoeldieinhetvorigehoofdstukwerdbeschrevenkonenkelstimulerenaanlage
(subthreshold) intensiteitenenraaktesneloververhit tijdensdestimulatie.Daarom
werd in samenwerking met MagVenture A/S (Farum, Denemarken) een nieuwe
geminiaturiseerde en actief gekoelde circulaire TMS-spoel voor ratten ontwikkeld.
Het geïnduceerde elektrische veld werd berekend door middel van
computersimulatiesendezetoondenaandatdezespoeldehersenenkanstimuleren
met intensiteitenbovende100V/m,met een ringvormigmaximaal elektrisch veld
net onder de spoelwindingen. Metingen van Motor Evoked Potentials (MEP)
bevestigden deze resultaten en toonden aan dat de spoelMEPs kon induceren bij
eengemiddeldedrempelintensiteit(deMotorThreshold–MT)vanslechts27%van
demaximaleoutputvandestimulator(MachineOutput–MO).Echterkondendeze
MEPsnietunilateraalwordengeïnduceerddooreengebrekaanvoldoendefocusvan
het ringvormig elektrisch veld. Bij een intensiteit van 27%MO konden lange rTMS
pulstreinengegenereerdwordenzonderdespoelhierbijteoververhitten.Zowasbij
dezeintensiteiteencontinuestimulatieaan1Hzmogelijkenkondenaan5Hz1000
pulsenenaan10en50Hz272pulsengegenereerdwordenvoordatdespoeltewarm
werd. Repetitieve TMSmet verschillende frequenties (1 Hz, 10 Hz en 50 Hz)werd
Samenvatting
145
gecombineerd met [18F]-FDG-µPET en toonde wijdverspreide bilaterale
veranderingen in [18F]-FDG uptake in vergelijking met sham stimulatie aan. Alle
geteste stimulatiefrequenties induceerden bilateraal hypometabolisme in dorsale
corticale regio’s onder de spoelwindingen en hypermetabolisme in de bilaterale
entorhinalecortex.Dezeresultatentonenaandatdezenieuwespoelvoorrattenkan
stimuleren aan hoge intensiteiten,maar datmeer onderzoek nodig is naar nieuwe
spoeldesignsomdefocusvandestimulatieverderteverbeteren.
Chapter9
146
Chapter10:
Listofpublications
Chapter10
148
Journalpapers
1. Parthoens, J.; Verhaeghe, J.; Servaes, S.; Miranda, A.; Stroobants, S.;Staelens, S. Performance characterization of an actively cooled repetitiveTranscranial Magnetic Stimulation coil for the rat. Neuromodulation:TechnologyattheNeuralInterface.5Feb2016.doi:10.1111/ner.12387.
2. Parthoens,J.;Servaes,S.;Verhaeghe,J.;Stroobants,S.;Staelens,S.Prelimbic
cortical injectionsofGABAagonist andantagonist: In vivoquantificationoftheeffect in the ratbrainusing [18F]-FDGmicroPET.Molecular ImagingandBiology.Vol17(6).2015.p.p856-864.
3. Parthoens, J.; Verhaeghe, J.; Stroobants, S.; Staelens, S. Deep brain
stimulation of the prelimbicmedial prefrontal cortex: quantification of theeffect on glucose metabolism in the rat brain using [18F]-FDG microPET.MolecularImagingandBiology.Vol16(6).2014.pp.838-845.
4. Parthoens, J.;Verhaeghe,J.;Wyckhuys,T.;Stroobants,S.;Staelens,S.Small
animalrepetitivetranscranialmagneticstimulationcombinedwith[18F]-FDGmicroPET to quantify the neuromodulation effect in the rat brain.Neuroscience.Vol275.2014.pp.436-443.
Abstracts
1. Servaes,S.;Verhaeghe,J.;Parthoens,J.;Miranda,A.;Staelens,S.Evaluationof a miniaturized circular coil for small animal repetitive TranscranialMagneticStimulation.BrainStimulation.2015.
2. Miranda,A.;Verhaeghe,J.;Servaes,S.;Parthoens,J.;Staelens,S.Calculationoftheinducedelectricfieldofadedicatedtranscranialmagneticstimulationcoilfortherat.BrainStimulation.2015.
3. Miranda, A.; Verhaeghe, J.;Parthoens, J.; Stroobants, S.; Staelens, S. Small
animal PET brain imaging of unconstrained and unanesthetized rats:implementation of a motion correction approach. European MolecularImagingMeeting.2014.
4. Parthoens, J.; Boonzaier, J.; Stroobants, S.; Staelens, S. Simultaneous high-
fieldsmallanimalrepetitivetranscranialmagneticstimulationand[18F]-FDGmicroPET to visualize regional neuromodulation effects on the rat brainmetabolism.EuropeanMolecularImagingMeeting.2014.
Listofpublications
149
5. Parthoens,J.;Wyckhuys,T.;wyffels,L.;Langlois,X.;Schmidt,M.;Stroobants,S.; Staelens, S. Evaluation of mGluR2 positive allosteric modulator JNJ-42153605 in an animalmodel of glutamatergic dysfunction using [18F]-FDGmicroPET.Neuroscience.2013.
6. Parthoens, J.; Servaes, S.;Wyckhuys, T.; Stroobants, S.; Staelens, S.Medial
prefrontalcorticalinjectionsofaGABAagonistandantagonist:quantificationof the effect on glucose metabolism in the rat brain using microPET.Neuroscience.2013.
7. Parthoens, J.; Engelen, V.; Wyckhuys, T.; Verhaeghe, J.; Stroobants, S.;Staelens, S. Dopaminergicmodulation in rats by Deep Brain Stimulation ofthemedialprefrontalcortex:quantificationof[11C]-racloprideD2Rbindinginthe caudate putamen using microPET.WorldMolecular Imaging Congress.2013.
8. Parthoens, J.; Engelen, V.; Wyckhuys, T.; Stroobants, S.; Staelens, S.
QuantifyingtheeffectofmedialprefrontalcorticaldeepbrainstimulationonglucosemetabolismintheratbrainusingmicroPET.InternationalConferenceonDeepBrainStimulation.2013.
9. Parthoens,J.;Wyckhuys,T.;Crevecoeur,G.;Stroobants,S.;Staelens,S.;Fast
screening of transcranial magnetic stimulation paradigms in the rat usingmicroPET.SocietyforNuclearMedicine.2012.
10. VanNieuwenhuyse,B.;Parthoens,J.;Wyckhuys,T.;Raedt,R.;Wadman,W.;
Boon, P.; Vonck, K. Poisson distributed deep brain stimulation (DBS) in theventral hippocampal commissure suppresses seizures in the kainic acid ratmodel.EpilepsyCurrents.2012.
Chapter10
150
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