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&paracingingulate&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.&parahim

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

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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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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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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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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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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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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

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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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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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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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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

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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

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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

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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.

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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

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