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A robust experimental protocol for pharmacological fMRI in rats and mice

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A robust experimental protocol for pharmacological fMRI in rats and mice
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  (This is a sample cover image for this issue. The actual cover is not yet available at this time.) This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institutionand sharing with colleagues.Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:http://www.elsevier.com/copyright  Author's personal copy  JournalofNeuroscienceMethods 204 (2012) 9–18 ContentslistsavailableatSciVerseScienceDirect  Journal   of    Neuroscience   Methods  journalh   omepage:www.elsevier.com/locate/jneumeth Basic   neuroscience A   robust   experimental   protocol   for   pharmacological   fMRI   inrats   andmice Livia   Ferrari a , 1 , Giuliano   Turrini a , 1 ,Valerio   Crestan a , 1 ,   Simone   Bertani a , 1 ,   PatriziaCristofori b ,Angelo   Bifone c ,   Alessandro   Gozzi c , ∗ a GlaxoSmithKlineMedicineResearchCentre,ViaFleming4,37135Verona,Italy b GlaxoSmithKlinePathologyDepartment,SAWare,Hertford,UK  c CentreforNanotechnologyInnovation@NEST,IstitutoItalianodi   Tecnologia,PiazzaSanSilvestro12,56127Pisa,Italy a   rtic   l   e   in   fo  Articlehistory: Received29July2011Receivedinrevisedform20   October2011Accepted22October2011 Keywords: fMRIAnaesthesiaRatMousephMRISurgery a   b   s   t   r   a   c   t Pharmacological   Magnetic   ResonanceImaging   (phMRI)methods   have   significantly   expanded   thestim-ulation   repertoire   available   topreclinical   fMRI   research,   by   allowing   toselectively   probetheactivity   of specific   braincircuitries   andneurotransmitter   systems.   However,   theapplication   ofphMRI   toanimalmodelsisconstrained   by   anumber   of    experimental   factors.Firstly,   in   ordertoprevent   motion   artefactsandreduce   restraint-induced   stress,   phMRIstudiesare   typicallyperformedunder   anaesthesia.   Moreover,severalpsychoactivedrugsproduce   blood   pressurechanges   and   alterations   in   respiratoryfrequencythatmayperturb   centralhaemodynamic   readoutsof    brainfunction.   Hence,   thequality   andoutcomeof    phMRIstudies   iscriticallydependenton   the   ability   tomonitor   andcontrol   peripheral   physiologicalparameters   (i.e.blood   pressure,   arterial   blood   gases)   thatcouldalterphMRIreadouts.Here   we   provide   athorough   methodologicaldescription   of    a   robust   protocol   tomeasuredrug-induced   cerebral   blood   vol-ume   changes   inanaesthetised   rats   and   mice.Weshow   thattheprotocol   ensuresstable   physiologicalparameters   and   robust   phMRIresponse   to   the   psychostimulantdrug d -amphetamine   inthree   differentratstrains.Wealso   document   thesuccessful   application   of    the   protocolto   map   thecentraleffects   pro-duced   by   d -amphetaminein   C57Bl/6Jmice,astrain   commonly   used   as   background   for   thegeneration   of transgeniclines,   thuspaving   thewaytothe   implementation   of    phMRIin   genetically   engineered   animals. © 2011 Elsevier B.V. All rights reserved. 1.Introduction FunctionalMagneticResonanceImaging(fMRI)hasbeenexten-sivelyappliedtostudythefunctionalorganizationofthehumanbraininhealthyanddiseasestates.Astrongrationaleexiststoexpandtheapplicationofthesemethodstolaboratoryanimalspecies.However,thevastrepertoireofparadigmsusedto   inves-tigatecognitiveoremotionalbrainfunctioninhumanscannotbestraightforwardlyappliedtorodentspeciesundertheconstrainedexperimentalconditionsofan   fMRIexperiment.Asaresult,despitetheconsiderabletranslationalpotentialoffMRIforbothbasicandappliedbrainresearch,theapplicationofthistechniquetolabora-toryspecieshasfailedtokeeppacewiththeclinic.AninterestingapproachtoovercomesomeoftheconstraintsrelatedtotheimplementationoffMRIinsmallanimalsreliesonpharmacologicalmanipulation,andhasbeendubbed“pharmaco-logicalMRI”(phMRI)(LeslieandJames,2000),i.e.fMRIappliedto ∗ Correspondingauthor.Tel.:+39050509032. E-mailaddress: alessandro.gozzi@iit.it(A.Gozzi). 1 Presentaddress:Aptuit,ViaFleming4,37135Verona,Italy. map   spatio-temporalpatternsofbrainactivityinducedbyphar-macologicalchallenges.Originallydevelopedtostudythecircuitalbasisofpharmacologicalactivity,theapproachhasdemonstratedtheabilitytoelicitreliablefMRIsignalsandtoprobemultiplefacetsofbrainfunctionthroughtheselectivestimulationofdifferentneu-rotransmittersystems(reviewedbyBifoneand   Gozzi,inpress).However,rodentfunctionalandpharmacologicalMRI   studiesaretypicallyperformedundergeneralanaesthesia,apracticethat   isrequiredtoreducemotionartefacts(Peetersetal.,2001;Sicardetal.,   2003),andtominimisethestressproducedbyrestraintandMRI   gradientnoise(forrecentreviewssee Jenkinsetal.,   2003;Silvaetal.,2011;Stewardetal.,2005;VanderLindenetal.,   2007).More-over,theuseofpsychoactivedrugsandpsychostimulantsinphMRIisoftenassociatedwithperipheralcardiovascularandrespiratoryeffectsthatmay   confoundcentralhaemodynamicmeasurementsofbrainfunction(Kalischetal.,2001;Tuoretal.,2002).Hence thesuccessofphMRIstudiescriticallyreliesontheimplemen-tationofexperimentalprocedurestomonitorvitalfunctionsandtopreventthe   onsetofconfoundingsupra-physiologicalstates.Bymeansofexample,artificialventilationcanbeemployedtopre-ventdrug-inducedalterationsintherespiratoryratewhichcouldalterbloodCO 2 levelsandproducestimulus-unrelatedvasodilatory 0165-0270/$–seefrontmatter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2011.10.020  Author's personal copy 10  L.Ferrarietal./JournalofNeuroscienceMethods  204 (2012) 9–18 responses( Jonesetal.,2005;Stewardetal.,2005).Likewise,periph- eralbloodpressuremonitoringisoftenemployedtomakesurethatthemappedphMRIresponsesarenotcontaminatedby   peripherallydrivencardiovascularchangesthatcouldovercomethe   autoregu-latorycerebralbloodflowrange(Gozzietal.,   2006,2007).Asaresult,animalpreparationandphysiologicalmonitoringinrodentphMRIstudiesimposesthe   applicationofmultiplecomplexproce-duresofcriticalimportanceforthesuccessfulimplementationof thetechnique.HereweprovideadetailedmethodologicaldescriptionofarobustphMRIprotocolthatwehavedevelopedandsuccessfullyappliedintheratandthemousetostudythebrainfunctionalresponsestoanumberofpsychoactivedrugs(Gozzietal.,2010b,2011a).Moreover,weshowthatthisprotocolensurescontrolof physiologicalparameters(arterialbloodgasesandbloodpressure)underdifferentanaestheticregimens(halothane,andisoflurane),andelicitsrobustphMRIresponsesindifferentratstrainsbyusingthepsychostimulantdrug d -amphetamine.Importantly,wedescribetheimplementationoftheprotocolinC57/Bl6Jmice,astraincommonlyusedasbackgroundforthegenerationoftrans-geniclines,andwedocumentitssuccessfulapplicationtomap   thecentraleffectsproducedbythe d -amphetamine.Thismethodologicalaccountisexpectedtopromotethe   dif-fusionofpre-clinicalphMRImethodsacrossdifferentareasof neuroscienceandpsychopharmacology. 2.Materialsandmethods AlltheexperimentswerecarriedoutinaccordancewithItal-ianregulationgoverninganimalwelfareandprotection(whichacknowledgestheEuropeanDirective86/609/EEC).  2.1.Anaestheticsandchemicals GeneralanaesthesiawasinducedAnimalswereanaesthetisedwithhalothane(HAL)(MerialBristol,UK)orisoflurane(IF)(AbbottLtd.,Italy).Neuromuscularblockadewasinducedwith d (+)-tubocurarinechloridehydrate(Tocris,Bristol,UK;0.25mg/mL    insalinesolution)orpancuroniumbromide(Sigma–Aldrich,Milano,Italy;0.25mg/mL    or0.0375mg/mL    salinesolutionforadminis-trationtoratsormice,respectively).Sodiumheparin(25UI/mL)(EparinaVister,Pfizer,Italy)wasaddedtothesesolutionstoensurearterialcatheterpatencythroughouttheMRI   experiment.Sen-sitisationofMRI   times-seriestocerebralbloodvolume(CBV)wasachievedbyusingblood-poolsuper-paramagneticironoxidenanoparticles(Endorem,Guebert,France).AsEndoremproduc-tionhasnowrecentlydiscontinuedbyGuerbert,thecontrastagentFerex(BiopalInc.,Worcester,MA,   USA)canbeusedasaneffectivereplacement.Thetwoagentshavecomparableironoxideconcentrations(11.2mg/mL    vs.10.0mg/mL,respectively)andmeanparticlesize(80–150nmvs.50–150nm,   respectively).Thepsychostimulant d -amphetaminesulphatewaspurchasedfromSigma–Aldrich(Milano,Italy).  2.2.Animals AdultmaleSpragueDawleyrats(268–364g)wereprovidedbyCharlesRiverItaly.MaleListerHoodedrats(305–357g)wereprovidedbyCharlesRiverUK.MaleWistarrats(385–437g)and C57BL6/J  mice(24–33g)wereprovidedbyCharlesRiverFrance.Animalswerehousedinstablegroupsoffiveindividualsinpolycar-bonatecageswithsawdustlitter,andfed   ad   libitum withstandardrodentchow.Animalsweremaintainedon   alight:darkcycleof 12:12h(lightsonat6a.m.),roomtemperaturewithintherange Fig.1. Schematicillustrationofthemainexperimentalstepsemployedforanimalpreparationaccordingtothe   protocolsdescribedinSection2. 20–22 ◦ C,andrelativehumidity45–65%.Afterarrival,animalswereacclimatizedforatleast5daysbeforeexperimentaluse.  2.3.Animalpreparation Partialaccountsofthesurgicalprotocoldescribedinthe   presentmanuscripthavebeengivenelsewhere(Gozzietal.,2006,2008a,b,2010a,2011a,b;Schwarzetal.,2008).Aschematicillustrationof themainexperimentalproceduresisillustratedinFig.1.Table1 summarisestheanaestheticlevelsusedduringthedifferentexper-imentalphasesinratsandmice.  2.3.1.Inductionofanaesthesia Anaesthesiawasinducedwith3%HALor5%IFinratsandwith4%IFinmice.TheuseofHALforsurgicalpreparationinmiceis  Table1 GasanaesthesialevelsemployedatdifferentstagesofanimalpreparationforphMRIstudyratandmouse.Anaestheticsweredeliveredthroughanintra-trachealcannula.Studyphases Ratanaesthesia MouseanaesthesiaHAL(%)IF   (%)HAL(%)IF(%)Induction 35   34Surgery 1.53   1.53Maintenance0.81.1   0.8   1.2  Abbreviations :HAL:halothane;IF:isoflurane.  Author's personal copy L.   Ferrarietal.    /JournalofNeuroscienceMethods  204 (2012) 9–18 11 notrecommendedasthiscanproducefatalhypotension.Anaes-theticgasesweredeliveredina1:1mixture(0.9L/min+   0.9L/min)ofO 2 /N 2 inaninductionchamberconnectedtoavaporizer(BurtonsMedicalEquipmentLtd.,UK).Theanimalwasthenplacedinsupinepositiononahomeothermic(37 ◦ C ±   1)heatingblanketconnectedtoarectalprobe(HarvardApparatus,UK),andgaseousanaesthesiawascontinuouslydeliveredthroughafacemask.  2.3.2.Tracheostomy Priortosurgicalincision,eachratreceivedasubcutaneous(SC)infiltrationoftetracainesolution0.1%ateachsurgicalsite(neckandfemoralarea)atavolumeof0.1mL/point(0.2mL/rat).MicereceivedaSC   infiltrationoftetracainesolution0.05%at   eachsurgicalsite(neckandfemoralarea)atvolumeof0.02mL/point(0.04mL/mouse).Thispracticeisintroducedtoprovidesupple-mentaryanalgesiaandpreventsub-thresholdactivationofpainpathwaysthatmay   confoundcentralmeasurementsofbrainfunc-tion(Ferrarietal.,   2010).Theneckandfemoralareaswerethenshavedwithanelectricalshaverandtheskindisinfected.Agauzerollwasplacedunderthenecktoslightlyextendthetracheaandfacilitateitssubsequentexposure.Amidlineskinincisionwasmadealongthelengthoftheneck,themuscularplanesseparatedtoexposethetrachea.Asmallincisioninthesublaryngealregionofthetracheawasperformed,andaG14(rat)ora   G22(mouse)bevelledbutterflycannula(Vygon,France)wasinsertedintothetracheaaspreviouslydescribed(RigalliandDiLoreto,2009).Tofacilitateconnectionwiththe   ventilator,thecannulawas   short-enedtoalengthofapproximately1and0.7cmforratsandmice,respectively.Thecannulawasthensecuredwitha3–0silksuturetread(Ethicon,Johnson-JohnsonIntl.,Belgium)passedthroughtheholesofitsbutterfly.Amodifiedintra-trachealcannula(depictedin   Fig.S1)   wasusedinmousestudiesas   an   alternativetothewholebutterflycannula,withtheaimtoreduceitsdimension,thusallow-inga   betterpositioningoftheanimalon   theimagingcradle.To   thispurpose,thedistal0.7cmofaG22butterflycannulawerecutoff,andjoinedtoa   Ytubingjunction(OD2mm,   2BiologicalInstru-ments,Italy)connectedtoaclosedtubingsystemconnectedtotheventilationpump.Aftertracheostomy,anaestheticdeliverywasswitchedfromthefacialmasktotheventilationpump(InspiraASV,HarvardApparatus,UK).Aventilationfrequencyof70bpmwasusedbothinratandmousestudies.Tidalventilationvolumeswereadjustedonthebasisoftheresultofarterialbloodgasanalysis.Inordertokeeptheanaesthesiaandanimalphysiologyapparatusoutsidethemagnetstrayfield,theventilatorwas   fittedwith4-m-longtubing.Asaresult,nominaltidalvolumeshadtobeadjustedto   compensatefortheincreasedflowresistanceassociatedto   theextendedlengthofthetubing.Thisresultedintheuseoftypically1.6–3.5ccforratstudies,and0.8–1ccformouseexperiments.AsarterialbloodgasanalysisinthemousewasperformedonlyattheendofthephMRIstudy,the   initialtidalvolumewas   setbasedonpilotexperimentswherearangeofinitialtidalvolumeswereexploredandtheircorrespondingbloodgaslevelswererecorded.Asaresultofthisactivity,wenotedthat,while5–20%tidalvolumeadjustmentswereoftennecessaryinratstudiestoaccountfordif-ferencesinanimalweight,thiswasnotthecasewithmice,wheretheuseofconstanttidalvolumesensuredstablebloodgasesacrosssubjectsofcomparableageandgeneticbackground.  2.3.3.Femoralvesselscannulation Aftertracheostomy,HALandIFanaesthesialevelsweredecreasedto1.5and3%,respectively(Table1).Thispracticeaccountsforthemoreeffectivedeliveryofanaestheticagentsachievedbytheuseofatracheotube,andpreventstheonsetof hypotensivestates.Theselevelsweremaintainedthroughoutthecannulationprocedure. Rat  .   Theleftlegwas   extended,tapedontothesurgicalsup-port,anda2-cmskinincisionwasmadewitha   scalpelintheventro-femoralareaaspreviouslydescribed(RigalliandDiLoreto,2009).Theleftfemoralarterywas   isolatedandcannulatedwithapolyethylenecatheter(PE50,OD0.97mm,   ID0.58mm,   ClayAdams,USA)filledwithheparinizedphysiologicsolution(25UI/mL)con-taining0.25g/mLof  d (+)-tubocurarine.ThecatheterwasconnectedtoaCRITIFLOTA4004device(flow/flushdevice;BectonDickinson,NJ,USA)coupledtoanarterialbloodpressuretransducer(TSD04,BiopacSystemsCorp.,Goweta,USA).Thisdevicewasinturncon-nectedviaa   PE50cathetertoaninfusionpump(KDS200,KDScientific,Massachusetts,USA)thatwasusedforcontinuousinfu-sionofasolutionofheparinandparalyzingagent(describedabove).An   intra-arterialbolusof1mL/kgof0.25mg/mL  d (+)-tubocurarinewasadministeredimmediatelyafterfemoralarterycannulationtoinducerapidneuromuscularblockade.Tubocurarinewasinfusedata   rateof1mL/kg/hthroughouttheexperiment.Pancuroniumbro-midewasusedas   neuromuscularblockerinListerHoodedrats.Inthiscase,an   intra-arterialbolusof1mL/kgofpancuroniumbromide0.25mg/mL    was   administeredimmediatelyafterfemoralarterycannulation,followedbya   continuousinfusionof1mL/kg/hthroughouttheimagingexperiment.Arterialbloodpressurewasrecordedwithaninvasivebloodpressuretransducerconnectedtoamulti-channelMP150Biopacsystem(BiopacSystemsCorp.,Goweta,USA).Theleftfemoralveinwas   cannulatedwithaPE50catheteraspreviouslydescribed(RigalliandDiLoreto,2009).Thecatheterwasconnectedtoa1mL    syringefilledwithsalinetopermittheadministrationofMRI   contrastagentandtestcompounds(e.g. d -amphetamine). Mouse .Therightfemoralarterywascannulatedtoallowforcontinuousmonitoringofbloodpressureandbloodsamplingforarterialgasanalysis.Thearterialaccesswas   alsousedforintravas-culardeliveryofsubstances(paralyzingagent,contrastagent,drugor   vehiclechallenge),asthefrailnatureoffemoralveinsinthisstrainmadethecannulationprocedurelengthyand   unre-liable.ThefemoralarterywascannulatedwithaPE10catheter(OD0.61mm,   ID0.28mm)   asdescribedaboveandconnectedtoacustom-madethree-wayplexiglasscubethatwasusedas   “switch”forintravascularaccess(Fig.S2).Uponcannulation,an   intra-arterialbolusofheparinizedphysiologicsolution(25UI/mL)containing0.0375mg/mLofpancuroniumbromidewasadministeredwithan   infusionvolumeof0.335mL/kg,whichresultedinanalmostimmediateneuromuscularblockade.Pancuroniumbromidewastheninfusedintra-arteriallyatrate   of6.7mL/kg/hthroughouttheexperiment.ThePE10catheterwasconnectedtotheplexiglass(Fig.S2)througha2-cmofPVC40(OD0.90mm,   ID0.50mm)    junc-tioninsertedintoapieceofSiliconetubing(Frenchsize3,e.g.outerdiameterapprox.1mm).   Thisthree-waysystemallowedsimulta-neousrecordingofMABPandinfusionofparalyzingagent(way1),aswellas   theinjectionofcompounds(way2),   upontemporaryclampingofthe   linetotheinfusionpump(way1)topreventrefluxofinjectionsolution.Instudiesrequiringintraperitoneal(IP)orsubcutaneous(SC)   of compounds(Gozzietal.,2008a,b;e.g.Gozzietal.,   2011b),a   G23infusioncannulawas   directlyinsertedinthedorsalskinoftheani-mal,orintheabdominalperitoneum.Thecannulawas   coupledtoaPE50catheterthroughashortsilasticjunctionandsecuredtotheskinwithafewdropsoftissueglue(Vetbond3M ® ).Theadmin-istrationofdenseorviscoussuspensionswasperformedviaG20cannulasconnectedtoa   PVC60catheter(OD1.22mm,   ID0.76mm).  2.3.4.Maintenancephase Attheendofsurgicalphase(25–35min   ofduration)theani-malswereplacedinpronepositionontoa   customizedstereotacticholder(Bruker,Ettlingen,Germany)andanaestheticlevelswere  Author's personal copy 12  L.Ferrarietal./JournalofNeuroscienceMethods  204 (2012) 9–18 decreasedtomaintenancelevel.Ratstudieswereperformedwith0.8%HALor1.1%IF(Table1).Mousestudieswereperformedeitherwith1.2%IF,orwith0.8%HAL(e.g.   with d -amphetamine).In   thelat-tercase,theIFvaporizerusedforsurgerywasreplacedwitha   HAL onepriortothemaintenancephase.AnMRI-compatiblethermo-coupleprobewas   usedtomeasurerectaltemperatureinallanimals.Thebodytemperatureofallsubjectswasmaintainedwithinphys-iologicalrange(37 ± 1.8 ◦ C)throughouttheexperimentbyusingawaterheatingsystemincorporatedinthestereotacticholder.  2.3.5.Arterialbloodgasmeasurements Abloodgasanalyzer(AVLOPTICCACriticalCareAnalyzer,Roswell,USA)wasusedtomeasurearterialoxygen(  p a O 2 )   andcar-bondioxide(  p a CO 2 )concentration.AninsulinsyringeconnectedwithaMirajectbluntneedle(0.9mm ×   22mm,   HagerWerken,Germany)wasusedtosamplearterialblood(ca.150  L)fromthe   femoralcannula.Inrats,measuresperformedatthebegin-ningofthemaintenancephasewereusedtoadjustventilationvolumesasappropriate.In   mousestudies,arterialsampling(ca.100  L)wasonlyperformedatthe   endoftheexperiment,withtheaimtoavoidhaemodynamicinterferencesrelatedtothesamplingofasignificantportion(ca.5%)oftheanimal’sbloodpool.TerminalmeasurementsperformedattheendofthephMRIacquisitioninratsandmicewereusedasasubjectexclusioncri-teriumwhensupra-physiologicalparameterswerefound(  p a O 2 levels<90mmHg   and  p a CO 2 >50mmHg).Terminalbloodsamplingwasperformedbyremovingthethree-wayarterialcubeandlettingthearterialcathetertospillbloodintoaninsulinsyringe(200  L)whichwasthenconnectedtothebloodgasanalyzer.Attheendof theexperimenttheanimalswereeuthanizedwithanoverdoseof anaestheticfollowedbycervicaldislocation.  2.4.Experimentalgroupsandprotocols Thecompositionoftheexperimentalgroupsandtreatmentsissummarisedbelow:(1)SD-HAL.SpragueDawley(SD)ratssurgicallypreparedandimagedunderHALanaesthesia.Paralyzingagenttubocurarine.Duringtheimagingsession,theratsreceivedanIVchallengewith d -amphetamine(0.5mg/rat,1mL/kginsaline, n =10).(2)LH-HAL.ListerHooded(LH)ratssurgicallypreparedandimagedunderHALanaesthesia.Paralyzingagenttubocurarine.Duringtheimagingsession,the   ratsreceivedanIV   challengewith d -amphetamine(0.5mg/rat, n =   10).(3)   Wistar-HAL.WistarratssurgicallypreparedandimagedunderHALanaesthesia.Paralyzingagentpancuroniumbromide.Dur-ingtheimagingsession,theratsreceivedanIVchallengewith d -amphetamine(0.5mg/rat, n =   14).(4)SD-IF.SDratssurgicallypreparedandimagedunderIFanaes-thesia.Paralyzingagentpancuroniumbromide( n =   15).Theseanimalsdidnotreceiveanystimulation,butwere   usedtoverifyphysiologicalstabilityunderisofluraneanaesthesia.(5)C57-HAL.C57BL6/JmicesurgicallypreparedwithIFandimagedunderHALanaesthesia.Paralyzingagentpancuroniumbro-mide.Themicereceivedan   intra-arterialadministrationof saline(5  L/g)followedbyanintra-arterialchallengewith d -amphetamine(3mg/kg)20min   later( n =   11).(6)   C57-IF.C57BL6/JmicesurgicallypreparedandmaintainedwithIF.Paralyzingagentpancuroniumbromide( n =8).Theseanimalsdidnotreceiveanystimulation,butwereusedtoverifyphysiologicalstabilityunderisofluraneanaesthesia.Thesameprotocolhasbeenrecentlyusedtoassessthe   functionalresponseproducedbytheserotoninantagonist8-OH-DPATintransgenicmouselines(Gozzietal.,2010b).(7)SDratssurgicallypreparedandimagedunderHALanaesthesia.Thesubjectsreceivedan   IV   vehicleinjection(saline,1mL/kg)andwereusedasbaselinephMRIreferenceforthestatisticalanalysisacrossallratcohorts.Differencesinthenumberofanimalsbetweengroupsare   notduetooutliersorabortedexperiments,butreflectthedifferentindependentexperiments.Thedoseof  d -amphetamineusedinratstudieshasbeenselectedbasedonpreviousinvivoworks(Gozzietal.,2011b;Michelietal.,2007;Schwarzetal.,   2007a).Thedoseusedinmicehasbeenselectedbasedontheresultsof pilotdose–responsephMRIstudies.  2.5.Relativecerebralvolume(rCBV)measurement Rat  .   MRI   datawereacquiredusingaBruker4.7Teslasystem.TherCBVmeasurementprotocolusedhasbeenpreviouslydescribedingreaterdetailelsewhere(Gozzietal.,2010a,2011b).Briefly,theMRacquisitionforeachsubjectcomprisedT2-weightedanatom-icalimagesusingthe   RAREsequencefollowedbyatimeseriesacquisitionwiththesamespatialcoverageandsimilarparame-ters,   butwithalowerin-planespatialresolution(128 ×   128).TotalMRI   time-seriesacquisitiontimewas60–80min   forallgroups.Followingsixreferenceimages,2.67mL/kgofthebloodpoolcon-trastagentEndorem(Guerbet,France)was   injectedthroughthefemoralveinofrattosensitiseimagestorCBV(Mandevilleetal.,1998;Schwarzetal.,2003).Intravenousadministrationof  d -amphetamine(0.5mg/kg)wasperformedafteran   equilibrationperiodof10–15min   andMRI   datawereacquiredovera   periodof 30minfollowingtheadministrationoftheamphetaminechallenge. Mouse .   TherCBVmeasurementprotocolusedinthephMRIandfMRIstudyhasbeendescribedingreaterdetailelsewhere(Gozzietal.,   2010b).Co-centredanatomicalimagesandfMRItimeserieswereacquiredusinga   RAREsequence(anatomical:TR  eff  =5597ms,TE eff  =   76ms,   RAREfactor8,FOV40mm,   256 ×   256 ×   24,16contigu-ous0.75mm   slices;fMRItimeseries:TR  eff  =5436ms,   TE eff  =   112ms,RAREfactor32,128 × 128;NA=   2,dt=   20s,   Nr=100,correspond-ingto70min   totalacquisitiontime).ImagesweresensitisedtoreflectalterationsinrCBVusing3.75  L/g   ofblood-poolcontrastagent(Endorem,Guerbet;Mandevilleetal.,1998).Fifteenminuteslatereachsubjectreceivedvehicleinjection(saline,5  L/g,i.a.)fol-lowed28min   laterby   achallengewithamphetamine(3mg/kgi.a.;Sigma–Aldrich).  2.6.DataanalysisRat  .   fMRIandphMRItimeserieswereanalyzedaspreviouslydescribed(Gozzietal.,   2008a,2011b).   Signalintensitychangesinthetimeserieswereconvertedintofractionalrelativecerebralbloodflow(rCBV)(Schwarzetal.,2003).IndividualsubjectswerespatiallynormalizedmappingtheirT2-weightedanatomicalimagestoa   stereotaxicratbrainMRI   templateset(Schwarzetal.,2006a)andapplyingtheresultingtransformationmatrixtotheaccompanyingrCBVtimeseries.rCBVtimeseriesforamphetamineorvehiclechallengewerenormalizedtoa   commoninjectiontimepoint.Imagebasedanalysiswas   carriedout   usingFEAT,partofFSL (FMRIB’sSoftwareLibrary,www.fmrib.ox.ac.uk/fsl),with0.8mmspatialsmoothingandusingaboxcarfunctiondepictingthepres-enceof  d -amphetamine(Gozzietal.,   2011b).   Higher-levelgroupcomparisons( d -amphetaminevs.vehicle)werecarriedoutusingFLAME(FMRIB’sLocalAnalysisofMixedEffects).  Z    (GaussianisedT/F)statisticimageswere   thresholdedusingclustersdeterminedby  Z  >1.6anda   correctedclustersignificancethresholdof  P  =   0.01(Worsleyetal.,   1992).VOItimecoursesfor d -amphetaminewereextractedfromunsmoothedrCBVtimeseriesdatausinga3D
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