Copyright by KathyM.Genzer 2008 COMMITTEE CERTIFICATION OF APPROVED VERSION ThecommitteeforKathyM.Genzercertifiesthatthis is theapprovedversionofthe followingthesis: DOPAMINE-INDUCED SYNAPTIC PLASTICITY IN THE AMYGDALA IN SALINE-AND COCAINE-TREATED ANIMALS UNDERGOING CONDITIONED PLACE PREFERENCE Committee: PatriciaShinnickGallagher,Ph.D. Supervisor GoldaAnneKevetter-Leonard,Ph.D. JosephC.Holt,Ph.D. __________________ Dean,GraduateSchool DOPAMINE-INDUCED SYNAPTIC PLASTICITY IN THE AMYGDALA IN SALINE-AND COCAINE-TREATED ANIMALS UNDERGOING CONDITIONED PLACE PREFERENCE by KathyM.Genzer,B.S. Thesis PresentedtotheFacultyofTheUniversityofTexasGraduate Schoolof Biomedical ScienceatGalveston InPartialFulfillmentoftheRequirements fortheDegreeof MasterofScience ApprovedbytheSupervisoryCommittee PatriciaShinnickGallagher,Ph.D. GoldaAnneKevetter-Leonard,Ph.D. JosephC.Holt,Ph.D. December,2008 Galveston,Texas Keywords:dopamine,PLD,conditionedplace preference ©2008,KathyM.Genzer TheUniversityofTexas MedicalBranch December,2008 Formyparents,whohavealwayssupportedme. ACKNOWLEDGEMENTS Iwanttothankmymentor,Dr.PatriciaShinnick-Gallagherforherwisdom, guidance,andpatience.Shenotonlysupportedmethroughoutmyentire project butalso encouragedmetofollow mytrue passionswhenIchangedmy plansanddecidedto pursuemedicalschool.Icouldnothaveaskedfora bettermentor.Ialsoexpressmy gratitudetomycommitteemembers,Dr.GoldaLeonardandDr.JosephHoltfortheir supportandknowledge. Dr.SebastianPollandtdevotedhis timetohelpingmelearnelectrophysiology,for whichIam grateful.I amalsothankfultoDr.JieLiuwhoaidedmeinmanyexperiments withhisexpertiseandpatience.Iwouldlike togivespecialthankstoDr.BalajiKrishnan whohas beenmyteachersince thefirstdayIwalkedintothe lab.Heguidedmenot only asalabmember,butasafriend. Finally,myfamily,ClaraandDavid,andmyfriends,especiallyKevinand Michelle,havealways beenmymostloyalsupporters.Their unflappable encouragement gavemethestrengthandconvictiontofollow myheartandpursuemydreams.Because ofthem,Iwasable tocompletemyMastersandattendmedicalschool. v DOPAMINE-INDUCED SYNAPTIC PLASTICITY IN THE AMYGDALA IN SALINE-AND COCAINE-TREATED ANIMALS UNDERGOING CONDITIONED PLACE PREFERENCE PublicationNo._____________ KathyM.Genzer,B.S. TheUniversityofTexas GraduateSchoolofBiomedicalSciencesatGalveston,2008 Supervisor: PatriciaShinnickGallagher Oneofthe biggest problems facingcocaineaddictsis relapse andcurrentlythere arenotreatmentsforrelapse.Thesestudieswill provideinsightintothe underlying mechanismsofLTP mediatingrelapsetococaine.A major mechanismof cocaine in addictionis toinhibitdopaminere-uptakeandglutamateisanexcitatoryneurotransmitter involvedinaddiction.Previousstudieshavedemonstratedalinkbetweenphospholipase D(PLD)andmetabotropicglutamatereceptors (mGluRs),whileotherstudiesshow an interactionbetweendopaminereceptors(DRs)andPLD.Therefore,weinvestigatedthe mechanismsofthedopamineagonist-inducedsynaptic plasticitythatisenhancedinthe amygdaladuetococaineexposure intheCentral-BasolateralAmygdala(CeA-BLA) pathway.Electrophysiologyrecordingsshowedthatthedopamineagonist-inducedLTP ismediatedviaD1receptorsandisdependentuponmGluR1andthemGluRlinkedto PLDandpartiallydependentuponmGluR5andphospholipaseC(PLC).Westernblots andcoimmunoprecipitationsrevealedincreasedexpressionofPLDaftercocaine administrationandpossible physical interactions betweengroupImGluRsandPLDand DRsandPLD.PLDactivityassaysshowedthat PLDactivitywasdecreasedby antagonists of mGluR1andthemGluRlinkedtoPLDandwasincreasedbytheagonist forthemGluRlinkedtoPLD.BasalPLDactivitymayalsobemediatedbyD1receptors andwasnot affectedbymGluR5antagonists. vi TABLE OF CONTENTS ACKNOWLEGEMENTS V ABSTRACT VI LIST OF FIGURES XI LIST OF ILLUSTRATIONS XIII LIST OF ABBREVIATIONS AND DRUGS XIV BACKGROUND 1 CocaineAddiction .....................................................................................................1 ConditionedPlacePreference....................................................................................2 AmygdalaandCocaine..............................................................................................2 Anatomyof theBLA-CeA pathway ..........................................................................3 DopamineandCocaine..............................................................................................3 MGluRs......................................................................................................................6 PhospholipaseD(PLD) .............................................................................................7 MATERIALS AND METHODS 10 Animals....................................................................................................................10 ConditionedPlacePreference..................................................................................10 Electrophysiology ....................................................................................................11 WesternBlotting ......................................................................................................11 Fractionation ............................................................................................................13 Coimmunoprecipitation..........................................................................................13 vii PLDActivityAssay .................................................................................................14 Injections..................................................................................................................15 Drugs........................................................................................................................15 RESULTS 16 ConditionedPlacePreference(CPP) withcocainewasdetectedonbothblackandwhite sidesofthe chamber.................................................................................................16 SKF81297inducedLTPinthe basolateraltocentral amygdala pathwayinthecocaine- butnotsaline-treatedgroup .........................................................................18 EffectofdopaminealsoinducedLTPintheBLA-CeA pathway ...........................20 SCH23390blockedthe dopamine-inducedLTPintheBLA-CeA pathway ...........22 Presenceof gamma-aminobutyric acid(GABA)inhibitionwasrequiredforSKF8297 inducedLTP.................................................................................................23 TheSKF81297inducedLTPwaslinkedtoametabotropic glutamatereceptor induced LTP ..............................................................................................................25 SKF81297inducedLTPwas mediatedthroughanmGluR1receptor ....................27 SKF81297inducedLTPwasalsodependentonmGluR5 ......................................29 InhibitingPLCattenuatedtheSKF81297inducedLTPintheBLA-CeA pathway....................................................................................................................31 PLD proteinexpressionintheamygdalawasincreasedduringwithdrawal incocaineCPP trainedanimals.............................................................................................32 Changesindopaminereceptorexpressionwerenotdetectedafterwithdrawalfrom cocaine .........................................................................................................34 MGluR proteinexpressionwasnotalteredaftercocainetreatment........................37 PLD1andPLD2werecoimmunoprecipitatedwithmGluR1andmGluR5antibodies in thecocaine CPPgroup.................................................................................39 PLD1butnotPLD2wereimmunoprecipitatedwithD1RandD5Rantibodies......40 PCCG-13andLY367385inhibitedandL-CSAstimulatedPLDactivityincocaine but viii notsalineslices ............................................................................................41 MGluR5antagonist,MPEP,alsoreducedPLDactivity..........................................43 SKF81297stimulated,andSCH23390blockedPLDactivity.................................44 EffectsofPCCG-13onCPP behavior.....................................................................46 DISCUSSION 48 D1agonist-inducedLTPintheBLA-CeA pathwaywasenhancedafterwithdrawalfrom cocaine .........................................................................................................48 TheD1agonist-inducedLTPwasdependentonGABAergic inhibitionsuggesting localizationofDAreceptorsonGABAergicinterneuronsintheBLA-CeA pathway........................................................................................................49 D1agonist-inducedLTPwas mediatedthroughmGluR1,mGluR5,thespecificmGluR1 linkedtoPLDandPLC................................................................................51 IncreasedPLDactivityinthecocainegroupisdependentuponDAreceptor activation......................................................................................................53 PLDactivitywasincreasedincocaine-treatedanimalsanddependent onmGluR1,andthespecificmGluR-linkedtoPLD,butnotmGluR5.......54 PLD,butnotPLD2,mGluR1,mGluR5,D1R,andD5Rwereunchangedinproteinfrom cocaine-animals........................................................................................................57 PLD1andPLD2expressionwasincreased,butmGluR1,mGluR5,D1R,andD5Rwere unchangedinfractionationproteinfromcocaine-comparedtosaline-treated animals .........................................................................................................57 PLD1,butnotPLD2,wascoimmunoprecipitatedwithD1RandD5Rnaïve protein ..........................................................................................................58