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RESEARCHARTICLEOpenAccess Geneexpressionchangesinthemedialprefrontal cortexandnucleusaccumbensfollowing abstinencefromcocaineself-administration WillardMFreeman 1,2* † ,MelindaELull 1 † ,KrutiMPatel 2 ,RobertMBrucklacher 2 ,DrakeMorgan 3 ,DavidCSRoberts 4 KentEVrana 1 Abstract Background: Manystudiesofcocaine-responsivegeneexpressionhavefocusedonchangesoccurringduring cocaineexposure,butfewstudieshaveexaminedthepersistenceofthesechangeswithcocaineabstinence. Persistentchangesingeneexpression,aswellasalterationsinducedduringabstinencemayunderlielong-lasting drugcravingandrelapseliability. Results: Whole-genomeexpressionanalysiswasconductedonaratcocainebinge-abstinencemodelthathas previouslybeendemonstratedtoengenderincreaseddrugseekingandtakingwithabstinence.Geneexpression changesintwomesolimbicterminalfields(mPFCandNAc)wereidentifiedinacomparisonofcocaine-naverats withratsafter10daysofcocaineself-administrationfollowedby1,10,or100daysofenforcedabstinence(n=611pergroup).Atotalof1,461genesinthemPFCand414genesintheNAcwerealteredbetweenatleasttwo timepoints(ANOVA,p<0.05;1.4fold-change).Thesegenescanbesubdividedinto:1)changeswithcocaine self-administrationthatdonotpersistintoperiodsofabstinence,2)changeswithcocaineself-administrationthat persistwithabstinence,3)andthosenotchangedwithcocaineself-administration,butchangedduringenforced abstinence.qPCRanalysiswasconductedtoconfirmgeneexpressionchangesobservedinthemicroarrayanalysis. Conclusions: Together,thesechangeshelptoilluminateprocessesandnetworksinvolvedinabstinence-induced behaviors,includingsynapticplasticity,MAPKsignaling,andTNFsignaling. Background Ahallmarkofcocaineaddictioniscontinueddrugcravingandrelapsepropensitydespitelong-termdrugabstinence.Developmentofeffectivecocaineaddiction treatmentsthereforerequirestherapiesthatdecreasethe likelihoodofrelapsetococaineabuseintherecovering addict.Acentralthemeofcocaineabuseresearchisthe roleofneurobio logicalchanges( e.g. ,electrophysiology, neurochemistry,neuroanatom y,epigenetic,transcriptomic,proteomic)inthedevelopmentandmaintenance oftheaddictedbehavioralphenotype(i.e.,increased drug-seekinganddrug-taking). Cocaineaddictiongenerally startswithrecreational useanddeterioratesovertimeintoacompulsiveand chronicallyrelapsingdrug-ta kingdisorder[1].Stress, environmentalcues,andconditionedstimulihavebeen demonstratedclinicallytoplayaroleincocainerelapse [2-4].Whileinitiatingdrugabstinencecanbeaccomplishedthroughin-patient treatment,maintaining cocaineabstinencehasprovendifficult[5].Incontrolled clinicaltrials,prolongedcocaineabstinenceisoften achievedbyonlyaminorityofpatients[6-8].Thismay beduetoincreasesincocainecravingduringdrugabstinence[9].Understandingthep ersistentneurobiological changesthatcontributetocontinueddrugcravingduringabstinenceandrelaps epotentialrepresentsan importantsteptowardsidentifyingtreatmentsthat reducethelikelihoodofrelapse[10]. Thereisagrowingunderstandingoftheacutegene/ proteinexpressionchangeswithcocaineadministration (eithernon-contingentlyorself-administered)thatmay beimportanttothedevelopmentandexpressionof *Correspondence:wfreeman@psu.edu † Contributedequally 1 DepartmentofPharmacology,PennStateCollegeofMedicine,Hershey,PA, 17033,USA Freeman etal BMCNeuroscience 2010, 11 :29 http://www.biomedcentral.com/1471-2202/11/29 2010Freemanetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited.

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cocaine-responsivebehavior[11],butonlyasmallnumberofstudieshaveexaminedwhetherthesechanges reverttonormallevelsorremainalteredwithcocaine abstinence.Observationsofmolecularchangespersisting intooroccurringduringthisabstinenceperiodprovide theopportunitytoidentifygenesandtheirprotein counterpartsthatcouldbeusedastherapeutictargetsto decreaserelapseliability. Thedevelopmentofanimalmodelsofcocaineabuse andabstinencehasledtotheidentificationofrodent behaviorssimilartothoseofhumancocaineabusers. Mostnotably,time-dependentincreasesincocaineseekingandtakingbehaviorshav ebeenobservedintherat modelofcocaineabuseandenforcedabstinence employedinthisstudy[12-14].Similarobservations havebeenmadeusingotheranimalmodelsofprolonged abstinencefromcocaine[15,16].Molecularanalysesof thesemodelshavenotonlyidentifiedchangesingene orproteinexpression[12,17-19],buthavealsocorrelatedgeneexpressionwithcocaine-responsivebehaviors [20,21].Manyoftheexistingreportsusedtargeted approachestoquantifyspecificgeneandproteinexpressionchangesduringabstinencefromcocaine.Largescalediscoverystudieswithlong-termenforcedabstinencefollowingcocaineselfadministrationarelimited andtranscriptomicstudies ,inparticular,havenotbeen conducted. Theself-administrationparadigmusedinthisstudy exhibitsincreasedreinforcingefficacy,drugseeking,and drugtakingwithatleast7days,andaslongas100 days,ofabstinenceafteraperiodofcocaineself-administration[12-14,22].Examinationofmesolimbicstructuresintheseanimalsiswarrantedbytherolesthat thesestructures,includingthemedialprefrontalcortex (mPFC)andnucleusaccumbens(NAc),playinreward andbehavioralresponsestostimuli.Bothofthesebrain regionshavebeenimplicatedincocaineabuseand withdrawalthroughimaging[23-25],behavioral [20,21,26],andmolecular[12,17-19]studies.Wehave conductedtargetedmRNAandepigeneticanalysisfrom thismodelpreviously[12].Theaimofthepresent studywastoextendthisinitialanalysisofmesolimbic dopaminergicterminalregionsbyprovidingagenomewidecharacterizationinboththemPFCandtheNAcof ratsfollowing10daysofcocaineself-administrationand afterincreasingperiodsofenforcedabstinencefrom cocaine(1,10,and100days).Identificationofgenes persistentlyalteredinexpressionbycocaineoraltered duringaperiodofcocaineab stinenceprovidesinsight intothemechanismsinvolvedinthelong-termbehavioralchangesthatoccurwithcocaineabuseandilluminatesnovelpotentialnewtargetsforpharmacological intervention.ResultsAnimalsBehavioralanalysesoftheratcocaineself-administration paradigmandtimepointsu sedinthisstudyhavebeen publishedpreviously[12,14,27].Thespecificanimals usedinthisstudyrepresentanindependentsetthatwas notbehaviorallytested(e.g.p rogressiveratioorextinctionresponding)toavoidanyconfoundingeffectsof behavioraltestingongeneexpression.Allcocaineselfadministeringgroupsweremaintainedonacontinuous access(24hours/day)discretetrials(DT)schedulefor 10days.Trialswerelimitedto4trialsperhour(DT4). After10daysofDT4responding,animalsweresubjectedto1,10,or100daysofenforcedabstinence.The cocaineintakedataforthespecificanimalsusedinthis studyispresentedinTable1.Nosignificantdifferences wereobservedinthetotalcocaineintakeofeachgroup ortheaveragenumberofdailyinjections.Thesimilarity intotalcocaineintakeandrespondingbetweengroups minimizesthepossibilitythatexposuretodiffering Table1CocaineintakedataforsamplesArraysqPCRConfirmation GroupNAverage TotalIntake AverageInjections PerSession NAverage TotalIntake AverageInjections PerSession mPFC Nave6-*-11-1-dayAbstinence6934108mg/kg6276934108mg/kg627 10-daysAbstinence7950105mg/kg637 100-daysAbstinence685642mg/kg573885539mg/kg573 NAc Nave5-*-11-1-dayAbstinence5932118mg/kg57286934108mg/kg627 10-daysAbstinence5951116mg/kg6387950105mg/kg637 100-daysAbstinence885539mg/kg573*Naveanimalswerenotexposedtococaine,andthereforehavezerointake.Therewerenosignificantdifferencesincocaineintakeornumberofinjec tionsper sessionbetweengroups. “ Arrays ” indicatesanimalsusedformicroarrayanalysisand “ qPCRConfirmationindicatesanimalsusedforconfirmatoryqPCRanalysis. AllofthesamplesfromthearrayanalysiswereincludedintheqPCRconfirmation.DataispresentedasMeanS.D.Freeman etal BMCNeuroscience 2010, 11 :29 http://www.biomedcentral.com/1471-2202/11/29 Page2of13

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amountsofcocaine,oradifferenceinself-administrationbehavior,couldaccountforgeneexpression changesobserved.MicroarraystudiesofthemPFCwere conductedonnave,1-dayabstinent,and100-daysabstinentanimals(n=6/group).Microarraystudiesofthe NAcwereconductedonnave,1-dayabstinent,and10daysabstinentanimals(n=5/group)fromthesame cohort.ForconfirmatoryqPCRanalysesofgeneexpressionlevels,alargernumberofsamplesweretested, includingthoseincludedinmicroarraystudies.Additionally,whilemicroarrayswereconductedonsamples fromthreetimepoints,allfourtimepointswereused fortheseconfirmationstudies(nave,n=11;1-day,n= 6;10-days,n=7;and100-days,n=8)toprovidefiner temporalresolution.MicroarrayanalysisInthemPFC,geneproductscorrespondingtoatotalof 21,814probes(ofthe44,000totalprobesonthearrays) wereconfidentlydetected,basedonsignalintensityata fixedvalueabovebackgroundlevels.IntheNAcanalysis, mRNAsforatotalof19,015probesweredetected.Differentiallyexpressedgeneswereidentifiedthrougha combinationofstatisticalsignificance(p<0.05,One-way ANOVAbetweengroups)andafoldchangefilterof=1.4 foldchange(Figure1A&1B).Thisilluminated1,461gene expressionchangesinthemP FC(representing6.7%of thetotalmRNAspeciesdetected)and414geneexpressionchangesintheNAc(2.2%ofthetotaldetected). Thesechangesdemonstratedthreetypesoftemporal profiles(Figure1C).Category1changesarethosethat occurredwithcocaineself-a dministration(eitherup-or down-regulatedat1-dayofabstinence),butthat didnot persist intolongerperiodsofabstinence(10-or100days).AmajorityofthemPFCgeneexpressionchanges belongedtothiscategory(793of1,461);however,onlya smallfractionofchangesintheNAc(68of414)exhibitedthisexpressionpattern.Category2changesarethose Figure1 Analysisofexpressionchangesfromthemicroarraystudies .Venndiagramsillustratethechangesidentifiedbymicroarrayanalysis ofnaveanimals,andanimalsfollowing1and10or100daysofabstinence.(A)InthemPFC,21,814probesweredetectedaspresent.Ofthese, 1,461werechangedbygreaterthan1.4foldwithp<0.05(ANOVA)andcanbesplitintothreecategoriesbasedonexpressionprofile:changed betweennaveand1-day(793),naveand100-days(480)orboth(188).(B)IntheNAc,19,015probesweredetectedaspresent.Ofthese,414were changedbygreaterthan1.4foldwithp<0.05(ANOVA),andcanbesplitintothesamethreecategories:changedbetweennaveand1-day(68), naveand10-days(314)orboth(32).(C)Thethreecategoriesofgeneexpressionchangescanbesegregatedinto(1)changesthatoccurwith cocaineself-administration(SA)thatdonotpersist,(2)changesthatoccurwithcocaineSAthat do persist,and(3)changesthat occurduring abstinence.Smallgraphsindicatetheexpressionprofileofchangesineachcategory,wherethex-axisrepresentsgroup(nave,1-dayand10-or 100-daysabstinent)andthey-axisrepresentspercent(%)ofnaveexpression.(D)Whencomparingthegeneexpressionchangedbetweenthe mPFCandNAc,amajority(97%)areuniquetoonebrainregionortheother.Onlyasmallfraction(50)ischangedinbothbrainregions. Freeman etal BMCNeuroscience 2010, 11 :29 http://www.biomedcentral.com/1471-2202/11/29 Page3of13

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thatoccurredwithcocaineself-administrationandpersistedintoperiodsofabstinence(changedat1-dayand remained changedwithenduringabstinence).Asmall numberofgeneexpressionchangesfromeachbrain region(188mPFC;32NAc)belongedtothiscategory. Finally,category3changesarethosethatdidnotoccur withcocaineself-administration,butchanged during the periodofabstinence(notchangedat1-day,butchanged at10-and/or100-days).Approximately33%ofthe changesinthemPFC(480of1,461)and76%ofthe changesintheNAc(314of414)belongtothiscategory. Acomparisonofthegeneschangedatanytimepoint betweenthemPFCandNAcrevealsthatalimitednumberofchangeswereobservedinbothbrainregions(Figure1D).Ofthetotalof1875geneexpressionchanges, lessthan3%weredetectedinboththemPFCandthe NAc.Afulllistofgeneexpressionchangesispresented inAdditionalFile1:TableS1,andhasbeenuploadedto theGeneExpressionOmnibusonlinedatabase.ConfirmatoryqPCRqPCRanalysiswasusedtoconfirmasubsetofgene expressionchangesobservedinthemicroarrayanalyses. GeneschosenforqPCRconfirmationwereselected basedonontologicalclasseswithpotentialrolesin drug-inducedchangesinthebrain.Additionalgenes wereincludedintheconfirmationstudiesbasedonpreviousreportsofcocaine-responsivegeneexpression(see AdditionalFile2:TableS2,forafulllistandgene expressionassayinformation). Differentiallyregulatedg enesconfirmedinthisstudy (13total;4mPFC,9NAc),belongtoeachofthe3categoriesdescribedabove(Figure2).InthemPFC,neurofilamentlight(Nefl)wastheonlycategory1change, witha20%decreaseinthemPFCat1-dayofabstinence (Figure2A).Category2changesincludeda23% decreaseinlevelsofCD47 at1-daythatpersisted through10-daysofabstinence(21%),andanincreasein levelsofdopaminereceptorD5(Drd5)at1-day(40%) Figure2 ConfirmedchangesinmPFCgeneexpression .FourchangesingeneexpressionwereconfirmedinthemPFCwithqPCRanalysis.(A) Neurofilamentlightisdecreasedby20%at1-dayofabstinence(p<0.05).(B)CD47expressionisdecreasedfromnaveat1-day(23%,p<0.05) and10-daysofabstinence(21%;p<0.01).(C)DopaminereceptorD5(Drd5)expressionisincreasedfromnaveat1-day(40%;p<0.01)and 100-days(41%;p<0.01).(D)AdenosineA2Breceptor(Adora2b)expressionisdecreasedfromnaveat10-daysofabstinence(20%;p<0.05). Statisticalanalysiswasperformedbyone-wayANOVAwithStudent-Newman-Keulsposthoctesting;*p<0.05,**p<0.01. Freeman etal BMCNeuroscience 2010, 11 :29 http://www.biomedcentral.com/1471-2202/11/29 Page4of13

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and100-daysofabstinence(41%)(Figure2B&2C). Finally,levelsofadenosineA2Breceptor(Adora2b) wereunchangedbycocaineadministration,butwere reducedby20%after10-daysofabstinence(Category3 change)(Figure2D). IntheNAc,ninegeneswereconfirmed,mostof whichdisplayedacategory3profileofchanging during abstinence.Theseincludebeta-catenin(32%increaseat 10-days),adenylatecyclaseassociatedprotein2(Cap2; 206%increaseat10-days),cysteine-richprotein2 (Crip2;17%increaseat10-days),dynamin2(Dnm2; 167%increaseat10-days),earlygrowthresponse2 (Egr2;55%decreaseat100-days),fucosyltransferase8 (Fut8;25%increaseat10-da ys),glialfibrillaryacidic protein(GFAP;87%increaseat10-days),andG-protein coupledreceptor88(Gpr88;277%increaseat10-days) (Figure3A).The5-hydroxytryptaminereceptor1d (Htr1d),whichwasdecreasedat1-day(28%)and remaineddecreasedat10-days(20%)representstheonly confirmedcategory2change(Figure3B). Consistentwithourpreviousfindingsinthisanimal modelandatthesametimepoints[12],themicroarray analysisperformedinthisstudydetectedsignificant changesinactivity-relatedcytoskeletalassociatedprotein (Arc),cocaineandamphetamine-relatedtranscript (CART),earlygrowthresponse1(Egr1),FBJosteosarcomaoncogene(Fos),neuropeptideY(NPY),and nuclearreceptorsubfamily4a1(Nr4a1)transcriptlevels. Priortothemicroarrayanalysis,wehadexaminedthese genesbyqPCRbasedontheirknownresponsivenessto cocaineandhavealreadyreportedtheseresults[12]. Persistentdecreases(Category2or3)inArc,Fos,and Nr4a1wereobservedinboththemPFCandNAc.Category1changesinCART,NPY(bothincreasedat1-day) andEgr1(decreasedat1-day)inthemPFCwerealso confirmedbyqPCR(Table2).OntologicalandNetworkanalysisAnalysisoftheGeneOntology(GO)categoriesof changesineachbrainregionidentifiedanumberof molecularfunctionssignificantlyregulatedwith cocaineself-administrati onandabstinence.Themolecularfunctionsalteredwereanatomicallydistinct.In themPFC,themost-representedclass,consistingof ~25%ofthechangesidentifiedbymicroarrayanalysis, wasproteinserine/threoninekinaseactivity,followed closelybystructuralcomponentsoftheribosomeand monovalentinorganiccationtransporteractivity (~15%ofthetotalchangeseach).ThemostrepresentedclassesamongthechangesinNAcgeneexpressionwerehydrolaseactivity(~40%),andphosphoric esterhydrolaseactivity(~10%). Networkanalysis,conductedusingIngenuityPathways Analysissoftware(Ingenui tySystems,RedwoodCity, CA),wasperformedtodeterminetherelationships betweenconfirmedgeneexpressionchangesfromthis studyandpreviouslyreportedchangesfromthismodel [12].TocomplementtheGOanalysis,additionalanalysiswasconductedtodeterminewhetherthesegenesare implicatedinspecificpathwaysand/orbiologicalfunctionsanddiseases.OnlythosegenesconfirmedbyqPCR wereusedinthisanalysis.InthemPFC,thesemolecules compriseanetworkofinteractionsinvolvedinsynaptic plasticity,calciumsignaling,andmitogenactivatedproteinkinase/extracellularsignal-regulatedkinase(MAPK/ ERK)signaling.IntheNAc,all12moleculeswerecomponentsofanetworkofinteractionswithmembers linkedtotheneuronalcytoskeleton,glialcells,andWnt andtumornecrosisfactor(TNF)signalingfunctions.DiscussionThisstudyrepresentsthefirstmicroarrayanalysisof mesolimbicgeneexpressionfollowinglong-term enforcedabstinencefromcocaineself-administration. Transcriptomicstudiesofcocaine-inducedgeneexpressionchangeshavebeenconducted,butthesehave focusedonnon-contingentcocaineadministrationand noorlimited(~1day)abstinence.Theworkconducted inthepresentstudyusedamodelwithwell-characterizedbehavioralchangesduringperiodsofabstinence, andusedanimalsnotsubjectedtobehavioraltesting duringabstinence(e.g.progressiveratioorextinction responding)sothatthegeneexpressionchanges observedarefreefromtheeffectselicitedbybehavioral testingconductedbeforesacrifice.Additionally,itis importanttonotethatallgroups(1,10,and100daysof abstinence)self-administeredequivalentamountsof cocaineoverthe10daysofdiscretetrialcocaineselfadministration.Thistim e-courseanalysisofgene expressionallowsfordiscrim inationofgeneexpression changesassociatedwithincreaseddrugseeking(10and 100daysofabstinence)fromthosethatoccurwith cocaineself-administration,butdonotpersistforas longasincreaseddrugseekingandtaking(1dayof abstinence). Theliteraturedescribesanumberofneurobiological changes(e.g.alteredgene/proteinexpression,neurotransmitterlevels,epigeneticevents)withdifferentmodelsofcocaineabuse,(forareviewsee[11,28]).Whether thesechangespersistintoperiodsofabstinence,however,hasgenerallynotbeendetermined.Inthisstudy, geneexpressionchangesthatoccurredasaresultof cocaineself-administrationandabstinencesegregated intothreecategoriesofexpressionpatterns.Category1 changesweredefinedasthosethatoccurwithcocaine use,but donot persistintoperiodsofabstinence.These wereobservedaschangesonlybetweennaveanimals and1-dayabstinentanimals.Afteronly1dayofFreeman etal BMCNeuroscience 2010, 11 :29 http://www.biomedcentral.com/1471-2202/11/29 Page5of13

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Figure3 ConfirmedchangesinNAcgeneexpression .NinechangesingeneexpressionwereconfirmedintheNAcwithqPCRanalysisin thisstudy.(A)Eightofthechangeshavetheprofileofchanginginexpression during abstinence(eitherat10or100-daysofabstinence).These genesarebeta-catenin(increasedby32%at10-days;p<0.001),adenylatecyclase-associatedprotein2(Cap2;increasedby206%at10-days;p< 0.001),cysteine-richprotein2(Crip2;increased17%at10-days;p<0.05),dynamin2(Dnm2;increased167%at10-days;p<0.001),earlygrowth response2(Egr2;decreasedby55%at100-days;p<0.05),fucosyltransferase8(Fut8;increased25%at10-days;p<0.01),glialfibrillaryacidic protein(GFAP;increased87%at10-days;p<0.05),andG-proteincoupledreceptor88(Gpr88;increased277%at10-days;p<0.01).(B) 5-hydroxytryptaminereceptor1d(Htrd1)isdecreasedby28%at1-day(p<0.001)andby20%at10-daysofabstinence(p<0.01).Statistical analysiswasperformedbyone-wayANOVAwithStudent-Newman-Keulsposthoctesting;*p<0.05,**p<0.01,#p<0.001. Freeman etal BMCNeuroscience 2010, 11 :29 http://www.biomedcentral.com/1471-2202/11/29 Page6of13

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abstinence,anincreaseindrugseekinganddrugtaking isnotobserved[12],sothechangesingeneexpression observedatthispointmaybenecessary,butarenotsufficient,tocausetheincubatedphenotypeandareprimarilyduetoexposuretoco caine.Category2changes arethosethatoccurwithcocaineusethat persist with periodsofabstinence.Thesewereobservedtobealtered inthecomparisonsbetweennaveand1-dayabstinent animals and betweennaveand10-or100-dayabstinent animals.Thesealterationsmayresultfromcocaine exposure,butdonotreturntonavelevelswithcessationofthecocainestimulus.Thepersistenceofthese changesmayindicatetheirpotentialroleinthedevelopment(10days)ormaintenance(100days)ofabstinence-persistentincreasesindrugseekinganddrug takingbehaviors.Category3changesconsistofgenes thatwereunchangedwithcocaineuse,butarealtered during theabstinenceperiod.Whilenotimmediately affectedbycocaineexposure,thissetofchangesmay resultfrominitiationorco ntinuationofabstinence. Thesemayfunctionsynergisticallywithother(category 2)changestocontributetothedevelopmentof increaseddrug-seekingand-taking.mPFCThemPFCmediatesexecutivefunctionanddecision makingprocessesandisthereforeakeyneuroanatomicalregioninaddictivebehaviors[29,30].Inresponseto cocaineadministration,changesinmetabolicactivity, neurotransmittersystems,andgeneorproteinexpressionoccurinthemPFC(forareviewsee[28]).Inthis study,alargenumberofgeneexpressionchangeswere observedinthemPFCbothasaresultofcocaineselfadministrationandwithsubsequentenforcedabstinence. Mostofthesechangesoccurredasadirectresultofthe cocaineself-administration(981)andamajority(793) returnedtococaine-navelevelswithcessationof cocaineself-administration.Asexpectedmanychanges (category1)ingeneexpressionrequirecontinued cocainestimulustoremainaltered,andreturntonormallevelsafterthestimulusisremoved.Asubsetof genes(188)remainedchangedafter100-daysof enforcedabstinence.Persistenceofgeneexpression changeswithabstinence(category2)requiresmaintenanceviaothermechanisms.Epigeneticchangesoccur inresponsetococaine,andmayconstitutearegulatory mechanismforpersistentchangesingeneexpression [12,31].Changes(480)thatdonotoccurduringcocaine self-administration,butareinducedwithabstinence (category3)mayreflectthewithdrawalofthecocaine stimulusanddevelopmentoftheincubatedphenotype. AlteredgeneexpressionofAdora2b,Arc,CART, Cd47,Drd5,Egr1,Fos,Nefl,NPY,andNr4a1wereconfirmedbyqPCR.Wehavepreviouslydescribedaltered expressionofArc,CART,Egr1,Fos,NPY,andNr4a1in thesesamplesinadirectedstudyofgeneswithknown relevancetodrugabuse[12].AnumberoftheqPCR confirmationanalysesthatdidnotreachstatisticalsignificancedemonstratedexp ressionprofilessimilarto thoseobservedinthemicroarray.Thismayreflectthe effectsofneuroanatomicalcomplexityonquantitation ofgeneexpressionendpointsandtheinclusionoflarger numbersofanimalsintheconfirmatoryexperiments. ThisworkidentifiedalteredexpressionoftwoG-proteincoupledreceptors(GPCRs;Adora2b,Drd5),acellsurfacesignalingmolecule(CD47),andacomponentof theneuronalcytoskeleton(Nefl).IncreasedDrd5,and signalingthroughthisreceptor,havebeenreportedto decreaseresponsivenesstoco caine[32,33].Similarly, adenosinesignalinghasbeenimplicatedindrugaddiction.Specifically,activationofAdora2breceptors attenuatescocaine-conditionedplacepreference[34]. Althoughthemechanismsunderlyingtheseeffectsare unclear,Drd5signalingisimplicatedinneuronal Table2Geneexpressionchangesreportedpreviously ExpressionLevel(%ofNaveControl) GeneName GeneID Nv1 Nv10 Nv100 mPFC Activity-relatedcytoskeleton-associatedproteinArc43.914**76.81251.810** CocaineandamphetamineregulatedtranscriptCART725.2130**191.955190.386 Earlygrowthresponse1Erg159.69.2*74.71574.55.3 FBJosteosarcomaoncogeneFos41.712**58.111**53.16.8** NeuropeptideYNpy158.421#94.59.5915.2 Nuclearreceptorsubfamily4,groupA,member1Nr4a135.111**53.412*49.47.7* NAc Activity-relatedcytoskeleton-associatedproteinArc83.52273.71249.56.4* FBJosteosarcomaoncogeneFos53.916**60.36.7**51.762** Nuclearreceptorsubfamily4,groupA,member1Nr4a165.72063.41148.15.1*Dataispresentedasmeanstandarderrorofthemean(SEM),ANOVAwithStudent-Newman-Keulsposthoctesting;*p<0.05,**p<0.01,#p<0.001.Data previouslyreportedin[12].Freeman etal BMCNeuroscience 2010, 11 :29 http://www.biomedcentral.com/1471-2202/11/29 Page7of13

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activitiesincludinglong-termpotentiation(LTP;[35]), andreinforcementlearning[36],whilebothDrd5[37] andAdora2b[38]appeartoaffectCa2+dynamics. ThedecreaseinCD47expressioninthismodelisa novelobservationandisofinterestduetoitsfunction inneuronaldevelopment[39,40].Neflfunctionsin cytoskeletalorganizationandcell-surfacereceptorremodeling[41,42],whichmaybeimpairedwiththeobserved decreaseinexpressionat1-dayofabstinence.Previously,changesinproteinlevelsandpost-translational modificationsofNefl,andotherneurofilamentisoforms, havebeenreportedwithcocaine,morphine,alcohol,and nicotineadministration[43-45]. Wehavepreviouslyreportedadirectedanalysisof immediateearlygenes(IEGs;Arc,Egr1,Fos,andNr4a1) andneuropeptides(NPYandCART)inthisanimal model[12].Thesegeneswerealsoidentifiedinthecurrentdiscoverymicroarrayanalysis,providingincreased confidenceinthemicroarrayfindings.Thesegenesplay importantrolesinanumberofneuronalprocesses includinglearningandmemory[46,47],synapticplasticity[48-50],Ca2+signaling[51,52],andMAPKsignaling [51]. NetworkanalysiswasconductedusingthesetofconfirmedmPFCgeneexpressionchanges,andrevealed thatCart,NPY,Nr4a1,Fos,Egr1,Adora2bandDrd5all interact(directlyorindirectly)withtheMAPK/ERK pathway.WhilealteredexpressionofMAPK/ERKpathwayelementswasnotdetectedinthisstudy,changesin expressionandactivitylevelsofMAPK/ERKgeneshave beenreported(forareviewsee[28])andthispathwayis thoughttoplayanimportantroleindrug-induced changesinthebrain[53,54].RegulatorsofCa2+dynamicswerealsoidentifiedinthenetworkanalysis. ThechangesinDrd5,Adora2b,CD47andCART expressionmayindicateadecreaseinintracellularCa2+signalingthatoccurswithco caineself-administration andpersistsintoperiodsofabstinence[40,52,55,56]. Additionally,thegeneexpr essionchangesidentified indicatethatsynapticplasticitymaybeaffectedby cocaineself-administrationa ndabstinence.Persistent reductionsinlevelsofCD47andArc,andinductionsin levelsofDrd5andNPYsuggestalteredsynapticplasticityprocessinvolvedinmemoryformationandremoval ofoldmemorytraces,respectively[50,57,58].ApotentialreductioninsynapticplasticityinthemPFCwith cocaineself-administration /abstinenceishypothesized basedonlevelsofCD47,Nefl,Arc,Egr1,andNPY [39,42,48-50].Thesedataareinagreementwithstudies ofthedirectroleofpsychostimulantsonmechanismsof synapticplasticity,includingLTPandLTD,inthemesolimbicsystem[59,60].Intotal,thesegeneexpression changesmaycontributetopers istentlyalteredsynaptic plasticityinthemPFC.NAcThecentralroleoftheNAcinpsychostimulantrewardis welldocumented[61].Wh ilecocaineexertscommon actionsontheNAcandmPFC[62],weobservedlittle overlap(50ofthe1875totalgeneexpressionchanges (mPFC+NAc))betweenthesebrainregions.TheregulatedgenescommontobothbrainregionsincludeIEGs reportedpreviously[12],varioussignalingmolecules,and genesinvolvedincellularmetabolism.Whenthemicroarraydatasetswereexaminedbyontologicalanalysisdistinctmolecularfunctionswereobservedineachbrain region.ThisindicatesthatthefunctionalchangesoccurringinthemPFCandNAcmaydifferandmayultimately playdifferentrolesinabstinence-dependentbehaviors. UnlikethemPFC,fewercategory1and2changes wereobservedintheNAc(100of414total),thancategory3changes(thosechangedspecificallyduringabstinence)(314).Ofthecocaine-inducedchanges,only32 persistedintoperiodsofab stinence(category2),while theremainderreturnedtopre-cocainelevels.Arc,Betacatenin,Cap2,Crip2,Dnm2,Egr2,Fos,Fut8,GFAP, Gpr88,Htr1d,andNr4a1wereallconfirmedbyqPCR tobedifferentiallyexpressed.Wehavepreviously demonstratedtheresponsivenessofArc,FosandNr4a1 inthisanimalmodel[12]. PublisheddataregardingCap2,Crip2,Fut8,and Gpr88inthebrainarelimited,withnopreviousreports ofcocaine-responsiveness.Crip2(aLIM-domainprotein),Cap2(anadenylatecyclase-associatedprotein)and Dnm2arecytoskeletalfunctionandorganizationgenes [63,64].Interestingly,Dnm2isregulatedbythetranscriptionfactorArc,alsoalteredintheNAcwith cocaine[12,65].Amongtheremainingchanges,Egr2 andGFAPhavebeenpreviouslydemonstratedtobe cocaine-responsive[66-68].Htr1dhasbeenlinkedwith anumberofpsychiatricdisorders[69,70].Changesin theexpressionofthesegenesmayalsoindicatecocaine inducedalterationsinreceptorsignaling,glialcellfunction,andsynapticplasticity. Beta-catenin,whichwasincreasedat10-daysofabstinenceinthisstudy,isawell-characterizedproteinthat regulatescellgrowthasa partoftheWntsignaling pathway.AsapartofWntsign aling,beta-cateninalso playsaroleinsynapticplasticity[71,72].Inresponseto chroniccocaine,beta-cateninhasbeenshownto increaseinanumberofbrainregions[73-75].Fut8,a fucosyltransferaseprotein,alsoincreasedat10-daysof abstinenceinthisstudy,hasbeenshowntoincrease uponWnt/beta-cateninactiva tion[76],indicatingthat theremaybeacoordinatedactivationofWntsignaling duringperiodsofabstinencefromcocaine. NetworkanalysisoftheconfirmedgenesintheNAc identifiedaTNF-centeredn etwork.Generallyinvolved ininflammatoryprocesses,TNFhasnotbeenFreeman etal BMCNeuroscience 2010, 11 :29 http://www.biomedcentral.com/1471-2202/11/29 Page8of13

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historicallyassociatedwi thbehavioralresponsesto cocaine.Studiesperformedontheeffectsofcocaineon macrophageshavereportedthatcocainesuppresses LPS-stimulatedTNFexpressi on[77,78].TNFinduction wasrecentlydemonstratedtoreduceconditionedplace preferenceandlocomotorsensitizationcausedby methamphetamineandmorphineadministration[79].If TNFdoesplayaroleinthebehavioralresponsesto cocaine,theseadditionalgenesmayrepresentregulatory andeffectorelementsofaTNFnetwork. Whilethesereportedchangesrepresentnewinsights intoabstinence-inducedchangesinthebrain,localization ofthesechangestospecificcelltypesisstilltobedetermined.Aswithotherfunctionalgenomicandproteomic approacheslookingatdissectedbrainregions,eventhese specificdissectionscontai naheterogeneouscellular population.Futuremolecularneurobiologystudiesthat seektoextendthese,andotherfindings,willneedtoutilizetechniques(e.g.lasercapturemicrodissectionand fluorescent in-situ hybridization)tolocalizechangesto specificcelltypesandneuronalnetworks[80].ConclusionsInadditiontoofferingfurthe revidenceoflong-lasting changesingeneexpressionfollowingabstinencefrom cocaineself-administration,theseresultsidentifycellular processesthatmayregulatethedevelopmentand/or maintenanceofincubationofdrug-seekinganddrug-taking.Anumberofadditionalchangesingeneexpression remaintobeexaminedinfuturestudies,buttheresults presentedheresupportthefindingthatpersistentshifts ingeneexpressioncanlastlongintoabstinence.Inthe mesolimbicrewardpathway,changesinthemPFCmay bemorepronouncedthanintheNAcandinvolvemostly distinctsetsofgenes.Thismayindicatedifferentmetaplasticprocessesoccurinthesebrainregionswiththe developmentandexpressionofabstinence-inducedbehaviors.InthemPFC,changesinMAPK/ERKandcalcium signalingandinsynapticplasticityoccur.Thealterations intheNAcsuggestapossibleroleofWntandTNFmediatedsignalingincocaine-associatedbehaviors. Togetherthefindingsofthisstudyhighlightanumberof pathwaysandprocessesinthebrainthatmayplayroles inthedevelopmentandmaintenanceofabstinenceinduceddrugseekinganddrugtaking.Aclearunderstandingofhowthesenovelchangescontributeto relapseliabilitywillnotonl yincreaseourknowledgeof theneurobiologyofaddiction,butwillprovidetargetsfor therapeuticdevelopment.MethodsCocaineself-administrationThesurgicalandcocaineself-administrationprocedures usedhavebeendescribedpreviously[12,18].Briefly, maleSprague-Dawleyrats(HarlanInc.,IN)were implantedwithachronicindwellingSilasticcannulain therightjugularvein,andt rainedtoself-administer cocainehydrochloridethroughexposuretoafixedratio 1(FR1)scheduleofreinforcementasdescribedpreviously[12,14].Afterestablishingastabledailyintakeof cocaine(40infusionswithin6hoursforatleast5days), accessconditionswerechangedtoadiscrete-trialsschedule.Duringthediscrete-trialschedule,ratsweregiven accesstococainefor10-mindiscretetrialsthatwere initiatedat15-minintervals.Aninfusion(1.5mg/kg/inj) ofcocainewasgivenfollowingaresponseonthelever, whichresultedinilluminationofastimuluslightfor20 sec.Theleverwasretractedandthetrialterminatedif aninjectionwascollectedorif10minuteshadelapsed. Ratsreceivedfourdiscretetrialsperhour(i.e.,DT4), 24hoursperday,for10days.Following10daysof self-administration,anim alswereplacedinstandard polycarbonatecagesfor1,10or100daysofenforced abstinence.AllresearchwasapprovedbytheWake ForestUniversitySchoolof MedicineandPennState CollegeofMedicineAnimalCareandUseCommittees andconductedaccordingtotheGuidefortheCareand UseofLaboratoryAnimals,aspromulgatedbythe NationalInstitutesofHealth.DissectionFollowing1,10,or100daysofdeprivation,ratswere sacrificedandthebrainswererapidlyremovedand cooledinice-chilledsaline.Brainswerethenplacedin anice-chilledASIbrainslicer(ASIInstruments,Warren MI).Themedialprefrontalcortex(mPFC)andnucleus accumbens(NAc)werecollectedasdescribedpreviously [12].Briefly,thesectionfromBregma+4.4to2.4mm [81]wascutalongtheforcepsminorandthecortex medialofthiscutwascollected.Thisisconsideredthe medialprefrontalcortex,andincludesthecingulate area,prelimbiccortex,andmedialorbitalcortex.The sectionfrom+2.2to0.2mmwascut0.5mmoneach sideofthemidline,onalineconnectingthetipofthe externalcapsuleandthepreviouscut,onalineconnectingthetipoftheexternalcapsuleandlateralventricle, andbetweentheventricle.Thisdissectionincludesboth thecoreandtheshelloftheNAc.SeeAdditionalFile3: FigureS1,forschematicsofthedissections.RNAisolationFollowingdissection,tissuesampleswerestoredat-80C untilRNAwasisolated.RNAisolationwasconductedas describedpreviously[74,82].TotalRNAfromcocaine naveratsandratsfollowing1,10,and100daysof enforcedabstinence(following10daysofDT4cocaine self-administrationasdescribedabove)wasisolated usingTri-Reagent(MolecularResearchCenterInc.,Freeman etal BMCNeuroscience 2010, 11 :29 http://www.biomedcentral.com/1471-2202/11/29 Page9of13

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Cincinnati,OH)[83].RNAquantityandqualitywas thenassessedusingtheAgilent2100Bioanalyzer(Agilent,PaloAlto,CA)followingfurtherRNApurification usinganRNeasyMiniKitforRNAclean-up(Qiagen Sciences,Maryland).MicroarrayanalysisMicroarrayanalysiswasperformedinthePennState CollegeofMedicineFunctionalGenomicsCoreFacility accordingtostandardprocedures.ForthemPFCarrays, nave,1-day,and100-dayabstinentsampleswereused (N=6).FortheNAcarrays,nave,1-dayand10-day abstinentsampleswereused(N=5;reducedsample numberduetoremovalofsamplesthatdidnotpass arrayqualitycontrol).MicroarraysfortheNAcwere performedafterthoseforthemPFC,andfortheNAc, the10-daytimepointwasusedinsteadofthe100-day timepointinordertodetectimportantchangesthat maynotlastto100daysofabstinence.Thepersistence ofthesechangesto100-dayscouldthenbetestedwith qPCR. MicroarrayanalysiswasperformedusingtheCodeLinkRatWholeGenomeBioarraysystem(GEHealthcare).Followingthemanufacturer ’ sprotocol,firststrand synthesiswasperformedwith2 gofRNAasstarting materialandwasfollowedbysecondstrandsynthesis andpurificationusingQiaq uickspincolumns(Qiagen, Valencia,CA).T7reactionbuffer,T7NTPs,10mM biotin-11-UTP,andT7polymerasewerethenaddedto thedsDNAfortheIVTreactionandincubatedat37C for14hours.TheresultingBiotin-labeledcRNAwas thenpurifiedusingRNEasycolumns(Qiagen),quantitated,andvolume-adjustedforatotalof10 L.The cRNAwasthenfragmentedanddenaturedbeforehybridizationfor18hoursat37C.Slideswerewashedand thenincubatedatroomtemperaturewithAlexaFluor 647labeledstreptavidinfor30minutesfollowedby washing. MicroarrayswerescannedonanAxon4000BscannerwithGenePix4v4.0softwareata5 mresolutionat 635nmwithlaserpowerat100%,PMTvoltageat600 V,focusposition0 m,andlinestoaverage=1.Images werethenimportedintoCode LinkExpressionAnalysis Softwarev4.1(GEHealthcare)andinitialqualitycontrol (positiveandnegativecont rols),exclusionofmanufacturingdefects(MSRspots),backgroundsubtraction,and intra-arraynormalizationwasperformed.DataanalysisFollowingimageanalysisonCodeLinkExpressionAnalysisSoftware,microarraydatawereimportedinto GeneSpringGX7.3(AgilentTechnologies)andsignal valueslessthan0.01weretransformedtoanintensityof 0.01.Normalizationwasperformedperchiptothe50thpercentile,andpergenetothemedian.Valueswere thennormalizedonapergenebasistothenavegroup foreachofthetwotimepoints(1and10,or1and100 daysofabstinence).Potentialdifferentialexpressionwas determinedwithaone-wayANOVA(variancesnot assumedtobeequal),p<0.05andfilteredfor1.4-fold orgreaterdifferencesinexpressioninaccordancewith standardsformicroarrayanalysis[84].Theuseofa combinationofstatisticalandfold-changecutoffsas opposedtotraditionalmultipletestingcorrections( e.g ., Bonferronipost-hoctesting)producegenelistswiththe lowestrateoftypeIandtypeIIerrors[85].Afoldchangecutoffof1.4foldwaschosen,asthismagnitude changeisatthelowerrangeofchangeshistoricallyconfirmedbyqPCRinthislaboratory.Lastly,probe sequencesonthearrayweresearchedagainstcurrent ratgenomesequencestoeliminateanyprobesfor sequencesremovedfromtheNCBIdatabase.QuantitativePCR(qPCR)analysisofgeneexpressioncDNAsynthesiswasperformedontotalRNAfromnave, 1,10,and100-dayabstinentanimalsusingSuperscriptIII ReverseTranscriptase(Invitrogen,Carlsbad,CA).1 g RNA,500ngOligo(dT),and10mMeachdNTP,were incubatedfor5minutesat65Candthenchilledonice for2minutes.5FirstStrandBuffer(250mMTris-HCl (pH8.3),375mMKCl,and15mMMgCl2),5mMDTT (finalconcentration),40URNaseOut,and200USuperscriptIIIRTwerethenadded.The20 lreactionwas incubatedfor60minutesat50Cfollowedbyafinal incubationat70Cfor15minutesfortermination.The resultingcDNAproductwasquantifiedand50ngofproductwasusedineachsubsequentqPCRreaction. QuantitativePCRwascarriedoutonareal-time detectioninstrument(ABI7900HTSequenceDetection System)in384-wellopticalplatesusingTaqManUniversalPCRMasterMixandAssayonDemandprimers andprobes(AppliedBiosystems,FosterCity,CA)as describedpreviously[86,8 7].Thisexaminationuseda largersetofanimalsthanthemicroarrayanalysis(Table 1).Primer/probesetsusedarelistedinAdditionalFile 2:TableS2.SDS2.2.2softwareandthe2Ctanalysis method[88].wereusedtoquantitaterelativeamounts ofproductusing b -actinasanendogenouscontrol.SignificancefromqPCRanalysiswasdeterminedwithSigmaStat3.5(SYSTATSoftware,Inc.)basedonone-way analysisofvariance(ANOVA)(p<0.05)witha posthoc StudentNewman-Keulstest(p<0.05).Ontological,pathway,andnetworkanalysisOntologicalanalysisusedGeneOntology(GO)categoriesanddifferentiallyexpressedprocessesorfunctionalcategoriesweredeterminedstatistically,as previouslydescribed[87]usingGeneSpringGXFreeman etal BMCNeuroscience 2010, 11 :29 http://www.biomedcentral.com/1471-2202/11/29 Page10of13

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software.Thisanalysisdeterminedthenumberofgenes inacategorypresentonthearrayandthenumberof expressionchangesthatwouldbepartofthatcategory byrandomchancegiventhenumberofdifferentially expressedgenes.Resultsfromtheseanalyseswereused tocompilealistofgenestoexaminebyqPCR.IngenuityPathwayAnalysis(IngenuitySystems,RedwoodCity CA)wasusedfornetworkandpathwaysanalysisofthe qPCRconfirmedgeneexpressionresults.Additionalfile1:Microarrayexpressiondata .Afulllistingof differentiallyexpressedgenesidentifiedbymicroarrayanalysis. Clickhereforfile [http://www.biomedcentral.com/content/supplementary/1471-2202-1129-S1.DOC] Additionalfile2:Geneexpressionassaynumbers .Genesymbols,full namesandgeneexpressionassaynumbers. Clickhereforfile [http://www.biomedcentral.com/content/supplementary/1471-2202-1129-S2.XLS] Additionalfile3:Dissectionschematics .Dissectiondiagrams. SchematicsofthemPFCandNAcdissectionsareprovidedusing modificationsoffiguresfromPaxinosandWatson.Numberedredlines arespecificdissectioncutsusingvisiblelandmarksasdescribedinthe methods.Theshadedarearepresentsthetissuecollectedformolecular analysis. Clickhereforfile [http://www.biomedcentral.com/content/supplementary/1471-2202-1129-S3.PDF] Abbreviations Adora2b:adenosinereceptorA2b;Arc:activity-relatedcytoskeletalassociated protein;Cap2:adenylatecyclase-associatedprotein2;CART:cocaineand amphetamine-relatedtranscript;Crip2:cysteine-richprotein2;Dnm2: dynamin2;Drd5:dopaminereceptorD5;DT:discretetrials;Egr1:early growthresponse1;Egr2:earlygrowthresponse2;Fos:FBJosteosarcoma oncogene;FR1:fixedratio1;Fut8;fucosyltransferase8;GFAP:glialfibrillary acidicprotein;GPR88:g-proteincoupledreceptor88;Htr1d:5hydroxytryptaminereceptor1d;MAPK/ERK:mitogenactivatedprotein kinase/extracellularsignal-regulatedkinase;mPFC:medialprefrontalcortex; NAc:nucleusaccumbens;Nefl:neurofilamentlight;NPY:neuropeptideY; Nr4a1:nuclearreceptorsubfamily4a1;TNF:tumornecrosisfactor Acknowledgements ThisworkwassupportedbygrantsR01-DA013770-08(KEV),F31-DA02281902 (MEL),R01DA14030(DCSR)andK01-DA13957(DM).Theauthorswishto thankthePennStateCollegeofMedicineFunctionalGenomicCoreFacility formicroarrayandqPCRanalysisandtechnicalassistanceandDr.Heather VanGuilderformanuscriptediting. Authordetails1DepartmentofPharmacology,PennStateCollegeofMedicine,Hershey,PA, 17033,USA.2FunctionalGenomicsFacility,PennStateCollegeofMedicine. Hershey,PA,17033,USA.3DepartmentofPsychiatry,UniversityofFlorida, Gainesville,FL,32611,USA.4DepartmentofPhysiology&Pharmacology, WakeForestUniversitySchoolofMedicine,Winston-Salem,NC,27157,USA. Authors ’ contributions WMFandKEVgeneratedtheexperimentaldesign.RMBandKMPconducted thearrayandRT-PCRanalyses,respectively.MELperformedthedataanalysis, preparedthefigures,archivedthedata,andwrotethemanuscript.WMF assistedwithdataanalysisandWMFandKEVcontributedtothedata interpretation.DMandDCSR(WakeForestUniversity)developedtheanimal modelusedandprovidedthesamplesforanalysis.Allauthorsreadand approvedthefinalmanuscript. Received:23September2009 Accepted:26February2010Published:26February2010 References1.O ’ BrienCP: Recentdevelopmentsinthepharmacotherapyofsubstance abuse. 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Abstract
Background
Many studies of cocaine-responsive gene expression have focused on changes occurring during cocaine exposure, but few studies have examined the persistence of these changes with cocaine abstinence. Persistent changes in gene expression, as well as alterations induced during abstinence may underlie long-lasting drug craving and relapse liability.
Results
Whole-genome expression analysis was conducted on a rat cocaine binge-abstinence model that has previously been demonstrated to engender increased drug seeking and taking with abstinence. Gene expression changes in two mesolimbic terminal fields (mPFC and NAc) were identified in a comparison of cocaine-naïve rats with rats after 10 days of cocaine self-administration followed by 1, 10, or 100 days of enforced abstinence (n = 6-11 per group). A total of 1,461 genes in the mPFC and 414 genes in the NAc were altered between at least two time points (ANOVA, p < 0.05; ± 1.4 fold-change). These genes can be subdivided into: 1) changes with cocaine self-administration that do not persist into periods of abstinence, 2) changes with cocaine self-administration that persist with abstinence, 3) and those not changed with cocaine self-administration, but changed during enforced abstinence. qPCR analysis was conducted to confirm gene expression changes observed in the microarray analysis.
Conclusions
Together, these changes help to illuminate processes and networks involved in abstinence-induced behaviors, including synaptic plasticity, MAPK signaling, and TNF signaling.
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p Gene expression changes in the medial prefrontal cortex and nucleus accumbens following abstinence from cocaine self-administration
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au ca yes id A1 ce snm Freemanmi Mfnm Willardinsr iid I1 I2 email wfreeman@psu.edu
A2 LullEMelindamlull@vcu.edu
A3 PatelMKrutikrutims@gmail.com
A4 BrucklacherMRobertrbrucklacher@hmc.psu.edu
A5 MorganDrakeI3 drakem@ufl.edu
A6 RobertsCSDavidI4 dcsrobts@wfubmc.edu
A7 VranaEKentkvrana@psu.edu
insg
ins Department of Pharmacology, Penn State College of Medicine, Hershey, PA, 17033, USA
Functional Genomics Facility, Penn State College of Medicine. Hershey, PA, 17033, USA
Department of Psychiatry, University of Florida, Gainesville, FL, 32611, USA
Department of Physiology & Pharmacology, Wake Forest University School of Medicine, Winston-Salem, NC, 27157, USA
source BMC Neuroscience
issn 1471-2202
pubdate 2010
volume 11
issue 1
fpage 29
url http://www.biomedcentral.com/1471-2202/11/29
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cpyrt 2010collab Freeman et al; licensee BioMed Central Ltd.note This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
abs
sec
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Abstract
Background
Many studies of cocaine-responsive gene expression have focused on changes occurring during cocaine exposure, but few studies have examined the persistence of these changes with cocaine abstinence. Persistent changes in gene expression, as well as alterations induced during abstinence may underlie long-lasting drug craving and relapse liability.
Results
Whole-genome expression analysis was conducted on a rat cocaine binge-abstinence model that has previously been demonstrated to engender increased drug seeking and taking with abstinence. Gene expression changes in two mesolimbic terminal fields (mPFC and NAc) were identified in a comparison of cocaine-naïve rats with rats after 10 days of cocaine self-administration followed by 1, 10, or 100 days of enforced abstinence (n = 6-11 per group). A total of 1,461 genes in the mPFC and 414 genes in the NAc were altered between at least two time points (ANOVA, p < 0.05; ± 1.4 fold-change). These genes can be subdivided into: 1) changes with cocaine self-administration that do not persist into periods of abstinence, 2) changes with cocaine self-administration that persist with abstinence, 3) and those not changed with cocaine self-administration, but changed during enforced abstinence. qPCR analysis was conducted to confirm gene expression changes observed in the microarray analysis.
Conclusions
Together, these changes help to illuminate processes and networks involved in abstinence-induced behaviors, including synaptic plasticity, MAPK signaling, and TNF signaling.
meta
classifications
classification refman subtype user_supplied_xml type bmc
bdy
Background
A hallmark of cocaine addiction is continued drug craving and relapse propensity despite long-term drug abstinence. Development of effective cocaine addiction treatments therefore requires therapies that decrease the likelihood of relapse to cocaine abuse in the recovering addict. A central theme of cocaine abuse research is the role of neurobiological changes (it e.g., electrophysiology, neurochemistry, neuroanatomy, epigenetic, transcriptomic, proteomic) in the development and maintenance of the addicted behavioral phenotype (i.e., increased drug-seeking and drug-taking).
Cocaine addiction generally starts with recreational use and deteriorates over time into a compulsive and chronically relapsing drug-taking disorder abbrgrp
abbr bid B1 1
. Stress, environmental cues, and conditioned stimuli have been demonstrated clinically to play a role in cocaine relapse
B2 2
B3 3
B4 4
. While initiating drug abstinence can be accomplished through in-patient treatment, maintaining cocaine abstinence has proven difficult
B5 5
. In controlled clinical trials, prolonged cocaine abstinence is often achieved by only a minority of patients
B6 6
B7 7
B8 8
. This may be due to increases in cocaine craving during drug abstinence
B9 9
. Understanding the persistent neurobiological changes that contribute to continued drug craving during abstinence and relapse potential represents an important step towards identifying treatments that reduce the likelihood of relapse
B10 10
.
There is a growing understanding of the acute gene/protein expression changes with cocaine administration (either non-contingently or self-administered) that may be important to the development and expression of cocaine-responsive behavior
B11 11
, but only a small number of studies have examined whether these changes revert to normal levels or remain altered with cocaine abstinence. Observations of molecular changes persisting into or occurring during this abstinence period provide the opportunity to identify genes and their protein counterparts that could be used as therapeutic targets to decrease relapse liability.
The development of animal models of cocaine abuse and abstinence has led to the identification of rodent behaviors similar to those of human cocaine abusers. Most notably, time-dependent increases in cocaine seeking and taking behaviors have been observed in the rat model of cocaine abuse and enforced abstinence employed in this study
B12 12
B13 13
B14 14
. Similar observations have been made using other animal models of prolonged abstinence from cocaine
B15 15
B16 16
. Molecular analyses of these models have not only identified changes in gene or protein expression
12
B17 17
B18 18
B19 19
, but have also correlated gene expression with cocaine-responsive behaviors
B20 20
B21 21
. Many of the existing reports used targeted approaches to quantify specific gene and protein expression changes during abstinence from cocaine. Large-scale discovery studies with long-term enforced abstinence following cocaine self-administration are limited and transcriptomic studies, in particular, have not been conducted.
The self-administration paradigm used in this study exhibits increased reinforcing efficacy, drug seeking, and drug taking with at least 7 days, and as long as 100 days, of abstinence after a period of cocaine self-administration
12
13
14
B22 22
. Examination of mesolimbic structures in these animals is warranted by the roles that these structures, including the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc), play in reward and behavioral responses to stimuli. Both of these brain regions have been implicated in cocaine abuse and withdrawal through imaging
B23 23
B24 24
B25 25
, behavioral
20
21
B26 26
, and molecular
12
17
18
19
studies. We have conducted targeted mRNA and epigenetic analysis from this model previously
12
. The aim of the present study was to extend this initial analysis of mesolimbic dopaminergic terminal regions by providing a genome-wide characterization in both the mPFC and the NAc of rats following 10 days of cocaine self-administration and after increasing periods of enforced abstinence from cocaine (1, 10, and 100 days). Identification of genes persistently altered in expression by cocaine or altered during a period of cocaine abstinence provides insight into the mechanisms involved in the long-term behavioral changes that occur with cocaine abuse and illuminates novel potential new targets for pharmacological intervention.
Results
Animals
Behavioral analyses of the rat cocaine self-administration paradigm and time points used in this study have been published previously
12
14
B27 27
. The specific animals used in this study represent an independent set that was not behaviorally tested (e.g. progressive ratio or extinction responding) to avoid any confounding effects of behavioral testing on gene expression. All cocaine self-administering groups were maintained on a continuous access (24 hours/day) discrete trials (DT) schedule for 10 days. Trials were limited to 4 trials per hour (DT4). After 10 days of DT4 responding, animals were subjected to 1, 10, or 100 days of enforced abstinence. The cocaine intake data for the specific animals used in this study is presented in Table tblr tid T1 1. No significant differences were observed in the total cocaine intake of each group or the average number of daily injections. The similarity in total cocaine intake and responding between groups minimizes the possibility that exposure to differing amounts of cocaine, or a difference in self-administration behavior, could account for gene expression changes observed. Microarray studies of the mPFC were conducted on naïve, 1-day abstinent, and 100-days abstinent animals (n = 6/group). Microarray studies of the NAc were conducted on naïve, 1-day abstinent, and 10-days abstinent animals (n = 5/group) from the same cohort. For confirmatory qPCR analyses of gene expression levels, a larger number of samples were tested, including those included in microarray studies. Additionally, while microarrays were conducted on samples from three time points, all four time points were used for these confirmation studies (naïve, n = 11; 1-day, n = 6; 10-days, n = 7; and 100-days, n = 8) to provide finer temporal resolution.
tbl Table 1caption Cocaine intake data for samples.tblbdy cols 8
r
c
left cspan 4
b Arrays
3
qPCR Confirmation
7
hr
center
Group
N
Average Total Intake
Average Injections Per Session
N
Average Total Intake
Average Injections Per Session
mPFC
Naïve
6
-*
-
11
-
-
1-day Abstinence
6
934 ± 108 mg/kg
62 ± 7
6
934 ± 108 mg/kg
62 ± 7
10-days Abstinence
7
950 ± 105 mg/kg
63 ± 7
100-days Abstinence
6
856 ± 42 mg/kg
57 ± 3
8
855 ± 39 mg/kg
57 ± 3
NAc
Naïve
5
-*
-
11
-
-
1-day Abstinence
5
932 ± 118 mg/kg
57 ± 28
6
934 ± 108 mg/kg
62 ± 7
10-days Abstinence
5
951 ± 116 mg/kg
63 ± 8
7
950 ± 105 mg/kg
63 ± 7
100-days Abstinence
8
855 ± 39 mg/kg
57 ± 3
tblfn
* Naïve animals were not exposed to cocaine, and therefore have zero intake. There were no significant differences in cocaine intake or number of injections per session between groups. "Arrays" indicates animals used for microarray analysis and "qPCR Confirmation indicates animals used for confirmatory qPCR analysis. All of the samples from the array analysis were included in the qPCR confirmation. Data is presented as Mean ± S.D.
Microarray analysis
In the mPFC, gene products corresponding to a total of 21,814 probes (of the 44,000 total probes on the arrays) were confidently detected, based on signal intensity at a fixed value above background levels. In the NAc analysis, mRNAs for a total of 19,015 probes were detected. Differentially expressed genes were identified through a combination of statistical significance (p < 0.05, One-way ANOVA between groups) and a fold change filter of =1.4 fold change (Figure figr fid F1 1A&1B). This illuminated 1,461 gene expression changes in the mPFC (representing 6.7% of the total mRNA species detected) and 414 gene expression changes in the NAc (2.2% of the total detected). These changes demonstrated three types of temporal profiles (Figure 1C). Category 1 changes are those that occurred with cocaine self-administration (either up- or down-regulated at 1-day of abstinence), but that did not persist into longer periods of abstinence (10- or 100-days). A majority of the mPFC gene expression changes belonged to this category (793 of 1,461); however, only a small fraction of changes in the NAc (68 of 414) exhibited this expression pattern. Category 2 changes are those that occurred with cocaine self-administration and persisted into periods of abstinence (changed at 1-day and remained changed with enduring abstinence). A small number of gene expression changes from each brain region (188 mPFC; 32 NAc) belonged to this category. Finally, category 3 changes are those that did not occur with cocaine self-administration, but changed during the period of abstinence (not changed at 1-day, but changed at 10- and/or 100-days). Approximately 33% of the changes in the mPFC (480 of 1,461) and 76% of the changes in the NAc (314 of 414) belong to this category. A comparison of the genes changed at any time point between the mPFC and NAc reveals that a limited number of changes were observed in both brain regions (Figure 1D). Of the total of 1875 gene expression changes, less than 3% were detected in both the mPFC and the NAc. A full list of gene expression changes is presented in Additional File supplr sid S1 1: Table S1, and has been uploaded to the Gene Expression Omnibus online database.
fig Figure 1Analysis of expression changes from the microarray studiestext
Analysis of expression changes from the microarray studies. Venn diagrams illustrate the changes identified by microarray analysis of naïve animals, and animals following 1 and 10 or 100 days of abstinence. (A) In the mPFC, 21,814 probes were detected as present. Of these, 1,461 were changed by greater than 1.4 fold with p < 0.05 (ANOVA) and can be split into three categories based on expression profile: changed between naïve and 1-day (793), naïve and 100-days (480) or both (188). (B) In the NAc, 19,015 probes were detected as present. Of these, 414 were changed by greater than 1.4 fold with p < 0.05 (ANOVA), and can be split into the same three categories: changed between naïve and 1-day (68), naïve and 10-days (314) or both (32). (C) The three categories of gene expression changes can be segregated into (1) changes that occur with cocaine self-administration (SA) that do not persist, (2) changes that occur with cocaine SA that do persist, and (3) changes that occur during abstinence. Small graphs indicate the expression profile of changes in each category, where the x-axis represents group (naïve, 1-day and 10- or 100-days abstinent) and the y-axis represents percent (%) of naïve expression. (D) When comparing the gene expression changed between the mPFC and NAc, a majority (97%) are unique to one brain region or the other. Only a small fraction (50) is changed in both brain regions.
graphic file 1471-2202-11-29-1 hint_layout single
suppl
Additional file 1
Microarray expression data. A full listing of differentially expressed genes identified by microarray analysis.
name 1471-2202-11-29-S1.DOC
Click here for file
Confirmatory qPCR
qPCR analysis was used to confirm a subset of gene expression changes observed in the microarray analyses. Genes chosen for qPCR confirmation were selected based on ontological classes with potential roles in drug-induced changes in the brain. Additional genes were included in the confirmation studies based on previous reports of cocaine-responsive gene expression (see Additional File S2 2: Table S2, for a full list and gene expression assay information).
Additional file 2
Gene expression assay numbers. Gene symbols, full names and gene expression assay numbers.
1471-2202-11-29-S2.XLS
Click here for file
Differentially regulated genes confirmed in this study (13 total; 4 mPFC, 9 NAc), belong to each of the 3 categories described above (Figure F2 2). In the mPFC, neurofilament light (Nefl) was the only category 1 change, with a 20% decrease in the mPFC at 1-day of abstinence (Figure 2A). Category 2 changes included a 23% decrease in levels of CD47 at 1-day that persisted through 10-days of abstinence (21%), and an increase in levels of dopamine receptor D5 (Drd5) at 1-day (40%) and 100-days of abstinence (41%) (Figure 2B&2C). Finally, levels of adenosine A2B receptor (Adora2b) were unchanged by cocaine administration, but were reduced by 20% after 10-days of abstinence (Category 3 change) (Figure 2D).
Figure 2Confirmed changes in mPFC gene expression
Confirmed changes in mPFC gene expression. Four changes in gene expression were confirmed in the mPFC with qPCR analysis. (A) Neurofilament light is decreased by 20% at 1-day of abstinence (p < 0.05). (B) CD47 expression is decreased from naïve at 1-day (23%, p < 0.05) and 10-days of abstinence (21%; p < 0.01). (C) Dopamine receptor D5 (Drd5) expression is increased from naïve at 1-day (40%; p < 0.01) and 100-days (41%; p < 0.01). (D) Adenosine A2B receptor (Adora2b) expression is decreased from naïve at 10-days of abstinence (20%; p < 0.05). Statistical analysis was performed by one-way ANOVA with Student-Newman-Keuls post hoc testing; p < 0.05, ** p < 0.01.
1471-2202-11-29-2
In the NAc, nine genes were confirmed, most of which displayed a category 3 profile of changing during abstinence. These include beta-catenin (32% increase at 10-days), adenylate cyclase-associated protein 2 (Cap2; 206% increase at 10-days), cysteine-rich protein 2 (Crip2; 17% increase at 10-days), dynamin 2 (Dnm2; 167% increase at 10-days), early growth response 2 (Egr2; 55% decrease at 100-days), fucosyltransferase 8 (Fut8; 25% increase at 10-days), glial fibrillary acidic protein (GFAP; 87% increase at 10-days), and G-protein coupled receptor 88 (Gpr88; 277% increase at 10-days) (Figure F3 3A). The 5-hydroxytryptamine receptor 1d (Htr1d), which was decreased at 1-day (28%) and remained decreased at 10-days (20%) represents the only confirmed category 2 change (Figure 3B).
Figure 3Confirmed changes in NAc gene expression
Confirmed changes in NAc gene expression. Nine changes in gene expression were confirmed in the NAc with qPCR analysis in this study. (A) Eight of the changes have the profile of changing in expression during abstinence (either at 10 or 100-days of abstinence). These genes are beta-catenin (increased by 32% at 10-days; p < 0.001), adenylate cyclase-associated protein 2 (Cap2; increased by 206% at 10-days; p < 0.001), cysteine-rich protein 2 (Crip2; increased 17% at 10-days; p < 0.05), dynamin 2 (Dnm2; increased 167% at 10-days; p < 0.001), early growth response 2 (Egr2; decreased by 55% at 100-days; p < 0.05), fucosyltransferase 8 (Fut8; increased 25% at 10-days; p < 0.01), glial fibrillary acidic protein (GFAP; increased 87% at 10-days; p < 0.05), and G-protein coupled receptor 88 (Gpr88; increased 277% at 10-days; p < 0.01). (B) 5-hydroxytryptamine receptor 1d (Htrd1) is decreased by 28% at 1-day (p < 0.001) and by 20% at 10-days of abstinence (p < 0.01). Statistical analysis was performed by one-way ANOVA with Student-Newman-Keuls post hoc testing; p < 0.05, ** p < 0.01, #p < 0.001.
1471-2202-11-29-3
Consistent with our previous findings in this animal model and at the same time points
12
, the microarray analysis performed in this study detected significant changes in activity-related cytoskeletal associated protein (Arc), cocaine and amphetamine-related transcript (CART), early growth response 1 (Egr1), FBJ osteosarcoma oncogene (Fos), neuropeptide Y (NPY), and nuclear receptor subfamily 4a1 (Nr4a1) transcript levels. Prior to the microarray analysis, we had examined these genes by qPCR based on their known responsiveness to cocaine and have already reported these results
12
. Persistent decreases (Category 2 or 3) in Arc, Fos, and Nr4a1 were observed in both the mPFC and NAc. Category 1 changes in CART, NPY (both increased at 1-day) and Egr1 (decreased at 1-day) in the mPFC were also confirmed by qPCR (Table T2 2).
Table 2Gene expression changes reported previously6
ul Expression Level (% of Naïve Control)
Gene Name
Gene ID
Nv1
Nv10
Nv100
mPFC
Activity-related cytoskeleton-associated protein
Arc
43.9 ± 14**
76.8 ± 12
51.8 ± 10**
5
Cocaine and amphetamine regulated transcript
CART
725.2 ± 130**
191.9 ± 55
190.3 ± 86
Early growth response 1
Erg1
59.6 ± 9.2*
74.7 ± 15
74.5 ± 5.3
FBJ osteosarcoma oncogene
Fos
41.7 ± 12**
58.1 ± 11**
53.1 ± 6.8**
Neuropeptide Y
Npy
158.4 ± 21sup #
94.5 ± 9.5
91 ± 5.2
Nuclear receptor subfamily 4, group A, member 1
Nr4a1
35.1 ± 11**
53.4 ± 12*
49.4 ± 7.7*
NAc
Activity-related cytoskeleton-associated protein
Arc
83.5 ± 22
73.7 ± 12
49.5 ± 6.4*
FBJ osteosarcoma oncogene
Fos
53.9 ± 16**
60.3 ± 6.7**
51.7 ± 62**
Nuclear receptor subfamily 4, group A, member 1
Nr4a1
65.7 ± 20
63.4 ± 11
48.1 ± 5.1*
Data is presented as mean ± standard error of the mean (SEM), ANOVA with Student-Newman-Keuls post hoc testing; p < 0.05, ** p < 0.01, # p < 0.001. Data previously reported in 12.
Ontological and Network analysis
Analysis of the Gene Ontology (GO) categories of changes in each brain region identified a number of molecular functions significantly regulated with cocaine self-administration and abstinence. The molecular functions altered were anatomically distinct. In the mPFC, the most-represented class, consisting of ~ 25% of the changes identified by microarray analysis, was protein serine/threonine kinase activity, followed closely by structural components of the ribosome and monovalent inorganic cation transporter activity (~ 15% of the total changes each). The most represented classes among the changes in NAc gene expression were hydrolase activity (~ 40%), and phosphoric ester hydrolase activity (~ 10%).
Network analysis, conducted using Ingenuity Pathways Analysis software (Ingenuity Systems, Redwood City, CA), was performed to determine the relationships between confirmed gene expression changes from this study and previously reported changes from this model
12
. To complement the GO analysis, additional analysis was conducted to determine whether these genes are implicated in specific pathways and/or biological functions and diseases. Only those genes confirmed by qPCR were used in this analysis. In the mPFC, these molecules comprise a network of interactions involved in synaptic plasticity, calcium signaling, and mitogen activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling. In the NAc, all 12 molecules were components of a network of interactions with members linked to the neuronal cytoskeleton, glial cells, and Wnt and tumor necrosis factor (TNF) signaling functions.
Discussion
This study represents the first microarray analysis of mesolimbic gene expression following long-term enforced abstinence from cocaine self-administration. Transcriptomic studies of cocaine-induced gene expression changes have been conducted, but these have focused on non-contingent cocaine administration and no or limited (~ 1 day) abstinence. The work conducted in the present study used a model with well-characterized behavioral changes during periods of abstinence, and used animals not subjected to behavioral testing during abstinence (e.g. progressive ratio or extinction responding) so that the gene expression changes observed are free from the effects elicited by behavioral testing conducted before sacrifice. Additionally, it is important to note that all groups (1, 10, and 100 days of abstinence) self-administered equivalent amounts of cocaine over the 10 days of discrete trial cocaine self-administration. This time-course analysis of gene expression allows for discrimination of gene expression changes associated with increased drug seeking (10 and 100 days of abstinence) from those that occur with cocaine self-administration, but do not persist for as long as increased drug seeking and taking (1 day of abstinence).
The literature describes a number of neurobiological changes (e.g. altered gene/protein expression, neurotransmitter levels, epigenetic events) with different models of cocaine abuse, (for a review see
11
B28 28
). Whether these changes persist into periods of abstinence, however, has generally not been determined. In this study, gene expression changes that occurred as a result of cocaine self-administration and abstinence segregated into three categories of expression patterns. Category 1 changes were defined as those that occur with cocaine use, but do not persist into periods of abstinence. These were observed as changes only between naïve animals and 1-day abstinent animals. After only 1 day of abstinence, an increase in drug seeking and drug taking is not observed
12
, so the changes in gene expression observed at this point may be necessary, but are not sufficient, to cause the incubated phenotype and are primarily due to exposure to cocaine. Category 2 changes are those that occur with cocaine use that persist with periods of abstinence. These were observed to be altered in the comparisons between naïve and 1-day abstinent animals and between naïve and 10- or 100-day abstinent animals. These alterations may result from cocaine exposure, but do not return to naïve levels with cessation of the cocaine stimulus. The persistence of these changes may indicate their potential role in the development (10 days) or maintenance (100 days) of abstinence-persistent increases in drug seeking and drug taking behaviors. Category 3 changes consist of genes that were unchanged with cocaine use, but are altered during the abstinence period. While not immediately affected by cocaine exposure, this set of changes may result from initiation or continuation of abstinence. These may function synergistically with other (category 2) changes to contribute to the development of increased drug-seeking and -taking.
mPFC
The mPFC mediates executive function and decision making processes and is therefore a key neuroanatomical region in addictive behaviors
B29 29
B30 30
. In response to cocaine administration, changes in metabolic activity, neurotransmitter systems, and gene or protein expression occur in the mPFC (for a review see
28
). In this study, a large number of gene expression changes were observed in the mPFC both as a result of cocaine self-administration and with subsequent enforced abstinence. Most of these changes occurred as a direct result of the cocaine self-administration (981) and a majority (793) returned to cocaine-naïve levels with cessation of cocaine self-administration. As expected many changes (category 1) in gene expression require continued cocaine stimulus to remain altered, and return to normal levels after the stimulus is removed. A subset of genes (188) remained changed after 100-days of enforced abstinence. Persistence of gene expression changes with abstinence (category 2) requires maintenance via other mechanisms. Epigenetic changes occur in response to cocaine, and may constitute a regulatory mechanism for persistent changes in gene expression
12
B31 31
. Changes (480) that do not occur during cocaine self-administration, but are induced with abstinence (category 3) may reflect the withdrawal of the cocaine stimulus and development of the incubated phenotype.
Altered gene expression of Adora2b, Arc, CART, Cd47, Drd5, Egr1, Fos, Nefl, NPY, and Nr4a1 were confirmed by qPCR. We have previously described altered expression of Arc, CART, Egr1, Fos, NPY, and Nr4a1 in these samples in a directed study of genes with known relevance to drug abuse
12
. A number of the qPCR confirmation analyses that did not reach statistical significance demonstrated expression profiles similar to those observed in the microarray. This may reflect the effects of neuroanatomical complexity on quantitation of gene expression endpoints and the inclusion of larger numbers of animals in the confirmatory experiments.
This work identified altered expression of two G-protein coupled receptors (GPCRs; Adora2b, Drd5), a cell-surface signaling molecule (CD47), and a component of the neuronal cytoskeleton (Nefl). Increased Drd5, and signaling through this receptor, have been reported to decrease responsiveness to cocaine
B32 32
B33 33
. Similarly, adenosine signaling has been implicated in drug addiction. Specifically, activation of Adora2b receptors attenuates cocaine-conditioned place preference
B34 34
. Although the mechanisms underlying these effects are unclear, Drd5 signaling is implicated in neuronal activities including long-term potentiation (LTP;
B35 35
), and reinforcement learning
B36 36
, while both Drd5
B37 37
and Adora2b
B38 38
appear to affect Ca2+ dynamics.
The decrease in CD47 expression in this model is a novel observation and is of interest due to its function in neuronal development
B39 39
B40 40
. Nefl functions in cytoskeletal organization and cell-surface receptor remodeling
B41 41
B42 42
, which may be impaired with the observed decrease in expression at 1-day of abstinence. Previously, changes in protein levels and post-translational modifications of Nefl, and other neurofilament isoforms, have been reported with cocaine, morphine, alcohol, and nicotine administration
B43 43
B44 44
B45 45
.
We have previously reported a directed analysis of immediate early genes (IEGs; Arc, Egr1, Fos, and Nr4a1) and neuropeptides (NPY and CART) in this animal model
12
. These genes were also identified in the current discovery microarray analysis, providing increased confidence in the microarray findings. These genes play important roles in a number of neuronal processes including learning and memory
B46 46
B47 47
, synaptic plasticity
B48 48
B49 49
B50 50
, Ca2+ signaling
B51 51
B52 52
, and MAPK signaling
51
.
Network analysis was conducted using the set of confirmed mPFC gene expression changes, and revealed that Cart, NPY, Nr4a1, Fos, Egr1, Adora2b and Drd5 all interact (directly or indirectly) with the MAPK/ERK pathway. While altered expression of MAPK/ERK pathway elements was not detected in this study, changes in expression and activity levels of MAPK/ERK genes have been reported (for a review see
28
) and this pathway is thought to play an important role in drug-induced changes in the brain
B53 53
B54 54
. Regulators of Ca2+ dynamics were also identified in the network analysis. The changes in Drd5, Adora2b, CD47 and CART expression may indicate a decrease in intracellular Ca2+ signaling that occurs with cocaine self-administration and persists into periods of abstinence
40
52
B55 55
B56 56
.
Additionally, the gene expression changes identified indicate that synaptic plasticity may be affected by cocaine self-administration and abstinence. Persistent reductions in levels of CD47 and Arc, and inductions in levels of Drd5 and NPY suggest altered synaptic plasticity process involved in memory formation and removal of old memory traces, respectively
50
B57 57
B58 58
. A potential reduction in synaptic plasticity in the mPFC with cocaine self-administration/abstinence is hypothesized based on levels of CD47, Nefl, Arc, Egr1, and NPY
39
42
48
49
50
. These data are in agreement with studies of the direct role of psychostimulants on mechanisms of synaptic plasticity, including LTP and LTD, in the mesolimbic system
B59 59
B60 60
. In total, these gene expression changes may contribute to persistently altered synaptic plasticity in the mPFC.
NAc
The central role of the NAc in psychostimulant reward is well documented
B61 61
. While cocaine exerts common actions on the NAc and mPFC
B62 62
, we observed little overlap (50 of the 1875 total gene expression changes (mPFC + NAc)) between these brain regions. The regulated genes common to both brain regions include IEGs reported previously
12
, various signaling molecules, and genes involved in cellular metabolism. When the microarray datasets were examined by ontological analysis distinct molecular functions were observed in each brain region. This indicates that the functional changes occurring in the mPFC and NAc may differ and may ultimately play different roles in abstinence-dependent behaviors.
Unlike the mPFC, fewer category 1 and 2 changes were observed in the NAc (100 of 414 total), than category 3 changes (those changed specifically during abstinence) (314). Of the cocaine-induced changes, only 32 persisted into periods of abstinence (category 2), while the remainder returned to pre-cocaine levels. Arc, Beta-catenin, Cap2, Crip2, Dnm2, Egr2, Fos, Fut8, GFAP, Gpr88, Htr1d, and Nr4a1 were all confirmed by qPCR to be differentially expressed. We have previously demonstrated the responsiveness of Arc, Fos and Nr4a1 in this animal model
12
.
Published data regarding Cap2, Crip2, Fut8, and Gpr88 in the brain are limited, with no previous reports of cocaine-responsiveness. Crip2 (a LIM-domain protein), Cap2 (an adenylate cyclase-associated protein) and Dnm2 are cytoskeletal function and organization genes
B63 63
B64 64
. Interestingly, Dnm2 is regulated by the transcription factor Arc, also altered in the NAc with cocaine
12
B65 65
. Among the remaining changes, Egr2 and GFAP have been previously demonstrated to be cocaine-responsive
B66 66
B67 67
B68 68
. Htr1d has been linked with a number of psychiatric disorders
B69 69
B70 70
. Changes in the expression of these genes may also indicate cocaine induced alterations in receptor signaling, glial cell function, and synaptic plasticity.
Beta-catenin, which was increased at 10-days of abstinence in this study, is a well-characterized protein that regulates cell growth as a part of the Wnt signaling pathway. As a part of Wnt signaling, beta-catenin also plays a role in synaptic plasticity
B71 71
B72 72
. In response to chronic cocaine, beta-catenin has been shown to increase in a number of brain regions
B73 73
B74 74
B75 75
. Fut8, a fucosyltransferase protein, also increased at 10-days of abstinence in this study, has been shown to increase upon Wnt/beta-catenin activation
B76 76
, indicating that there may be a coordinated activation of Wnt signaling during periods of abstinence from cocaine.
Network analysis of the confirmed genes in the NAc identified a TNF-centered network. Generally involved in inflammatory processes, TNF has not been historically associated with behavioral responses to cocaine. Studies performed on the effects of cocaine on macrophages have reported that cocaine suppresses LPS-stimulated TNF expression
B77 77
B78 78
. TNF induction was recently demonstrated to reduce conditioned place preference and locomotor sensitization caused by methamphetamine and morphine administration
B79 79
. If TNF does play a role in the behavioral responses to cocaine, these additional genes may represent regulatory and effector elements of a TNF network.
While these reported changes represent new insights into abstinence-induced changes in the brain, localization of these changes to specific cell types is still to be determined. As with other functional genomic and proteomic approaches looking at dissected brain regions, even these specific dissections contain a heterogeneous cellular population. Future molecular neurobiology studies that seek to extend these, and other findings, will need to utilize techniques (e.g. laser capture microdissection and fluorescent in-situ hybridization) to localize changes to specific cell types and neuronal networks
B80 80
.
Conclusions
In addition to offering further evidence of long-lasting changes in gene expression following abstinence from cocaine self-administration, these results identify cellular processes that may regulate the development and/or maintenance of incubation of drug-seeking and drug-taking. A number of additional changes in gene expression remain to be examined in future studies, but the results presented here support the finding that persistent shifts in gene expression can last long into abstinence. In the mesolimbic reward pathway, changes in the mPFC may be more pronounced than in the NAc and involve mostly distinct sets of genes. This may indicate different metaplastic processes occur in these brain regions with the development and expression of abstinence-induced behaviors. In the mPFC, changes in MAPK/ERK and calcium signaling and in synaptic plasticity occur. The alterations in the NAc suggest a possible role of Wnt and TNF-mediated signaling in cocaine-associated behaviors. Together the findings of this study highlight a number of pathways and processes in the brain that may play roles in the development and maintenance of abstinence-induced drug seeking and drug taking. A clear understanding of how these novel changes contribute to relapse liability will not only increase our knowledge of the neurobiology of addiction, but will provide targets for therapeutic development.
Methods
Cocaine self-administration
The surgical and cocaine self-administration procedures used have been described previously
12
18
. Briefly, male Sprague-Dawley rats (Harlan Inc., IN) were implanted with a chronic indwelling Silastic cannula in the right jugular vein, and trained to self-administer cocaine hydrochloride through exposure to a fixed ratio 1 (FR1) schedule of reinforcement as described previously
12
14
. After establishing a stable daily intake of cocaine (40 infusions within 6 hours for at least 5 days), access conditions were changed to a discrete-trials schedule. During the discrete-trial schedule, rats were given access to cocaine for 10-min discrete trials that were initiated at 15-min intervals. An infusion (1.5 mg/kg/inj) of cocaine was given following a response on the lever, which resulted in illumination of a stimulus light for 20 sec. The lever was retracted and the trial terminated if an injection was collected or if 10 minutes had elapsed. Rats received four discrete trials per hour (i.e., DT4), 24 hours per day, for 10 days. Following 10 days of self-administration, animals were placed in standard polycarbonate cages for 1, 10 or 100 days of enforced abstinence. All research was approved by the Wake Forest University School of Medicine and Penn State College of Medicine Animal Care and Use Committees and conducted according to the Guide for the Care and Use of Laboratory Animals, as promulgated by the National Institutes of Health.
Dissection
Following 1, 10, or 100 days of deprivation, rats were sacrificed and the brains were rapidly removed and cooled in ice-chilled saline. Brains were then placed in an ice-chilled ASI brain slicer (ASI Instruments, Warren MI). The medial prefrontal cortex (mPFC) and nucleus accumbens (NAc) were collected as described previously
12
. Briefly, the section from Bregma +4.4 to 2.4 mm
B81 81
was cut along the forceps minor and the cortex medial of this cut was collected. This is considered the medial prefrontal cortex, and includes the cingulate area, prelimbic cortex, and medial orbital cortex. The section from + 2.2 to 0.2 mm was cut 0.5 mm on each side of the midline, on a line connecting the tip of the external capsule and the previous cut, on a line connecting the tip of the external capsule and lateral ventricle, and between the ventricle. This dissection includes both the core and the shell of the NAc. See Additional File S3 3: Figure S1, for schematics of the dissections.
Additional file 3
Dissection schematics. Dissection diagrams. Schematics of the mPFC and NAc dissections are provided using modifications of figures from Paxinos and Watson. Numbered red lines are specific dissection cuts using visible landmarks as described in the methods. The shaded area represents the tissue collected for molecular analysis.
1471-2202-11-29-S3.PDF
Click here for file
RNA isolation
Following dissection, tissue samples were stored at -80°C until RNA was isolated. RNA isolation was conducted as described previously
74
B82 82
. Total RNA from cocaine naïve rats and rats following 1, 10, and 100 days of enforced abstinence (following 10 days of DT4 cocaine self-administration as described above) was isolated using Tri-Reagent (Molecular Research Center Inc., Cincinnati, OH)
B83 83
. RNA quantity and quality was then assessed using the Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA) following further RNA purification using an RNeasy Mini Kit for RNA clean-up (Qiagen Sciences, Maryland).
Microarray analysis
Microarray analysis was performed in the Penn State College of Medicine Functional Genomics Core Facility according to standard procedures. For the mPFC arrays, naïve, 1-day, and 100-day abstinent samples were used (N = 6). For the NAc arrays, naïve, 1-day and 10-day abstinent samples were used (N = 5; reduced sample number due to removal of samples that did not pass array quality control). Microarrays for the NAc were performed after those for the mPFC, and for the NAc, the 10-day timepoint was used instead of the 100-day timepoint in order to detect important changes that may not last to 100 days of abstinence. The persistence of these changes to 100-days could then be tested with qPCR.
Microarray analysis was performed using the CodeLink Rat Whole Genome Bioarray system (GE Healthcare). Following the manufacturer's protocol, first strand synthesis was performed with 2 μg of RNA as starting material and was followed by second strand synthesis and purification using Qiaquick spin columns (Qiagen, Valencia, CA). T7 reaction buffer, T7 NTPs, 10 mM biotin-11-UTP, and T7 polymerase were then added to the dsDNA for the IVT reaction and incubated at 37°C for 14 hours. The resulting Biotin-labeled cRNA was then purified using RNEasy columns (Qiagen), quantitated, and volume-adjusted for a total of 10 μL. The cRNA was then fragmented and denatured before hybridization for 18 hours at 37°C. Slides were washed and then incubated at room temperature with Alexa Fluor 647 labeled streptavidin for 30 minutes followed by washing.
Micro arrays were scanned on an Axon 4000 B scanner with GenePix4 v4.0 software at a 5 μm resolution at 635 nm with laser power at 100%, PMT voltage at 600 V, focus position 0 μm, and lines to average = 1. Images were then imported into CodeLink Expression Analysis Software v4.1 (GE Healthcare) and initial quality control (positive and negative controls), exclusion of manufacturing defects (MSR spots), background subtraction, and intra-array normalization was performed.
Data analysis
Following image analysis on CodeLink Expression Analysis Software, microarray data were imported into GeneSpring GX 7.3 (Agilent Technologies) and signal values less than 0.01 were transformed to an intensity of 0.01. Normalization was performed per chip to the 50th percentile, and per gene to the median. Values were then normalized on a per gene basis to the naïve group for each of the two time points (1 and 10, or 1 and 100 days of abstinence). Potential differential expression was determined with a one-way ANOVA (variances not assumed to be equal), p < 0.05 and filtered for 1.4-fold or greater differences in expression in accordance with standards for microarray analysis
B84 84
. The use of a combination of statistical and fold-change cutoffs as opposed to traditional multiple testing corrections (e.g., Bonferroni post-hoc testing) produce gene lists with the lowest rate of type I and type II errors
B85 85
. A fold-change cutoff of 1.4 fold was chosen, as this magnitude change is at the lower range of changes historically confirmed by qPCR in this laboratory. Lastly, probe sequences on the array were searched against current rat genome sequences to eliminate any probes for sequences removed from the NCBI database.
Quantitative PCR (qPCR) analysis of gene expression
cDNA synthesis was performed on total RNA from naïve, 1, 10, and 100-day abstinent animals using Superscript III Reverse Transcriptase (Invitrogen, Carlsbad, CA). 1 μg RNA, 500 ng Oligo (dT), and 10 mM each dNTP, were incubated for 5 minutes at 65°C and then chilled on ice for 2 minutes. 5× First Strand Buffer (250 mM Tris-HCl (pH8.3), 375 mM KCl, and 15 mM MgClsub 2), 5 mM DTT (final concentration), 40 U RNaseOut, and 200 U Superscript III RT were then added. The 20 μl reaction was incubated for 60 minutes at 50°C followed by a final incubation at 70°C for 15 minutes for termination. The resulting cDNA product was quantified and 50 ng of product was used in each subsequent qPCR reaction.
Quantitative PCR was carried out on a real-time detection instrument (ABI 7900HT Sequence Detection System) in 384-well optical plates using TaqMan Universal PCR Master Mix and Assay on Demand primers and probes (Applied Biosystems, Foster City, CA) as described previously
B86 86
B87 87
. This examination used a larger set of animals than the microarray analysis (Table 1). Primer/probe sets used are listed in Additional File 2: Table S2. SDS 2.2.2 software and the 2-ΔΔCt analysis method
B88 88
. were used to quantitate relative amounts of product using β-actin as an endogenous control. Significance from qPCR analysis was determined with SigmaStat 3.5 (SYSTAT Software, Inc.) based on one-way analysis of variance (ANOVA) (p < 0.05) with a post hoc Student Newman-Keuls test (p < 0.05).
Ontological, pathway, and network analysis
Ontological analysis used Gene Ontology (GO) categories and differentially expressed processes or functional categories were determined statistically, as previously described
87
using GeneSpring GX software. This analysis determined the number of genes in a category present on the array and the number of expression changes that would be part of that category by random chance given the number of differentially expressed genes. Results from these analyses were used to compile a list of genes to examine by qPCR. Ingenuity Pathway Analysis (Ingenuity Systems, Redwood City CA) was used for network and pathways analysis of the qPCR confirmed gene expression results.
Abbreviations
Adora2b: adenosine receptor A2b; Arc: activity-related cytoskeletal associated protein; Cap2: adenylate cyclase-associated protein 2; CART: cocaine and amphetamine-related transcript; Crip2: cysteine-rich protein 2; Dnm2: dynamin 2; Drd5: dopamine receptor D5; DT: discrete trials; Egr1: early growth response 1; Egr2: early growth response 2; Fos: FBJ osteosarcoma oncogene; FR1: fixed ratio 1; Fut8; fucosyltransferase 8; GFAP: glial fibrillary acidic protein; GPR88: g-protein coupled receptor 88; Htr1d: 5-hydroxytryptamine receptor 1d; MAPK/ERK: mitogen activated protein kinase/extracellular signal-regulated kinase; mPFC: medial prefrontal cortex; NAc: nucleus accumbens; Nefl: neurofilament light; NPY: neuropeptide Y; Nr4a1: nuclear receptor subfamily 4a1; TNF: tumor necrosis factor
Authors' contributions
WMF and KEV generated the experimental design. RMB and KMP conducted the array and RT-PCR analyses, respectively. MEL performed the data analysis, prepared the figures, archived the data, and wrote the manuscript. WMF assisted with data analysis and WMF and KEV contributed to the data interpretation. DM and DCSR (Wake Forest University) developed the animal model used and provided the samples for analysis. All authors read and approved the final manuscript.
bm
ack
Acknowledgements
This work was supported by grants R01-DA013770-08 (KEV), F31-DA02281902 (MEL), R01 DA14030 (DCSR) and K01-DA13957 (DM). The authors wish to thank the Penn State College of Medicine Functional Genomic Core Facility for microarray and qPCR analysis and technical assistance and Dr. Heather VanGuilder for manuscript editing.
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Gene expression changes in the medial prefrontal cortex and nucleus accumbens following abstinence from cocaine self-adm...
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Title: Gene expression changes in the medial prefrontal cortex and nucleus accumbens following abstinence from cocaine self-administration
Series Title: BMC Neuroscience
Physical Description: Book
Language: English
Creator: Freeman,Willard
Lull,Melinda
Patel,Kruti
Brucklacher,Robert
Morgan,Drake
Roberts,David
Vrana,Kent
Publisher: BMC Neuroscience
Publication Date: 2010
 Notes
Abstract: BACKGROUND:Many studies of cocaine-responsive gene expression have focused on changes occurring during cocaine exposure, but few studies have examined the persistence of these changes with cocaine abstinence. Persistent changes in gene expression, as well as alterations induced during abstinence may underlie long-lasting drug craving and relapse liability.RESULTS:Whole-genome expression analysis was conducted on a rat cocaine binge-abstinence model that has previously been demonstrated to engender increased drug seeking and taking with abstinence. Gene expression changes in two mesolimbic terminal fields (mPFC and NAc) were identified in a comparison of cocaine-naïve rats with rats after 10 days of cocaine self-administration followed by 1, 10, or 100 days of enforced abstinence (n = 6-11 per group). A total of 1,461 genes in the mPFC and 414 genes in the NAc were altered between at least two time points (ANOVA, p < 0.05; ± 1.4 fold-change). These genes can be subdivided into: 1) changes with cocaine self-administration that do not persist into periods of abstinence, 2) changes with cocaine self-administration that persist with abstinence, 3) and those not changed with cocaine self-administration, but changed during enforced abstinence. qPCR analysis was conducted to confirm gene expression changes observed in the microarray analysis.CONCLUSIONS:Together, these changes help to illuminate processes and networks involved in abstinence-induced behaviors, including synaptic plasticity, MAPK signaling, and TNF signaling.
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Freeman et al. BMC Neuroscience 2010, 11:29
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BMeuroscience
Neuroscience


Gene expression changes in the medial prefrontal

cortex and nucleus accumbens following

abstinence from cocaine self-administration

Willard M Freeman '2*t, Melinda E Lullit, Kruti M Patel2, Robert M Brucklacher 2, Drake Morgan3, David CS Roberts4,
Kent E Vranal


Abstract
Background: Many studies of cocaine-responsive gene expression have focused on changes occurring during
cocaine exposure, but few studies have examined the persistence of these changes with cocaine abstinence.
Persistent changes in gene expression, as well as alterations induced during abstinence may underlie long-lasting
drug craving and relapse liability.
Results: Whole-genome expression analysis was conducted on a rat cocaine binge-abstinence model that has
previously been demonstrated to engender increased drug seeking and taking with abstinence. Gene expression
changes in two mesolimbic terminal fields (mPFC and NAc) were identified in a comparison of cocaine-naive rats
with rats after 10 days of cocaine self administration followed by 1, 10, or 100 days of enforced abstinence (n = 6-
11 per group). A total of 1,461 genes in the mPFC and 414 genes in the NAc were altered between at least two
time points (ANOVA, p < 0.05; 1.4 fold-change). These genes can be subdivided into: 1) changes with cocaine
self administration that do not persist into periods of abstinence, 2) changes with cocaine self administration that
persist with abstinence, 3) and those not changed with cocaine self administration, but changed during enforced
abstinence. qPCR analysis was conducted to confirm gene expression changes observed in the microarray analysis.
Conclusions: Together, these changes help to illuminate processes and networks involved in abstinence-induced
behaviors, including synaptic plasticity, MAPK signaling, and TNF signaling.


Background
A hallmark of cocaine addiction is continued drug crav-
ing and relapse propensity despite long-term drug absti-
nence. Development of effective cocaine addiction
treatments therefore requires therapies that decrease the
likelihood of relapse to cocaine abuse in the recovering
addict. A central theme of cocaine abuse research is the
role of neurobiological changes (e.g., electrophysiology,
neurochemistry, neuroanatomy, epigenetic, transcrip-
tomic, proteomic) in the development and maintenance
of the addicted behavioral phenotype (i.e., increased
drug-seeking and drug-taking).
Cocaine addiction generally starts with recreational
use and deteriorates over time into a compulsive and

* Correspondence' wfreeman@psu edu
t Contributed equally
1Department of Pharmacology, Penn State College of Medicine, Hershey, PA,
17033, USA


chronically relapsing drug-taking disorder [1]. Stress,
environmental cues, and conditioned stimuli have been
demonstrated clinically to play a role in cocaine relapse
[2-4]. While initiating drug abstinence can be accom-
plished through in-patient treatment, maintaining
cocaine abstinence has proven difficult [5]. In controlled
clinical trials, prolonged cocaine abstinence is often
achieved by only a minority of patients [6-8]. This may
be due to increases in cocaine craving during drug absti-
nence [9]. Understanding the persistent neurobiological
changes that contribute to continued drug craving dur-
ing abstinence and relapse potential represents an
important step towards identifying treatments that
reduce the likelihood of relapse [10].
There is a growing understanding of the acute gene/
protein expression changes with cocaine administration
(either non-contingently or self-administered) that may
be important to the development and expression of


2010 Freeman et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
nBiollled Central Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.







Freeman et al. BMC Neuroscience 2010, 11:29
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cocaine-responsive behavior [11], but only a small num-
ber of studies have examined whether these changes
revert to normal levels or remain altered with cocaine
abstinence. Observations of molecular changes persisting
into or occurring during this abstinence period provide
the opportunity to identify genes and their protein
counterparts that could be used as therapeutic targets to
decrease relapse liability.
The development of animal models of cocaine abuse
and abstinence has led to the identification of rodent
behaviors similar to those of human cocaine abusers.
Most notably, time-dependent increases in cocaine seek-
ing and taking behaviors have been observed in the rat
model of cocaine abuse and enforced abstinence
employed in this study [12-14]. Similar observations
have been made using other animal models of prolonged
abstinence from cocaine [15,16]. Molecular analyses of
these models have not only identified changes in gene
or protein expression [12,17-19], but have also corre-
lated gene expression with cocaine-responsive behaviors
[20,21]. Many of the existing reports used targeted
approaches to quantify specific gene and protein expres-
sion changes during abstinence from cocaine. Large-
scale discovery studies with long-term enforced absti-
nence following cocaine self-administration are limited
and transcriptomic studies, in particular, have not been
conducted.
The self-administration paradigm used in this study
exhibits increased reinforcing efficacy, drug seeking, and
drug taking with at least 7 days, and as long as 100
days, of abstinence after a period of cocaine self-admin-
istration [12-14,22]. Examination of mesolimbic struc-
tures in these animals is warranted by the roles that
these structures, including the medial prefrontal cortex
(mPFC) and nucleus accumbens (NAc), play in reward
and behavioral responses to stimuli. Both of these brain
regions have been implicated in cocaine abuse and


Table 1 Cocaine intake data for samples
Arrays


Group

mPFC Naive
1-day Abstinence
10-days Abstinence
100-days Abstinence
NAc Nalve
1-day Abstinence
10-days Abstinence
100-days Abstinence


N Average Average Injections
Total Intake Per Session
6
6 934 + 108 mq/kq 62 + 7


6 856 + 42 mc


932 + 118 mc
951 + 116 mc


57 + 28
63 + 8


withdrawal through imaging [23-25], behavioral
[20,21,26], and molecular [12,17-19] studies. We have
conducted targeted mRNA and epigenetic analysis from
this model previously [12]. The aim of the present
study was to extend this initial analysis of mesolimbic
dopaminergic terminal regions by providing a genome-
wide characterization in both the mPFC and the NAc of
rats following 10 days of cocaine self-administration and
after increasing periods of enforced abstinence from
cocaine (1, 10, and 100 days). Identification of genes
persistently altered in expression by cocaine or altered
during a period of cocaine abstinence provides insight
into the mechanisms involved in the long-term beha-
vioral changes that occur with cocaine abuse and illumi-
nates novel potential new targets for pharmacological
intervention.

Results
Animals
Behavioral analyses of the rat cocaine self-administration
paradigm and time points used in this study have been
published previously [12,14,27]. The specific animals
used in this study represent an independent set that was
not behaviorally tested (e.g. progressive ratio or extinc-
tion responding) to avoid any confounding effects of
behavioral testing on gene expression. All cocaine self-
administering groups were maintained on a continuous
access (24 hours/day) discrete trials (DT) schedule for
10 days. Trials were limited to 4 trials per hour (DT4).
After 10 days of DT4 responding, animals were sub-
jected to 1, 10, or 100 days of enforced abstinence. The
cocaine intake data for the specific animals used in this
study is presented in Table 1. No significant differences
were observed in the total cocaine intake of each group
or the average number of daily injections. The similarity
in total cocaine intake and responding between groups
minimizes the possibility that exposure to differing


qPCR Confirmation
N Average Average Injections
Total Intake Per Session


6 934 + 108 mc
7 950 + 105 mc
8 855 + 39 mg
11
6 934 + 108 mc
7 950 + 105 mc
8 855 + 39 mq


62 + 7
63 + 7
57 + 3


* Naive animals were not exposed to cocaine, and therefore have zero intake. There were no significant differences in cocaine intake or number of injections per
session between groups. "Arrays" indicates animals used for microarray analysis and "qPCR Confirmation indicates animals used for confirmatory qPCR analysis.
All of the samples from the array analysis were included in the qPCR confirmation. Data is presented as Mean + S.D.


Page 2 of 13







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amounts of cocaine, or a difference in self-administra-
tion behavior, could account for gene expression
changes observed. Microarray studies of the mPFC were
conducted on naive, 1-day abstinent, and 100-days absti-
nent animals (n = 6/group). Microarray studies of the
NAc were conducted on naive, 1-day abstinent, and 10-
days abstinent animals (n = 5/group) from the same
cohort. For confirmatory qPCR analyses of gene expres-
sion levels, a larger number of samples were tested,
including those included in microarray studies. Addi-
tionally, while microarrays were conducted on samples
from three time points, all four time points were used
for these confirmation studies (naive, n = 11; 1-day, n =
6; 10-days, n = 7; and 100-days, n = 8) to provide finer
temporal resolution.

Microarray analysis
In the mPFC, gene products corresponding to a total of
21,814 probes (of the 44,000 total probes on the arrays)


were confidently detected, based on signal intensity at a
fixed value above background levels. In the NAc analysis,
mRNAs for a total of 19,015 probes were detected. Dif-
ferentially expressed genes were identified through a
combination of statistical significance (p < 0.05, One-way
ANOVA between groups) and a fold change filter of =1.4
fold change (Figure 1A&1B). This illuminated 1,461 gene
expression changes in the mPFC (representing 6.7% of
the total mRNA species detected) and 414 gene expres-
sion changes in the NAc (2.2% of the total detected).
These changes demonstrated three types of temporal
profiles (Figure 1C). Category 1 changes are those that
occurred with cocaine self-administration (either up- or
down-regulated at 1-day of abstinence), but that did not
persist into longer periods of abstinence (10- or 100-
days). A majority of the mPFC gene expression changes
belonged to this category (793 of 1,461); however, only a
small fraction of changes in the NAc (68 of 414) exhib-
ited this expression pattern. Category 2 changes are those


Naive vs 1-day


mPFC
Naive vs 100-days


NAc
Naive vs 1-dav


Naive vs 10-days


188 480



20353
Unchanged

Category 2
Changes with cocaine
SA that do persist


N 1 10/100 N 1 10/100


Category 1
Changes with cocaine
SA that do not persist


32 ) 314 I


18601
Unchanged


Common Changes
mPFC NAc


Category 3
. Changes that occur
i i, during abstinence


100 L I II I I I

N 1 10/100 N 1 10/100 N 1 10/100 N 1 10/100
Figure 1 Analysis of expression changes from the microarray studies. Venn diagrams illustrate the changes identified by microarray analysis
of naive animals, and animals following I and 10 or 100 days of abstinence. (A) In the mPFC, 21,814 probes were detected as present. Of these,
1,461 were changed by greater than 1.4 fold with p < 0.05 (ANOVA) and can be split into three categories based on expression profile: changed
between naive and 1-day (793), naive and 100-days (480) or both (188). (B) In the NAc, 19,015 probes were detected as present. Of these, 414 were
changed by greater than 1.4 fold with p < 0.05 (ANOVA), and can be split into the same three categories: changed between naive and 1-day (68),
nalve and 10-days (314) or both (32). (C The three categories of gene expression changes can be segregated into (1) changes that occur with
cocaine self-administration (SA) that do not persist, (2) changes that occur with cocaine SA that do persist, and (3) changes that occur during
abstinence. Small graphs indicate the expression profile of changes in each category, where the x-axis represents group (nalve, 1-day and 10- or
100-days abstinent) and the y-axis represents percent (%) of naive expression. (D) When comparing the gene expression changed between the
mPFC and NAc, a majority (97%) are unique to one brain region or the other. Only a small fraction (50) is changed in both brain regions.


Page 3 of 13


50 1 364







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that occurred with cocaine self-administration and per-
sisted into periods of abstinence (changed at 1-day and
remained changed with enduring abstinence). A small
number of gene expression changes from each brain
region (188 mPFC; 32 NAc) belonged to this category.
Finally, category 3 changes are those that did not occur
with cocaine self-administration, but changed during the
period of abstinence (not changed at 1-day, but changed
at 10- and/or 100-days). Approximately 33% of the
changes in the mPFC (480 of 1,461) and 76% of the
changes in the NAc (314 of 414) belong to this category.
A comparison of the genes changed at any time point
between the mPFC and NAc reveals that a limited num-
ber of changes were observed in both brain regions (Fig-
ure ID). Of the total of 1875 gene expression changes,
less than 3% were detected in both the mPFC and the
NAc. A full list of gene expression changes is presented
in Additional File 1: Table S1, and has been uploaded to
the Gene Expression Omnibus online database.


A 120



- 100-


z
15

80



60,
04


120-


Neurofilament Light


Confirmatory qPCR
qPCR analysis was used to confirm a subset of gene
expression changes observed in the microarray analyses.
Genes chosen for qPCR confirmation were selected
based on ontological classes with potential roles in
drug-induced changes in the brain. Additional genes
were included in the confirmation studies based on pre-
vious reports of cocaine-responsive gene expression (see
Additional File 2: Table S2, for a full list and gene
expression assay information).
Differentially regulated genes confirmed in this study
(13 total; 4 mPFC, 9 NAc), belong to each of the 3 cate-
gories described above (Figure 2). In the mPFC, neurofi-
lament light (Nefl) was the only category 1 change,
with a 20% decrease in the mPFC at 1-day of abstinence
(Figure 2A). Category 2 changes included a 23%
decrease in levels of CD47 at 1-day that persisted
through 10-days of abstinence (21%), and an increase in
levels of dopamine receptor D5 (Drd5) at 1-day (40%)


CD47


I I 0
0

8-


*


Naive 1 day 10 days 100 days


0-









Naive 1 day 10 days 100 days
Naive I ay l0 days ToOdays


C 180.


140.


6,
8100

60


D 120-


**_*
J -_ -T


Adora2b


I II 1 1 01 I I I
Naive 1 day 10 days 100 days Naive 1 day 10 days 100 days
Figure 2 Confirmed changes in mPFC gene expression. Four changes in gene expression were confirmed in the mPFC with qPCR analysis. (
Neurofilament light is decreased by 20% at 1-day of abstinence (p < 0.05). (B) CD47 expression is decreased from naive at 1-day (23%, p < 0.05)
and 10-days of abstinence (21%; p < 0.01). (C) Dopamine receptor D5 (Drd5) expression is increased from naive at 1-day (40%; p < 0.01) and
100-days (41%; p < 0.01). (D) Adenosine A2B receptor (Adora2b) expression is decreased from naive at 10-days of abstinence (20%; p < 0.05).
Statistical analysis was performed by one-way ANOVA with Student-Newman-Keuls post hoc testing; p < 0.05, ** p < 0.01.


Page 4 of 13






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and 100-days of abstinence (41%) (Figure 2B&2C).
Finally, levels of adenosine A2B receptor (Adora2b)
were unchanged by cocaine administration, but were
reduced by 20% after 10-days of abstinence (Category 3
change) (Figure 2D).
In the NAc, nine genes were confirmed, most of
which displayed a category 3 profile of changing during
abstinence. These include beta-catenin (32% increase at
10-days), adenylate cyclase-associated protein 2 (Cap2;
206% increase at 10-days), cysteine-rich protein 2
(Crip2; 17% increase at 10-days), dynamin 2 (Dnm2;
167% increase at 10-days), early growth response 2
(Egr2; 55% decrease at 100-days), fucosyltransferase 8
(Fut8; 25% increase at 10-days), glial fibrillary acidic
protein (GFAP; 87% increase at 10-days), and G-protein
coupled receptor 88 (Gpr88; 277% increase at 10-days)
(Figure 3A). The 5-hydroxytryptamine receptor ld
(Htrld), which was decreased at 1-day (28%) and
remained decreased at 10-days (20%) represents the only
confirmed category 2 change (Figure 3B).
Consistent with our previous findings in this animal
model and at the same time points [12], the microarray
analysis performed in this study detected significant
changes in activity-related cytoskeletal associated protein
(Arc), cocaine and amphetamine-related transcript
(CART), early growth response 1 (Egrl), FBJ osteosar-
coma oncogene (Fos), neuropeptide Y (NPY), and
nuclear receptor subfamily 4al (Nr4al) transcript levels.
Prior to the microarray analysis, we had examined these
genes by qPCR based on their known responsiveness to
cocaine and have already reported these results [12].
Persistent decreases (Category 2 or 3) in Arc, Fos, and
Nr4al were observed in both the mPFC and NAc. Cate-
gory 1 changes in CART, NPY (both increased at 1-day)
and Egrl (decreased at 1-day) in the mPFC were also
confirmed by qPCR (Table 2).

Ontological and Network analysis
Analysis of the Gene Ontology (GO) categories of
changes in each brain region identified a number of
molecular functions significantly regulated with
cocaine self-administration and abstinence. The mole-
cular functions altered were anatomically distinct. In
the mPFC, the most-represented class, consisting of
~ 25% of the changes identified by microarray analysis,
was protein serine/threonine kinase activity, followed
closely by structural components of the ribosome and
monovalent inorganic cation transporter activity
(~ 15% of the total changes each). The most repre-
sented classes among the changes in NAc gene expres-
sion were hydrolase activity (~ 40%), and phosphoric
ester hydrolase activity (~ 10%).
Network analysis, conducted using Ingenuity Pathways
Analysis software (Ingenuity Systems, Redwood City,


CA), was performed to determine the relationships
between confirmed gene expression changes from this
study and previously reported changes from this model
[12]. To complement the GO analysis, additional analy-
sis was conducted to determine whether these genes are
implicated in specific pathways and/or biological func-
tions and diseases. Only those genes confirmed by qPCR
were used in this analysis. In the mPFC, these molecules
comprise a network of interactions involved in synaptic
plasticity, calcium signaling, and mitogen activated pro-
tein kinase/extracellular signal-regulated kinase (MAPK/
ERK) signaling. In the NAc, all 12 molecules were com-
ponents of a network of interactions with members
linked to the neuronal cytoskeleton, glial cells, and Wnt
and tumor necrosis factor (TNF) signaling functions.

Discussion
This study represents the first microarray analysis of
mesolimbic gene expression following long-term
enforced abstinence from cocaine self-administration.
Transcriptomic studies of cocaine-induced gene expres-
sion changes have been conducted, but these have
focused on non-contingent cocaine administration and
no or limited (~ 1 day) abstinence. The work conducted
in the present study used a model with well-character-
ized behavioral changes during periods of abstinence,
and used animals not subjected to behavioral testing
during abstinence (e.g. progressive ratio or extinction
responding) so that the gene expression changes
observed are free from the effects elicited by behavioral
testing conducted before sacrifice. Additionally, it is
important to note that all groups (1, 10, and 100 days of
abstinence) self-administered equivalent amounts of
cocaine over the 10 days of discrete trial cocaine self-
administration. This time-course analysis of gene
expression allows for discrimination of gene expression
changes associated with increased drug seeking (10 and
100 days of abstinence) from those that occur with
cocaine self-administration, but do not persist for as
long as increased drug seeking and taking (1 day of
abstinence).
The literature describes a number of neurobiological
changes (e.g. altered gene/protein expression, neuro-
transmitter levels, epigenetic events) with different mod-
els of cocaine abuse, (for a review see [11,28]). Whether
these changes persist into periods of abstinence, how-
ever, has generally not been determined. In this study,
gene expression changes that occurred as a result of
cocaine self-administration and abstinence segregated
into three categories of expression patterns. Category 1
changes were defined as those that occur with cocaine
use, but do not persist into periods of abstinence. These
were observed as changes only between naive animals
and 1-day abstinent animals. After only 1 day of


Page 5 of 13








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A
o160- Bela-catenin 400 CAP2

140,













CRP2 Dnm2
T T2










I I I 1 I I
Naive 1 day 10 daym 100 days Naive 1 day 10 days 10O days

140 1
Egr2 FUT8


1'0 140







00
S300






I- I I I








Naive 1 day 0 days 1 00 days Na e 1 dy 1- days 6 odays

140.
Htrld











I I dy 0 s a

Figure 3 Confirmed changes in NAc gene expression. Nine changes in gene expression were confirmed in the NAc with qPCR analysis in
this study. (A) Eight of the changes have the profile of changing in expression during abstinence (either at 10 or 100-days of abstinence). These
genes are beta-catenin (increased by 32% at 10-days; p < 0.001), adenylate cyclase-associated protein 2 (Cap2; increased by 206% at 10-days; p <
0.001), cysteine-rich protein 2 (Crip2; increased 17% at 10-days; p < 0.05), dynamin 2 (Dnm2; increased 167% at 10-days; p < 0.001), early growth
response 2 (Egr2; decreased by 55% at 100-days; p < 0.05), fucosyltransferase 8 (Fut8; increased 25% at 10-days; p < 0.01), glial fibrillary acidic

protein (GFAP; increased 87% at 10-days; p < 0.05), and G-protein coupled receptor 88 (Gpr88; increased 277% at 10-days; p < 0.01). (B)
5-hydroxytryptamine receptor Id (Htrdl) is decreased by 28% at 1-day (p < 0.001) and by 20% at 10-days of abstinence (p < 0.01). Statistical
analysis was performed by one-way ANOVA with Student-Newman-Keuls post hoc testing; p < 0.05, ** p < 0.01, #p < 0.001.







Freeman et al. BMC Neuroscience 2010, 11:29
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Table 2 Gene expression changes reported previously


Gene Name
mPFC Activity-related cytoskeleton-associated protein
Cocaine and amphetamine regulated transcript
Early growth response 1
FBJ osteosarcoma oncogene
Neuropeptide Y
Nuclear receptor subfamily 4, group A, member 1
NAc Activity-related cytoskeleton-associated protein
FBJ osteosarcoma oncogene
Nuclear receptor subfamily 4, group A, member 1


Gene ID Nvl
Arc 43.9 + 14**
CART 725.2 + 130*
Ergl 59.6 + 9.2*
Fos 41.7 + 12**
Npy 158.4 + 21'
Nr4al 35.1 + 11**
Arc 83.5 + 22
Fos 53.9 + 16**
Nr4al 65.7 + 20


Data is presented as mean + standard error of the mean (SEM), ANOVA with Student-Newman-Keuls post hoc testing; p < 0.05, ** p < 0.01, # p < 0.001. Data
previously reported in [12].


abstinence, an increase in drug seeking and drug taking
is not observed [12], so the changes in gene expression
observed at this point may be necessary, but are not suf-
ficient, to cause the incubated phenotype and are pri-
marily due to exposure to cocaine. Category 2 changes
are those that occur with cocaine use that persist with
periods of abstinence. These were observed to be altered
in the comparisons between naive and 1-day abstinent
animals and between naive and 10- or 100-day abstinent
animals. These alterations may result from cocaine
exposure, but do not return to naive levels with cessa-
tion of the cocaine stimulus. The persistence of these
changes may indicate their potential role in the develop-
ment (10 days) or maintenance (100 days) of absti-
nence-persistent increases in drug seeking and drug
taking behaviors. Category 3 changes consist of genes
that were unchanged with cocaine use, but are altered
during the abstinence period. While not immediately
affected by cocaine exposure, this set of changes may
result from initiation or continuation of abstinence.
These may function synergistically with other (category
2) changes to contribute to the development of
increased drug-seeking and -taking.

mPFC
The mPFC mediates executive function and decision
making processes and is therefore a key neuroanatomi-
cal region in addictive behaviors [29,30]. In response to
cocaine administration, changes in metabolic activity,
neurotransmitter systems, and gene or protein expres-
sion occur in the mPFC (for a review see [28]). In this
study, a large number of gene expression changes were
observed in the mPFC both as a result of cocaine self-
administration and with subsequent enforced abstinence.
Most of these changes occurred as a direct result of the
cocaine self-administration (981) and a majority (793)
returned to cocaine-naive levels with cessation of


cocaine self-administration. As expected many changes
(category 1) in gene expression require continued
cocaine stimulus to remain altered, and return to nor-
mal levels after the stimulus is removed. A subset of
genes (188) remained changed after 100-days of
enforced abstinence. Persistence of gene expression
changes with abstinence (category 2) requires mainte-
nance via other mechanisms. Epigenetic changes occur
in response to cocaine, and may constitute a regulatory
mechanism for persistent changes in gene expression
[12,31]. Changes (480) that do not occur during cocaine
self-administration, but are induced with abstinence
(category 3) may reflect the withdrawal of the cocaine
stimulus and development of the incubated phenotype.
Altered gene expression of Adora2b, Arc, CART,
Cd47, Drd5, Egrl, Fos, Nefl, NPY, and Nr4al were con-
firmed by qPCR. We have previously described altered
expression of Arc, CART, Egrl, Fos, NPY, and Nr4al in
these samples in a directed study of genes with known
relevance to drug abuse [12]. A number of the qPCR
confirmation analyses that did not reach statistical sig-
nificance demonstrated expression profiles similar to
those observed in the microarray. This may reflect the
effects of neuroanatomical complexity on quantitation
of gene expression endpoints and the inclusion of larger
numbers of animals in the confirmatory experiments.
This work identified altered expression of two G-pro-
tein coupled receptors (GPCRs; Adora2b, Drd5), a cell-
surface signaling molecule (CD47), and a component of
the neuronal cytoskeleton (Nefl). Increased Drd5, and
signaling through this receptor, have been reported to
decrease responsiveness to cocaine [32,33]. Similarly,
adenosine signaling has been implicated in drug addic-
tion. Specifically, activation of Adora2b receptors
attenuates cocaine-conditioned place preference [34].
Although the mechanisms underlying these effects are
unclear, Drd5 signaling is implicated in neuronal


Page 7 of 13


Expression Level (% of Naive Control)


Nv10
76.8 + 12
191.9 + 55
74.7 + 15
58.1 + 11*
94.5 + 9.5
53.4 + 12*
73.7 + 12
60.3 + 6.71
63.4 + 11


Nv1 00
51.8 + 10**
190.3 + 86
74.5 + 5.3
53.1 + 6.8*
91 + 5.2
49.4 + 7.7*
49.5 + 6.4*
51.7 + 62*
48.1 + 5.1*






Freeman et al. BMC Neuroscience 2010, 11:29
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activities including long-term potentiation (LTP; [35]),
and reinforcement learning [36], while both Drd5 [37]
and Adora2b [38] appear to affect Ca"2 dynamics.
The decrease in CD47 expression in this model is a
novel observation and is of interest due to its function
in neuronal development [39,40]. Nefl functions in
cytoskeletal organization and cell-surface receptor remo-
deling [41,42], which may be impaired with the observed
decrease in expression at 1-day of abstinence. Pre-
viously, changes in protein levels and post-translational
modifications of Nefl, and other neurofilament isoforms,
have been reported with cocaine, morphine, alcohol, and
nicotine administration [43-45].
We have previously reported a directed analysis of
immediate early genes (IEGs; Arc, Egrl, Fos, and Nr4al)
and neuropeptides (NPY and CART) in this animal
model [12]. These genes were also identified in the cur-
rent discovery microarray analysis, providing increased
confidence in the microarray findings. These genes play
important roles in a number of neuronal processes
including learning and memory [46,47], synaptic plasti-
city [48-50], Ca2, signaling [51,52], and MAPK signaling
[51].
Network analysis was conducted using the set of con-
firmed mPFC gene expression changes, and revealed
that Cart, NPY, Nr4al, Fos, Egrl, Adora2b and Drd5 all
interact (directly or indirectly) with the MAPK/ERK
pathway. While altered expression of MAPK/ERK path-
way elements was not detected in this study, changes in
expression and activity levels of MAPK/ERK genes have
been reported (for a review see [28]) and this pathway is
thought to play an important role in drug-induced
changes in the brain [53,54]. Regulators of Ca2+
dynamics were also identified in the network analysis.
The changes in Drd5, Adora2b, CD47 and CART
expression may indicate a decrease in intracellular Ca 2
signaling that occurs with cocaine self-administration
and persists into periods of abstinence [40,52,55,56].
Additionally, the gene expression changes identified
indicate that synaptic plasticity may be affected by
cocaine self-administration and abstinence. Persistent
reductions in levels of CD47 and Arc, and inductions in
levels of Drd5 and NPY suggest altered synaptic plasti-
city process involved in memory formation and removal
of old memory traces, respectively [50,57,58]. A poten-
tial reduction in synaptic plasticity in the mPFC with
cocaine self-administration/abstinence is hypothesized
based on levels of CD47, Nefl, Arc, Egrl, and NPY
[39,42,48-50]. These data are in agreement with studies
of the direct role of psychostimulants on mechanisms of
synaptic plasticity, including LTP and LTD, in the meso-
limbic system [59,60]. In total, these gene expression
changes may contribute to persistently altered synaptic
plasticity in the mPFC.


NAc
The central role of the NAc in psychostimulant reward is
well documented [61]. While cocaine exerts common
actions on the NAc and mPFC [62], we observed little
overlap (50 of the 1875 total gene expression changes
(mPFC + NAc)) between these brain regions. The regu-
lated genes common to both brain regions include IEGs
reported previously [12], various signaling molecules, and
genes involved in cellular metabolism. When the micro-
array datasets were examined by ontological analysis dis-
tinct molecular functions were observed in each brain
region. This indicates that the functional changes occur-
ring in the mPFC and NAc may differ and may ultimately
play different roles in abstinence-dependent behaviors.
Unlike the mPFC, fewer category 1 and 2 changes
were observed in the NAc (100 of 414 total), than cate-
gory 3 changes (those changed specifically during absti-
nence) (314). Of the cocaine-induced changes, only 32
persisted into periods of abstinence (category 2), while
the remainder returned to pre-cocaine levels. Arc, Beta-
catenin, Cap2, Crip2, Dnm2, Egr2, Fos, Fut8, GFAP,
Gpr88, Htrld, and Nr4al were all confirmed by qPCR
to be differentially expressed. We have previously
demonstrated the responsiveness of Arc, Fos and Nr4al
in this animal model [12].
Published data regarding Cap2, Crip2, Fut8, and
Gpr88 in the brain are limited, with no previous reports
of cocaine-responsiveness. Crip2 (a LIM-domain pro-
tein), Cap2 (an adenylate cyclase-associated protein) and
Dnm2 are cytoskeletal function and organization genes
[63,64]. Interestingly, Dnm2 is regulated by the tran-
scription factor Arc, also altered in the NAc with
cocaine [12,65]. Among the remaining changes, Egr2
and GFAP have been previously demonstrated to be
cocaine-responsive [66-68]. Htrld has been linked with
a number of psychiatric disorders [69,70]. Changes in
the expression of these genes may also indicate cocaine
induced alterations in receptor signaling, glial cell func-
tion, and synaptic plasticity.
Beta-catenin, which was increased at 10-days of absti-
nence in this study, is a well-characterized protein that
regulates cell growth as a part of the Wnt signaling
pathway. As a part of Wnt signaling, beta-catenin also
plays a role in synaptic plasticity [71,72]. In response to
chronic cocaine, beta-catenin has been shown to
increase in a number of brain regions [73-75]. Fut8, a
fucosyltransferase protein, also increased at 10-days of
abstinence in this study, has been shown to increase
upon Wnt/beta-catenin activation [76], indicating that
there may be a coordinated activation of Wnt signaling
during periods of abstinence from cocaine.
Network analysis of the confirmed genes in the NAc
identified a TNF-centered network. Generally involved
in inflammatory processes, TNF has not been


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historically associated with behavioral responses to
cocaine. Studies performed on the effects of cocaine on
macrophages have reported that cocaine suppresses
LPS-stimulated TNF expression [77,78]. TNF induction
was recently demonstrated to reduce conditioned place
preference and locomotor sensitization caused by
methamphetamine and morphine administration [79]. If
TNF does play a role in the behavioral responses to
cocaine, these additional genes may represent regulatory
and effector elements of a TNF network.
While these reported changes represent new insights
into abstinence-induced changes in the brain, localization
of these changes to specific cell types is still to be deter-
mined. As with other functional genomic and proteomic
approaches looking at dissected brain regions, even these
specific dissections contain a heterogeneous cellular
population. Future molecular neurobiology studies that
seek to extend these, and other findings, will need to uti-
lize techniques (e.g. laser capture microdissection and
fluorescent in-situ hybridization) to localize changes to
specific cell types and neuronal networks [80].

Conclusions
In addition to offering further evidence of long-lasting
changes in gene expression following abstinence from
cocaine self-administration, these results identify cellular
processes that may regulate the development and/or
maintenance of incubation of drug-seeking and drug-tak-
ing. A number of additional changes in gene expression
remain to be examined in future studies, but the results
presented here support the finding that persistent shifts
in gene expression can last long into abstinence. In the
mesolimbic reward pathway, changes in the mPFC may
be more pronounced than in the NAc and involve mostly
distinct sets of genes. This may indicate different meta-
plastic processes occur in these brain regions with the
development and expression of abstinence-induced beha-
viors. In the mPFC, changes in MAPK/ERK and calcium
signaling and in synaptic plasticity occur. The alterations
in the NAc suggest a possible role of Wnt and TNF-
mediated signaling in cocaine-associated behaviors.
Together the findings of this study highlight a number of
pathways and processes in the brain that may play roles
in the development and maintenance of abstinence-
induced drug seeking and drug taking. A clear under-
standing of how these novel changes contribute to
relapse liability will not only increase our knowledge of
the neurobiology of addiction, but will provide targets for
therapeutic development.

Methods
Cocaine self-administration
The surgical and cocaine self-administration procedures
used have been described previously [12,18]. Briefly,


male Sprague-Dawley rats (Harlan Inc., IN) were
implanted with a chronic indwelling Silastic cannula in
the right jugular vein, and trained to self-administer
cocaine hydrochloride through exposure to a fixed ratio
1 (FR1) schedule of reinforcement as described pre-
viously [12,14]. After establishing a stable daily intake of
cocaine (40 infusions within 6 hours for at least 5 days),
access conditions were changed to a discrete-trials sche-
dule. During the discrete-trial schedule, rats were given
access to cocaine for 10-min discrete trials that were
initiated at 15-min intervals. An infusion (1.5 mg/kg/inj)
of cocaine was given following a response on the lever,
which resulted in illumination of a stimulus light for 20
sec. The lever was retracted and the trial terminated if
an injection was collected or if 10 minutes had elapsed.
Rats received four discrete trials per hour (i.e., DT4),
24 hours per day, for 10 days. Following 10 days of
self-administration, animals were placed in standard
polycarbonate cages for 1, 10 or 100 days of enforced
abstinence. All research was approved by the Wake
Forest University School of Medicine and Penn State
College of Medicine Animal Care and Use Committees
and conducted according to the Guide for the Care and
Use of Laboratory Animals, as promulgated by the
National Institutes of Health.

Dissection
Following 1, 10, or 100 days of deprivation, rats were
sacrificed and the brains were rapidly removed and
cooled in ice-chilled saline. Brains were then placed in
an ice-chilled ASI brain slicer (ASI Instruments, Warren
MI). The medial prefrontal cortex (mPFC) and nucleus
accumbens (NAc) were collected as described previously
[12]. Briefly, the section from Bregma +4.4 to 2.4 mm
[81] was cut along the forceps minor and the cortex
medial of this cut was collected. This is considered the
medial prefrontal cortex, and includes the cingulate
area, prelimbic cortex, and medial orbital cortex. The
section from + 2.2 to 0.2 mm was cut 0.5 mm on each
side of the midline, on a line connecting the tip of the
external capsule and the previous cut, on a line connect-
ing the tip of the external capsule and lateral ventricle,
and between the ventricle. This dissection includes both
the core and the shell of the NAc. See Additional File 3:
Figure S1, for schematics of the dissections.

RNA isolation
Following dissection, tissue samples were stored at -80C
until RNA was isolated. RNA isolation was conducted as
described previously [74,82]. Total RNA from cocaine
naive rats and rats following 1, 10, and 100 days of
enforced abstinence (following 10 days of DT4 cocaine
self-administration as described above) was isolated
using Tri-Reagent (Molecular Research Center Inc.,


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Cincinnati, OH) [83]. RNA quantity and quality was
then assessed using the Agilent 2100 Bioanalyzer (Agi-
lent, Palo Alto, CA) following further RNA purification
using an RNeasy Mini Kit for RNA clean-up (Qiagen
Sciences, Maryland).

Microarray analysis
Microarray analysis was performed in the Penn State
College of Medicine Functional Genomics Core Facility
according to standard procedures. For the mPFC arrays,
naive, 1-day, and 100-day abstinent samples were used
(N = 6). For the NAc arrays, naive, 1-day and 10-day
abstinent samples were used (N = 5; reduced sample
number due to removal of samples that did not pass
array quality control). Microarrays for the NAc were
performed after those for the mPFC, and for the NAc,
the 10-day timepoint was used instead of the 100-day
timepoint in order to detect important changes that
may not last to 100 days of abstinence. The persistence
of these changes to 100-days could then be tested with
qPCR.
Microarray analysis was performed using the Code-
Link Rat Whole Genome Bioarray system (GE Health-
care). Following the manufacturer's protocol, first strand
synthesis was performed with 2 pg of RNA as starting
material and was followed by second strand synthesis
and purification using Qiaquick spin columns (Qiagen,
Valencia, CA). T7 reaction buffer, T7 NTPs, 10 mM
biotin-11-UTP, and T7 polymerase were then added to
the dsDNA for the IVT reaction and incubated at 37C
for 14 hours. The resulting Biotin-labeled cRNA was
then purified using RNEasy columns (Qiagen), quanti-
tated, and volume-adjusted for a total of 10 pL. The
cRNA was then fragmented and denatured before hybri-
dization for 18 hours at 37C. Slides were washed and
then incubated at room temperature with Alexa Fluor
647 labeled streptavidin for 30 minutes followed by
washing.
Micro arrays were scanned on an Axon 4000 B scan-
ner with GenePix4 v4.0 software at a 5 pm resolution at
635 nm with laser power at 100%, PMT voltage at 600
V, focus position 0 pm, and lines to average = 1. Images
were then imported into CodeLink Expression Analysis
Software v4.1 (GE Healthcare) and initial quality control
(positive and negative controls), exclusion of manufac-
turing defects (MSR spots), background subtraction, and
intra-array normalization was performed.

Data analysis
Following image analysis on CodeLink Expression Ana-
lysis Software, microarray data were imported into
GeneSpring GX 7.3 (Agilent Technologies) and signal
values less than 0.01 were transformed to an intensity of
0.01. Normalization was performed per chip to the 50th


percentile, and per gene to the median. Values were
then normalized on a per gene basis to the naive group
for each of the two time points (1 and 10, or 1 and 100
days of abstinence). Potential differential expression was
determined with a one-way ANOVA (variances not
assumed to be equal), p < 0.05 and filtered for 1.4-fold
or greater differences in expression in accordance with
standards for microarray analysis [84]. The use of a
combination of statistical and fold-change cutoffs as
opposed to traditional multiple testing corrections (e.g.,
Bonferroni post-hoc testing) produce gene lists with the
lowest rate of type I and type II errors [85]. A fold-
change cutoff of 1.4 fold was chosen, as this magnitude
change is at the lower range of changes historically con-
firmed by qPCR in this laboratory. Lastly, probe
sequences on the array were searched against current
rat genome sequences to eliminate any probes for
sequences removed from the NCBI database.

Quantitative PCR (qPCR) analysis of gene expression
cDNA synthesis was performed on total RNA from naive,
1, 10, and 100-day abstinent animals using Superscript III
Reverse Transcriptase (Invitrogen, Carlsbad, CA). 1 pg
RNA, 500 ng Oligo (dT), and 10 mM each dNTP, were
incubated for 5 minutes at 65C and then chilled on ice
for 2 minutes. 5x First Strand Buffer (250 mM Tris-HCl
(pH8.3), 375 mM KC1, and 15 mM MgCl2), 5 mM DTT
(final concentration), 40 U RNaseOut, and 200 U Super-
script III RT were then added. The 20 pl reaction was
incubated for 60 minutes at 50C followed by a final
incubation at 70C for 15 minutes for termination. The
resulting cDNA product was quantified and 50 ng of pro-
duct was used in each subsequent qPCR reaction.
Quantitative PCR was carried out on a real-time
detection instrument (ABI 7900HT Sequence Detection
System) in 384-well optical plates using TaqMan Uni-
versal PCR Master Mix and Assay on Demand primers
and probes (Applied Biosystems, Foster City, CA) as
described previously [86,87]. This examination used a
larger set of animals than the microarray analysis (Table
1). Primer/probe sets used are listed in Additional File
2: Table S2. SDS 2.2.2 software and the 2-AACt analysis
method [88]. were used to quantitate relative amounts
of product using f3-actin as an endogenous control. Sig-
nificance from qPCR analysis was determined with Sig-
maStat 3.5 (SYSTAT Software, Inc.) based on one-way
analysis of variance (ANOVA) (p < 0.05) with a post hoc
Student Newman-Keuls test (p < 0.05).

Ontological, pathway, and network analysis
Ontological analysis used Gene Ontology (GO) cate-
gories and differentially expressed processes or func-
tional categories were determined statistically, as
previously described [87] using GeneSpring GX


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Freeman et al. BMC Neuroscience 2010, 11:29
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software. This analysis determined the number of genes
in a category present on the array and the number of
expression changes that would be part of that category
by random chance given the number of differentially
expressed genes. Results from these analyses were used
to compile a list of genes to examine by qPCR. Ingenu-
ity Pathway Analysis (Ingenuity Systems, Redwood City
CA) was used for network and pathways analysis of the
qPCR confirmed gene expression results.


Additional file 1: Microarray expression data A full listing of
differentially expressed genes identified by microarray analysis
Click here for file
[http'//wwwbiomedcentral com/content/supplementary/1 471 -2202-11-
29-51 DOC]
Additional file 2: Gene expression assay numbers Gene symbols, full
names and gene expression assay numbers
Click here for file
[http'//wwwbiomedcentral com/content/supplementary/1 471 -2202-11-
29-52 XLS
Additional file 3: Dissection schematics Dissection diagrams
Schematics of the mPFC and NAc dissections are provided using
modifications of figures from Paxinos and Watson Numbered red lines
are specific dissection cuts using visible landmarks as described in the
methods The shaded area represents the tissue collected for molecular
analysis
Click here for file
[http'//wwwbiomedcentral com/content/supplementary/1 471 -2202-11-
29-53 PDF]




Abbreviations
Adora2b' adenosine receptor A2b; Arc' activity-related cytoskeletal associated
protein; Cap2' adenylate cyclase-associated protein 2; CART cocaine and
amphetamine-related transcript; Crip2' cysteine-rich protein 2; Dnm2'
dynamin 2; Drd5' dopamine receptor D5; DT discrete trials; Egrl' early
growth response 1; Egr2' early growth response 2; Fos' FBJ osteosarcoma
oncogene; FR1 fixed ratio 1; Fut8; fucosyltransferase 8; GFAP glial fibrillary
acidic protein; GPR88' g-protein coupled receptor 88; Htrld' 5-
hydroxytryptamine receptor Id; MAPK/ERK mitogen activated protein
kinase/extracellular signal-regulated kinase; mPFC medial prefrontal cortex;
NAc' nucleus accumbens; Nefl' neurofilament light; NPY' neuropeptide Y;
Nr4al nuclear receptor subfamily 4al; TNF' tumor necrosis factor

Acknowledgements
This work was supported by grants R01-DA013770-08 (KEV), F31-DA02281902
(MEL), R01 DA14030 (DCSR) and K01-DA13957 (DM) The authors wish to
thank the Penn State College of Medicine Functional Genomic Core Facility
for microarray and qPCR analysis and technical assistance and Dr Heather
VanGuilder for manuscript editing

Author details
1Department of Pharmacology, Penn State College of Medicine, Hershey, PA,
17033, USA 2Functional Genomics Facility, Penn State College of Medicine
Hershey, PA, 17033, USA Department of Psychiatry, University of Florida,
Gainesville, FL, 32611, USA 4Department of Physiology & Pharmacology,
Wake Forest University School of Medicine, Winston-Salem, NC, 27157, USA

Authors' contributions
WMF and KEV generated the experimental design RMB and KMP conducted
the array and RT-PCR analyses, respectively MEL performed the data analysis,
prepared the figures, archived the data, and wrote the manuscript WMF
assisted with data analysis and WMF and KEV contributed to the data
interpretation DM and DCSR (Wake Forest University) developed the animal
model used and provided the samples for analysis All authors read and
approved the final manuscript


Received: 23 September 2009
Accepted: 26 February 2010 Published: 26 February 2010

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doi:10.1186/1471-2202-11-29
Cite this article as: Freeman et al Gene expression changes in the
medial prefrontal cortex and nucleus accumbens following abstinence
from cocaine self-administration. BMC Neuroscience 2010 11 29


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