effects of repeated long‐term psychosocial stress and
TRANSCRIPT
CNS Neurosci Ther. 2018;1–11. wileyonlinelibrary.com/journal/cns | 1
Received:9November2017 | Revised:2January2018 | Accepted:4January2018DOI: 10.1111/cns.12810
O R I G I N A L A R T I C L E
Effects of repeated long- term psychosocial stress and acute cannabinoid exposure on mouse corticostriatal circuitries: Implications for neuropsychiatric disorders
Jordi Tomas-Roig1,2,3 | Fabiana Piscitelli4 | Vanesa Gil5,6 | Ester Quintana3 | Lluís l. Ramió-Torrentà3 | Jose Antonio del Río5,6 | Timothy Patrick Moore1,2,7 | Hope Agbemenyah8 | Gabriela Salinas9 | Claudia Pommerenke9 | Stephan Lorenzen10,11 | Tim Beißbarth10 | Sigrid Hoyer-Fender12 | Vincenzo Di Marzo4 | Ursula Havemann-Reinecke1,2
1DepartmentofPsychiatryandPsychotherapy,UniversityofGöttingen,Göttingen,Germany2CenterNanoscaleMicroscopyandMolecularPhysiologyoftheBrain(CNMPB),Göttingen,Germany3GironaNeuroimmunologyandMultipleSclerosisUnit(UNIEMTG),Dr.JosepTruetaUniversityHospitalandNeurodegenerationandNeuroinflammationResearchGroup,GironaBiomedicalResearchInstitute(IDIBGI),Girona,Spain4EndocannabinoidResearchGroup,InstituteofBiomolecularChemistry,ConsiglioNazionaledelleRicerche,Pozzuoli,Italy5InstituteforBioengineeringofCatalonia(IBEC),TheBarcelonaInstituteofScienceandTechnology,CentrodeInvestigaciónBiomédicaenReddeEnfermedadesNeurodegenerativas(CIBERNED),Barcelona,Spain6DepartmentofCellBiology,PhysiologyandImmunology,FacultyofBiology,UniversityofBarcelona,Barcelona,Spain7DepartmentofChildandAdolescentPsychiatry,UniversityHospitalMünster,Münster,Germany8LaboratoryforAgingandCognitiveDiseases,EuropeanNeuroscienceInstitute,Goettingen,Germany9DepartmentofDevelopmentalBiochemistry,Georg-August-UniversitätGöttingen,Göttingen,Germany10DepartmentofMedicalStatistics,UniversityMedicalCenterGöttingen,Göttingen,Germany11DepartmentofMolecularMedicine,BernhardNochtInstituteforTropicalMedicine,Hamburg,Germany12Johann-Friedrich-BlumenbachInstituteforZoologyandAnthropology,DevelopmentalBiology,Göttingen,Germany
Thefirstandlastauthorshareseniorauthorship.Thesecondandthirdauthorcontributedequallytothiswork.
CorrespondenceJordiTomas-Roig,NeurodegenerationandNeuroinflammationResearchGroup,GironaBiomedicalResearchInstitute(IDIBGI),Girona,Spain.Email:[email protected]
Funding informationDeutscheForschungsgemeinschaft,Grant/AwardNumber:CNMPBC1-6andTO977/1-1
SummaryIntroduction:Vulnerabilitytopsychiatricmanifestationsisachievedbytheinfluenceofgeneticandenvironmentincludingstressandcannabisconsumption.Here,weusedapsychosocialstressmodelbasedonresident-intruderconfrontationstostudythebraincorticostriatal-function,sincederegulationofcorticostriatalcircuitrieshasbeenreportedinmanypsychiatricdisorders.CB1receptorsarewidelyexpressedinthecen-tralnervoussystemandparticularly,inbothcortexandstriatumbrainstructures.Aims and methods:Theinvestigationpresentedhereisaddressedtoassesstheimpactofrepeatedstressfollowingacutecannabinoidexposureonbehaviorandcorticostri-atalbrainphysiologybyassessingmicebehavior,theconcentrationofendocannabi-noidandendocannabinoid-likemoleculesandchangesinthetranscriptome.
ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttribution-NonCommercial-NoDerivsLicense,whichpermitsuseanddistributioninanymedium,providedtheoriginalworkisproperlycited,theuseisnon-commercialandnomodificationsoradaptationsaremade.©2018TheAuthors.CNS Neuroscience & TherapeuticsPublishedbyJohnWiley&SonsLtd.
2 | TOMAS- ROIG eT Al.
1 | INTRODUCTION
Vulnerability to psychiatric disorders is determined by the inter-actionofgeneticandenvironmental factors1 suchaspsychosocialstressandcannabisconsumption.Thedysregulationofcorticostri-atal circuitries is involved in the pathophysiology of psychoticdisorders.2Psychosocialstresshasaremarkable influenceoncen-tral nervous system (CNS) and animal behavior.3When an organ-ism is exposed to a stressor, biological mechanisms such as thehypothalamic-pituitary-adrenalaxis(HPA)andcardiovascularread-justmentscomeintoplaytorestorehomeostasis.4Understandardconditions,HPAaxisfunctionismainlyinfluencedbystress,whichenhances its activity, and the circadian rhythm.5 However, whenstress is prolonged over the time and the activation exceeds thecapacitytokeepbody’shomeostasis,psychopathologicalsequelaecanappear.Amongthebroadspectrumofbrainstructurescloselyinvolvedinanxietyandstressdisorders(forreviewseeRef.[6]),wedirectedoureffortstowardthedorsalstriatum(dorsalCPu)andtheprefrontal cortex (PFC) due to the fact that these areas are criti-cally involved insocial relationships.7,8There isagrowingbodyofthe literature that suggests that glucocorticoid receptor(s) involveG protein-dependent mechanisms.9 In particular, corticostriatalactivity ismodulated by a variety of G protein-coupled receptorssuch as the cannabinoid CB1 receptor, which is highly abundant
in the CNS.10 CB1 receptors are the main target for endogenousendocannabinoids lipid signaling molecules and mediate bothΔ9-tetrahydrocannabinol (THC) and synthetic cannabinoid drugspharmacological actions.11 Themain endocannabinoidsmoleculesfor CB1 receptors are N-arachidonylethanolamine (AEA)12 and 2-arachidonoylglycerol (2-AG).13 N-arachidonoylphospatidylethanolamine-specific phospholipaseD (NAPE-PLD) is responsible fortheproductionofAEA,while2-AGismostlysynthesizedbydiacyl-glycerollipase(DAGL-α)intheCNS.14Afteractivatingcannabinoidreceptors,theendocannabinoidAEAandrelatedendocannabinoid-likeNAEsPEAandOEAareenzymaticallyhydrolyzedbythefattyacidamidehydrolase(FAAH)while2-AGisprimarilymetabolizedbymonoacylglycerollipase(MAGL).15
Interestingly,theendocannabinoidsystemhasbeendescribedtohaveadirecteffectonbothneurotransmission16andglucocorticoidsignaling.17Despitethelackofknowledgeconcerningtheconcretecorticostriatalmechanisms underlying anxiety, both pharmacologic(syntheticcannabinoiddrugs)andbehavioraltestinghavebeenap-plied.Thus,theaimofthisstudywastoinvestigatetheinterplaybe-tweenrepeatedlong-termexposuretopsychosocialstressandacutechallengewithcannabinoiddrugsonmicecorticostriatalcircuitries.Forthispurpose,weevaluatedtheanimalsbybehavioraltestingandthen quantified endocannabinoid and endocannabinoid-like mole-culesandalsochangesinwhole-genomegeneexpressionprofile.
Results:Stressedanimalsurinatedfrequently;showedexacerbatedscratchingactivity,lowerstriatalN-arachidonylethanolamine(AEA)levelsandhighercorticalexpressionofcholinergicreceptornicotinicalpha6.ThecannabinoidagonistWIN55212.2dimin-ishedlocomotoractivitywhiletheinverseagonistincreasedthedistancetravelledinthe center of the open field. Upon CB1 activation, N-oleoylethanolamide and N-palmitoylethanolamide,twoAEAcongenersthatdonotinteractdirectlywithcannabi-noid receptors, were enhanced in the striatum. The co-administration with bothcannabinoidsinducedanup-regulationofstriatalFK506bindingprotein5.Theinverseagonist in controls reversed the effects of WIN55212.2 on motor activity. WhenRimonabantwas injected under stress, the cortical levels of 2-arachidonoylglycerolweremaximum.Theagonistandtheantagonistinfluencedthecorticalexpressionofcholinergicreceptornicotinicalpha6andserotonintransporterneurotransmittertype4inoppositedirections,whiletheirco-administrationtendedtoproduceanulleffectunderstress.Conclusions:Theendocannabinoidsystemhadadirecteffectonserotoninergicneu-rotransmissionandglucocorticoidsignaling.Cholinergicreceptornicotinicalpha-6wasshown tobederegulated in response to stressand followingsyntheticcannabinoiddrugsthuscouldconfervulnerabilitytocannabisaddictionandpsychosis.Targetingthe receptors of endocannabinoids and endocannabinoid-likemediatorsmight be avaluableoptionfortreatingstress-relatedneuropsychiatricsymptoms.
K E Y W O R D S
CB1,Chrna6andSlc6a4,Fkbp5,psychosocialstress
| 3TOMAS- ROIG eT Al.
2 | MATERIALS AND METHODS
AllanimalexperimentalprocedureswereapprovedbyUniversityofGöttingenInstitutionalAnimalCareandUseCommitteeandalsowereinaccordancewithNIHguidelinesfortheuseofanimalsinresearchandtheEuropeanCommunitiesCouncilDirective(86/609/EEC).
2.1 | Animals
Atotalof120C57Bl6/Jmalemiceaged7-8weekswerepurchasedfromCharlesRiverLaboratories(Sulzfeld,Germany).Onarrival,theywere kept five per cage andmaintained under standard conditions(12-hourlight/darkcyclewith6:00/18:00lightson/off,aroomtem-peratureof21±2°C,andfoodandwateradlibitum).Afterone-weekhabituationperiod,miceweresubjectedtotheexperiment.One-year-oldFVB/Nmalemice(CharlesRiverLaboratories,Sulzfeld,Germany)werekept individuallyandusedas residents.FVB/Nmicewerese-lectedasresidentsbecausetheyaremoreoffensivethantheC57Bl/6strain.3TheFVB/Nstrainwaskeptunderthesameprotocolcondi-tionsastheC57Bl/6Jcolonybuthousedinaseparateroomtopre-venttheC57Bl/6Jmicehabituationtotheodoroftheresidents.
2.2 | Drugs
TheCB1/CB2 receptor agonistWIN55212.2 (Sigma-Aldrich, Seelze,Germany) and the selective cannabinoid CB1 receptor antagonistRimonabant(SequoiaResearchProductsLtd,Pangbourne,UK)weredissolved in a vehicle solution consisting of 10% DMSO (Sigma-Aldrich,Seelze,Germany)and0.1%Tween80(Sigma-Aldrich,Seelze,Germany)in0.9%salineandpreparedonthe21daysoftheexperi-ment.Avolumeof200μLofdrugand/orvehiclewasadministeredintraperitoneally(WIN55212.2andRimonabantwereadministeredataconcentrationof3mg/kg)beforethebehavioraltesting.
2.3 | Experiment design and experimental groups
TheC57Bl6/Jmalemiceweresortedintotwogroups:Thoseexposeddaily to psychosocial stress for 1hour (stress) and those who wereleftundisturbed(control).Thestressprotocolwasperformeddailyfor3weeks.Onday21,theanimalsreceivedanappropriatedrugorvehicleinjectionandthenevaluatedbybehavioraltesting.Indeed,controlandstressedmiceweresplitintofoursubgroupseach(15animalspersub-group):micetreatedtwicewithvehicle(Veh),micesubjectedfirsttove-hicleandthenWIN55212.2(WIN),micetreatedfirstwithRimonabantand thenwithWIN55212.2 (Rim+WIN), andmice injected firstwithRimonabantandthenvehicle(Rim).Followingthelastinjectionwithei-thervehicleorcannabinoiddrug,theanimalsweretestedbydifferentbehavioralparadigmsduringtherodentactivephase(ie,after8pm).
2.4 | Social stress procedure
An intruder (C57Bl/6J mouse) was placed in the home cage of aresident(FVB/Nmouse),andthentheyfreelyinteracteduntilthefirst
aggressionwasachieved.Afterthefirstattack,theC57Bl/6Jmousewasisolatedbytheuseofasmallplasticwire-meshcagewithintheFVB/Nmouse’scageprotectingtheintruderagainstdirectaggression;however,olfactory,visual,andsomevibrissaecontactwithresidentsweremaintained.After1hour,theC57Bl/6Jmousewasplacedagaininitshomecage.Topreventhabituation,everydayintrudermicehadencounterswithdifferentresidents.Thepsychosocialstressprotocolwasperformedonceperdayatasimilartime(after8pm)toenhancethe stress prediction factor in intruders. In contrast, controls wereleft undisturbed in an empty cage everyday for 1hour. Therefore,controlsweresubjectedtothesameexperimentalprotocolintermsofhandlingandexposuretoanewenvironment(differentcage),buttheydidnotinteractwiththeresidents.
2.5 | Behavioral assessment
Mice were exposed to psychosocial stress or left undisturbedfor 21days. On day 21, they were acutely treated with differentcannabinoid drugs or vehicle and finally evaluated by behavioraltesting. The stress paradigm, the administration of drugs, and thebehavioral assessment took place during the dark phase (activephase).Wedirectedthebehavioralanalysesbymeasuringlocomotorandanxiety-likebehaviorintheopen-field(OF)testandtestingalsoCNS activity and excitability by useof the functional observationalbattery(FOB).
2.5.1 | Functional observational battery
TheFOBhas beenwidely used and adapted for general behavioralstudiesinmice.18Inthisstudy,theFOBtestcomprised28parametersbywhich the investigatorevaluatedCNSactivityandexcitabilitybyrecording neuromuscular and autonomic effects, and sensorimotorreactivity. There were four consecutive testing situations: (i) in thehomecage,(ii)intheobserver’shand,(iii)intheOF,and(iv)manipula-tiontests.Afterabriefassessmentinitshomecage,eachmousewasremovedandhandledby theobserver to evaluate ease-of-removal,handling reactivity, and general appearance. Then, eachmousewasassessedinanOFarena(60cm×90cm)whiletheobserveranalyzedCNSactivity,autonomiceffects,muscletone,equilibrium,andsenso-rimotorreactivity.Gaitconditionwasscoredfrom1to8.Thegaitwasscoredasfollows:normalgait,ataxia,splayedhindlimbs,feetmarkedlysplayed outward from the body, fore-limbdrag,walking on tiptoes,hunchedbody,andbodydrag.Theseverityofgaitabnormalitywasalsoevaluatedonascalefrom1(normal)to4(severelyabnormal).18
2.5.2 | Open- field activity
IntheOFtest,weevaluatedthespontaneouslocomotorandexplora-toryactivityofmice.Theexperimentwasconducted inaPlexiglassarena(45×45×55cm),andeachmousewasleftinthesamestart-ingposition.AnimalswereallowedtoexaminetheOFfor10minuteswithouthabituation.Weregisteredthetotaldistancetravelled,per-centoftimemoving,timespentinthecenter(definedas70%ofarea),
4 | TOMAS- ROIG eT Al.
hyperactivity(forwardmovementwithspeedgreaterthan20cm/s),andthenumberofrearingsbyuseoftheActiMotsoftware(TSE,BadHomburg,Germany)asdescribed.3Theapparatuswascleanedwith70%ethanolp.a.betweeneachtest.
2.6 | Brain sample collection and tissue evaluation
AnimalsweresacrificedimmediatelyafterOFtaskfinished,andbraintissuewascollectedaccordingly.Allmiceweredeeplyanesthetizedby intraperitoneal injectionof2,2,2-tribromoethanol (Sigma-Aldrich,Hamburg,Germany)andthentranscardiallyperfusedwithcold0.1%phosphate buffer saline (PBS). Finally, the PFC and the dorsal CPuwere freshly isolatedand frozen in liquidnitrogen for LC-MS,RNAseq,andquantitativeRT-PCR.
2.6.1 | Extraction and measurement of AEA, 2- AG, OEA, and PEA levels
The endogenous lipid signaling molecules AEA and 2-AG, andendocannabinoid-related moleculesN-palmitoylethanolamide (PEA),and N-oleoylethanolamide(OEA)werepurifiedfromthePFCandthedorsalCPuandthenquantifiedasdescribedelsewhere.19First,sam-plesweredounce-homogenizedfollowingwithchloroform/methanol/Tris-HCl50mmol/LpH7.5(2:1:1,v/v)containinginternaldeuteratedcontrolssolutionforAEA,2-AG,PEA,andOEAmeasurementbyiso-topedilution([2H]8AEA,[2H]52AG,[2H]4PEA,[2H]4OEA(CaymanChemicals,MI,USA).Thelipidorganicphasewasdrieddown,weighed,andprepurifiedonsilicagel.Fractionswerecollectedbyelutingthecolumnwith99:1,90:10,and50:50(v/v)chloroform/methanol.The90:10fractioncontainedAEA,2-AG,PEA,andOEA,anditwasusedfor isotopic dilution liquid chromatography-atmospheric pressurechemical ionization-massspectrometryquantification(LC-APCI-MS),aspreviouslydescribedandusing selected ionmonitoring atM+1valuesforthefourcompoundsandtheirdeuteratedhomologues,asdescribedinRef.[20]N=4mice/group.
2.6.2 | RNA extraction
TotalRNAwasextractedfrom80samples (controlandstress;Veh,WIN, Rim+WIN, and Rim; PFC and dorsal CPu; N=5 mice/group)usingtheTRIzolreagentaccordingtothemanufacturer’sinstructions(Invitrogen,NY,USA).TheRNAwasdigestedwithRNase-freeDNase(Qiagen,Düsseldorf,Germany)andchecked for integrityusingcap-illary gel electrophoresis (Bioanalyzer, Agilent Technologies, SantaClara,USA).
2.6.3 | cDNA Library Preparation and RNA Seq
ThelibrarywaspreparedusingtheTruSeqRNASamplePreparationKit(Illumina,SanDiego,USA)startingfrom500ngoftotalRNA.ThecDNAlibrarieswerequantifiedusingtheQuantiFluordsDNASystem(Promega,Madison,USA).ThesizerangeofthefinalcDNAlibrarieswasdeterminedbytheBioanalyzer2100(Agilent,SantaClara,USA).
The cDNA libraries were amplified and sequenced using cBot andHiSeq2000(Illumina,SanDiego,USA).
ThesequenceimageswereconvertedwiththeIlluminaBaseCallersoftware tobcl files,whichweredemultiplexedtoFASTQfileswithCASAVA version 1.8.2. Quality was checked via FastQC (version0.10.0,BabrahamBioinformatics).
2.6.4 | Gene expression analyses
SequenceswerealignedtotheRefSeq21mousetranscriptomeusingbwa,22 and raw “hits”per transcriptweremergedgenewise.DESeqwas used to analyze the counts per gene.23 The resultingP-valueswerecorrectedusingBenjamini-Hochbergadjustment.24
2.6.5 | Quantitative RT- PCR
ThemostrelevanttophitsderegulatedbystressandcannabinoiddrugswerefurtherevaluatedbyquantitativeRT-PCR.First-strandcDNAwassynthesized from1μgof totalRNAusing theHighCapacityRNA-to-cDNAKit(AppliedBiosystems,Darmstadt,Germany).mRNAexpressionwasquantifiedbyRT-PCRusingtheCXF96TMReal-timePCRsystem(Bio-Rad,Hercules,USA).WeusedGAPDHmRNAasanendogenouscontrol. TaqMan assays formouseChrna6,Slc6a4, and Fkbp5 cDNAswere selected from validated and predesigned Assays-on-Demand(AppliedBiosystems,Darmstadt,Germany) andused in real-timePCRamplifications todetect theexpressionsofChrna6,Slc6a4, and Fkbp5. The reactions were performed in triplicate using 2μL of cDNA in a10 μL reaction volume. mRNA expression was determined using thecomparativecyclethreshold(Ct)methodwith2−ΔΔCt,accordingtothemanufacturer’sinstructions(AppliedBiosystems,Darmstadt,Germany).cDNAsweremeasuredrelativetoa“calibrator”controlsample.The2−ΔΔCtforthis“calibrator”wasarbitrarilysetto1.
2.7 | Statistical analysis
Atwo-wayANOVAwasusedtoexaminetheeffectsofstressandpharmacologicaltreatmentonourcohortofanimals.Themeandif-ferences among the levelsofone factorweredeterminedbyone-way ANOVA or Brown-Forsythe test when applicable. Bonferronior nonparametric Tamhane post hoc testwas applied for pairwisecomparisons.Analysisofsimplemaineffectswasperformedwhethertherewasasignificantinteractionofthetwomainfactors.Individualcomparisonsweremadeusing theStudent’s t test.ANOVAfor re-peatedmeasureswas used to test the equality ofmeans formicebodyweight.SignificancelevelwassettoP<0.05.Dataarerepre-sentedasmean±SEM.Significant effectswere identifiedbySPSS(IBMCorp,Chicago,USA).
3 | RESULTS
Resultsaresummarizedinthreemaincategories:(i)effectsofstress,(ii)effectsofdrugs,and(iii)effectsofstressunderdruginfluence.
| 5TOMAS- ROIG eT Al.
3.1 | Effects of stress
Werevealedasignificanteffectofpsychosocialstressonbodyweightthroughout the experimental period (F(1, 123)=4.84, P<0.05).Indeed, exposure to2-week social defeat paradigm significantly di-minished body weight (t(122)=2.78, P<0.01), which returned tocontrol levels by the end of the stress period (Figure1, panel A).Socialdefeatanimalsdisplayedapronouncedscratchingactivity(F(1,123)=4.06, P<0.05) (Figure1, panel B), urinatedmore frequently(F(1, 123)=4.97, P<0.05) (Figure1, panel C), and showed lowerlevelsofAEA(F(1,18)=36.81,P<0.01,inthedorsalCPu)(Figure1,panelD).Transcriptomeanalysisoftheselectedbrainstructuresre-vealed62tophitsasderegulatedgenesbychronicpsychosocialstressbefore Benjamini-Hochberg adjustment (FigureS1). The cholinergicreceptornicotinicalpha6(Chrna6),asacandidategene,wasfurtheranalyzed by quantitative RT-PCR. Thus, we determined that long-termexposuretostressincreasedtheexpressionofChrna6gene(F(1,43)=38.32,P<0.01,inthePFC)(Figure1,panelE)whencomparedtothenonstressedgroup.
3.2 | Effects of drugs
Two-wayANOVAshowedasignificantmaineffectofthedrugtreat-ment on total distance traveled (F(3, 121)=8.36, P<0.001), dis-tance traveled in center (F(3,121=3.72,P<0.05), rearing activity
(F(3, 121)=3.07,P<0.05), PEA levels (F(3, 17)=7.72,P<0.01, inthedorsalCPu),andOEAlevels(F(3,22)=5.29,P<0.01,inthedor-salCPu).Drug administrationhada remarkableeffecton the rela-tive fold change expression forFkbp5 (F(3, 44)=11.44,P<0.001,inthedorsalCPu).Themaineffectsofdrugtreatmentwerefurtherevaluatedbyone-wayANOVAorBrown-Forsythetestwhenappli-cablefollowedbymultiplecomparisonsposthoctests(TableS1).IntheOF, thecannabinoidagonistWINdecreased the totaldistancetraveled(P<0.01)(Figure2,panelA)whiletheadministrationoftheinverseagonistRimsignificantlyantagonizedsucheffects(P<0.01)(Figure2, panel A). Coadministration with both cannabinoid drugsclearlyreducedthetotaldistancetraveledincomparisonwiththoseanimalsonlytreatedwithRim(P<0.05)(Figure2,panelA).Thedis-tancetraveledincenter,ameasureofanxiety-likebehavior,wassta-tisticallyelevatedinanimalstreatedwithRimalonerelativetomiceonlytreatedwiththeagonist (P<0.01) (Figure2,panelB).Animalswere also observed in their homecage by registering the verticalmovements (rearings). We found that animals treated simultane-ouslywithbothdrugsshowedmorefrequentrearingbehaviorthanWIN-treatedanimals(P<0.05)(Figure2,panelC).Thequantificationoftheendocannabinoid-likemolecules,PEAandOEA,revealedthehighestamountsfortheselipidsinmicesubjectedtoacuteadminis-trationwiththeagonist(P<0.05,inthedorsalCPu)(Figure2,panelD&E).GenomewidetranscriptionalprofilingrevealedtwotophitsasderegulatedgenesbyWINand32tophitsasderegulatedgenes
F IGURE 1 Effectsofrepeatedexposuretopsychosocialstress.Dataareexpressedasmean±SEM.Exposureto2-wksocialdefeatparadigmsignificantlydiminishedbodyweight(P<0.01),whichreturnedtocontrollevelsbytheendofthestressperiod(panelA).Socialdefeatanimalsdisplayedapronouncedscratchingactivity(P<0.05)(panelB),urinatedmorefrequently(P<0.05)(panelC),andshowedlowerlevelsofAEAcontent(P<0.01,inthedorsalCPu)(panelD).Wedeterminedthatlong-termexposuretostressincreasedtheexpressionofChrna6gene(P<0.01,inthePFC)(panelE)whencomparedtocontrols.An*indicatessignificantdifferencesbetweenstressgroupandtheirrespectivecontrolgroup.OneortwosymbolsindicateP < 0.05; P<0.01,respectively.N=15forbehavioraltesting,n=4forendocannabinoidsquantification;n=5forwhole-genomegeneexpression.PFC,prefrontalcortex;dorsalCPu,dorsalstriatum;CTR,control;STS,stress;AEA,N-arachidonylethanolamine
6 | TOMAS- ROIG eT Al.
by Rim before Benjamini-Hochberg adjustment (FigureS1). Thecandidategene (Fkbp5)wasvalidatedbyquantitativeRT-PCR, andmainresultsaredepictedinFigure2(panelF).WemeasuredhighertranscriptionrateforFkbp5afteradministrationwithRim+WINthanvehicle (P<0.01, in the dorsal CPu) or when compared to eitherWINorRimalone(P<0.05,P<0.01;respectively,inthedorsalCPu)(Figure2,panelF).
3.3 | Effects of stress under drug influence
A2-wayANOVAreportedasignificantinteractionbetweenthefactors(stress×drug)ontotaldistancetraveledintheOFarena(F(3,121)=2.76,P<0.05),2-AGcontent(F(3,23)=18.91,P<0.001,inthePFC),andfoldchangeexpressionforChrna6(F(3,43)=7.38,P<0.001,inthePFC)and
Slc6a4gene(F(3,43)=3.18,P<0.05,inthePFC).SimplemaineffectsoftheinteractionbetweenthefactorsaredepictedinTableS2.
In an OF arena, repeated long-term exposure to psychosocialstressexacerbatedgeneralmotoractivity followingvehicleadmin-istration (Figure3,panelA). Indeed, socialdefeatanimalsexposedto vehicle traveled longer distances than their controls (P<0.01)(Figure3, panel A). However, the pharmacological treatmentwithWINreducedthetotaldistancetraveledintheOFwhencomparedto Rim-treated animals under either control or stress conditions(P<0.01)(Figure3,panelA).Likewise,psychosociallystressedmiceexposed to theCB1 agonistWIN alone traveled smaller distancesthanthevehiclegroup(P<0.01)(Figure3,panelA).Theadministra-tionof the inverseagonist incontrolsenhancedthe totaldistancetraveled when compared to vehicle-exposed animals (P<0.05)
F IGURE 2 Effectsofcannabinoiddrugsadministration.Dataareexpressedasmean±SEM.IntheOF,thecannabinoidagonistWINdecreasedtandthetotaldistancetraveled(P<0.01)(panelA)whiletheadministrationoftheinverseagonistRimsignificantlyantagonizedsucheffects(P<0.01)(panelA).CoadministrationwithbothcannabinoiddrugsclearlyreducedthetotaldistancetraveledincomparisontothoseanimalssingletreatedwithRim(P<0.05)(panelA).ThedistancetraveledincenterwasstatisticallyhigherinanimalstreatedwithRimalonethanthosemicesingletreatedwiththeagonist(P<0.01)(panelB).WereportedthatanimalstreatedsimultaneouslywithbothdrugsshowedmorefrequentrearingactivityintheirhomecagethanthoseanimalstreatedwithWINalone(P<0.05)(panelC).WefoundbiggeramountofPEAandOEAinmicesubjectedtosingleadministrationwiththeagonistincomparisonwiththeremainingdrug-treatedgroups(P<0.05,inthedorsalCPu)(panelD&E).WemeasuredhighertranscriptionrateforFkbp5genefollowingadministrationwithRim+WINthanthevehiclegroup(P<0.01,inthedorsalCPu)orwhencomparedtoeitherWINorRimalone(P<0.05,P<0.01;respectively,inthedorsalCPu)(panelF).An*indicatessignificantdifferencesbetweendrug-treatedmiceandtheirrespectivevehiclegroup.ComparisonsbetweenWIN-treatedmiceandremainingdrugtreatmentsareindicatedbyan§.Otherwiseunderlined+indicatedothersignificantcomparisonintragroup.OneortwosymbolsindicateP < 0.05; P<0.01,respectively.N=15forbehavioraltesting,n=4forendocannabinoidsquantification;n=5forwhole-genomegeneexpression.PFC,prefrontalcortex;dorsalCPu,dorsalstriatum;OF,openfield
| 7TOMAS- ROIG eT Al.
(Figure3, panelA).Upon long-lasting effects of stress, coadminis-trationwithRim+WINreducedthe totaldistance traveled incom-parisonwith vehicle (P<0.01) and Rim-treated animals (P<0.01)(Figure3,panelA).
Ontheotherhand,controlsonlytreatedwithRimexhibitedhigher2-AGlevelsthaneithervehicleorRim+WIN-treatedmice(P<0.05,inthePFC)(Figure3,panelB).However,socialdefeatmicetreatedwithRim+WIN displayed higher amounts of 2-AG than their nonstressedcounterparts(P<0.01,inthePFC)butalsowhencomparedtoeithervehicle,WIN,orRimaloneunderstress(P<0.01,inthePFC)(Figure3,panelB).TheadministrationoftheinverseagonistRimbytheendofthestressperiodreducedthelevelsof2-AGwhencomparedtocontrolstreatedwiththesamedrug(P<0.01,inthePFC)(Figure3,panelB).
Whole-genome gene expression revealed 10 top hits genes de-regulated simultaneously by stress and cannabinoid administrationbeforeBenjamini-Hochbergadjustment(FigureS1)(Table1).Threeofthem(Fkbp5,Chrna6, and Slc6a4)werefurtheranalyzedbyquantitative
RT-PCR.Two-wayANOVAshowedasignificanteffectoftheinterac-tiononSlc6a4 (F(3,43)=3.18,P<0.05)andChrna6 (F(3,43)=7.38,P<0.001)inthePFCbutdidnotrevealdifferencesinthedorsalCPu.TheuseofsyntheticcannabinoiddrugsdiminishedtheexpressionofChrna6 incontrols (P<0.05, in thePFC) (Figure3,panelC)andalsodidso,butnotsignificantly,whenWINwasadministered instressedanimals. In contrast, the inverse agonistRim clearly antagonized theeffectsofWINonChrna6expressionunderstress(P<0.01,inthePFC)(Figure3,panelC).Furthermore,Rim+WINadministrationinstressedmiceslightlydecreased theexpressionofChrna6whencompared toRim alone, althoughChrna6 expressionwas higher thanwith singleWINtreatment(P<0.05,inthePFC)(Figure3,panelC).Bytheendofthestressprotocol,acuteinjectionwithRimincreasedtheexpressionofChrna6incontrasttovehicle(P<0.01,inthePFC)(Figure3,panelC).SocialdefeatanimalsexposedtoeitherRim+WINorRimaloneun-derwentanupregulationofChrna6expressionwhencomparedtotheirnonstressedcounterparts (P<0.01, inthePFC)(Figure3,panelC). It
F IGURE 3 Effectsofrepeatedexposuretopsychosocialstressfollowingacutecannabinoiddrugsadministration.Dataareexpressedasmean±SEM.(1)Effects on mice behavior:Inanopen-fieldarena,exposuretopsychosocialstressexacerbatedgeneralmotoractivityfollowingvehicleadministration(panelA-B).However,psychosociallystressedmiceexposedtotheagonistWINalonetraveledsmallerdistancesthanthevehiclegroup(P<0.01)(panelA).TheadministrationofinverseagonistRimincontrolsenhancedthetotaldistancetraveledwhencomparedtovehicle(P<0.05)(panelA).(2)Effects on endocannabinoids levels:ControlstreatedwithRimaloneunderwentanincreasein2-AGlevelswhencomparedtoeithervehicleorRim+WIN-treatedmice(P<0.05,inthePFC)(panelB).TheadministrationoftheinverseagonistRimbytheendofstressperiodreducedthelevelsoftheendocannabinoid2-AGwhencomparedtocontrolsthatreceivedthesamedrugtreatment(P<0.01,inthePFC)(panelB).(3)Effects on gene expression:TheuseofsyntheticcannabinoiddrugsdecreasedtheexpressionofChrna6 in controls(P<0.05,inthePFC)(panelC)andalsodidbutnotsignificantlywhenWINwasadministereduponstressexposurewhiletheinverseagonistRimclearlyantagonizedsucheffects(P<0.01,inthePFC)(panelC).SocialdefeatanimalscotreatedwithRim+WINortreatedwithRimaloneunderwentaremarkableupregulationofChrna6expressionwhencomparedtotheirnonstressedcounterparts(P<0.01,inthePFC)(panelC).WeassessedahighertranscriptionrateforSlc6a4geneincontrolsuponinjectionwiththeinverseagonistincomparisonwiththeremainingcontrolgroups(P<0.01,inthePFC)andalsowhenthisdrugwasadministeredinstressedanimals(P<0.01,inthePFC)(panelD).Theremainingcomparisonsaredepictedinthegraph.An*indicatessignificantdifferencesbetweenstressgroupsandtheirrespectivecontrolgroup.Controlorstressintragroupcomparisonsbetweenvehicleandtreatedmiceareindicatedbyan§.Otherwiseunderlined+indicatedintragroupcomparisonsbetweentreatmentgroupsincontrolorstressconditions.OneortwosymbolsindicateP < 0.05; P<0.01,respectively.N=15forbehavioraltesting,n=4forendocannabinoidsquantification;n=5forwhole-genomegeneexpression.PFC,prefrontalcortex;CTR,control;STS,stress
8 | TOMAS- ROIG eT Al.
wasgenerallyobservedthatinthePFC,theexpressionofSlc6a4tran-scriptswashigherinthecontrolgrouptreatedwiththeinverseagonistRimcomparedtoallthegroups(P<0.01,inthePFC,Figure3,panelD).
4 | DISCUSSION
Long-term stress confers risk to develop psychiatric disorders andsymptoms.25 Inresponsetoprolongedstress,glucocorticoidreleasehasbeenshowntohavedeleteriouseffectsondifferentbrainregionsandalsoisassociatedwiththeregressionofsynapsesanddendriticspines.26 Remarkably, dysfunctions in corticostriatal connectivityhavebeeninvolvedinthepathophysiologyofpsychiatricdisorders.27 Moreover, theGprotein-coupledCB1 is extremely abundant and akeymodulatorofthecorticostriatalpathways.10
4.1 | Effects of stress
After 2weeks of daily psychosocial stress, animals showed loss ofbodyweightinlinewithIniguezetal.28Ourdatarevealedthatchronicpsychosocial stress exacerbates scratching activity.29 The endocan-nabinoidsystemhasbeendescribedtohaveadirecteffectonsero-tonergicneurotransmissionwhichinturn,modulatesseveralbehaviorsandphysiologicalfunctions16suchasitchsensation.30Moreover,thepronouncedscratchingphenotypereportedherecouldalsobeattrib-utabletothelowerlevelsofAEAobservedinthePFC.
In fact, pharmacologicalmanipulationof thedegradingenzymesleadingtoincreasedlevelsofAEAhasshownapromisingtoolforre-ducingitchsensation.31Additionally,micewithdefectiveendocanna-binoidsignalingatCB1 receptorsdisplayedapronouncedscratchingbehavior(forreviewseeRef.[32]).
Stressiswellknowntoaffecturinarybladderfunctionandexac-erbatessignsofurinarybladderdysfunction.Wedemonstratedthatsociallydefeatedanimalsurinatedmorefrequentlyprobablycausedbyreducedbladdercapacityasdescribed.33
Adecreaseinmotoractivityisamongthechangesmostcommonlynotedinsubordinatesanddefeatedanimals.3However,wefoundthatsocialdefeatanimalstraveledlongerdistancesintheOFarena.Thesediscrepancies could be attributable to methodological differencessuchas:(i)theuseofmiceratherthanrats;(ii)housingconditions(fivemicetoacageinsteadofjustone);and(iii)thetimeattesting,asherethe stress protocol and drug administrationwere performed duringthe activephase (after 8pm) insteadof the sleepphase. Finally,wemeasuredanoverexpressionofChrna6understress.StudiesofgeneticlinkagereportedthatthecodingChrna6regionconfersrisktodevelopneuropsychiatric disorders.34 Likewise, Kimbrel etal35 reported thattwo SNPs linked to CHRNA6 conferred risk for developing post-traumaticstressdisorder.Thus,theresultsdescribedherepointoutaplausiblecontributionofChrna6tothelong-lastingeffectsobservedinsocialdefeatanimals.
4.2 | Effects of drug administration
Single administration of the agonist WIN reduced total distancetraveledwhileacutetreatmentwiththeinverseagonistRimslightlyincreaseditwhencomparedtocontrolsanobservationcorroboratedbyBrzozkaetal.3
IntheOF,micetreatedwiththeinverseagonistRimtraveledlon-gerdistancesinthecenterthantheremaininggroups,whichsupportananxiolyticeffectmediatedbythedrug,36eventhoughCB1antag-onism can also produce anxiogenic actions.37 Animals treatedwithRim+WINshowedmorefrequentrearingbehaviorthanWIN-treatedanimals. Indeed, cannabis smoke exposure induced lower rearingactivity,while theuseofRimonabantprevented thesmoke-induced decreaseinrearing.38
UponCB1activationbyWIN,weobservedanincreaseinthelevelsofbothOEAandPEA,twomediatorsmetabolicallyrelatedtoAEA.Inlinewithourresults,Bardouetal39reportedanincreaseinOEAandPEAcontentsafterinhibitingtheenzymefattyacidamidehydrolase.DespitethefactthatOEAandPEAlevelsoften,butnotalways,change
TABLE 1 Differentiallyderegulatedtop10genesinresponsetochronicpsychosocialstressandacutecannabinoidtreatment
Genes PFC DS FC Stress FC Rim FC WIN q value Stress q value Rim q value WIN
Chrna6 33.8 45.6 13.5 −13.2 −15.2 6.46E-11 0 0
Slc6a4 64.2 102.3 31.7 −34.1 −32.5 1 1 0
Ldlr 7.5 7.7 −0.2 −0.3 −0.1 0.7 0.01 1
Sdf2l1 5.6 5.3 0.5 0.5 0.2 0.002 0.02 1
Lrg1 4.1 2.5 0.5 0.7 0.5 0.1 0.02 1
Fkbp5 7.8 8.0 0.5 0.4 0.2 4.81E-06 0.03 1
Dok3 5.2 4.9 0.4 0.5 0.1 0.1 0.03 1
Hspa5 10.7 10.8 0.3 0.3 0.2 0.01 0.05 1
Manf 7.9 7.9 0.2 0.2 0.1 0.01 0.05 1
Hspa1a 6.8 6.0 0.3 0.4 0.2 0.03 0.05 1
Columntitlesfromlefttoright:officialgenesymbol;PFC,prefrontalcortex;dorsalCPu,dorsalstriatum;FCstress,foldchangestress;FCRim,foldchangeRimonabant;FCWIN,foldchangeWIN55212.2;qvalueStress,PadjustvalueStress;qvalueRim,PadjustvalueRimonabant;qvalueWIN,PadjustvalueWIN55212.2.
| 9TOMAS- ROIG eT Al.
inasimilarmannertothoseofAEAunderphysiologicalorpathologicalconditions,wedidnotobservehereanysimilarchangesinAEAlevelsfollowingchronicpsychosocialstress.Thisfindingiscompatiblewiththegeneralconcept thatOEAandPEAarenoteCBs inasmuchastheydonotactivatedirectlyCB1andCB2receptors,butinsteadmod-ulatenoncannabinoidreceptors,suchasGprotein-coupledreceptor55 (GPR55), peroxisome proliferator-activated receptor-α (PPAR-α),and transient receptor potential vanilloid type-1 (TRPV1) channels.Recently,MusellaetalreportedthatsignificantlevelsofPEAarepres-entinthestriatum,wherethismediatorenhancesGABAergicneuro-transmissionviaGPR55.Interestingly,PEAwaspreviouslyreportedtoexhibitgreaterbindingaffinityforGPR55thanCB1,CB2,PPAR-α,andTRPV1(forreviewseeRef.[40]).Thus,it isnoteworthytospeculatethatexposuretocannabinoiddrugs,byalteringPEAlevels,modifiesGPR55receptoractivity.
It iswell known that theglucocorticoid receptor is regulatedbyseveralchaperonesandcochaperonesincludingtheFKBP5protein.41 Inparticular,wefoundthattheadministrationoftheCB1inverseag-onist Rimonabant potentiated the expression of Fkbp5 when com-paredtotheremaininggroups.Thisisinagreementwithdatashowingthat FKBP5 regulates glucocorticoid receptor sensitivity, increasesitsresistance,anddecreasesitsefficiencyatcontrollingthenegativefeedbackinresponsetoelevatedlevelsofcorticosterone,42whereasRimonabant canaggravate thehyperactivityof theHPAaxisduringstress.43SeveralstudiesattesttotheideathatFKBP5iscrucialforthedevelopmentofstress-relatedmentaldisorders (44,45).However, it iscurrentlyunclearhowtheactivationoftheendocannabinoidsystemthrough theuseof syntheticcannabinoiddrugsmay regulateeitherFkbp5orglucocorticoid signalingaspointedoutbyWangetal.46 In conclusion,ourunderstandingofFKBP5functionsisstill incompleteandthus,furtherworkmustaddressthisquestion.
4.3 | Effects of stress under drug influence
TheinverseagonistRimonabantinnonstressconditionsantagonizedtheeffectsofWIN55212.2ontotaldistancetravelled.3Underexperi-mentallyinducedpsychosocialstress,micetreatedwitheitherWINorRim+WINshowedlesstotaldistancetraveledthanthevehiclegroup,while single Rimonabant administration antagonized the effects ofWIN.3Wealsoobservedthatstressedmiceexposedtovehicledis-playedanexacerbatedtotaldistancetraveledwhencomparedtotheirnonstressedcounterparts.Againstthepresentfindings,Brzozkaetal3 foundthatrepeatedexposuretosocialdefeatparadigmreducedthetotaldistancetraveledintheOF.Thedifferencesreportedherecouldbeexplainedbytheinfluenceofthecircadianrhythmandthehous-ingconditions(5micepercageinsteadofjustone).Indeed,micearenocturnalanimalsmainlyactiveduringthedarkperiod.5Furthermore,theabsenceofsocialsupport(individualhousing)amplifiestheanxietyinducedbypsychosocialstress.47
It is widely accepted that upon HPA activation, the content ofendocannabinoid lipid molecules varies according to the duration/typeof stress stimuli and thebrain structure.We found that socialdefeat animals exposed tovehicle showed similar 2-AG levels than
theircontrolsinagreementwith.48Incontrast,Gorzalkaetal49deter-mined that after chronic stress, the endocannabinoid signalingwascompromised because endocannabinoid levels were reduced in alllimbicstructures.Chronicrestraintstress,forexample,progressivelyincreases2-AGcontentwithindistinctanatomicalregionssuchasthemedialPFC, limbicforebrain,amygdala,hippocampus,andhypothal-amus.50Thesedifferencescouldbeexplainedbytheinfluenceofthecircadianrhythmandalsorelatedtothestressprotocolused.Infact,thereisastrongevidencetosuggestthat inrodentscircadianalter-ationsoccurinendocannabinoidbrainlevels,51CB1expression,
52 and theactivityofenzymescontrolling themetabolismofendocannabi-noids.51TheadministrationofRimonabant incontrolsaswellasthepharmacological blockadeofMAGLenzymehasbeen reported in apowerful increaseof2-AG levels in theCNS.53Here, such increasewasalsoobservedwhenbothcannabinoidswereadministeredbutnotwhenRimwasinjectedalone.Thus,theuseofsyntheticcannabinoidsinthismodelwarrantsfurtherinvestigationinordertoelucidatetheunderlyingmechanismsassociatedwiththemetabolismof2-AG.
Notsurprisingly,wefoundthatthecannabinoidagonistWINandthe inverse agonist Rim influenced the expressions of Chrna6 and Slc6a4 in opposite directions, while their coadministration tendedto produce a net null effect under experimentally induced psycho-social stress. Nicotinic receptor containing α6 subunit (CHRNA6) isexpressedindistinctbrainregionsimportantforaddictionbehaviorsandalsoisidentifiedtoconfersusceptibilitytoneuropsychiatricdisor-ders.34Inlinewiththisreport,emergingdatapointoutthatthecodingregionforCHRNA6confersvulnerabilitytodrugsofabuseandtheirrelatedbehavioralphenotypes.54Interestingly,thepresentinvestiga-tionisthefirsttodemonstratethatsimultaneousexposuretodistinctenvironmental factors suchaschronic stressandacutecannabinoiddrugadministrationcan regulate theexpressionof theChrna6genewhichinturnandcouldbecrucialforthedevelopmentofcannabisad-dictionandpsychosis.Long-termexposuretostressconstitutesakeyenvironmentalriskcomponentfordevelopingstress-relateddisordersin susceptible individuals. The underlying mechanisms disrupted inthesedisordersareserotonergic55 and endocannabinoid56dependent.Particularly,serotoninreleaseinhibitionisattributabletothe lackofCB1 receptorswhich in turn, promotes higher concentration of thisneurotransmitterinthesynapticcleft.57Then,wecanspeculatethatCB1blockagebytheadministrationofRimcouldcounteracttheex-cessiveserotoninneurotransmitterbyincreasingtheexpressionoftheserotonintransporterprotein(Slc6a4).58Furthermore,SLC6A4singlenucleotide polymorphisms have been associatedwith stress-relatedpsychiatricdisorders,59whichareconsistentwithourfindings.
5 | CONCLUSIONS
Insummary,thedatadescribedherehighlighttheeffectsoflong-termexposuretostressonmousecorticostriatalcircuitriesfollowingacutechallengewithdistinctcannabinoiddrugs(ForfurtherdetailsseeFigureS2).Particularly,wefoundthatpsychologicalstressplayedaroleintheexacerbationofmicturitionfrequency,anxiety-relatedbehavior,58 and
10 | TOMAS- ROIG eT Al.
scratchingphenotype,29whiletheuseofsyntheticcannabinoidsdrugsinterferedwithlocomotoractivity,rearing,andanxiety-likebehavior.38 The glucocorticoid receptor is regulated by several chaperones andcochaperonesincludingtheFKBP5protein41butalsoisinfluencedbyendocannabinoid system.17Additionally,wedemonstrated thatpsy-chosocially stressed animals displayed changes in the serotonergicsystem upon acute exposure to synthetic cannabinoid drugs.55 ThecodingregionforCHRNA6confersvulnerabilitytodrugsofabuseandtheirrelatedbehavioralphenotypes,54whichinturn,couldbecrucialforthedevelopmentofcannabisaddictionandpsychosis.OurfindingsinbothPFCanddorsalCPuneedtobecorroboratedbyfurtherstudiesifwewanttounderstandtheroleofCB1 and endocannabinoids and relatedmediatorsincorticostriatalsignalingunderpsychosocialstress.
ACKNOWLEDGMENTS
WethankProf.LutzfromUniversityMedicalCenteroftheJohannesGutenbergUniversity(Germany)forsupplyingtheCB1riboprobe.TheauthorswouldliketothankProf.Colomina,Dr.Yang,andMr.Ludewigforexcellenttechnicalsupport.Dr.GilwassupportedbyJuande laCierva postdoctoral fellowship of MINECO. Dr. Tomas-Roig wassupported by Deutsche Forschungsgemeinschaft fellowship (GrantTO 977/1-1). The work was supported by DFG Research Centerfor Nanoscale Microscopy and Molecular Physiology of the Brain(CNMPB)andtheGerman-Research-Foundation(DFG)grantCNMPBC1-6toProf.Havemann-Reinecke.
CONFLICTS OF INTEREST
Theauthorreportsnopotentialconflictofinterests.
ORCID
Jordi Tomas-Roig http://orcid.org/0000-0002-8997-3436
REFERENCES
1. vanOsJ,KenisG,RuttenBPF.Theenvironmentandschizophrenia.Nature.2010;468:203-212.
2. FornitoA,HarrisonBJ,GoodbyE,et al.Functionaldysconnectivityof corticostriatal circuitry as a risk phenotype for psychosis. JAMA Psychiatry [Internet].2013;70:1143-1151.
3. BrzozkaMM,FischerA,FalkaiP,Havemann-ReineckeU.Acutetreat-ment with cannabinoid receptor agonist WIN55212.2 improvesprepulse inhibition inpsychosociallystressedmice.Behav Brain Res. 2011;218:280-287.
4. SapolskyR.Tamingstress.Sci Am.2003;289:86-95. 5. deKloetER.Stressinthebrain.Eur J Pharmacol.2000;405:187-198. 6. DuvalER,JavanbakhtA,LiberzonI.Neuralcircuitsinanxietyandstress
disorders:afocusedreview.Ther Clin Risk Manag.2015;11:115-126. 7. BlairKS,GeraciM,OteroM,etal.Atypicalmodulationofmedialpre-
frontalcortextoself-referentialcommentsingeneralizedsocialpho-bia. Psychiatry Res.2011;193:38-45.
8. SripadaC,AngstadtM,LiberzonI,McCabeK,PhanKL.Aberrantre-wardcenterresponsetopartnerreputationduringasocialexchangegameingeneralizedsocialphobia.Depress Anxiety.2013;30:353-361.
9. BoychukCR,ZsombokA,TaskerJG,SmithBN.Rapidglucocorticoid-induced activation of TRP and CB1 receptors causes biphasicmodulation of glutamate release in gastric-related hypothalamicpreautonomicneurons.Front Neurosci. 2013;7:3.
10. HerkenhamM,LynnAB,JohnsonMR,MelvinLS,deCostaBR,RiceKC. Characterization and localization of cannabinoid receptors inrat brain: a quantitative in vitro autoradiographic study. J Neurosci [Internet].1991;11:563-583.
11. Lovinger DM, Davis MI, Costa RM. Handbook of Basal Ganglia Structure and Function. Steiner H, Tseng K, eds. London:AcademicPress;2010:693.
12. DevaneWA,Hanus L, BreuerA, et al. Isolation and structure of abrain constituent that binds to the cannabinoid receptor. Science. 1992;258:1946-1949.
13. Sugiura T, Kondo S, Sukagawa A, et al. 2-arachidonoylglycerol: apossibleendogenous cannabinoid receptor ligand inbrain.Biochem Biophys Res Commun [Internet].1995;215:89-97.
14. BasavarajappaBS.Criticalenzymesinvolvedinendocannabinoidme-tabolism.Protein Pept Lett.2007;14:237-246.
15. DiMarzoV.Endocannabinoids:synthesisanddegradation.Rev Physiol Biochem Pharmacol.2008;160:1-24.
16. LazaryJ,JuhaszG,HunyadyL,BagdyG.Personalizedmedicinecanpavethewayfor thesafeuseofCB1receptorantagonists.Trends Pharmacol Sci.2011;32:270-280.
17. Tasker JG, Di S, Malcher-Lopes R. Minireview: rapid glucocorti-coid signaling via membrane-associated receptors. Endocrinology. 2006;147:5549-5556.
18. Golub MS, Germann SL, Lloyd KC. Behavioral characteristics of anervous system-specific erbB4 knock-out mouse. Behav Brain Res. 2004;153:159-170.
19. Matias I, PetrosinoS,RacioppiA,CapassoR, IzzoAA,DiMarzoV.Dysregulationofperipheralendocannabinoidlevelsinhyperglycemiaandobesity:effectofhighfatdiets.Mol Cell Endocrinol.2008;286(1–2suppl1):S66-S78.
20. BisognoT,MartireA,PetrosinoS,PopoliP,DiMarzoV.Symptom-relatedchangesofendocannabinoidandpalmitoylethanolamidelev-els inbrainareasofR6/2mice,atransgenicmodelofHuntington’sdisease. Neurochem Int.2008;52:307-313.
21. Pruitt KD, Brown GR, Hiatt SM, et al. RefSeq: an update onmammalian reference sequences. Nucleic Acids Res [Internet]. 2014;42:D756-D763.
22. LiH,DurbinR.FastandaccurateshortreadalignmentwithBurrows-Wheelertransform.Bioinformatics.2009;25:1754-1760.
23. Anders S, HuberW. Differential expression analysis for sequencecountdata.Genome Biol. 2010;11:R106.
24. BenjaminiY,HochbergY.Controllingthefalsediscoveryrate:aprac-tical and powerful approach tomultiple testing. J R Stat Soc Ser B [Internet].1995;57:289-300.
25. HoltzmanCW,TrotmanHD,GouldingSM,etal.Stressandneurode-velopmentalprocessesintheemergenceofpsychosis.Neuroscience. 2013;249:172-191.
26. McEwenBS.Theever-changingbrain:cellularandmolecularmech-anisms for the effects of stressful experiences. Dev Neurobiol. 2012;72:878-890.
27. ShepherdGMG.Corticostriatal connectivity and its role indisease.Nat Rev Neurosci [Internet].2013;14:278-291.
28. IñiguezSD,RiggsLM,NietoSJ,etal.Socialdefeatstress inducesadepression-likephenotype inadolescentmalec57BL/6mice.Stress [Internet].2014;17:247-255.
29. ZhaoP,HiramotoT,AsanoY,KuboC,SudoN.Chronicpsychologicalstressexaggeratesthecompound48/80-inducedscratchingbehaviorofmice.Pharmacol Biochem Behav.2013Apr;105:173-176.
30. WeisshaarE,ZiethenB,RöhlFW,GollnickH.Theantipruriticeffectofa5-HT3receptorantagonist(tropisetron)isdependentonmastcelldeple-tion—anexperimentalstudy.Exp Dermatol [Internet].1999;8:254-260.
| 11TOMAS- ROIG eT Al.
31. TosunNC,GunduzO,UlugolA.Attenuationofserotonin-induceditchresponses by inhibition of endocannabinoid degradative enzymes,fatty acid amide hydrolase and monoacylglycerol lipase. J Neural Transm.2014;122:363-367.
32. CaterinaMJ.TRPchannelcannabinoidreceptorsinskinsensation,ho-meostasis,andinflammation.ACS Chem Neurosci.2014;5:1107-1116.
33. MinginGC,PetersonA,EricksonCS,NelsonMT,VizzardM.Socialstressinduceschangesinurinarybladderfunction,bladderNGFcon-tent,andgeneralizedbladderinflammationinmice.Am J Physiol Regul Integr Comp Physiol.2014;307:R893-R900.
34. Tabarés-Seisdedos R, Rubenstein JLR. Chromosome 8p as a po-tential hub for developmental neuropsychiatric disorders: implica-tions forschizophrenia,autismandcancer.Mol Psychiatry [Internet]. 2009;14:563-589.
35. KimbrelNA,GarrettME,DennisMF,etal.Effectofgeneticvariationinthenicotinicreceptorgenesonriskforposttraumaticstressdisor-der. Psychiatry Res.2015;229:326-331.
36. LafenêtreP,ChaouloffF,MarsicanoG.Theendocannabinoidsystemin the processing of anxiety and fear and howCB1 receptorsmaymodulatefearextinction.Pharmacol Res.2007;56:367-381.
37. KomakiA,AbdollahzadehF,SarihiA,ShahidiS,Salehi I. Interactionbetween antagonist of cannabinoid receptor and antagonist ofadrenergic receptor on anxiety in male rat. Basic Clin Neurosci. 2014;5:218-224.
38. Bruijnzeel AW, Qi X, Guzhva LV, et al. Behavioral characterizationoftheeffectsofcannabissmokeandanandamideinrats.PLoS ONE. 2016;11:e0153327.
39. Bardou I, DiPatrizio N, Brothers HM, et al. Pharmacological ma-nipulation of cannabinoid neurotransmission reduces neuroinflam-mation associated with normal aging. Health (Irvine Calif) [Internet]. 2012;4:679-684.
40. MusellaA,FresegnaD,RizzoFR,etal.AnovelcrosstalkwithintheendocannabinoidsystemcontrolsGABAtransmissioninthestriatum.Sci Rep. 2017;7:7363.
41. Wochnik GM, Rüegg J, Abel GA, Schmidt U, Holsboer F, Rein T.FK506-bindingproteins51and52differentiallyregulatedynein in-teractionandnucleartranslocationoftheglucocorticoidreceptorinmammalian cells. J Biol Chem.2005;280:4609-4616.
42. BinderEB.TheroleofFKBP5,aco-chaperoneoftheglucocorticoidreceptorinthepathogenesisandtherapyofaffectiveandanxietydis-orders. Psychoneuroendocrinology.2009;34(suppl1):S186-S195.
43. BlasioA, IemoloA,SabinoV,etal.Rimonabantprecipitatesanxietyinratswithdrawnfrompalatablefood:roleofthecentralamygdala.Neuropsychopharmacology [Internet].2013;38:2498-2507.
44. Xie P, Kranzler HR, Poling J, et al. Interaction of FKBP5 withchildhood adversity on risk for post-traumatic stress disorder.Neuropsychopharmacology.2010;35:1684-1692.
45. ZimmermannP,BrücklT,NoconA,etal.InteractionofFKBP5genevariants andadverse life events inpredictingdepressiononset: re-sultsfroma10-yearprospectivecommunitystudy.Am J Psychiatry. 2011;168:1107-1116.
46. Wang M, Hill MN, Zhang L, Gorzalka BB, Hillard CJ, Alger BE.Acute restraint stress enhances hippocampal endocannabinoidfunction via glucocorticoid receptor activation. J Psychopharmacol. 2012;26:56-70.
47. AdamcioB,Havemann-ReineckeU,EhrenreichH.Chronicpsychoso-cialstressintheabsenceofsocialsupportinducespathologicalpre-pulseinhibitioninmice.Behav Brain Res.2009;204:246-249.
48. WangW,SunD,PanB,etal.Deficiencyinendocannabinoidsignalingin thenucleus accumbens inducedbychronicunpredictable stress.Neuropsychopharmacology.2010;35:2249-2261.
49. GorzalkaBB,HillMN,HillardCJ.Regulationofendocannabinoidsig-naling by stress: implications for stress-related affective disorders.Neurosci Biobehav Rev.2008;32:1152-1160.
50. PatelS,KingsleyPJ,MackieK,MarnettLJ,WinderDG.Repeatedho-motypic stresselevates2-arachidonoylglycerol levelsandenhancesshort-termendocannabinoidsignalingatinhibitorysynapsesinbaso-lateralamygdala.Neuropsychopharmacology.2009;34:2699-2709.
51. ValentiM,ViganòD,CasicoMG,etal.Differentialdiurnalvariationsofanandamideand2-arachidonoyl-glycerollevelsinratbrain.Cell Mol Life Sci.2004;61:945-950.
52. Rueda-OrozcoPE,Soria-GomezE,Montes-RodriguezCJ,etal.Apo-tentialfunctionofendocannabinoidsintheselectionofanavigationstrategybyrats.Psychopharmacology.2008;198:565-576.
53. ChandaPK,GaoY,MarkL,etal.Monoacylglycerollipaseactivityisacriticalmodulatorofthetoneand integrityoftheendocannabinoidsystem.Mol Pharmacol [Internet].2010;78:996-1003.
54. SadlerB,HallerG,AgrawalA,etal.VariantsnearCHRNB3-CHRNA6areassociatedwithDSM-5cocaineusedisorder:evidenceforpleiot-ropy.Sci Rep [Internet].2014;4:4497.
55. InoueT,TsuchiyaK,KoyamaT.Regionalchangesindopamineandse-rotoninactivationwithvariousintensityofphysicalandpsychologicalstressintheratbrain.Pharmacol Biochem Behav.1994;49:911-920.
56. BurokasA,Martín-GarcíaE,Gutiérrez-CuestaJ,etal.Relationshipsbe-tweenserotonergicandcannabinoidsystemindepressive-likebehav-ior:aPETstudywith[11C]-DASB.J Neurochem.2014;130:126-135.
57. RudnickG.Serotonintransporters—structureandfunction.J Membr Biol.2006;213:101-110.
58. KargK,BurmeisterM,SheddenK,SenS.Theserotonintransporterpromoter variant (5-HTTLPR), stress, and depressionmeta-analysisrevisited: evidence of genetic moderation. Arch Gen Psychiatry. 2011;68:444-454.
59. SmithAL, Leung J, Kun S, et al. The effects of acute and chronicpsychologicalstressonbladderfunctioninarodentmodel.Urology. 2011;78:967.e1-7.
SUPPORTING INFORMATION
Additional Supporting Information may be found online in thesupportinginformationtabforthisarticle.
How to cite this article:Tomas-RoigJ,PiscitelliF,GilV,etal.Effectsofrepeatedlong-termpsychosocialstressandacutecannabinoidexposureonmousecorticostriatalcircuitries:Implicationsforneuropsychiatricdisorders.CNS Neurosci Ther. 2018;00:1–11. https://doi.org/10.1111/cns.12810