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:jtomas@idibgi.org

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.

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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

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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),

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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.

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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

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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

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(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

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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