The endocannabinoid system in mental disorders: Evidence from human brain studies

BiochemicalPharmacology157(2018)97–107 Review Theendocannabinoidsysteminmentaldisorders:Evidencefromhumanbrainstudies InésIbarra‐Lecuea,b,1,FuencislaPilar‐Cuéllarb,c,d,1,CarolinaMuguruzaa,b,EvaFlorensa‐Zanuyb,c,d, ÁlvaroDíazb,c,d,LeyreUrigüena,b,e,ElenaCastrob,c,d,AngelPazosb,c,d,LuisF.Calladoa,b,e, aDepartmentofPharmacology,UniversityoftheBasqueCountryUPV/EHU,Leioa,Spain bCentrodeInvestigaciónBiomédicaenReddeSaludMentalCIBERSAM,Spain cInstitutodeBiomedicinayBiotecnologíadeCantabria(IBBTEC);UniversidaddeCantabria‐ CSIC,Santander,Spain dDepartamentodeFisiologíayFarmacología,FacultaddeMedicina,UniversidaddeCantabria, Santander,Spain eBiocrucesHealthResearchInstitute,Bizkaia,Spain Correspondingauthorat:DepartmentofPharmacology,MedicalSchool,Universityofthe BasqueCountry(UPV/EHU),BarrioSarrienas/n,48940Leioa,Bizkaia,Spain. E‐mailaddress:L[email protected](L.F.Callado). 1Theseauthorscontributedequallytothiswork. https://doi.org/10.1016/j.bcp.2018.07.009Received29May2018;Accepted12July2018 Keywords:Humanbrain,Depression,Anxiety,Schizophrenia,Endocannabinoids,Cannabinoid receptors ABSTRACT Mentaldisordershaveahighprevalencecomparedwithmanyotherhealthconditionsandare theleadingcauseofdisabilityworldwide.Severalstudiesperformedinthelastyearssupport theinvolvementoftheen‐docannabinoidsystemintheetiopathogenesisofdifferentmental disorders.Thepresentreviewwillsummarizethelatestinformationontheroleofthe endocannabinoidsysteminpsychiatricdisorders,specificallydepression,anxiety,and schizophrenia.Wewillfocusonthefindingsfromhumanbrainstudiesregardingalterationsin endocannabinoidlevels,cannabinoidreceptorsandendocannabinoidmetabolizingenzymesin patientssufferingmentaldisorders. Studiescarriedoutinhumanshaveconsistentlydemonstratedthattheendocannabinoidsystem isfunda‐mentalforemotionalhomeostasisandcognitivefunction.Thus,deregulationofthe differentelementsthatarepartoftheendocannabinoidsystemmaycontributetothe pathophysiologyofseveralmentaldisorders.However,theresultsreportedarecontroversial. Inthissense,differentalterationsingeneand/orproteinex‐pressionofCB1receptorshave beenshowndependingonthetechnicalapproachusedorthebrainregionstudied.Despitethe currentdiscrepanciesregardingcannabinoidreceptorschangesindepressionandschizo‐ phrenia,presentfindingspointtotheendocannabinoidsystemasapivotalneuromodulatory pathwayrelevantinthepathophysiologyofmentaldisorders. This is a postprint of an article published by Elsevier . The final version of Inés Ibarra-Lecue, Fuencisla Pilar-Cuéllar, Carolina Muguruza, Eva Florensa-Zanuy, Álvaro Díaz, Leyre Urigüen, Elena Castro, Angel Pazos, Luis F. Callado, The endocannabinoid system in mental disorders: Evidence from human brain studies, Biochemical Pharmacology 157 : 97-107 (2018) is available at https://doi.org/10.1016/j.bcp.2018.07.009 © 2018 This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 1. Introduction Mentaldisordersareresponsibleforthelargestproportionoftheglobalburdenofdisease worldwide.Ithasbeensuggestedthatby2030depressionwillbetheleadingcauseofdisease burdenglobally.Inthisway,mood‐relateddisorderscontributemostofthenon‐fatalburdenof mentalillnessfollowedbyanxiety‐relateddisorders,substanceabuseandschizophrenia[1]. Theypresentamajormedical,societalandeconomicburdenthathasalargeimpacton individuals,familiesandcommunities. Actualknowledgeabouttheetiologyandpathophysiologyofmentaldisordersismainlyaresult ofaninteractionbetweenthedevelopmentofnewtechnologyandthedirectstudyofthebrain tissueofpatients.Thus,thedescriptionofmorphologicaldifferences,functionaldeficitsand molecularalterationsiswidelyacceptedtodayasexistinginthebrainofpsychiatricpatientsdue totheadvanceofinvivoneuroimagingtechniques,geneticandgenomicdevelopment,andthe useofpost‐mortembraintissueasakeysubstrateofthedisease[2].Nevertheless,despitethe hugeeconomicandscientificeffortdevelopedinthelastdecades,thepathophysiologyof mentaldisordersremainselusive.Inthiscontext,manystudieshavefocusedinthepossible involvementofalterationsoftheendocannabinoidsystem(ECS)inthepathophy‐ siologyof mentaldisorderssuchasdepressionorschizophrenia.TheECSparticipates,inpart,inthe controlofemotionalbehaviorandmoodthroughafunctionalcouplingwithmonoaminergic systemsinthebrain[3].ThesefunctionalinteractionshavesuggestedapotentialroleforECS signalingintheneurobiologyofvariouspsychiatricdisorders[4–7].TheECSiscomposedoftwo inhibitoryG‐proteincoupledre‐ceptors(GPCRs),cannabinoidreceptor1and2(CB1andCB2, respec‐ tively),andtwomajorendogenousligands,N‐arachidonoylethanola‐ mine (anandamide/AEA)and2‐arachidonoylglycerol(2‐AG).TheECSalsoincludestwomain metabolicenzymes,thefattyacidamidehy‐drolase(FAAH)andthemonoacylglycerollipase (MAGL)whichhy‐drolyzeAEAand2‐AG,respectively;andtwomainsynthetizingen‐zymes,N‐ acylphosphatidylethanolamine‐phospholipaseD(NAPE‐PLD)andthediacylglycerollipase (DAGL)whichsynthesizeAEAand2‐AG,respectively.Thecorrectinterplaybetweenallthese ECSelementsplaysanimportantroleincentralnervoussystem(CNS)development,sy‐naptic plasticity,andthehomeostaticmaintenanceofcognitive,beha‐ vioral,emotional, developmental,andphysiologicalprocesses[8,9].Inthebrain,CB1receptorsarepresentin GABAergicandglutamatergicneurons,exertingapresynapticinhibitoryfunctionwhentheyare ac‐tivatedbythereleasedendocannabinoids[10,11].TheyarethemostabundantG‐protein coupledreceptorsandarewidelyexpressedallthroughoutthebrain,beinglocatedincortical, subcortical,cerebellarandbrainstemstructures[8].TheCB2receptorsarelessnumerousand wereinitiallythoughttobelocatedmainlyintheimmunesystem;however,currentlytheyseem tobewidelydistributedintheCNS,takingpartinimmune‐mediatedresponsesandsupporting aneuro‐protectiveroleagainstinflammation[12].Thetwomainendogenousligands,AEAand 2‐AG,areeicosanoidneuromodulatorylipidsderivedfrommembranephospholipids, synthesizedwhenandwheretheyarerequired,andactingpresynapticallyonbothtypeof cannabinoidre‐ceptors[8]. Inthepresentreview,wewillsummarizedataobtainedfromhumanstudiesprovidingevidence abouttheroleofthedifferentECScompo‐nents(endocannabinoids,metabolizing/synthetizing enzymesandcannabinoidreceptors)inthepathophysiologyandtreatmentofseveral psychiatricdisorders,withafocusonresultsfrompostmortemandlivinghumanbrainstudies. Wewillreviewfindingsfrompatientssuf‐feringamood‐relateddisorder(depression,anxiety, posttraumaticstressdisorder(PTSD))orschizophreniacomparedtohealthysubjects. 2. Theendocannabinoidsystemandtheemotionalhomeostasis TheECSinfluencestheactivityofmultiplebrainareasinvolvedintheregulationofthe hypothalamic‐pituitary‐adrenalsystem(HPA),mood,anxietyandotherrelatedbehaviors(i.e. extinctionoffearlearning,reward…).Indeed,theECSenablestheefficientinteractionwithin andbetweenbrainregionsthatmodulatecognitiveandbeha‐vioralfunctioning. Aconsiderablenumberofstudiessuggesttherelationshipbetweenchangesinoneormore componentsoftheECSandsomeofthesymptomsthatarepresentindepressionandanxiety‐ relateddisorders.TheECSmodulatesfearandanxiety‐relatedbehaviorsinbothhumansand rodents[13–15].AugmentedECSsignalingisusuallyfollowedbyreducedconditionedfearand anxiety,whereastheoppositeeffectisobservedwhenitisinhibited[16–19].Thisisnot surprisingsincetheECSispresentinkeystructureswithinthebrainsuchasprefrontalcortex (PFC),amygdala,andhippocampus[20–25]. TherearealsoanimalstudiesshowingthecorrelationbetweenCB1receptor‐deficiencyand depressive/comorbidsymptoms(anhedonia,anxiety,andheightenedstress‐response)[26–28]. Inlinewiththisre‐lationship,chronicstress,asapathogenicfactorfordepressive‐beha‐vior, hasbeenassociatedwithadysfunctionalendocannabinoidsig‐nalinginthebrain[29].Thus, strategiesthataredirectedtotheaugmentationoftheendocannabinoidsignalingarereported tomiti‐gatemanyoftheadverseeffectsofchronicstress,suchasanhedoniaandanxiety[30– 32].Thereadersaredirectedtocomprehensivere‐viewsonthistopicthatisbeyondthescope ofthisreview[33–38]. 3. Theendocannabinoidsystemanddepression Depressionisoneofthemostprevalentmajorneuropsychiatricdiseases,affecting20%ofthe population,beingalmosttwiceascommoninfemalesthanmales[39].Therearetwomain challengestofightagainstdepression.First,westillpoorlyunderstanditsneuro‐biologicaland pathologicalbases.Second,thereneedstobemoreef‐fectiveantidepressantdrugsovercoming thetherapeuticlagbetweendrugadministrationandtheonsetofclinicalimprovement,thelack ofresponseinsomepatientsandsafety/tolerabilityissues[40,41]. TheimplicationoftheECSindepressioncomesfromobservationalfindingsregardingthemood‐ relatedeffectsofcannabisinhumans,thoughcontradictoryresultsarereported.Ononehand, theheavyuseofcannabisisassociatedtoahigherincidenceofdepressivedisorders[42].A recentmeta‐analysisshowedapositivecorrelationbetweencannabisuseanddepression,being moreevidentamongheavycan‐nabisusers[43].Moreover,theabuseofcannabishasbeen linkedtoahigherriskofsuicideinpatientswithmooddisorders[44].Ontheotherhand,other authorsindicatethattheuseofmarijuana,oritsmainpsychoactivecomponentΔ9‐ tetrahidrocannabinol(THC),reducesthedepressivebehavior[45–48].Inaddition,otherauthors describethattheadministrationofTHCtopatientswithmoderatetoseverede‐pressionshows alackofeffectonmood[49],oronthesuicidalideation[50],butanincreasedanxiety[49]. Curiously,thepharmacologicalblockadeofCB1receptorsusingantagonistsorinverseagonists wasinitiallysuggestedasapotentialnoveltargetforantidepressanttreatment,accordingto differentevi‐dencesinpreclinicalstudies[51].However,furtherstudiesrevealedthatdrugsas theCB1receptorantagonistrimonabant(SR141716A)thatwasmarketedtotreatobesity, induceddepressedmood[52,53].Inanimalstudies,thepharmacologicalactivationorblockade ofCB1re‐ ceptorsalsogiverisetocontradictoryresults,sincebothapproachesleadtoan antidepressant‐likeeffect[5,51,54–58]. Althoughthesestudiesdrawconflictingfindings,theypointtotheinvolvementofthebrainECS inthemodulationofmoodand,espe‐ cially,thecontributionofCB1receptorstomajor depressionhavere‐ceivedparticularattention[59].Theactivationofcannabinoidre‐ceptors producesthereleaseofstresshormonesasACTHandcortisolintheHPAaxis[60],whichhas beenobservedfollowingacutemarijuanaadministration[61].However,thispresentstolerance afterchronicadministration[62],astheTHC‐inducedcortisolreleaseisbluntedinfrequent marijuanausers[63]. Neuroimagingstudiesinnon‐cannabisusersshowthattheadmin‐istrationofTHCproducesa reducedactivationofsomebrainareasinresponsetoanegativecontent[64,65].Conversely, thereisanin‐creasedactivationinresponsetoapositivecontent,mediatedbytheactivation ofareassuchasprefrontalandoccipitalcortices,amygdala,hippocampusandorbitofrontal gyrus[65],whichisassociatedtoareductioninthenegativeattentionalbias,andthe potentiationofpo‐sitiveattentionalbias[65].Thistypeofstudieshavealsoshownthatthe activationofcannabinoidreceptorsleadstomorphologicalchanges,suchasthereductionin whitematter(WM)observedinmarijuanausersthatnegativelycorrelateswiththeseverityof thedepressivedisorder[66,67].ThisdecreaseinWMvolumehasbeenassociatedwiththe presenceofcannabinoidreceptorsinoligodendrocytes,themyelin‐formingcells[68]. 3.1. Cannabinoidreceptorsindepression HumanstudieshavecorroboratedtheexistenceofanalteredECSactivityassociatedtomajor depression[34,69].TheCB1receptordensity[70]andmRNA[71]isincreasedinthedorsolateral prefrontalcortexofpatientswithmajordepression,inparalleltoCB1receptorfunctionality (evaluatedby[35S]GTPγSbindingstudies)[70](Matoetal.,inthisissue).However,nochanges inCB1immunoreactivityinthedorsolateralprefrontalcortex[72],orareductionin CB1immunoreactivityinglialcellsintheanteriorcingulatecortex[73]havebeenreported (Table1).RegardingCB2receptor,nochangeshavebeendetectedinitsmRNAlevelsin prefrontalcortexofdepressedpa‐tients[71](Table1). TheroleoftheECSintheeffectofantidepressantdrugshasalsobeenevidencedinstudies reportinganincreasedCB1receptorexpres‐sion[74],andalackofchangesinCB1‐mediated activationofGi/oproteinsinprefrontalcortex(Matoetal.,inthisissue)intheanti‐depressant‐ treatedgroup.OtherareassuchasthehippocampusonlyelicitedincreasedCB1receptor densityafterchronicmonoamineoxi‐ daseinhibitors(MAOI)treatment[74].Oncontrast, studiesinhumanbrainsampleshaveshownareductioninCB1receptorim‐munoreactivityin theanteriorcingulatecortexofpatientstreatedwithserotoninselectivereuptakeinhibitors (SSRIs)[73]. Thepresenceofdifferentsinglenucleotidepolymorphismsinthecannabinoidreceptor1(CNR1) geneappearstomodulateeitherthedepressivephenotype,and/ortheresponseto antidepressanttreatment.ThecarriersofCNR1genevariantsinfluencesthevulnerabilityto suffermentaldisorders,includingmajordepression[75].ThefrequencyoftheGalleleofthe CNR1genepolymorphismrs806371ishigherinpatientswithmajordepressionshowing comorbidpsychoticsymptoms,whilethehaplotypeC‐G‐T(rs806368,rs1049353,rs806371)is associatedwithanincreasedriskformelancholicandpsychoticsymptomsofmajordepression [76].Thisisconsistentwiththemelancholicde‐ pressive‐likesymptomsobservedinanimal modelswithpharmacolo‐gicalorgeneticblockadeoftheECS[4].Otherstudiesreportalower incidenceofdepressioninParkinsons’diseasepatientscarryingtwolongallelesoftheCNR1 genepolymorphism(AAT)n[77].TheCallelecarriersoftheCNR1genepolymorphism rs2023239presentalowerincidenceofmajordepressionwithinagroupofmethadone‐ responderpatients[78]. InpatientswiththeCNR1genepolymorphismrs1049353,carriersofoneormorecopiesofthe minorallele(AA/AG)exhibitabufferingeffecttoanhedoniaanddepressionafterearlychildhood trauma[79].Moreover,theGalleleofthers1049353polymorphismisassociatedtothe resistancetotheantidepressanttreatmentinfemalesdiagnosedwithmajordepressionthat presentcomorbidanxiety[80].PatientswiththeGalleleoftheCNR1rs1049353alsopresenta subcorticalhypo‐responsivenessinthebilateralamygdala,putamen,andpallidumactivityand leftlateralizedcaudateandthalamusactivity,tospecificcues,whichmightbelinkedtoa deficienteffectontheprocessingofemotionalandsocialbehavior[80].Oncontrast,other authorsreportabetterresponsetotreatmentofmalepresentingtheGGgenotype[76].In patientstreatedwithcitalopram,TThomozygouscarriersforthers806368andrs806371 polymorphisms,showahigherincidenceofnoremission,comparedtotheGcarriers[76]. Moreover,theresponseinthers806368Gcarrierswasdifferentdependingonthegender,pre‐ sentingabetterantidepressantoutcomeinmenthaninwomen[76]. AlthoughtheCB2receptorsubtypeislessabundantthanCB1re‐ceptorsubtypeinthebrain, somestudiesalsodescribeanassociationwithmentaldisorders.Inthissense,theRRgenotype oftheQ63RpolymorphismintheCNR2genepresentsahigherassociationwithdepressionin theJapanesepopulation[81].ThisQ63Rpolymorphismpresentsalsoahighincidenceinpatients witheatingdisorders(anor‐ exianervosaandbulimianervosa)[82]andschizophrenia[83]. StudiesincellsexpressingthismutatedformoftheCNR2geneshowedthatthefunctional relevanceofthispolymorphismisduetochangesinCB2ligandaffinity,constitutiveactivityand areduced2‐AG‐inducedade‐ nylylcyclaseinhibition[84].RegardingtheCNR2gene polymorphismrs2501431,theAAcarrierspresentahigherseverityofthedisease,comparedto theGcarriers[85]. Theactivityofcannabinoidreceptorsisassociatedtothecross‐ regulationthattheCB1 receptorsexertovertheNMDAreceptorsmediatedbytheirinteractionviathehistidinetriad nucleotidebindingprotein1(HINT‐1),inwhichareductioninthenumberofCB1re‐ceptors maybeassociatedtotheNMDAhyperfunctionobservedindepression[86].Inthissense, molecularstudieshaveshownanin‐creaseintheHINT‐1proteinandtheNR1subunitofthe NMDAre‐ceptorsintheprefrontalcortexofdepressedpatients,inparalleltoresultsobtained inCB1receptorknockoutmice[86]. Table1 StudiesaboutalterationsofthedifferentcomponentsoftheECSinthebrainofpatientswith depressionoranxiety‐relateddisorders. ↑Increase;↓decrease;≈nosignificantchange.ACC:anteriorcingulatecortex;BA:Brodmann’sarea;CB1,CNR1: cannabinoidreceptor1;CB2:cannabinoidreceptor2;Co‐IP:co‐immunoprecipitation;Ct:control;DLPC:dorsolateral prefrontalcortex;FAAH:fattyacidamidehydrolase;fMRI:functionalmagneticresonanceimage;GM:greymatter; HINT‐1:histidinetriadnucleotidebindingprotein1;IHC:immunohistochemistry;MDD:majordepressiondisorder; PET:positronemissiontomography;PFC:prefrontalcortex;PMBT:post‐mortembraintissue;PTSD:posttraumatic stressdisorder;SSRI:serotoninselectivereuptakeinhibitor;TC:traumacontrols;WB:westernblot. 3.2. Endocannabinoidmetabolizingenzymesindepression OneofthemostfrequentpolymorphismofFAAHinhumansisafunctionalnon‐synonymous single‐nucleotidepolymorphism(C385A;rs324420)associatedwithareducedcellular expressionofthisenzyme.TheC385ApolymorphismoftheFAAHgene,presentsagreaterasso‐ ciationinAallelecarrierswithpathologiessuchasdepressionandbi‐ polardisorder[87]. Moreover,thepresenceofthispolymorphismconstitutesasusceptibilityfactortodevelop depressiveandanxiousphenotypesinadultindividualsthathavebeenexposedtochildhood trauma[88].ThehighAEAlevelsbecausethereducedFAAHactivityintheAallelecarriers, inducethedesensitizationofCB1receptors.ThisreductioninCB1receptorspromotesa glutamatergichyperactivitythat,togetherwithhighcortisollevelsduetochildhoodtraumain criticneurodevelopmentalperiods,resultsinanxiousand/ordepressivedis‐orders[88].TheCC carriersofthers324420polymorphismintheFAAHgenepresentareductionintheWM integrityoffibersthatcon‐nectwiththeanteriorcingulatecortexandtheorbitalcortex,and greaterincidenceofself‐reporteddepressivesymptoms[67]. 3.3. Endocannabinoidslevelsindepression Theserumcontentofendocannabinoidsisalsoalteredinmajordepression.Someauthors reportlowerlevelsofthecirculatingen‐ docannabinoidsAEAand2‐AGinpatientswith depression[89,90].Moreover,the2‐AGlevelsarelowerinpatientswithalongerdurationof thedepressiveepisode,whilepatientswithminordepressionpresenthigherlevelsofAEA[89]. Incontrast,otherauthorsreportnochangesinAEAand2‐AGlevelsindepressedwomen[91]. ThelevelsoftheendocannabinoidsAEAand2‐AGwerenotmod‐ifiedinresponsetoSSRIssuch asfluoxetine,whilethechronicad‐ministrationofMAOIsinducedareductioninareassuchas ECS element Finding (% change) Brain region Cohort (n: disease-Ct) Method/sample References CB1 (mRNA) ↑ (60%) DLPC (BA46) MDD:Ct 26:46 Gene expression microarray/PMBT [71] CB1 (protein) ≈ DLPC (BA46) MDD:Ct 14:14 IHC/PMBT [72] ↑ (38%) DLPC (BA9) MDD with suicide:Ct 10:10 WB/PMBT [70] ↓ (22.1%) Glial + cells in ACC GM MDD:Ct 15:15 IHC/PMBT [73] ≈ Neurons + cells in ACC MDD:Ct 15:15 IHC/PMBT [73] CB1 (density) ↓ ( ∼ 8.5–9.5%) ↑ (31%) Neurons + cells in ACC DLPC (BA9) MDD+SSRI:MDD no-SSRI MDD with suicide:Ct 10:10 IHC/PMBT [ 3 H]CP-55,940 binding/PMBT [73] [70] CB1 (functionality) ↑ (45%) DLPC (BA9) MDD with suicide:Ct 10:10 [ 3 H]CP-55,940 and [ 35 S]GTPγS [70] binding/PMBT CB1 (availability) ↑ (19.5%) Brain-wide PTSD:Ct 25:23 In vivo brain PET scan [ 11 C]OMAR [100] ↑ (14.5%) Brain-wide PTSD:TC 25:12 In vivo brain PET scan [ 11 C]OMAR [100] ↑ Amygdala PTSD:TC:Ct 12:4:4 In vivo brain PET scan [ 11 C]OMAR [101] CNR1 gene rs1049353 (A) ↑ activity associated to emotional Bilateral amygdala, putamen MDD(AG) MDD(GG) 13:20 Genetic association study with [80] processing and pallidum fMRI/peripheral cells CB1-HINT1 (protein) ≈ PFC (BA9) MDD:Ct 24:24 Co-IP/PMBT [86] CB1-NR1 C1 (protein) ≈ PFC (BA9) MDD:Ct 24:24 Co-IP/PMBT [86] CB2 (mRNA) ≈ DLPC (BA46) MDD:Ct 26:46 Gene expression microarray/PMBT [71] prefrontalcortex,hippocampusandhypothalamus[74].Thesedataareconsistentwithastudy thatassociatesthebeneficialeffectofexerciseindepres‐sionwithanincreaseintheplasma levelsofAEA,BDNFandcortisol[92]. 4. Theendocannabinoidsystemandanxiety‐relateddisorders Fewneurochemical,moleculargeneticsandneuroimagingstudiessuggestapotentiallink betweendysregulationoftheendocannabinoidsignalingandanxiety‐relatedbehaviorinboth healthyandpatientswithmentaldisordersinwhichanxietyisacoresymptom(PTSD,social phobia,agoraphobia,etc…). 4.1. Cannabinoidreceptorsinanxiety Twosinglenucleotidepolymorphism(SNP)variants(C‐AandC‐G)ofthehaplotypeformedby thepolymorphismsrs806368andrs1049353attheCNR1geneshowedasignificantassociation withPTSD[93].Moreover,ahigherrisktosufferanxietyisobservedwhenhomozygous‘SS’of thepolymorphismofserotonintransporter(5‐HTTLPR)intheSLC6A4promoteriscombined withthehomozygous’GG’rs2180619ofCNR1gene[94].Thishighlightsthestronginter‐action betweentheserotonergicsystemandtheECSonanxietydis‐ordersasextensivelydescribedin manypreclinicalandclinicalstudies,usingpharmacologicalandgeneticapproaches[37,95,96]. Moreover,Heitlandetal.[97]publishedthefirstevidence,inhealthymedication‐freehuman subjects,oftheimplicationofECSinthefearextinctionphenomenon,arelevantmechanism underlyingthepathophysiologyofhumananxietydisorders.Theseauthorsdescribetheeffect oftheCNR1genepolymorphismrs2180619intheresponsetofearconditioningandextinction. Theyfoundthatbothhomozygote(G/ G) andheterozygote(A/G)G‐allelecarriersofthispolymorphismshowedaclearextinctionof fear,whereasthisresponsewasabsentinhomozygotes(A/A). Recentfindingssuggestthatduringchildhoodandadolescence,theECSiscriticaltomediatea correctbalancebetweenexcitatoryandinhibitoryneurotransmission,especiallywithinthe prefrontalcortex[98].Thismakestheendocannabinoidsignalingquitesensitivefor developmentalfluctuationsduetoenvironmentalcauses,whichmayincreasetheriskofanxiety andotherstress‐relateddisorders.Inter‐ estingly,arecentstudyshowstheimpactofthe variationintheCNR1,CNR2,andFAAHgenesinasampleofchildrenwithaprimaryanxiety disorderdiagnosis[99].TheseauthorsnicelyreportedanassociationbetweentwoSNPs (rs12133557andrs6454676)intheCNR1geneandthechangeinsymptomseverityinboththe entiresampleandasubsetofpatientswithfearbaseddiagnoses[99].Moreover,afavorable andapoorerresponseduringtheactivetreatmentperiodwereassociatedwithminoralleleof rs12133557andrs6454676,respectively.Re‐gardingtheCNR2gene,unliketopreviousfindings indepression[81],thers2501431genotypewasnotassociatedwithanxietysymptomsor treatmentresponse[99]. Invivoneuroimagingstudiesalsosupporttheexistenceofanab‐normalCB1receptor‐mediated signalingespeciallyinPTSD.Usingpositronemissiontomography(PET)with[11C]OMAR,aCB1‐ selectivetracer,Neumeisterandcolleagues[100]reportedahigherCB1receptoravailabilityin untreatedindividualswithPTSD,relativetocontrolsubjects(withorwithoutlifetimehistories oftrauma),whichwasmostpronouncedinwomen.Thisup‐regulationofCB1receptorswas presentinanxiety‐relatedbrainareas,especiallytheamygdala‐hippocampal‐ cortico‐striatal neuralcircuit.Inalaterreport,thesamegroup[101]assessedtheattentionalbiastothreat, whichisconsideredoneofthemainendophenotypiccharacteristicsoftrauma‐relatedmental dis‐orders.Inlinewiththeirpreviousfindings,theyreportedapositivecorrelationbetweenan increasedCB1receptoravailability([11C]OMARbinding)intheamygdalaandincreasedinboth attentionalbiastothreatandtheseverityofthreat.Interestingly,thisgreaterCB1re‐ceptor availabilityintheamygdalawasassociatedwithlowerplasmalevelsofAEA. 4.2. Endocannabinoidmetabolizingenzymesinanxiety PharmacologicalstrategiesthatreducetheactivityofeitherFAAHorMAGLhavebeenreported toreduceanxiety‐likebehaviorsinro‐ dentsandhumans[37];however,dualFAAH/MAGL inhibitorsdidnotreducestress‐relatedaffectivedysfunctionregardlessoftreatmenttiming [102].ThereareseveralstudieslinkingtheactivityofFAAH,especiallyintheamygdala,with stress‐reactivityandrisktosufferan‐xiety‐disorders. Inhealthyvolunteers,therearesomestudiesexaminingtheimpactoftheFAAHgene polymorphismC385A(rs324420)onthreat‐andre‐ward‐relatedhumanbrainfunction.Using imaginggenetics,ade‐creasedthreat‐relatedamygdalareactivitybutincreasedreward‐related ventralstriatalreactivitywasdetectedincarriersoftheAalleleoftheFAAHenzymegene[103]. Inalaterstudy,aquickerhabituationofamygdalareactivitytothreatandlowerscoresonthe personalitytraitofstress‐reactivitywasfoundtobeassociatedwithcarriersofalow‐expressing FAAHvariant(385Aallele;rs324420)[104].Morerecently,anenhancedfearextinctionwas demonstratedinbothmouseandhumanA‐allelecarriersofthisFAAHgeneC385A polymorphism,highlightingagaintheassociationoftheincreasedfronto‐amygdalaconnectivity withenhancedstress‐reactivity[105].Thisgenetical‐ terationappearstohavefunctional consequencessinceamarkedlyre‐ ducedFAAHproteinexpressionwasdetectedinmany cortico‐limbicareasusingthefirstavailablePETradiotracer([11C]CURB)inhumanbrain[106]. RegardingtheroleofFAAHonPTSD,Pardinietal.[107]reportedanassociationofthers2295633 SNPofFAAHgenewithPTSDdiagnosisinmaleVietnamWarveteranswithoutlesionsintheven‐ tromedialprefrontalcortex.Evenmore,theCallelewaspresentinsubjectsthathadamore negativereportedexperienceoftrauma. 4.3. Endocannabinoidlevelsinanxiety PreclinicalfindingssuggestthatthepharmacologicalmanipulationofendogenouseitherAEAor 2‐AGlevelsunderstressfulconditionscouldrepresentagoodstrategyfortreatmentofanxiety‐ relateddis‐ orders[108].Thus,itisplausibletohypothesizetheexistenceofaltered endocannabinoidlevelsinthebrainofpatientsdiagnosedofpsychiatricdiseasesinwhich anxietyiseithercoreoracomorbidsymptom. DecreasedplasmaAEAlevelsarefoundinPTSDpatientsrelativetohealthycontrolsubjects withouttraumahistory[109],thougheleva‐tionsarealsoreportedinchronicPTSD[110].The AEAdeficiencyseemstobespecificofPTSDpatientssincehealthysubjectswithlife‐ time historiesoftrauma,butwithoutPTSD,exhibitnormalAEAplasmalevels[100].Asmentioned above,apositivecorrelationbetweenlowerplasmalevelsofAEAandincreasedCB1receptor availabilityintheamygdalahasbeenreported;inaddition,AEAlevelswerenegatively associatedwithattentionalbiastothreat[101].Intriguingly,thisin‐verserelationshipbetween AEAlevelsandanxietyappearstobedis‐ease‐specificsinceacutestressincreasesthecirculating levelsofAEAandotherendocannabinoids,inparallelwithcortisol[111].Regardingthe2‐AG, reduced[112]andincreased[110]levelsamongindividualsmeetingdiagnosticcriteriaforPTSD weredescribed.Healthyhumansthatweresubjectedtoprolongedstress(520‐dayisolation periodandsimulatingaflighttoMars)showedreducedbloodlevelsof2‐AG,butnotAEA[113]. AllthesefindingssuggesttheinteractionbetweentheECSandHPAaxisactivityinresponseto stress,especiallyattheleveloftheamygdala[19].Inlinewiththisrelationship,arecentimaging geneticsstudy[114]revealedamolecularinteractionbetweengeneticpolymorphisms associatedwithdifferentialAEAlevels(FAAHrs324420)andcortico‐trophinreleasinghormone (corticotropin‐releasinghormonereceptor1,CRHR1rs110402)signaling.andamygdalafunction. However,inaveryrecentstudy,thelevelsofcirculatingendocannabinoidsweremeasuredin subjectswithandwithouthistoryofpsychiatricdisorderandtherelationshipswithcategorically DSM‐5defineddisordersorstatedimensionalmeasuresofdepressionoranxietywerestudied. Surprisingly,neitherAEAnor2‐AGlevelsdifferedasafunctionofanysyndromal/personality disorderandneithercorrelatedsignificantlywithstatedepressionorstateanxietyscores[115]. Therefore,wemustbecautioussinceperipheralendocannabinoidlevelsmaybenotwell correlatedwithbrainconcentrations.  5. Theendocannabinoidsystemandschizophrenia SchizophreniaisoneofthemainpsychiatricsyndromestogetherwithMajorDepression.Itisa chronicanddevastatingdisorderaffecting1%ofthepopulationworldwide.Individuals diagnosedwithschizo‐phreniahaveimpairedsocialandoccupationalfunctioning.Thus,the combinedeconomicandsocialcostsofschizophreniaplaceitastheworld’s15thcauseof disease‐relateddisability[116].Theclinicalfeaturesofschizophreniaareclusteredinpositive symptoms(i.e.hal‐lucinationsanddelusions);negativesymptoms(i.e.socialwithdrawaland bluntedaffect)andcognitivedeficits(i.e.impairedworkingmemoryandcognitiveflexibility). Currentantipsychoticdrugs,whicharethemaintreatmentforschizophrenia,arenoteffective inallpa‐tientsandtheirbenefitsarerestrictedtotheameliorationofthepositivesymptoms, havingnoneorlimitedimpactinnegativesymptomsandcognitiveimpairment. Despitetheeffortsofthescientificcommunityinthelastdecadestoelucidatetheetiological basisofschizophrenia,theetiopathogenesisofthediseaseremainsunknown.Sincemanyyears, thepredominantfocusofstudiesconcerningthebiologicalsubstratesofschizophreniahasbeen primarilycenteredonuniqueneurotransmittersincludingdopa‐mine,serotonin,glutamateand γ‐aminobutyricacid(GABA).Nonetheless,thelimitedefficacyofcurrentantipsychoticdrugsto treatsomeofthesymptomsofschizophreniahasleadupresearcherstoin‐vestigateother potentialneurotransmittersystemsthatmaybealteredinthisdisease. Inthissense,theECShasbecomeahot‐topicinschizophreniare‐searchinthelastyears.Several studiesstartingfromthe40suptonowadaysagreethattheECSrepresentsamajor neuromodulatorysystemparticipatingintonesofphysiologicalprocesses[117,118].Thereby, deregulationoftheECShasbeenspeculatedtobeaproximalpathologyinsomeformsof schizophrenia.Inthissense,two‘canna‐ binoidhypotheses’ofschizophreniahavebeen proposed[119].TheendogenoushypothesisreferstothefactthatderegulationoftheECSmay contributetothepathophysiologyofschizophrenia,whereastheexogenoustheoryreferstothe ItisalsonoteworthythatCSFendocannabinoidlevelshavebeenshowntobeaffected dependingonthehistoryofcannabisuse.Thus,markedlyalteredAEAconcentrations(>10‐fold higher)werereportedinCSFofasubgroupofschizophrenicpatientswhohadlowfrequency cannabisusecomparedtocontrols(withhighandlowfrequencyuse),aswellascomparedto schizophrenichigh‐frequencyusers[161].However,thisimpactofcannabisusewasnot observedinotherstudies[160,162]. Compilingtheinformationavailablefromthesestudiesisevidentthattherelationshipbetween levelsofendocannabinoidsmeasuredintheCSF,peripheralbloodandconcentrationsof endocannabinoidsinbraintissueisnotclearyet.Thus,theunderstandingoffunctionalim‐ plicationsofalteredlevelsofendocannabinoidsineachtypeofsampleofschizophrenicpatients remainstobeelucidated.  6. Conclusions Severalevidencessuggesttherelationshipbetweenchangesinoneormorecomponentsofthe ECSandsomeofthesymptomsthatarepresentindepression,anxiety‐relateddisordersand schizophrenia.Indeed,recenthumanpostmortemandinvivoneuroimagingstudiesare providingmoreknowledgeabouttheimplicationoftheECSinthesementaldisorders.Mostof thefindingsindepressionandanxietyarerelatedtotheexpressionand/orfunctionalityofCB1 receptorsandFAAHinbrainareasbelongingtotheamygdala‐hippocampal‐cortico‐ striatal neuralcircuit,especiallythefrontalcortexindepressionandtheamygdalainanxietydisorders. Regardingschizophrenia,thefindingsinpostmortemandlivinghumanbrainshighlighta deregulationofCB1receptorinspecificbrainareasthatarehighlyaffectedinthisdisease.The findingsonperipheralendocannabinoidlevelsareingoodcon‐sonancewiththeseadaptive changes.However,wemustbecautioussinceperipheralendocannabinoidlevelsmaynotbe wellcorrelatedwithbrainconcentrations. ThepharmacologicalmanipulationoftheECSisenvisagedasanattractivealternativetreatment forthesementaldisorders.Forinstance,drugsasthephytocannabinoidcompoundCBDhave beenreportedtobeeffectivetotreatschizophrenia. Theadvanceinthisfield,togetherwiththetranslationalpreclinicalresearchisopeningan attractiveresearchscenarioforthedevelopmentofpromisingnewpharmacologicalstrategies basedondrugstargetingtheECStotreatmentaldisorders.  Acknowledgements ThisstudywassupportedbytheSpanishMinistryofEconomyandCompetitiveness(SAF2015‐ 67457‐R,MINECO/FEDER),thePlanEstataldeI+D+i2013‐2016,theInstitutodeSaludCarlosIII‐ SubdirecciónGeneraldeEvaluaciónyFomentodelaInvestigación,SpanishMinistryofEconomy, FEDER(PI13/01529)andtheBasqueGovernment(IT616/13).II‐LisarecipientofaPredoctoral FellowshipfromtheBasqueGovernment.EF‐ZisarecipientofaPredoctoralFellowshipfrom theUniversityofCantabria.CMisarecipientofaPostdoctoralMarieSkłodowska‐Curie IndividualFellowship(H2020‐MSCA‐IF‐2016,ID747487).   Conflictofinterests Thereisnoconflictofinteresttodeclare.  References [1]H.A.Whiteford,L.Degenhardt,J.Rehm,A.J.Baxter,A.J.Ferrari,H.E.Erskine,etal.,Global burdenofdiseaseattributabletomentalandsubstanceusedisorders:findingsfromthe GlobalBurdenofDiseaseStudy2010,Lancet382(9904)(2013)1575–1586. [2]J.J.Meana,L.F.Callado,B.Morentin,Dopost‐mortembrainstudiesprovideuseful informationforPsychiatry?Rev.Psiquiatr.SaludMent(Barc.)7(2014)3. [3]C.H.Ashton,P.B.Moore,Endocannabinoidsystemdysfunctioninmoodandre‐ lated disorders,ActaPsychiatr.Scand.124(4)(2011)250–261. 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