Training-Associated Emotional Arousal Shapes Endocannabinoid Modulation of Spatial Memory ...

13 Pages·2015·2.19 MB·English

13962•TheJournalofNeuroscience,October14,2015•35(41):13962–13974
Behavioral/Cognitive
Training-Associated Emotional Arousal Shapes
Endocannabinoid Modulation of Spatial Memory Retrieval
in Rats
XMariaMorena,1,2,3ValentinaDeCastro,1J.MeganGray,2,3MauraPalmery,1VivianaTrezza,4BennoRoozendaal,5,6
MatthewN.Hill,2,3andPatriziaCampolongo1
1DepartmentofPhysiologyandPharmacology,SapienzaUniversityofRome,00185Rome,Italy,2HotchkissBrainInstituteand3DepartmentsofCell
BiologyandAnatomyandPsychiatry,UniversityofCalgary,Calgary,AlbertaT2N4N1,Canada,4DepartmentofScience,SectionofBiomedicalSciencesand
Technologies,UniversityRomaTre,00146Rome,Italy,and5DepartmentofCognitiveNeuroscience,RadboudUniversityMedicalCenterand6Donders
InstituteforBrain,CognitionandBehaviour,RadboudUniversityNijmegen,6525EZNijmegen,TheNetherlands
Variationsinenvironmentalaversivenessinfluenceemotionalmemoryprocessesinrats.Wehavepreviouslyshownthatcannabinoid
effectsonmemoryaredependentonthestresslevelatthetimeoftrainingaswellasontheaversivenessoftheenvironmentalcontext.
Here, we investigated whether the hippocampal endocannabinoid system modulates memory retrieval depending on the training-
associatedarousallevel.MaleadultSpragueDawleyratsweretrainedonawatermazespatialtaskattwodifferentwatertemperatures
(19°Cand25°C)toeliciteitherhigherorlowerstresslevels,respectively.Ratstrainedunderthehigherstressconditionhadbetter
memoryandhighercorticosteroneconcentrationsthanratstrainedatthelowerstresscondition.Thecannabinoidreceptoragonist
WIN55212-2(10–30ng/side),the2-arachidonoylglycerol(2-AG)hydrolysisinhibitorJZL184(0.1–1(cid:2)g/side),andtheanandamide
(AEA)hydrolysisinhibitorURB597(10–30ng/side)wereadministeredbilaterallyintothehippocampus60minbeforeprobe-trial
retentiontesting.WIN55212-2orJZL184,butnotURB597,impairedprobe-trialperformancesonlyofratstrainedatthehigherstressful
condition.Furthermore,ratstrainedunderhigherstresslevelsdisplayedanincreaseinhippocampal2-AG,butnotAEA,levelsatthetime
ofretentiontestingandadecreasedaffinityofthemain2-AG-degradingenzymeforitssubstrate.Thepresentfindingsindicatethat
the endocannabinoid 2-AG in the hippocampus plays a key role in the selective regulation of spatial memory retrieval of stressful
experience,sheddinglightontheneurobiologicalmechanismsinvolvedintheimpactofstresseffectsonmemoryprocessing.
Keywords: cannabinoidreceptors;emotionalarousal;endocannabinoids;memoryforemotionalexperiences;stress
SignificanceStatement
Endogenouscannabinoidsplayacentralroleinthemodulationofmemoryforemotionalevents.Herewedemonstratethatthe
endocannabinoid2-arachidonoylglycerolinthehippocampus,abrainregioncruciallyinvolvedintheregulationofmemory
processes,selectivelymodulatesspatialmemoryrecallofstressfulexperiences.Thus,ourfindingsprovideevidencethatthe
endocannabinoid2-arachidonoylglycerolisakeyplayerinmediatingtheimpactofstressonmemoryretrieval.Thesefindingscan
pavethewaytonewpotentialtherapeuticinterventionforthetreatmentofneuropsychiatricdisorders,suchaspost-traumatic
stressdisorder,whereapreviousexposuretotraumaticeventscouldaltertheresponsetotraumaticmemoryrecallleadingto
mentalillness.
Introduction 2011).Endocannabinoids,mainlyN-arachidonoylethanolamine
Extensive evidence indicates that the endocannabinoid system (anandamide, AEA) and 2-arachidonoyl glycerol (2-AG), are
regulatescognitivefunction(Wotjak,2005;Campolongoetal., synthesized on demand and travel retrogradely to presynaptic
2009a;Kanoetal.,2009;MarsicanoandLafeneˆtre,2009;Akirav, sites to bind G-protein-coupled cannabinoid receptors type 1
ReceivedMay22,2015;revisedAug.5,2015;acceptedAug.28,2015. ThisworkwassupportedbyHumanFrontierScienceProgramGrantRGY0077andItalianMinistryofEducation
Authorcontributions:M.M.,V.T.,B.R.,M.N.H.,andP.C.designedresearch;M.M.,V.D.C.,J.M.G.,M.P.,V.T., MIURGrantRBFR10XKHS(2010BN3MXM_003)toP.C.,CanadianInstitutesofHealthResearchoperatingfundsto
M.N.H.,andP.C.performedresearch;M.M.,V.D.C.,M.N.H.,andP.C.analyzeddata;M.M.,B.R.,andP.C.wrotethe M.N.H.,ItalianSocietyofPharmacologytoM.M.,andAlbertaInnovatesHealthSolutionssalarysupporttoJ.M.G.We
paper. thankAndreaPelosoandHaleyVecchiarellifortechnicalhelp.
Morenaetal.•Cannabinoids,Stress,andSpatialMemoryRetrieval J.Neurosci.,October14,2015•35(41):13962–13974•13963
(CB1)(Herkenhametal.,1990;Matsudaetal.,1990;Devaneet els.Ratsweretrainedonawatermazespatialtaskattwodifferent
al.,1992;Sugiuraetal.,1995;Kanoetal.,2009).AEAand2-AG watertemperatures(19°Cand25°C)toeliciteitherahigheror
aresubsequentlydegradedmainlybyfattyacidamidehydrolase lowerlevelofstress.Beforeretentiontesting,wepharmacologi-
(FAAH) and monoacylglycerol lipase (MAGL), respectively callymanipulatedtheendocannabinoidsystembyadministering
(Kano et al., 2009). CB1 receptors are highly expressed within thecannabinoidreceptoragonistWIN55212-2,the2-AGhydro-
corticolimbicbrainareas,suchasthehippocampus,basolateral lysisinhibitorJZL184,ortheAEAhydrolysisinhibitorURB597
complexoftheamygdala(BLA),andprefrontalcortex(Herken- directly into the dorsal hippocampus. Behavioral experiments
ham et al., 1991; Tsou et al., 1998), where they predominately wereparalleledbybiochemicalmeasurementofplasmacortico-
modulatebothexcitatoryandinhibitorysignalingwithinspecific steronelevels,hippocampal2-AGandAEAcontent,andthecan-
neuronalcircuitsinvolvedinlearningandmemoryprocessesfor nabinoidhydrolyticenzymaticmachinery.
emotionallyarousingexperiences(Wotjak,2005;Campolongoet
al.,2009a;Kanoetal.,2009;MarsicanoandLafeneˆtre,2009;Aki- MaterialsandMethods
rav,2011).Althoughevidenceregardingcannabinoideffectson Animals.MaleadultSpragueDawleyrats(320–370gatthetimeofbe-
memory retrieval is limited, the majority of studies show that havioralexperiments;CharlesRiverBreedingLaboratories)werehoused
exogenous cannabinoid agonists induce detrimental effects on individuallyinatemperature-controlled(20(cid:2)1°C)vivariumroomand
memory retrieval when either administered systemically maintainedundera12h/12hlight/darkcycle(7:00A.M.to7:00P.M.
lightson).Foodandwaterwereavailableadlibitum.Trainingandtesting
(Mishimaetal.,2001;Niyuhireetal.,2007;Wiseetal.,2009)or
wereperformedduringthelightphaseofthecyclebetween10:00A.M.
intodiscretebrainareas,suchasthehippocampus(PiriandZar-
and4:00P.M.Allexperimentalprocedureswereincompliancewiththe
rindast,2011;SegevandAkirav,2011;Atsaketal.,2012)orBLA
EuropeanUnionDirectiveontheprotectionofanimalsusedforscien-
(SegevandAkirav,2011).
tificpurposes(2010/63/EU),theItalianlaw(D.L.26/2014),theDeclara-
Theendocannabinoidsystemplaysakeyroleinthecontrolof tionofHelsinki,andtheGuidefortheCareandUseofMammalsin
emotional responses to environmental challenges (Zanettini et Neuroscience and Behavioral Research (National Research Council,
al.,2011;Ruehleetal.,2012;Gunduz-Cinaretal.,2013;Morena 2004).
andCampolongo,2014).Wehavepreviouslyreportedthatcan- Surgery. The rats were anesthetized with sodium pentobarbital (50
nabinoidsinducedifferenteffectsonmemorydependingonthe mg/kg,i.p.)andgivenatropinesulfate(0.4mg/kg,i.p.)tomaintainres-
aversivenessoftheenvironmentalconditionsandthestresslevel piration.Subsequently,theywereinjectedwith3mlofsaline(s.c.)to
facilitateclearanceofthesedrugsandpreventdehydration.Theratswere
at the time of training (Campolongo et al., 2013). Further, we
thenpositionedinastereotaxicframe(DavidKopfInstruments),and
haverecentlydemonstratedthatendocannabinoidsarephysio-
two stainless-steel guide cannulae (23 gauge, 11-mm-long) were im-
logicallyreleasedinlimbicbrainregionsonlywhenanimalsare
plantedbilaterallywiththecannulatips1.5mmabovetheCA1regionof
trained under high arousal conditions (Morena et al., 2014). thedorsalhippocampus(coordinates:anteroposterior,(cid:3)3.4mm;me-
Variationsinenvironmentalaversivenessdifferentiallyinfluence diolateral,(cid:2)1.8mm;dorsoventral,(cid:3)2.7mm)accordingtotheatlasof
spatialmemoryprocessesinrats(Sandietal.,1997;Akiravetal., PaxinosandWatson(2005).Thecannulaewereaffixedtotheskullwith
2004; Salehi et al., 2010). Salehi et al. (2010) induced different twoanchoringscrewsanddentalcement.Stylets(11-mm-long00insect
stresslevelsbytrainingratsonaradial-armwatermazetaskat dissectionpins)wereinsertedintoeachcannulatomaintainpatency.
three different water temperatures (25°C, 19°C, and 16°C). An Aftersurgery,theratswereretainedinanincubatoruntilrecoveredfrom
inverted-Ushapeeffectwasfound,withanimalstrainedat19°C anesthesiaandwerethenreturnedtotheirhomecages.Ratswereallowed
torecoverfromsurgeryfor10dbeforetrainingandwerehandled1min
showingbettermemorythananimalstrainedateitherthehigher
perdayfor3dbeforetraining.
(16°C)orlower(25°C)stresscondition(Salehietal.,2010).Once
Watermazespatialtaskandexperimentalprocedures.Theexperimental
memoriesareconsolidated,theefficacyoraccuracyoftheinfor-
apparatuswasacircularblackgalvanizedtank,1.83mindiameterand
mationretrievedisvulnerabletostresseffectsatthetimeofrecall.
0.6minheight,filledwithwatertoadepthof20cm.Themazewas
Considerableevidencepointstothehippocampusasabrain locatedinaroomcontainingmanysalient,visual,extra-mazecues.A
structure of crucial importance for retrieval of spatial memory rectangularplatform(20cm(cid:4)25cm)wasplacedatafixedlocation25
(Hirsh, 1974; Moser and Moser, 1998; Holt and Maren, 1999; cmawayfromtheedgeofthepooland2.5cmbelowthewatersurface.A
Riedeletal.,1999;Eldridgeetal.,2000;Brunetal.,2002;Matus- slightlymodifiedprocedureofthosedescribedbyAkiravetal.(2004)and
Amatetal.,2004).Stressorexogenousglucocorticoidadminis- Campolongoetal.(2009b)wasused.Separategroupsofanimalswere
trationtypicallyimpairsmemoryretrievalofcontextual/spatial trainedatawatertemperatureofeither19°Cor25°C,whichwereprevi-
ouslyshowntoelicitdifferentstresslevelsandcognitiveperformancein
informationinratsanddeclarativeinformationinhumans(de
rats(Akiravetal.,2004;Salehietal.,2010).Oneachdayoftraining,the
Quervainetal.,1998;Roozendaaletal.,2003,2004a;deQuervain
ratswerecarriedfromthevivariumtotheexperimentalroom,andtrain-
etal.,2009).Theendocannabinoid2-AGinthedorsalhippocam-
ingbegan120minlater.Forspatialtraining,theratsweregivenfourtrials
pusplaysanintermediaryroleinmediatingglucocorticoideffects
oneachdailysessionfortwoconsecutivedays.Thisrelativelysmallnum-
ontheimpairmentofcontextualmemoryretrieval(Atsaketal., beroftrialswaschosensuchthattheanimalsacquiredthetaskbutthat
2012). theirretentionperformancewasmoderatetoavoidanypotentialceiling
Here,weinvestigatedwhetherthehippocampalendocannabi- orflooreffects.Beforethefirsttrainingtrial,eachratwasplaceddirectly
noidsystemisdifferentiallyinvolvedinregulatingspatialmem- ontothesubmergedplatformfor15s.Foreachtrial,theratwasplaced
oryretrievalofratstrainedattwoexperimentalconditionsthat intothetankatoneofthefourdesignatedstartingpointsandallowedto
differedwithrespecttotheirenvironment-associatedstresslev- findtheplatform.Ifananimalfailedtofindtheplatformwithin60s,it
wasmanuallyguidedtotheplatform.Aftermountingtheplatform,the
rat was allowed to remain there for 10 s and was then placed into a
holdingcagefor25suntilthestartofthenexttrial.Thetimeeachrat
Theauthorsdeclarenocompetingfinancialinterests.
spent searching for the platform was recorded as the escape latency.
CorrespondenceshouldbeaddressedtoeitherDr.MariaMorenaorDr.PatriziaCampolongo,Departmentof
Retentionofthespatialtrainingwasassessed24hafterthelasttraining
PhysiologyandPharmacology,SapienzaUniversityofRome,P.leAldoMoro5,00185Rome,Italy.E-mail:
[email protected]@uniroma1.it. sessionwitha60sfree-swimprobetrialunderequaltemperaturecon-
DOI:10.1523/JNEUROSCI.1983-15.2015 ditions(22(cid:2)0.5°C).Duringtheprobetrial,theplatformwasremoved
Copyright©2015theauthors 0270-6474/15/3513963-13$15.00/0 fromthetank,andanewstartingpositionwasused.Trainingandprobe
13964•J.Neurosci.,October14,2015•35(41):13962–13974 Morenaetal.•Cannabinoids,Stress,andSpatialMemoryRetrieval
trialswerevideotaped,andanautomatedtrackingsystem(Smart-BS, homogenizedwithaglassrod.Tissuewassonicatedfor30minonice
PanlabS.L.U.,Barcelona,Spain)wasusedtoanalyzetheswimpathof waterandincubatedovernightat(cid:3)20°Ctoprecipitateproteins,then
eachsubjectandcalculateseveralcorrespondingdependentmeasures, centrifugedat1500(cid:4)gtoremoveparticulates.Thesupernatantswere
suchastimespentinthequadrantcontainingtheplatformduringtrain- transferredtoanewglasstubeandevaporatedtodrynessunderN
2
ing(targetquadrant),timespentinthequadrantoppositetothetarget gas.Thesampleswerereconstitutedin300(cid:2)lofacetonitrileanddried
quadrant(oppositequadrant),initiallatencytocrosstheplatformloca- againunderN gas.Lipidextractsweresuspendedin20(cid:2)lofaceto-
2
tion,numberofcrossingsthroughtheplatformlocation,andtotalswim nitrileandstoredat(cid:3)80°Cuntilanalysis.AnalysisofAEAand2-AG
distance.Thetargetandoppositequadrantswereequidistantfromthe wasperformedbyliquidchromatographymassspectrometryanalysis
startingpositionontheprobetrial. aspreviouslydetailed(Dinchevaetal.,2015).
Drugtreatment.TheCB1receptoragonistWIN55212-2(10or30ngin Membranepreparation.Immediatelyaftertheprobetrial,following
0.5(cid:2)l),the2-AGhydrolysisinhibitorJZL184(0.1or1(cid:2)gin0.5(cid:2)l),or rapiddecapitation,thehippocampiweredissectedfromnonoperated
theAEAhydrolysisinhibitorURB597(10or30ngin0.5(cid:2)l)wasadmin- ratstrainedatawatertemperatureof19°Cor25°Candfromcontrol
isteredintothedorsalhippocampus60minbeforetheprobetrial.To ratsthatwerehandledbutnottrainedortested.Brainsampleswere
examinewhetherWIN55212-2andJZL184effectsweremediatedviaan stored at (cid:3)80°C. Membranes were collected by homogenization of
activationofCB1receptors,othergroupsofratswereadministeredthe frozentissuein10volumesofTMEbuffer(50mMTrisHCl,pH7.4;1
effectivedoseofWIN55212-2(10ngin0.5(cid:2)l)orJZL184(1(cid:2)gin0.5(cid:2)l) mMEDTA,and3mMMgCl2)(Hilletal.,2009).Homogenateswere
togetherwiththeCB1receptorantagonistAM251atadosenonaltering thencentrifugedat18,000(cid:4)gfor20min,andtheresultingcrude
memoryperformanceperse(0.28ng).Doseswereselectedonthebasisof membranefraction-containingpelletwasresuspendedin10volumes
previousandpilotexperimentsperformedinourlaboratory(Morenaet of TME buffer. Protein concentrations were determined using the
al.,2014).Alldrugsweredissolvedinavehiclecontaining5%polyethyl- Bradfordmethod(Bio-Rad).MembraneswereusedforMAGLand
eneglycol,5%Tween80,and90%saline.Bilateralinfusionsofdrugsor FAAHactivityassays.
anequivalentvolumeofvehicleintothedorsalhippocampusweremade MAGLactivityassay.MAGLactivitywasmeasuredbyconversionof
byusinga30-gaugeinjectionneedleconnectedbypolyethylenetubing 2-oleoylglycerollabeledwith[3H]([3H]2-OG)intheglycerolpor-
(PE-20)toa10(cid:2)lHamiltonmicrosyringedrivenbyaminipump(KD tionofthemoleculeto[3H]glycerolpreparations.Aslightlymodi-
Scientific).Theinjectionneedleprotruded1.5mmbeyondthetipofthe fied procedure of that described by Rademacher et al. (2008) was
cannula,anda0.5(cid:2)linjectionvolumeperhemispherewasinfusedover used. Membranes were incubated in a final volume of 0.5 ml TME
aperiodof50s(Roozendaaletal.,2004b;Atsaketal.,2012).Theinjec- buffer(50mMTris-HCl,3.0mMMgCl2,1.0mMEDTA,and300nM
tionneedleswereretainedwithinthecannulaeforanadditional20safter URB597,pH7.4)thatcontained1.0mg/mlfattyacid-freeBSAand
druginfusiontomaximizediffusionandtopreventbackflowofdruginto 100,000dpm[3H]2-OG.Isothermswereconstructedusingsixcon-
thecannulae.AlldrugswerekindlydonatedbytheNationalInstituteof centrationsof2-OGatconcentrationsbetween10and500(cid:2)M.Incu-
Mental Health, and drug solutions were freshly prepared before each bation was performed at 30°C, and the enzymatic reaction was
experiment. stoppedbytheadditionof2mlofchloroform/methanol(1:2).After
Histology.Theratswereanesthetizedwithanoverdoseofsodium remainingatroomtemperaturefor30minwithfrequentmixing,0.67
pentobarbital(100mg/kg,i.p.)andperfusedtranscardiallywith0.9% mlofchloroformand0.6mlofwaterwereadded,andtheaqueous
saline.Thebrainswerethenremovedandimmersedina4%formal- andorganicphaseswereseparatedbycentrifugationat1000rpmfor
dehyde solution. At least 48 h before sectioning, the brains were 10min.Theamountof[3H]in0.5mlofeachoftheaqueousand
transferred to a 20% sucrose solution in saline for cryoprotection. organic phases was determined by liquid scintillation counting
Coronal sections of 35 (cid:2)m were cut on a cryostat, mounted on andconversionof[3H]2-OGto[3H]glycerolwascalculated.The
gelatin-coatedslides,andstainedwithcresylviolet.Thesectionswere bindingaffinity(Km)andmaximalhydrolyticactivity(Vmax)values
examined under a light microscope (Microscope Nikon 801), and forthisconversionweredeterminedbyfittingthedatatoasingle-site
determinationofthelocationofinfusionneedletipswithinthehip- Michaelis–MentenequationusingGraphPadPrism(GraphPad).
pocampus was made according to the standardized atlas plates of FAAHactivityassay.FAAHactivityfromhippocampalmembranes
PaxinosandWatson(2005)byanobserverblindtodrugtreatment wasmeasuredbyconversionofAEAlabeledwith[3H]intheetha-
condition.Forallexperiments,onlyratswithneedletipsterminating nolamineportionofthemoleculeto[3H]ethanolaminepreparations
withintheCA1regionofthehippocampuswereincludedinthedata asreportedpreviously(Hilletal.,2009).TheKmofAEAforFAAH
analysis.Approximately5%oftheanimalswereexcludedbecauseof andVmaxofFAAHforthisconversionweredeterminedbyfittingthe
eithercannulamisplacementordamagetothetargetedtissue. data to the Michaelis–Menten equation using GraphPad Prism
Plasmacorticosteronelevels.Plasmacorticosteronelevelsweredeter- (GraphPad).
minedinparallelgroupsofratshandledfor3dandsubsequentlytrained Statistics.Dataaremean(cid:2)SEM.Watermazetrainingdatawereana-
at a water temperature of either 19°C or 25°C. Control animals were lyzed with one- or two-way ANOVAs with training day as repeated
handledfor3dbutwerenottrainedortested.Ratswereeuthanized20 measure.Probe-trialretentiondatawereanalyzedwithunpairedttests,
minafterthetwotrainingsessionsorimmediatelyaftertheprobetrial. one-,two-,orthree-wayANOVAs,whenappropriate.Plasmacortico-
Afterdecapitation,trunkbloodwascollectedandsampleswerecentri- steronelevels,hippocampalendocannabinoidcontent,andMAGLand
fugedat1900(cid:4)gfor20minat4°C.Plasmawasstoredat(cid:3)20°Cand FAAHactivityparameterswereanalyzedwithunpairedttestsorone-way
analyzedforcorticosteroneusinganELISAkit(AssayDesigns)according ANOVAs. Tukey–Kramer post hoc tests were used to determine the
tothemanufacturer’sinstructions.Thekitsensitivityanddetectionlimit sourceofthedetectedsignificances,whenappropriate.pvaluesof(cid:5)0.05
are18.6and16.9pg/ml,respectively.TheCVvaluesforintra-assayand wereconsideredstatisticallysignificant.Thenumberofratspergroupis
interassayprecisionare5.2%and8.7%,respectively. indicatedinthefigurelegends.
Endocannabinoidextractionandanalysis.Toexaminewhether2-AG
and AEA are physiologically released into the hippocampus during Results
memoryretrieval,separategroupsofnonoperatedratsweretrainedat Learningunderstressenhancesprobe-trialretention
thetwodifferentwatertemperatures(19°Cor25°C)andeuthanized
performance
immediatelyaftertheprobetrial.Controlanimalswerehandledbut
Wefirstsoughttodeterminewhetherthedifferentwatertemper-
nottrainedortested.Afterrapiddecapitation,hippocampiwererap-
idlydissectedandstoredat(cid:3)80°C.Thelipidextractionprocesswas atures at training influenced spatial memory performance of
vehicle-treatedrats.
performedbyusingaslightlymodifiedprocedureofthatdescribedby
Dincheva et al. (2015). Brain tissue was weighed and placed into Repeated-measuresANOVAforescapelatenciestofindthe
borosilicateglassculturetubescontaining2mlofacetonitrilewith5 hidden platform during training, before drug treatment, re-
pmol of [2H ] AEA and 5 nmol of [2H ] 2-AG for extraction, and vealedasignificanteffectoftrainingday(F (cid:6)203.03,p(cid:5)
8 8 (1,74)
Morenaetal.•Cannabinoids,Stress,andSpatialMemoryRetrieval J.Neurosci.,October14,2015•35(41):13962–13974•13965
by significantly longer search times in
the vicinity of the platform location
(i.e.,targetquadrant)thanintheoppo-
sitequadrant(p(cid:5)0.01;Fig.1D).How-
ever, rats trained with the lower water
temperature spent significantly less
timeintheoppositequadrantthanrats
trained with the higher water tempera-
ture(p(cid:5)0.01;Fig.1D).Nogroupdif-
ferences were found for total swim
distanceontheprobetrial(t (cid:6)0.35;
(74)
p (cid:6) 0.73; data not shown), indicating
thatthetwogroupsdidnotdifferinmo-
torperformance.
Watermazetrainingunderthetwo
stressconditionsdifferentiallyaffects
plasmacorticosteronelevels
Toassesswhetherthetwowatertemper-
atures differentially affected plasma
corticosteronelevels,parallelgroupsof
ratsweretrainedatawatertemperature
ofeither19°Cor25°C,andtrunkblood
wascollected20minaftereitherdayof
trainingorimmediatelyaftertheprobe
trial.Homecagecontrolratswereonly
handled.AsshowninTable1,one-way
ANOVA for plasma corticosterone
levels 20 min after the first or second
Figure1. Effectofwatertemperatureontrainingandprobeperformancesofvehicle-treatedrats.A,Ratstrainedateither19°C
training day revealed a significant ex-
or25°CacquiredthebehavioraltaskasindicatedbyasignificantdifferencebetweenmeanescapelatenciesduringTraining1and
Training2.Ratstrainedat19°Cshowedabettermemoryperformancethanratstrainedat25°CduringTraining2.**p(cid:5)0.01 perimental condition effect (training
versusTraining1.(cid:2)(cid:2)p(cid:5)0.01,meanescapelatenciesat19°Cversusmeanescapelatenciesat25°C.B–D,Thehigherstress day1:F (cid:6)28.47;p(cid:5)0.0001;train-
(2,20)
condition(19°C)enhancedprobe-trialretentionperformanceinrats.Ratstrainedatthehigherstresscondition(19°C)spentless ing day 2: F (cid:6) 21.41; p (cid:5) 0.0001).
(2,19)
timetofirstcrosstheplatformlocation(B)andperformedahighernumberoftargetcrossings(C)duringprobecomparedwiththe Rats trained with either the higher or
grouptrainedunderthelowerstresscondition(25°C).D,Bothexperimentalgroupsspentmoretimeinthetargetquadrantthan lower water temperature had higher
intheoppositequadrantinsearchingoftheplatformlocation.Ratstrainedatthehigherstressconditionspentlesstimeinthe corticosterone levels than home cage
oppositequadrantthanratstrainedatthelowerstresscondition.E,Illustrationofratcoronalsectionsshowinginfusionneedle controlrats(p(cid:5)0.01forbothcompar-
terminationsitesoftheratsincludedinthefinalanalyses.F,Representativeprobe-trialswimpathsofthe19°Candthe25°C
isons). More importantly, rats trained
groups.**p(cid:5)0.01versus25°Cgroup.##p(cid:5)0.01versusthecorrespondentoppositequadrant.§p(cid:5)0.05versusopposite
quadranttimeofthe25°Cgroup.Dataaremean(cid:2)SEM(n(cid:6)36–40pergroup). with the lower water temperature had
higherplasmacorticosteronelevelsthan
thosetrainedwiththehigherwatertem-
0.0001),confirmingthatthetwogroupsprogressivelylearned perature(p(cid:5)0.05),implyingthatthelowerwatertempera-
tolocatetheplatformacrossthetwotrainingsessions.Italso turerepresentedahigherstresscondition.Asexpected,one-
revealedasignificanttrainingday(cid:4)watertemperatureeffect way ANOVA for plasma corticosterone levels measured
(F(1,74)(cid:6)7.38,p(cid:6)0.008).Posthocanalysisindicatedthatrats immediatelyafterthe60sprobetrialdidnotrevealanyexper-
trained at the higher stress condition (19°C) had shorter es- imental condition effect (F (cid:6) 1.29; p (cid:5) 0.30; Table 1)
(2,22)
capelatenciesontheseconddayoftrainingthanthosetrained
because it is well documented that plasma corticosterone
at 25°C (p (cid:5) 0.01; Fig. 1A). Thus, rats trained at the higher
peaks20minafterthestressonset.Thus,atthetimeofmem-
stressconditionacquiredthebehavioraltaskbetterthanrats
oryretrieval,bothgroupsofanimalspresentedthesamelevels
trainedat25°C.
ofplasmacorticosterone.
AsshowninFigure1B–D,ratstrainedatthehigherstress
condition had also better probe-trial retention performance
ThecannabinoidreceptoragonistWIN55212-2impairs
comparedwithratstrainedat25°C.Unpairedttestsrevealed
probe-trialretentionperformanceofratstrainedatthe
thatratstrainedatthehigherstressconditiontooklesstimeto
higherstresscondition
initiallycrosstheplatformlocation(t (cid:6)(cid:3)3.72;p(cid:6)0.0004;
(74) Thisexperimentinvestigatedwhetherthecannabinoidreceptor
Fig. 1B) and had a higher number of crossings through the agonist WIN55212-2 (10 or 30 ng in 0.5 (cid:2)l) infused into the
platformlocation(t (cid:6)2.97;p(cid:6)0.004;Fig.1C).Two-way
(74) dorsalhippocampus60minbeforetheprobetrialwouldaffect
ANOVA for time spent searching for the platform location
retentionperformanceandwhetherWIN55212-2effectsdepend
revealednowatertemperatureeffect(F (cid:6)0.85,p(cid:6)0.36)
(1,148) onthestresscondition.
butasignificantquadrant(F (cid:6)171.18,p(cid:5)0.0001)and
(1,148)
water temperature (cid:4) quadrant interaction effect (F (cid:6) 19°Cwatertemperature
(1,148)
4.30,p(cid:6)0.04).Bothgroupsofvehicle-treatedratsexhibited Allanimalsprogressivelylearnedtolocatetheplatformacrossthe
memoryoftheplatformpositionduringtraining,asindicated trainingsessions,beforedrugtreatment,asindicatedbydecreasing
13966•J.Neurosci.,October14,2015•35(41):13962–13974 Morenaetal.•Cannabinoids,Stress,andSpatialMemoryRetrieval
Table1.Plasmacorticosteronelevelsofratstrainedunderhigher(19°C)andlower fects(F (cid:6)4.20;p(cid:6)0.04;Fig.2G).Ratsadministeredvehicle
(1,88)
stress(25°C)conditionandhomecageratsa intothehippocampusspentsignificantlymoretimeinthetarget
Corticosterone(ng/ml) quadrantthanintheoppositequadrant(p(cid:5)0.01;Fig.2G).Asin
Timepoint Homecage 19°C 25°C the previous experiment, WIN55212-2 infused into the hip-
pocampus60minbeforetheprobetrialreducedthetimespentin
20minafterTraining1 76.5(cid:2)16.6 490.2(cid:2)24.0(cid:2),** 339.6(cid:2)66.4** the target quadrant (p (cid:5) 0.01; Fig. 2G). Administration of
20minafterTraining2 57.6(cid:2)26.5 451.4(cid:2)36.4(cid:2),** 309.1(cid:2)41.5**
Immediatelyafterprobe 120.3(cid:2)45.2 174.9(cid:2)29.7 101.8(cid:2)31.2 AM251aloneintothehippocampusdidnotaffectthetimespent
ineitherthetargetoroppositequadrant,butAM251blockedthe
aDataaremean(cid:2)SEM(n(cid:6)5–9pergroup).
effectsinducedbyWIN55212-2.Ratsgivencombinedinfusions
**p(cid:5)0.01versusthecorrespondinghomecagegroup;(cid:2)p(cid:5)0.05versusthecorresponding25°Cgroup.
ofAM251andWIN55212-2spentsignificantlymoretimeinthe
targetquadrantthanratsgivenWIN55212-2alone(p(cid:5)0.01;Fig.
meanescapelatenciesastrainingprogressed(F (cid:6)140.88,p(cid:5) 2G).Two-wayANOVAfortotalswimdistancedidnotreveala
(1,31)
0.0001;datanotshown).Figure2A–CshowsthatWIN55212-2ad- significant WIN55212-2 (F (cid:6) 0.006; p (cid:6) 0.94), AM251
(1,44)
ministeredintothedorsalhippocampus60minbeforetheprobe (F (cid:6)0.34;p(cid:6)0.56),orWIN55212-2(cid:4)AM251interaction
(1,44)
trialimpairedretentionperformanceofratstrainedatthishigher effect(F (cid:6)0.13;p(cid:6)0.72;datanotshown).
(1,44)
stresscondition.One-wayANOVAsforcrossinglatenciesandtarget
crossingsduringtheprobetrialrevealedasignificantWIN55212-2 25°Cwatertemperature
effect(crossinglatencies:F (cid:6)3.53,p(cid:6)0.04;targetcrossings: WIN55212-2 infused into the hippocampus 60 min before the
(2,31)
F (cid:6)5.78,p(cid:6)0.007;Figure2A,B).Posthocanalysesindicated probetrialdidnotaffectretentionperformanceofratstrainedat
(2,31)
thatthe10ngdoseofWIN55212-2,butnotthehigherdose,signif- this lower stress condition (Fig. 2I–K). One-way ANOVAs for
icantlyincreasedcrossinglatencies(p(cid:5)0.05;Fig.2A)and,con- initiallatencytocrosstheplatformlocationandthenumberof
versely, decreased the number of crossings through the platform crossingsthroughtheplatformlocationdidnotrevealasignifi-
location(p(cid:5)0.01;Fig.2B).Two-wayANOVAforquadrantsearch cantWIN55212-2effect(F(2,32)(cid:6)0.50,p(cid:6)0.62;F(2,32)(cid:6)0.52,
times during the probe trial showed a significant quadrant effect p(cid:6)0.60;respectively,Figure2I,J).Two-wayANOVAforquad-
(F (cid:6)109.23,p(cid:5)0.0001),noWIN55212-2effect(F (cid:6)9.23, rant search times during the probe trial showed a significant
p ((cid:6)1,62)0.56), and a significant interaction between b(2o,6th2) factors quadranteffect(F(1,64)(cid:6)59.30,p(cid:5)0.0001)butnoWIN55212-2
m(Fe(2n,6t2a)l(cid:6)gro7u.3p7s,psp(cid:6)en0t.0m0o1r)e.Ptiomstehioncathnealtyasrigsertevqeuaaldedratnhtatthaallnexinpetrhie- teoffresc(tF((F2,(624,6)4(cid:6))(cid:6)0.605.1,3p,(cid:6)p(cid:6)0.502.8;F8)igo.r2Kin)t.eAralcsoti,onnobgertowuependibffoetrhenfacce-s
oppositequadrant(p(cid:5)0.01;Fig.2C),indicatingthatallgroups were found for total swim distance during the probe trial
exhibitedmemoryoftheplatformlocationduringtraining.How- (F(2,32)(cid:6)0.77;p(cid:6)0.47;datanotshown).
ever,WIN55212-2significantlyreducedthetimespentsearchingfor
theplatformlocationinthetargetquadrant(p(cid:5)0.05;Fig.2C).No The2-AGhydrolysisinhibitorJZL184impairsprobe-trial
WIN55212-2effectwasfoundfortotalswimdistanceduringthe retentionperformanceofratstrainedatthehigherstress
probetrial(F (cid:6)0.46;p(cid:6)0.63;datanotshown). condition
(2,31)
ToinvestigatewhethertheCB1receptormediatestheimpair- We examined whether an increased endogenous tone of 2-AG
ingeffectofWIN55212-2,theCB1receptorantagonistAM251 couldbeinvolvedinthemodulationofprobe-trialperformance
(0.28ngin0.5(cid:2)l)wasadministeredtogetherwiththeeffective atthehigherstresscondition.Tothisaim,the2-AGhydrolysis
doseofWIN55212-2(10ng)intothedorsalhippocampus60min inhibitorJZL184(0.1and1(cid:2)gin0.5(cid:2)l)wasinfusedbilaterally
before the probe trial. As shown in Figure 2E, F, two-way intothedorsalhippocampus60minbeforetheprobetrialofrats
ANOVAsforcrossinglatenciesandthenumberoftargetcross- trainedwiththetwodifferentwatertemperatures.
ingsduringtheprobetrialrevealedasignificantmaineffectof
WIN55212-2(crossinglatencies:F (cid:6)8.99;p(cid:6)0.004;target 19°Cwatertemperature
(1,44)
crossings: F (cid:6) 5.37; p (cid:6) 0.03), no AM251 effect (crossing Allanimalsprogressivelylearnedtolocatetheplatformacrossthe
(1,44)
latencies:F (cid:6)3.43;p(cid:6)0.07;targetcrossings:F (cid:6)2.81; twotrainingsessions,beforedrugtreatment,asindicatedbyde-
(1,44) (1,44)
p(cid:6)0.10),andasignificantinteractionbetweenWIN55212-2and creasingmeanescapelatenciesastrainingprogressed(F (cid:6)
(1,26)
AM251(crossinglatencies:F (cid:6)5.59;p(cid:6)0.02;targetcross- 119.11, p (cid:5) 0.0001; data not shown). Similar to the effects in-
(1,44)
ings: F (cid:6) 7.22; p (cid:6) 0.01). Tukey’s post hoc comparisons ducedbyWIN55212-2,wefoundthatthe2-AGhydrolysisinhib-
(1,44)
showedthatWIN55212-2alonesignificantlyincreasedtheinitial itorJZL184impairedprobe-trialretentionperformanceofrats
timetocrosstheplatformlocation(p(cid:5)0.01;Fig.2E)andde- trained at this higher stress condition. One-way ANOVAs for
creasedthenumberoftargetcrossings(p(cid:5)0.01;Fig.2F).These crossinglatenciesandtargetcrossingsduringtheprobetrialre-
effectsweremediatedbyCB1receptoractivationbecauseinitial vealed a significant JZL184 effect (crossing latencies: F (cid:6)
(2,26)
crossing latencies of rats given WIN55212-2 together with 4.28,p(cid:6)0.03;targetcrossings:F (cid:6)5.99,p(cid:6)0.007;Figure
(2,26)
AM251 were significantly shorter than those of rats given 3A,B).The1(cid:2)gdoseofJZL184,butnotthelowerdose,signifi-
WIN55212-2alone(p(cid:5)0.05;Fig.2E)andequivalenttothoseof cantlyincreasedcrossinglatencies(p(cid:5)0.05;Fig.3A),andboth
ratsgiveneithervehicleorAM251alone.Consistently,thenum- dosesofJZL184decreasedthenumberofcrossingsthroughthe
beroftargetcrossingsofratsgivenWIN55212-2togetherwith platformlocation(both,p(cid:5)0.05;Fig.3B).AsshowninFigure
AM251 was significantly higher than those of rats given 3C,two-wayANOVAforquadrantsearchtimesduringtheprobe
WIN55212-2alone(p(cid:5)0.05;Fig.2F)andequivalenttothose trial showed a significant quadrant effect (F (cid:6) 59.30, p (cid:5)
(1,52)
of rats given vehicle or AM251 alone. Three-way ANOVA for 0.0001),noJZL184effect(F (cid:6)0.17,p(cid:6)0.84),andasignif-
(2,52)
quadrantsearchtimesduringtheprobetrialrevealednomain icantinteractionbetweenbothfactors(F (cid:6)7.05,p(cid:6)0.002).
(2,52)
effectofWIN55212-2(F (cid:6)1.18;p(cid:6)0.28)butsignificant Posthocanalysisindicatedthatbothcontrolratsandratstreated
(1,88)
AM251(F (cid:6)4.95;p(cid:6)0.03),quadrant(F (cid:6)191.75;p(cid:5) withthelowerdoseofJZL184(0.1(cid:2)g)spentsignificantlymore
(1,88) (1,88)
0.0001),andWIN55212-2(cid:4)AM251(cid:4)quadrantinteractionef- timeinthetargetquadrantthanintheoppositequadrant(p(cid:5)
Morenaetal.•Cannabinoids,Stress,andSpatialMemoryRetrieval J.Neurosci.,October14,2015•35(41):13962–13974•13967
Figure2. IntrahippocampalinfusionsofthecannabinoidagonistWIN55212-2(WIN)impairedprobe-trialretentionperformance,andtheCB1receptorantagonistAM251(0.28ng/0.5
(cid:2)l)blockedthememoryimpairingeffectinducedbyWIN(10ng/0.5(cid:2)l;WIN10),onlyinthehigherstressconditiontrainedgroup(19°C).Incontrast,intrahippocampalinfusionsofWIN
didnotaffectspatialmemoryperformanceatthelowerstresscondition(25°C).WINeffectsonthelatencytofirstcrosstheplatformlocation(A),thenumberoftargetcrossings(B),and
thetimespentinthetargetandoppositequadrants(C)duringthe1minprobetrialat19°C.D,IllustrationofratcoronalsectionsshowinginfusionneedleterminationsitesofWIN-treated
ratsandtheirvehiclecontrolgroupatthehigherstressfulconditionincludedinthefinalanalyses.EffectsofWIN(cid:7)AM251administrationonthelatencytofirstcrosstheplatformlocation
(E),thenumberoftargetcrossings(F),andthetimespentinthetargetandoppositequadrants(G)duringthe1minprobetrialat19°C.H,Illustrationofratcoronalsectionsshowing
infusionneedleterminationsitesofWIN-,WIN(cid:7)AM251-,AM251-treatedratsandtheirvehiclecontrolgroupatthehigherstressfulconditionincludedinthefinalanalyses.WINeffects
onthelatencytofirstcrosstheplatformlocation(I),thenumberoftargetcrossings(J),andthetimespentinthetargetandoppositequadrants(K)duringthe1minprobetrialat25°C.
L,IllustrationofratcoronalsectionsshowinginfusionneedleterminationsitesofWIN-treatedratsandtheirvehiclecontrolgroupatthelowerstressfulconditionincludedinthefinal
analyses.M,Representativeprobe-trialswimpaths.*p(cid:5)0.05versusthecorrespondentvehicle(Veh)group.**p(cid:5)0.01versusthecorrespondentvehicle(Veh)group.##p(cid:5)0.01
versusthecorrespondentoppositequadrant.(cid:2)p(cid:5)0.05versustargetquadranttimeofvehiclegroup.(cid:2)(cid:2)p(cid:5)0.01versustargetquadranttimeofvehiclegroup.(cid:3)p(cid:5)0.05versus
thecorrespondentWIN(cid:7)AM251group.(cid:3)(cid:3)p(cid:5)0.01versusthecorrespondentWIN(cid:7)AM251group.Dataaremean(cid:2)SEM(n(cid:6)9–14pergroup).
0.01;Fig.3C),thusindicatingthattheyrememberedtheplatform ThenextexperimentexaminedwhethertheJZL184effecton
location.Thisdifferencewasnotfoundinanimalstreatedwith probe-trialperformanceismediatedbyCB1receptoractivation.
the1(cid:2)gdoseofJZL184(Fig.3C),indicatingthattheydidnot Tothisaim,theCB1receptorantagonistAM251(0.28ng/0.5(cid:2)l)
exhibitmemoryfortheplatformlocation.JZL184didnotaffect andtheeffectivedoseofJZL184(1(cid:2)g)wereinfusedtogetherinto
total swim distance during the probe trial (F (cid:6) 2.44; p (cid:6) thedorsalhippocampus60minbeforeretentiontesting.Figure
(2,26)
0.11;datanotshown). 3E–GshowstheeffectofJZL184togetherwithAM251onprobe-
13968•J.Neurosci.,October14,2015•35(41):13962–13974 Morenaetal.•Cannabinoids,Stress,andSpatialMemoryRetrieval
Figure3. Intrahippocampalinfusionsofthe2-AGhydrolysisinhibitorJZL184(JZL)impairedprobe-trialretentionperformance,andtheCB1receptorantagonistAM251(0.28ng/0.5(cid:2)l)blocked
thememoryimpairingeffectinducedbyJZL(1(cid:2)g/0.5(cid:2)l;JZL1),onlyinthehigherstressconditiontrainedgroup(19°C).Incontrast,intrahippocampalinfusionsofJZLdidnotaffectspatialmemory
performanceatthelowerstresscondition(25°C).JZLeffectsonthelatencytofirstcrosstheplatformlocation(A),thenumberoftargetcrossings(B),andthetimespentinthetargetandopposite
quadrants(C)duringthe1minprobetrialat19°C.D,IllustrationofratcoronalsectionsshowinginfusionneedleterminationsitesofJZL-treatedratsandtheirvehiclecontrolgroupatthehigher
stressfulconditionincludedinthefinalanalyses.EffectsofJZL(cid:7)AM251administrationonthelatencytofirstcrosstheplatformlocation(E),thenumberoftargetcrossings(F),andthetimespent
inthetargetandoppositequadrants(G)duringthe1minprobetrialat19°C.H,IllustrationofratcoronalsectionsshowinginfusionneedleterminationsitesofJZL-,JZL(cid:7)AM251-,AM251-treated
ratsandtheirvehiclecontrolgroupatthehigherstressfulconditionincludedinthefinalanalyses.EffectsofJZLonthelatencytofirstcrosstheplatformlocation(I),thenumberoftargetcrossings
(J),andthetimespentinthetargetandoppositequadrants(K)duringthe1minprobetrialat25°C.L,IllustrationofratcoronalsectionsshowinginfusionneedleterminationsitesofJZL-treated
ratsandtheirvehiclecontrolgroupatthelowerstressfulconditionincludedinthefinalanalyses.M,Representativeprobe-trialswimpaths.*p(cid:5)0.05versusthecorrespondentvehicle(Veh)group.
**p(cid:5)0.01versusthecorrespondentvehicle(Veh)group.##p(cid:5)0.01versusthecorrespondentoppositequadrant.(cid:2)p(cid:5)0.05versustargetquadranttimeofvehiclegroup.§p(cid:5)0.05versus
oppositequadranttimeofvehiclegroup.(cid:3)(cid:3)p(cid:5)0.01versusthecorrespondentJZL(cid:7)AM251group.Dataaremean(cid:2)SEM(n(cid:6)8–14pergroup).
trialretentionperformance.Two-wayANOVAsforthecrossing F (cid:6) 9.79; p (cid:6) 0.003; target crossings: F (cid:6) 10.55; p (cid:6)
(1,41) (1,41)
latenciesandthenumberoftargetcrossingsrevealedsignificant 0.002).JZL184alonesignificantlyincreasedtheinitiallatencyto
JZL184 (crossing latencies: F (cid:6) 9.56; p (cid:6) 0.004; target crosstheplatformlocation(p(cid:5)0.01;Fig.3E)andreducedthe
(1,41)
crossings:F (cid:6)5.90;p(cid:6)0.02),AM251(crossinglatencies: numberoftargetcrossings(p(cid:5)0.01;Fig.3F).Initialcrossing
(1,41)
F (cid:6)8.24;p(cid:6)0.007;targetcrossings:F (cid:6)4.28;p(cid:6)0.04), latenciesofratsgivenJZL184togetherwithAM251weresignifi-
(1,41) (1,41)
and JZL184 (cid:4) AM251 interaction effects (crossing latencies: cantlyshorterthanthoseofratsgivenJZL184alone(p(cid:5)0.01;
Morenaetal.•Cannabinoids,Stress,andSpatialMemoryRetrieval J.Neurosci.,October14,2015•35(41):13962–13974•13969
Table2.Hippocampalendocannabinoid2-AGandAEAlevelsofratsgiven aim,theAEAhydrolysisinhibitorURB597wasinfusedbilaterally
intrahippocampaladministrationofJZL184oritsvehiclea intothehippocampus60minbeforetheprobetrialofratstrained
Treatment 2-AG(nmol/gtissue) AEA(pmol/gtissue) withthetwowatertemperatures.
Vehicle 7.5(cid:2)0.6 17.7(cid:2)1.9
19°Cwatertemperature
JZL184(1(cid:2)g/side) 10.7(cid:2)1.0* 15.9(cid:2)2.1
Repeated-measuresANOVAforescapelatenciestofindthehid-
aDataaremean(cid:2)SEM(n(cid:6)7–10pergroup). denplatformonthetwotrainingdaysrevealedasignificanteffect
*p(cid:5)0.05versusthecorrespondingvehiclegroup. of training day (F (cid:6) 84.23, p (cid:5) 0.0001; data not shown),
(1,28)
confirmingthatratsprogressivelylearnedtolocatetheplatform.
Fig. 3E) and equivalent to those of rats given either vehicle or
Figure4A–CshowsthatURB597infusedintothehippocampus
AM251alone.Consistently,thenumberoftargetcrossingsofrats
60 min before the probe trial did not affect retention of rats
givenJZL184andAM251wassignificantlyhigherthanthoseof
trainedwiththishigherstresscondition.One-wayANOVAdid
rats given JZL184 alone (p (cid:5) 0.01; Fig. 3F) and equivalent to
not reveal any significant drug effect for crossing latencies
thoseofratsgivenvehicleorAM251alone.Three-wayANOVA (F (cid:6)0.36,p(cid:6)0.70;Fig.4A)ortargetcrossingsduringthe
fortimespentinthetargetandoppositequadrantrevealedno pr(o2b,2e8)trial(F (cid:6)0.62,p(cid:6)0.55;Fig.4B).Two-wayANOVA
maineffectsofJZL184(F (cid:6)0.76;p(cid:6)0.39)orAM251(F (2,28)
(1,82) (1,82) forquadrantsearchtimesduringtheprobetrialshowedasignif-
(cid:6)1.52;p(cid:6)0.22)onquadrantsearchtimesduringtheprobetrial icant quadrant effect (F (cid:6) 82.64, p (cid:5) 0.0001), but no
(Fig.3G).However,therewasasignificantquadrant(F(1,82)(cid:6) URB597 (F (cid:6) 0.084(,1,p56)(cid:6) 0.92), or quadrant (cid:4) URB597
141.89;p(cid:5)0.0001)andJZL184(cid:4)AM251(cid:4)quadrantinterac- interaction(e2f,5fe6)ct (F (cid:6) 1.56, p (cid:6) 0.22). Post hoc analysis
tioneffect(F (cid:6)7.18;p(cid:6)0.009).Controlratsspentsignifi- (2,56)
(1,82) revealed that all experimental groups spent more time in the
cantly more time in the target quadrant than in the opposite targetquadrantthanintheoppositequadrant(p(cid:5)0.01;Fig.4C).
quadrant(p(cid:5)0.01;Fig.3G).JZL184decreasedthetimespentin
Also, no group differences were found for total swim distance
the target quadrant (p (cid:5) 0.05; Fig. 3G). AM251 alone did not duringtheprobetrial(F (cid:6)0.52;p(cid:6)0.60;datanotshown).
affectthetimespentineitherthetargetoroppositequadrantbut (2,28)
blockedtheeffectsinducedbyJZL184.RatstreatedwithAM251 25°Cwatertemperature
andJZL184spentsignificantlymoretimeinthetargetquadrant URB597infusedintothehippocampus60minbeforetheprobe
(p(cid:5)0.01;Fig.3G)thanratsgivenJZL184aloneanddidnotdiffer trialalsodidnotaffectretentionofratstrainedatthelowerstress
significantly from those given either vehicle or AM251 alone. condition (Fig. 4E–G). One-way ANOVA indicated no signifi-
Two-wayANOVAfortotalswimdistancedidnotrevealasignif- cantURB597effectforinitiallatencytocrosstheplatformloca-
icantJZL184effect(F (cid:6)0.008;p(cid:6)0.93),AM251(F (cid:6) tion (F (cid:6) 0.08, p (cid:6) 0.93; Fig. 4E) or for the number of
(1,41) (1,41) (2,33)
1.69;p(cid:6)0.20),orJZL184(cid:4)AM251interactioneffect(F (cid:6) crossingsthroughtheplatformlocation(F (cid:6)0.69,p(cid:6)0.51;
(1,41) (2,33)
1.21;p(cid:6)0.28;datanotshown). Fig.4F).Two-wayANOVAforquadrantsearchtimesduringthe
probetrialshowedasignificantquadranteffect(F (cid:6)112.69,
25°Cwatertemperature p(cid:5)0.0001),butnoURB597effect(F (cid:6)0.16(1,,6p6)(cid:6)0.86)or
JZL184 administered into the hippocampus 60 min before the (2,66)
interaction between both factors (F (cid:6) 2.35, p (cid:6) 0.10; Fig.
probetrialdidnotaffectmemoryretentionofratstrainedatthis (2,66)
4G). No group differences were found for total swim distance
lower stress condition (Fig. 3I–K). One-way ANOVAs did not
revealasignificantJZL184effectforcrossinglatencies(F (cid:6) (F(2,66)(cid:6)0.053;p(cid:6)0.95;datanotshown).
(2,36)
0.24,p(cid:6)0.79;Fig.3I)ortargetcrossings(F (cid:6)0.37,p(cid:6)0.69;
(2,36)
Fig.3J).Two-wayANOVAforquadrantsearchtimesindicateda Watermazetrainingatthehigherstressconditionincreases
significantquadranteffect(F (cid:6)47.31,p(cid:5)0.0001)butno hippocampal2-AGlevelsduringprobe-trialretentiontesting
(1,72)
JZL184effect(F (cid:6)0.45,p(cid:6)0.64)orinteractionbetween Thisexperimentinvestigatedwhetherthedifferentstresslevels
(2,72)
bothfactors(F (cid:6)0.19,p(cid:6)0.83;Fig.3K).Nogroupeffects associatedtothetrainingproceduresinducedanyalterationin
(2,72)
werefoundfortotalswimdistance(F (cid:6)0.008;p(cid:6)0.99;data endocannabinoid 2-AG and/or AEA hippocampal levels at the
(2,36)
notshown). timeofretentiontesting.Parallelgroupsofratsweretrainedata
AstheeffectivenessofJZL184inratsremainscontroversial, watertemperatureofeither19°Cor25°Candeuthanizedimme-
withsomestudiesreportingunchangedbrain2-AGlevelsfollow- diatelyaftertheprobetrialforbraintissuedissection.Homecage
ingsystemicorlocaladministrationofJZL184(Longetal.,2009; controlratswerehandledonly.AsshowninFigure5A,one-way
Wiskerkeetal.,2012;Kerretal.,2013)andotherstudiesreport- ANOVAforhippocampal2-AGlevelsindicatedasignificantex-
ingbothbiochemicalandbehavioraleffects(Sciolinoetal.,2011; perimentalconditioneffect(F(2,24)(cid:6)4.68;p(cid:6)0.02).Ratstrained
Olesonetal.,2012;Seillieretal.,2014),weperformedanaddi- at19°Chadhigher2-AGlevelsimmediatelyaftertheprobetrial
tionalexperimenttorelatethebehavioraleffectsofJZL184with thanratstrainedat25°Corhomecagecontrolrats(p(cid:5)0.05for
any change in endocannabinoid levels. Two parallel groups of both comparisons; Fig. 5A). Conversely, one-way ANOVA for
rats,handledfor3d,werebilaterallyadministered,onday4,with hippocampal AEA levels did not reveal any group differences
thebehavioraleffectivedoseofJZL184(1(cid:2)gin0.5(cid:2)l)orvehicle (F(2,25)(cid:6)0.94;p(cid:6)0.40;Fig.5B).
into the dorsal hippocampus and killed 60 min later for hip-
pocampaldissection.AsshowninTable2,JZL184significantly Watermazetrainingatthehigherstressconditionalters
increased2-AGlevelscomparedwithcontrols(t (cid:6)2.41,p(cid:6) hippocampalMAGLactivityduringprobe-trialretention
(15)
0.03)butdidnotaffectAEAlevels(t (cid:6)(cid:3)59,p(cid:6)0.56). testing
(15)
Thisexperimentinvestigatedwhetherthedifferentstresslevelsasso-
TheAEAhydrolysisinhibitorURB597doesnotalterprobe- ciatedtothetrainingprocedure(19°Cor25°Cwatertemperature)
trialretentionperformance inducedanyalterationinhippocampalMAGLandFAAHactivityat
We next examined whether an increased endogenous tone of thetimeofretentiontesting.Ratswereeuthanizedimmediatelyafter
AEAwouldmodulateprobe-trialretentionperformance.Tothis theprobetrialforhippocampaltissuedissection.
13970•J.Neurosci.,October14,2015•35(41):13962–13974 Morenaetal.•Cannabinoids,Stress,andSpatialMemoryRetrieval
Figure4. IntrahippocampalinfusionsoftheAEAhydrolysisinhibitorURB597(URB)didnotaffectprobe-trialretentionperformanceineitherofthetwodifferentstressconditions.URBeffectson
thelatencytofirstcrosstheplatformlocation(A),thenumberoftargetcrossings(B),andthetimespentinthetargetandoppositequadrants(C)duringthe1minprobetrialat19°C.D,Illustration
ofratcoronalsectionsshowinginfusionneedleterminationsitesofURB-treatedratsandtheirvehiclecontrolgroupatthehigherstressfulconditionincludedinthefinalanalyses.URBeffectsonthe
latencytofirstcrosstheplatformlocation(E),thenumberoftargetcrossings(F),andthetimespentinthetargetandoppositequadrants(G)duringthe1minprobetrialat25°C.H,Illustrationof
ratcoronalsectionsshowinginfusionneedleterminationsitesofURB-treatedratsandtheirvehiclecontrolgroupatthelowerstressfulconditionincludedinthefinalanalyses.##p(cid:5)0.01versus
thecorrespondentoppositequadrant.Dataaremean(cid:2)SEM(n(cid:6)9–13pergroup).
AsshowninFigure5C,one-wayANOVAforV ofMAGLdid thananimalstrainedunderlowerstresscondition.Furthermore,
max
not reveal any significant experimental condition effect onlythehigherstresstrainingprocedureinducedanincreasein
(F (cid:6)1.12;p(cid:6)0.37).Interestingly,one-wayANOVAforK of hippocampal2-AGlevelsandareductionofMAGLaffinityforits
(2,8) m
MAGLindicatedasignificantexperimentalconditioneffect(F (cid:6) substrate,whenassessedatthetimeofmemoryretrieval,without
(2,8)
7.04; p (cid:6) 0.02; Fig. 5D). Post hoc comparison indicated that rats alteringeitherAEAlevelsorFAAHactivityinthehippocampus.
trainedatthehigherstressconditionhadanincreaseinK valuefor Thesefindingsareinlinewithevidencereportingincreased2-AG
m
MAGLcomparedwiththosetrainedatthelowerstressconditionor levelsafterrepeatedexposuretostressfulexperiencesinlimbic
homecagecontrolrats(p(cid:5)0.05,forbothcomparisons),indicating brain regions, including the hippocampus (Patel et al., 2005b,
thatthishigherstressconditioninducedadecreaseintheaffinityof 2009;Rademacheretal.,2008;Hilletal.,2010;Dubreucqetal.,
MAGLforitssubstrate.Noexperimentalconditioneffectwasfound 2012).Severallinesofevidenceindicatethatthesignalingcapac-
fortheV (F (cid:6)0.85;p(cid:6)0.45;Fig.5E)orK (F (cid:6)0.28;
max (2,15) m (2,15) ityof2-AGbecomesprogressivelyincreasedunderconditionsof
p(cid:6)0.76;Fig.5F)ofFAAH.
repeatedstress,whereitisthoughttomediatechangesinsynaptic
Discussion transmission and neural excitability (Patel et al., 2005b, 2009;
PatelandHillard,2008;Hilletal.,2010;Sumislawskietal.,2011;
Thepresentfindingsshowthatthehippocampalendocannabi-
Dubreucqetal.,2012).Thepresentfindingsaddtothisnotionby
noid2-AGselectivelymodulatesspatialmemoryperformanceof
demonstratinganincreasein2-AGlevelsafterrepeatedexposure
higherstressfultrainingexperience.Indeed,wefoundthatbilat-
tothehigherstressfultrainingconditionandanaugmentedbe-
eralintrahippocampalinfusionsofthecannabinoidreceptorag-
havioralresponsetotheincreased2-AGsignaling.Themecha-
onistWIN55212-2orthe2-AGhydrolysisinhibitorJZL184,but
nisms by which stress increases the signaling capacity of 2-AG
not the AEA hydrolysis inhibitor URB597, impaired spatial
remain unknown, deserving further investigation; however,
memoryperformanceofanimalstrainedunderahigherstressful
condition(19°Cwatertemperature)inaspatialwatermazepar- within the amygdala, it has been demonstrated that, following
adigm, without inducing any effect in animals trained at the exposuretorepeatedstress,MAGLexpressiontranslocatesfrom
lowerstressfulcondition(25°Cwatertemperature).Asanimals themembranetothecytosolwhereitwouldlessefficientlycatab-
wereinjected60minbeforememoryretentiontesting,inthetime olize2-AG(Sumislawskietal.,2011).
windowduringwhichthetrainingperformancewasretrieved,we Extensivecognitiveandneurobiologicalresearchonanimals,
demonstratedthatboosting2-AGsignalingselectivelyimpaired healthyhumansubjects,andamnesicpatientsindicatesthatthe
spatialmemoryretrievalinratstrainedunderthehigherstress hippocampusisanimportantbrainregioninvolvedinmemory
conditionthroughtheactivationofhippocampalCB1receptors. retrieval(Hirsh,1974;MoserandMoser,1998;Riedeletal.,1999;
Interestingly,vehicle-treatedanimalstrainedatthehigherstress Eldridgeetal.,2000;Squireetal.,2001;Brunetal.,2002;Matus-
conditionhadabetterretention(probe)memoryperformance Amatetal.,2004)andrepresentsaprimarytargetforstresshor-
Morenaetal.•Cannabinoids,Stress,andSpatialMemoryRetrieval J.Neurosci.,October14,2015•35(41):13962–13974•13971
Consistentwithourresults,previousfindingsdemonstrated
that rats trained on a water maze task at 19°C exhibited better
memoryacquisitionandretrievalthanratstrainedat25°C(Sandi
etal.,1997;Akiravetal.,2001,2004).Amorerecentstudyre-
ported that animals trained on a radial-arm water maze task
showedaninvertedU-shapedmemoryfunctionaccordingwith
thestressfulnessoftheexperimentalconditionsused(Salehiet
al.,2010).Alsoconsistentwithourcurrentfindings,ithasbeen
shownthatratstrainedat19°Chavehigherplasmacorticoste-
rone levels than rats trained at 25°C and lower than animals
trainedat16°C(Sandietal.,1997;Akiravetal.,2001,2004;Salehi
etal.,2010).Thisfacilitationofspatiallearningbystress,occur-
ringinawatermazeatlowwatertemperature,hasbeenshownto
be coupled with increased hippocampal synaptic expression of
theGluA2AMPAreceptorsubunit(ConboyandSandi,2010),
which has a critical role in controlling various properties of
AMPA receptors and in promoting dendritic spine formation
andgrowth(Sagliettietal.,2007)occurringduringmemorycon-
solidation (Diamond et al., 2006). Interestingly, the increased
expressionofGluA2subunitsiscorticosterone-dependent,and
preventingcorticosteronereleaseunderthehigherstresscondi-
tionalsointerfereswiththestress-inducedfacilitationofmemory
(ConboyandSandi,2010).
Our findings also show that intrahippocampal infusions of
WIN55212-2orJZL184impairspatialmemoryretrievalofrats
trainedunderthehigherstressconditionandthatthiseffectis
mediated by the activation of CB1 receptors. Conversely, the
samepharmacologicalmanipulationsdonotinduceanyeffecton
memoryofratstrainedunderthelowerstresscondition.There-
fore,wedemonstratethat2-AGisnotonlyinvolvedintheregu-
lationofthishippocampus-relatedmemoryprocessbutalsothat
itcanselectivelymodulatememoryretrievalofhighstressfulex-
periences.Inlinewithourfindings,intrahippocampalinfusions
ofWIN55212-2havebeenreportedtoimpairmemoryretrieval
ofhigharousingtestsituations(PiriandZarrindast,2011;Segev
and Akirav, 2011; Atsak et al., 2012). A pharmacologically in-
ducedenhancementofhippocampal2-AGexertsacomparable
effect(Atsaketal.,2012),whereasexogenousAEAadministra-
tioninthesamebrainregionhasbeenshowntonotaltermemory
Figure5. Effectsofwatertemperatureonhippocampallevelsasfollows:A,2-AGlevels
retrievalperformance(DeOliveiraAlvaresetal.,2008).
(nmol/gtissue).B,AEAlevels(pmol/gtissue).C,MAGLmaximalhydrolyticactivity(V ;
nmol/mgprotein/min).D,MAGLbindingaffinity(Km;(cid:2)M).E,FAAHmaximalhydrolyticactimviatxy ThefindingthatstimulatinghippocampalCB1receptors,di-
(Vmax;pmol/mgprotein/min).F,FAAHbindingaffinity(Km;(cid:2)M),asassessedimmediatelyafter rectlywithWIN55212-2orindirectlywithJZL184,impairsmem-
probe.*p(cid:5)0.05versusthecorrespondinghomecagegroup.(cid:2)p(cid:5)0.05versusthecorre- ory retrieval only of rats trained at the higher stress condition
sponding25°Cgroup.Dataaremean(cid:2)SEM(n(cid:6)3–10pergroup). suggeststhattherearedifferentcircuitsimplicatedintheprocess-
ingofspatialinformationdependingonthelevelofstressassoci-
atedtothelearningexperience.Previousstudieshavesuggested
mones(ReulanddeKloet,1985).Itiswellknownthatstressand thatspatiallearninginawatermazeundercoldwateractivates
glucocorticoidscanfacilitatelearningperformance(Akiravetal., theBLA,whereasincreasedactivationofthehippocampusisde-
2004; Salehi et al., 2010) and enhance memory consolidation tectedfollowingspatiallearningunderwarmwater(Akiravetal.,
(OitzlanddeKloet,1992;deKloetetal.,1999;Roozendaal,2000; 2001; Kogan and Richter-Levin, 2008). It is well known that
Joe¨ls and Baram, 2009). Accordingly, we found higher plasma intra-amygdalaprocessespromotememoryformationofstress-
corticosteronelevelsinratstrainedatthehigherstresscondition fulexperiencesinotherbrainregions,includingthehippocam-
(19°C)thanratstrainedunderthelowerstresscondition(25°C) pus(RoozendaalandMcGaugh,1997;McGaugh,2000;Packard
20minaftertraining.Furthermore,ratstrainedunderthehigher andWingard,2004;Morenaetal.,2014).Moreover,ithasbeen
stressconditionshowedastrongeracquisitionofthebehavioral demonstrated that, once established, the spatial memory trace
task,togetherwithabettermemoryretentionofastronglyac- formedunderhighstresscondition(i.e.,watermazeat19°C)is
quired training, relative to rats in the lower stress condition. strongercomparedwiththetraceformedunderlowerlevelsof
Thus,thetwoexperimentalconditionsattrainingelicitedadif- stress(Sandietal.,1997;KoganandRichter-Levin,2010;Salehiet
ferentreleaseofcorticosteronedependingonthelevelofstress al.,2010).However,thisstrongmemorytraceismorecomplex,
associatedtothewatertemperature;andsubsequently,suchhor- thusmorevulnerabletointerference(KoganandRichter-Levin,
monemayhavedifferentiallyaffectedretentionprocessesofthe 2010). The dual effect of emotionality may reflect the relative
twogroupsofrats,leadingtodiversememoryretrievalperfor- contributionofdifferentneuronalcircuitsandbrainregionsre-
mancesontheprobetrial. cruited to process stressful information (Adolphs et al., 1997;