Spatial memory is impaired by peripubertal GnRH agonist treatment and testosterone replacement in sheep

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Psychoneuroendocrinology

jou rn a l h om ep ag e :w w w . e l s e v i e r . c o m / l o c a t e / p s y n e u e n

Spatial memory is impaired by peripubertal GnRH agonist treatment and testosterone replacement in sheep

D. Hough

^a

, M. Bellingham

^a

, I.R.H. Haraldsen

^b

, M. McLaughlin

^c

, M. Rennie

^a

, J.E. Robinson

^a

, A.K. Solbakk

^b,d,e

, N.P. Evans

^a,∗

aInstituteofBiodiversityAnimalHealthandComparativeMedicine,CollegeofMedicalVeterinaryandLifeSciences,UniversityofGlasgow,Glasgow,G61 1QH,UK

bDepartmentofMedicalNeurobiology,DivisionofClinicalNeuroscience,OsloUniversityHospital—Rikshospitalet,0027,Oslo,Norway

cDivisionofVeterinaryBioscienceandEducation,SchoolofVeterinaryMedicine,CollegeofMedicalVeterinaryandLifeSciences,UniversityofGlasgow, Glasgow,G611QH,UK

dDepartmentofPsychology,UniversityofOslo,Pb1094Blindern,0317Oslo,Norway

eDepartmentofNeuropsychology,HelgelandHospital,Mosjøen,Norway

a r t i c l e i n f o

Articlehistory:

Received12August2016

Receivedinrevisedform19October2016 Accepted19October2016

Keywords:

Spatialorientation Spatialmemory Hippocampus GnRH Puberty Genderdysphoria

a b s t r a c t

Chronicgonadotropin-releasinghormoneagonist(GnRHa)isusedtherapeuticallytoblockactivitywithin thereproductiveaxisthroughdown-regulationofGnRHreceptorswithinthepituitarygland.GnRH receptorsarealso expressedin non-reproductivetissues,including areasof thebrainsuchas the hippocampusandamygdala.Theimpactoflong-termGnRHa-treatmentonhippocampus-dependent cognitivefunctions,suchasspatialorientation,learningandmemory,isnotwellstudied,particularly whentreatmentencompassesacriticalwindowofdevelopmentsuchaspuberty.Thecurrentstudyused anovinemodeltoassessspatialmazeperformanceandmemoryoframsthatwereuntreated(Controls), hadbothGnRHandtestosteronesignalingblocked(GnRHa-treated),orspeciﬁcallyhadGnRHsignaling blocked(GnRHa-treatedwithtestosteronereplacement)duringtheperipubertalperiod(8,27and41 weeksofage).Theresultsdemonstratethatemotionalreactivityduringspatialtaskswascompromised bytheblockadeofgonadalsteroidsignaling,asseenbytherestorativeeffectsoftestosteronereplace- ment,whiletraversetimesremainedunchangedduringassessmentofspatialorientationandlearning.

TheblockadeofGnRHsignalingalonewasassociatedwithimpairedretentionoflong-termspatialmem- oryandthiseffectwasnotrestoredwiththereplacementoftestosteronesignaling.Theseresultsindicate thatGnRHsignalingisinvolvedintheretentionandrecollectionofspatialinformation,potentiallyvia alterationstospatialreferencememory,andthattherapeuticmedicaltreatmentsusingchronicGnRHa mayhaveeffectsonthisaspectofcognitivefunction.

1. Introduction

Gonadotropin-releasing hormone (GnRH) is a hypothalamic decapeptidethatbindstoGnRHreceptors(GnRHR)intheante- riorpituitaryglandtostimulatethereleaseofthegonadotropins;

luteinizinghormone(LH)andfolliclestimulatinghormone(FSH).

WhileGnRHneuronalcellbodiesareprincipallylocatedinthepre- opticareaof thehypothalamus andthemajorityoftheiraxons project tothe median eminence, some GnRH axons extendto otherregionsofthecentralnervoussystem(CNS),includingthe limbicsystem(Silvermanetal.,1987).GnRHcancrosstheblood- brainbarrier,fromthemedianeminence,intothethirdventricle

∗Correspondingauthor.

E-mailaddress:[email protected](N.P.Evans).

cerebrospinal ﬂuid (Caraty and Skinner, 2008), soGnRH could haveeffectsonbrainfunction.CaratyandSkinner(2008)reported thatonlyextremelyhighintravenousdosesofexogenousGnRH (2.5␮gand1mginjectionintothejugularveinofewes)resulted inelevatedGnRHin thethirdventricle toa physiologicallyrel- evant level. Chronic GnRH agonist (GnRHa)treatment leads to GnRHRdesensitizationviareceptor-Gproteinuncoupling,inter- nalizationandrecyclingofGnRHRinthepituitarygland(Ferguson et al., 1996; Armstrong et al.,2011), which suppresses activity withinthehypothalamic-pituitary-gonadal(HPG)axis,including gonadotropinand gonadal steroid signaling.Long-term GnRHa- treatment is therefore used in pediatric human medicine as a co-treatmentforconditionssuchasearlyonset genderdysphoria, centralprecociouspuberty,idiopathicshortstature, growth hormone deﬁciency,congenital adrenalhyperplasia, andsevere hypothyroidism(Careletal.,2009;Hembreeetal.,2009).GnRHR http://dx.doi.org/10.1016/j.psyneuen.2016.10.016

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174 D.Houghetal./Psychoneuroendocrinology75(2017)173–182

expressionandGnRHbindingarepresent inextra-pituitarytis- sues(Hapgood etal.,2005), includingbrainregionssuchasthe hippocampus and other limbic structures (Jennes et al., 1997;

Albertsonetal.,2009;Skinneret al.,2009;Schangetal.,2010).

Therefore,long-termGnRHa-treatmentmayhaveadditionalcog- nitiveandbehavioraleffects,duetoablockadeofGnRHsignaling outsidetheHPG axis(Carel etal., 2009;Hembreeetal., 2009).

Indeed,agrowingbodyofevidencesupportsnumerousanddiverse autocrine, paracrine and endocrine roles for GnRH/GnRHa and GnRHRoutsidethepituitary gland(Hsueh and Schaeffer,1985;

Hapgoodetal.,2005;Prange-Kieletal.,2008).

PeripubertalGnRHa-treatment raises additional concerns, as thisisacriticalwindowforneuronaldevelopmentandprogram- ming(Berenbaum and Beltz, 2011). In humans, adolescence is associatedwithmarkedchangesinbehaviorandcognitionasso- ciatedwithneurocognitivematurationand/orchangesinpubertal hormones(Ernstetal.,2009).Adolescentstypicallydemonstrate increasedrisk-taking,exploratoryandobsessivebehaviors(Ernst et al., 2009) and emotional reactivity, e.g. heightened anxiety (reviewedinBuchananetal.,1992)andresponsivenesstostressful situations(Walkeretal.,1995;Hascoetetal.,1999).Adolescence isalsoatimewhenmanyneuropsychiatricdisorders,aswellas behavioralandemotionalproblems,becomeapparent(Eatonetal., 2008;CaseyandJones,2010).Whetherdevelopmentalchangesin cognitionandbehavioraredirectlyaffectedbypatternsofGnRH secretionseenatthistime,isnotknown.

Previousstudieswithanovinemodelhavedemonstratedthat pharmacologicalblockadeofthepubertaltransitionwithaGnRHa (goserelin acetate) results in sex speciﬁc changes in cognition andbehavior.GnRHa-treatedmalesweremorelikelytodisplay risk-takingbehavior in a foodacquisitiontask(Wojniuszet al., 2011), and heightened emotional reactivity (i.e.emotional and behavioralresponsestoafearfulsituation)inearlyadulthood,but decreasedemotionalreactivityatalaterage(Evansetal.,2012).

Furthermore,peripubertal GnRHa-treatment is accompanied by changesin amygdalavolume (Nuruddin et al.,2013a)and hip- pocampalgeneexpression(Nuruddinetal.,2013b).Inthelatter study,changeswereobservedinmRNAexpressionforgenesrelat- ingtoendocrinesignalingandsynapticplasticity,butthesewere notaccompaniedbysignificantdifferencesinspatialorientation (Wojniuszetal.,2013),whichisahippocampus-dependentfunc- tion(Burgess et al.,2002).A tendencywasnoted,however, for GnRHa-treatedanimalsof bothsexestotraverseaspatialmaze slowerthanuntreatedcontrols(Nuruddinetal.,2013b;Wojniusz etal.,2013).Itispossiblethattheabilitytomeasuresucheffects onspatialorientationwerelimited,inthat study,bytheuseof toosimplisticamazedesignandanassociatedceilingeffect.Fur- thermore,Robinsonetal.(2014)notedthattheinterpretationof behavioraldata(e.g.novelty-seeking)inthisovinemodeliscom- plex,becausetheassessedbehaviorsmaybepre-andperinatally programmedand/orstronglyinfluencedbytheemotionalreactiv- ityoftheanimalasitadaptstotheenvironmentinwhichitfinds itself.

Inthisstudy,theaimistoinvestigatetherisksinvolvedwith peripubertalGnRHa-treatmentforthedevelopmentofspatialori- entation,learningandmemory.Wefocusedonthemaleandextend ourinitialresults,byinvestigatingtheeffectsofchronicperipuber- talGnRHa-treatmentonage-relatedchangesinspatialorientation inamorecomplexmazedesign,whilealsotakingintoaccountany associatedchangesinemotionalreactivity.Leeetal.(2006)demon- stratedthatsheeparecapableofretainingspatialinformation,or solvingstrategies,foratleast6weeks.Spatialmemoryisalsosexu- allydifferentiated(Jonasson,2005)andinﬂuencedbytestosterone (Celecetal.,2015).Thus,inthisstudywetestedthehypothesis thatchronicperipubertalGnRHa-treatmentwouldaffectlong-term spatialmemory,andweattemptedtodissociatebetweentwocom-

ponents oflong-term spatialmemory, namelyspatialreference memory(i.e.recollectionoffamiliarspatialcues)andspatialwork- ingmemory(i.e.solvingstrategybasedonsequenceofspatialcues).

Finally,weincludedanadditionalgroupthatreceivedtestosterone replacement,inconjunctionwithGnRHa-treatment,toallowdif- ferentiationbetweentheeffectsofblockingGnRHand/orgonadal steroidsignaling.

2. Materialsandmethods

2.1. Animalsandtreatment

ThisstudywasconductedattheUniversityofGlasgowCochno Farmand ResearchCentre(55^◦ 55N)in accordancewithHome OfﬁceRegulations(ProjectLicense:60/4422).ScottishMuleTexel crossesbornbetween23Marchand12April2013werekeptwith damsuntilweaningat21weeksofage.Sheepweregrazedonpas- ture,exceptduringlambingandbehavioraltrials,whentheywere housedindoorwithadlibitumaccesstohayorsilage,andsupple- ments(i.e.proteinormineralconcentrates)accordingtostandard management practices.Malelambs fromsinglesexlitterswere usedtoruleoutpotentialeffectsoftheprenatalsteroidenviron- mentandtwins ortripletswererandomlyassigned todifferent treatmentgroupstominimizematernaleffects.Treatmentgroups consistedof:1)untreated(Control);2)GnRHa-treated(GnRHa);

and3)GnRHa-treatedramsthatalsoreceivedtestosteronereplacement(GnRHa+T).GnRHa-treatmentconsistedofasubcutaneous implantofgoserelin acetate(Zoladex3.6mg,kindlydonatedby AstraZeneca,Macclesﬁeld,UK)every4weeks,from8to44weeks ofage,astheaverageageofpubertalonsetinmalesheepis10 weeksofage(WoodandFoster,1998).Testosteronereplacement consisted of intramuscular injections of testosterone cypionate (A6960-000,Steraloids,Newport,USA)dissolvedinvegetableoil at dosesestimated toreplicate endogenous proﬁles in controls (assumedtobeasreportedbyRobinsonetal.,2014).Testosterone cypionatewasadministered onceevery2weeks asfollows: 16 weeksofage,50mg/mL;18–24weeksofage,120mg/mL;26–30 weeksofage,160mg/mL;32–44weeksofage,240mg/mL;and46 weeksofage,136.4mg/mL.Initially,139ramswereassignedtothe project,namelyControln=60,GnRHan=55andGnRHa+Tn=24, ofwhich12(Controln=4,GnRHa,n=6,Teston=2)wereremoved fromthestudyduetoillness.ThereweretwiceasmanyControland GnRHaramsasTestorams,becausehalfoftheControlandGnRHa groupscontinuedintoanotherstudy.

2.2. Assessmentofspatialorientationandlearning 2.2.1. Spatialmazedesign

Thespatialmazewasamodificationofthedesigndescribedby Wojniuszetal.(2013)andLeeetal.(2006)inthatthecomplexityof themazewasincreasedbyinclusionof‘traps’thatforcedanimalsto moveawayfromtheirconspecificsintheaudiencepen,toprogress throughthemaze(Fig.1,layout1).Themazewasconstructedin thesamebarnwheresheepwerehoused.Themazewasbordered bymetalfencingcoveredwithblackplasticsheeting,toobstruct theviewofactivityoutsidethemazearea.Theinternalwallsofthe mazeremaineduncovered,thustheaudiencepenwasvisibleatall timestoutilizetheflockinginstinctasmotivationtomovethrough themazeandreunitewiththeirmothers(8weeksofage)andflock members(8,27and41weeksofage).Oneachtestday,approxi- mately30randomlyselectedsheepwerekeptintheaudiencepen withadlibitumaccesstohayandwater.

2.2.2. Assessmentofspatialorientationandlearning

Changesin spatialorientation wereassessedas theperfor- manceofsheepintheﬁrstmazeattemptoftheday,ateachage.

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Fig.1. Spatialmazelayoutsthatwereusedfortheassessmentofspatialorientation,learningandlong-termspatialmemory(Layout1),aswellasthenovelmaze(Layout 2)inwhichtrapswithinthesame-letteredzoneswererearranged.

Spatiallearningwasassessedastheperformanceofsheepover threeseparatemazeattemptswithinthesameday(eachattempt separatedby∼2h).Duringanattempt,eachsheepwascalmlyush- eredfromtheaudiencepentothestartofthemaze.Oftherams thatdidnotcompletethemazewithinthe5mintimelimit,some remainedneartheentrancethroughout,whereasotherstraversed someofthemazebeforereturningtotheentranceinanattempt toexitthemazeviatheirpointofentry.Itwasobservedthatsome sheeppausedincertainareaswithinthemaze,asindicatedbygrey circlesinFig.1.Sheepfailingtocompletethemazewithin5min wereusheredbacktotheaudiencepenviathemazeentrance,so thatthecorrectrouteremainedunknown.Onthelastattemptof theday,unsuccessfulsheepproceededtotheaudiencepenviathe quickestroute.

2.2.3. Recordedobservations

Spatialperformancewasindividuallyassessedat8weeks(pre- pubertal,prior tohormone treatment),27weeks(post-pubertal forControls,duringthebreedingseason,19weeksafterthestart ofhormonetreatment),and41weeksofage(post-pubertal,dur- ing non-breeding season,33 weeks after thestart of hormone treatment).Traversetime(min:s:ms)wasrecordedasthetime taken to move from theentrance to ﬁnish line(line E in lay- out1ofFig.1);judgedontheplacementofthefrontlegsacross the line(5min=incomplete). Progressthrough the mazewas recordedasthetimedifferencemovingbetweenlinesAtoE.Emo- tional reactivity wasrecorded as thenumber ofvocalizations, escapeattempts, urinationsanddefecations. Anescapeattempt wasdeﬁnedasanyproactiveefforttomovethrough,overorunder awall.

2.3. Assessmentoflong-termspatialmemory 2.3.1. Training

Long-termspatialmemorytrainingwasdone,withagroupof randomlyselectedanimals(Controln=19,GnRHan=22,GnRHa+T n=22)overtwodays,shortlyafterthe41-weekspatialorientation

andlearningassessment,usingthesamemazelayout(Fig.1,Layout 1).Trainingwasatwo-stageprocess,whichconsistedofeducation andconﬁrmationruns.Foreducationruns,eachsheepwasgiven 10consecutiveattemptstocompletethemazewithin1min.After eachunsuccessfulattempt,theramwascalmlyescortedthrough theremainderofthemaze,viatheﬁnishline,totheaudiencepen.

Sheepcompletedtheeducationrunwhentheywereabletotra- versethemazewithin1minontwosuccessiveattempts.Whenall animalshadcompletedtheeducationrun,theyunderwentacon- firmationrun,whichconsistedoftwoattemptstocompletethe mazewithin1min.Ifsuccessful,trainingwascompleted.Ifunsuc- cessful,thesheephadafurther8attemptstocompletethemaze within1min,i.e.asecondsetofeducationruns,followedbytwo furtherconfirmationruns.Amaximumof3setsofeducationruns wereconductedwithinthesameday.Thetotalnumberofattempts duringeducationandconfirmationrunswasrecordedforeachram, togetherwiththequickesttraversetime,toserveasameasureof theeaseoftraining.

2.3.2. Assessmentoflong-termmemory

Retentionoflong-termspatialmemorywasassessed4weeks aftertrainingwascompleted(45weeksofage).Eachsheepwas given one maze attempt(Fig. 1,Layout 1) withtraversetimes (incomplete=5min)andprogressthroughmazezonesrecorded.

2.3.3. Assessmentoffamiliarityinanovelmazedesign

Immediatelyafterassessmentoflong-termmemory,eachsheep wasgivenoneattempttotraverseanewspatialmazelayout,which containedthesame‘traps’butinadifferentorderororientation (Fig.1,Layout2).Mazetraversetimes(incomplete=5min) and progressthroughmazezoneswererecorded.

2.4. Statisticalanalysis

Foreachanimal,theemotionalreactivityparameterswerenor- malizedbyexpressionrelativetothetimespentinthemaze.The proportionof timespentin eachzone wascalculatedas aper-

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Fig.2.Spatialorientation(Attempt1only)andspatiallearning(Attempts1–3)performancewasmeasuredbythemean±s.e.m.mazetraversetimesoframsastheyaged.

At8weeksofage,allramsremaineduntreated,whereasat27and41weeksofage,GnRHa-treatmenthadbeenadministeredfor19and33weeksrespectively.Different lettersontopofbarsindicatesigniﬁcantdifferencesbetweentreatmentgroupmeansatthatparticularage,asdeterminedbyaTukeyposthoctestfromatwo-wayANOVA (Treatment×Attempt).Control:untreatedrams;GnRHa:GnRHa-treatedrams;GnRHa+T:GnRHa-treatedramsthatalsoreceivedtestosteronereplacement.

centageoftotaltimespentinthemaze.Datawereexcludedfrom analysiswhere performance wasjudged tohavebeen compro- misedbecauseof temporary incapacity,i.e. health concerns. In addition,datawereexcludedfromanalysiswhereanimalsescaped fromthemazeareaorjumpedoverinternalmazewalls.Exclusion ofdatawasdonebyspecifying amissingvaluefortherelevant responsevariable(s)inthatparticularmazeattempt(nspeciﬁed inFig.2).Urinationanddefecationfrequenciesweretoolowfor statisticalanalysestoyieldmeaningfulresults.

AllstatisticalanalyseswereperformedwithRsoftware(Version 3.2.1,©2015TheRFoundationforStatisticalComputingPlatform) usingtheRStudiointerface(Version0.99.467,©2009–2015RStu- dioInc.).Wheredatawerenotnormallydistributed,theresponse variablewaslog-transformed.Responsevariableswereanalyzed usingthegeneralizedlinearmodel(GLM)functionusingaGaussian distribution;ramidentitywasincludedasanexplanatoryvariable toaccountforindividualvariationacrosstimeorrespectivemaze attempts.Effectsofageandtreatmentonspatialorientationwere assessedwithdatafromtheﬁrstattemptofthemaze,acrossall ages,usingatwo-wayANOVA(Treatment×Age).Effectsoftreat- mentonspatiallearning,overthreeconsecutivemazeattempts, wereassessedwithtwo-wayANOVA(Treatment×Mazeattempt) ateachrespectiveage.One-wayANOVAwasusedtoassessthe effectsoftreatmentontheeaseofmazetraining(numberoftrain- ingattempts),aswellastraversetimesuponcompletionoftraining.

Effectsoftreatmentonlong-termspatialmemoryweretestedby comparisonoftraversetimesat:1)45weeksofageonly(one-way ANOVA);2)41(lasttrainingattempt)versus45(theassessment attempt)weeksofage(two-wayANOVA:Treatment×Time).The effectof mazedesign familiaritywas examinedby comparison ofthetraversetimeofmazelayouts1and2(two-wayANOVA:

Treatment×Maze Layout). Tukey Honest Significant Difference (TukeyHSD)posthoctestwasusedtoassesswheresignificantdif- ferencesexistedbetweentreatmentgroups.Allgraphsrepresent meansandstandarderrorsofthemean.StatisticalP-values<0.05 wereconsideredsignificant.

3. Results

3.1. Spatialorientation-Completionofmazeattheﬁrst attempt

3.1.1. Traversetime

Themeantraversetimedecreasedsigniﬁcantly(P<0.001)with age (Fig. 2 & Table 1)and was accompanied by a progressive increaseintheproportionoframsthatsuccessfullycompletedthe maze(23,71,85%at8,27and41weeks,respectively).Therewere nosigniﬁcanteffectsofeithertheGnRHaorGnRHa+Ttreatments

onthetimetakentocompletethemazeattheﬁrstattempt,at either27or41weeksofage(Fig.2&Table1).

3.1.2. Progressthroughmazezones

Theproportionoftimespentinthedifferentmazezonesvaried significantlyasafunctionofage(Fig.3,Attempt1&Table1).Specif- ically,whileramsspentthegreatestproportionoftimeinmaze zoneC,regardlessofage,theproportionoftimespentinzones A,BandDdecreased,andzoneEincreasedsignificantly(P<0.05) withage.Typically,thelargestchangeswereseenbetween8and 27weeksofage.Therewerenosignificanteffectsoftreatmenton theproportionoftimespentinthedifferentmazezones.

3.1.3. Emotionalreactivity

Emotional reactivity improved with age, as seen by the progressivedecreaseinvocalizationrateandescapeattemptfre- quency.Theoverallmeanvocalizationratedecreasedsignificantly (P<0.001) (Fig. 4&Table 1)from14.3±0.50 vocalizations/min at 8weeks, to 3.3±0.23 and 1.0±0.13 at 27 and 41 weeks of age,respectively.Therewasnosignificanteffectoftreatmenton vocalizationrate,regardlessofage(Table1).Escapeattemptfre- quency also decreased significantly (P<0.001) with age (Fig. 4

&Table1),fromanoverallaverageof1.23±0.14 attempts/min at8 weeks, toonly 0.06±0.018and 0.14±0.035 attempts/min at27 and41weeksof age,respectively.Therewasa signiﬁcant (P<0.05)Treatment×Ageinteraction,asescapeattemptfrequency decreasedtoagreaterextentinGnRHa+TcomparedtoControland GnRHaanimals,astheyaged(Fig.4,Attempt1).

Table1

SpatialOrientation.Summaryoftwo-wayANOVAP-valuestoassesstheeffectsof treatmentonperformanceduringtheﬁrstmazeattemptatages8,27and41weeks ofage.

ResponseVariable Treatment Age Treatment×Age

Traversetime 0.356 <0.001 0.754

Proportionoftime

ZoneA 0.648 <0.001 0.902

ZoneB 0.713 0.013 0.583

ZoneC 0.602 0.371 0.081

ZoneD 0.737 <0.001 0.895

ZoneE 0.697 <0.001 0.866

Emotionalreactivity

Vocalizationrate 0.148 <0.001 0.783

Escapeattempts 0.373 <0.001 0.014

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Fig.3.Proportionatetimespentineachzoneofthemazeduringspatialorientation(Attempt1only)andspatiallearning(Attempts1–3)assessmentsasramsaged.At8 weeksofage,allramsremaineduntreated,whereasat27and41weeksofage,GnRHa-treatmenthadbeenadministeredfor19and33weeksrespectively.Control:untreated rams;GnRHa:GnRHa-treatedrams;GnRHa+T:GnRHa-treatedramsthatalsoreceivedtestosteronereplacement.*P<0.05.

Fig.4. Measuresofemotionalreactivity,expressedasthemean±s.e.m.ratesofvocalizationsandescapeattempts,duringspatialorientation(Attempt1only)andspatial learning(Attempts1–3)assessmentsasramsaged.At8weeksofage,allramsremaineduntreated,whereasat27and41weeksofage,GnRHa-treatmenthadbeen administeredfor19and33weeksrespectively.Differentlettersontopofbarsindicatesigniﬁcantdifferencesbetweentreatmentgroupmeans,asdeterminedbyaTukey posthoctestfromatwo-wayANOVA(Treatment×Attempt)atthatparticularage.Control:untreatedrams;GnRHa:GnRHa-treatedrams;GnRHa+T:GnRHa-treatedrams thatalsoreceivedtestosteronereplacement.

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Table2

Spatiallearning.Summaryoftwo-wayANOVAP-valuestoassesstheeffectsoftreatmentonperformanceacrossallthreemazeattemptswithinthesamedayatages8,27 and41weeks.

ResponseVariable 8wks 27wks 41wks

Attempt Treatment Attempt Treatment×Attempt Treatment Attempt Treatment×Attempt

Traversetime <0.001 0.281 <0.001 0.391 0.164 <0.001 0.807

Proportionoftime

ZoneA <0.001 0.099 <0.001 0.786 0.715 0.094 0.498

ZoneB 0.466 0.678 0.004 0.423 0.672 0.007 0.859

ZoneC 0.657 0.312 0.045 0.490 0.342 <0.001 0.267

ZoneD 0.205 0.856 0.354 0.428 0.025 <0.001 0.944

ZoneE 0.028 0.331 0.936 0.879 0.224 0.506 0.749

Emotionalreactivity

Vocalizationrate <0.001 <0.001 0.802 0.019 <0.001 0.966 0.909

Escapeattempts <0.001 0.316 0.378 0.497 0.646 0.391 0.326

3.2. Spatiallearning-Completionofmazewithsame-day repeatedattempts

3.2.1. Traversetime

Themeantraversetimes,acrossthethreemazeattemptsateach age,areshowninFig.2withtheassociatedtwo-wayANOVAP- valuesummaryinTable2.At8weeksofage,therewasasigniﬁcant (P<0.001)reductionintheaveragetraversetime;18.2%between attempts1and2,and30.5%between2and3.At27and41weeks ofagetherewasalsoasigniﬁcant(P<0.001)overallreductionin traversetimeacrossthethreeattempts;at27weeksitwas41.2%

betweenattempts1to2,and19.5%between2and3,whereasat 41weeksitwas5.4%betweenattempts1to2,and17.1%between 2and3.

Whiletherewasnooveralleffectoftreatmentonthetraverse timesacrossthethreeattemptsat27and41weeksofage,when onlytheGnRHaandGnRHa+Tgroupswascomparedat41weeks ofage,atrend(P=0.054)wasnotedfortraversetimetobelonger inGnRHa+Tcompared toGnRHarams;aneffectthat wasmost pronouncedinattempts1and2.

Themeanproportionaltimesspentinthemazezonesacrossall threeattemptsat8,27and41weeksofage,areshowninFig.3with theassociatedtwo-wayANOVAP-valuesummaryinTable2.At8 weeksofage,theproportionoftimespentinzoneAsignificantly (P<0.001)decreasedandZoneEsignificantly(P<0.05)increased, acrossthe three maze attempts. At 27 weeks of age, although thepattern ofzoneusage,acrossthethreemazeattempts,was moreconsistentcomparedto8weeksofage,significant(P<0.001) changesintheproportionoftimespentineachmazezonewere observed.Specifically,animalsspentincreasinglymore(P<0.001) timeinzoneA,lesstimeinzoneC(P<0.005),whereasanimalsonly spentlesstime(P<0.05)inzoneBduringattempt2.At41weeks ofage,whiletheproportionoftimespentintheindividualmaze zoneswasdominatedbyzoneCandEduringattempt1,itbecame moreequallyspreadacrosszonesduringattempts2and3.This resultedinanoverallsignificant(P<0.001)effectofmazeattempt, withasignificantincreaseintheproportionoftimespentinzones B(P<0.01)andD(P<0.001),andasignificant(P<0.001)decrease intheproportionoftimespentinzoneC.

Theonlystatisticallysigniﬁcant(P<0.05)effectoftreatmenton theproportionaltimesspentinanyofthemazezones,ateither27 or41weeksofage,wasinzoneD.Thiswasbecause,at41weeksof age,theGnRHa+Tgroupspentahigherproportionoftimeinzone DthantheControlandGnRHagroups,particularlyduringmaze attempt2.At27weeksofage,theGnRHaanimalstendedtospend thegreatest,andtheGnRHa+Tanimalstheleast,proportionoftime inzoneA(statisticalcomparisonofGnRHaandGnRHa+Tgroups

only:TreatmentP=0.081,AttemptP<0.001,Treatment×Attempt P=0.578).

3.2.3. Emotionalreactivity

Meanvocalization and escape attemptrates acrossall three attemptsateachageareshowninFig.4withtheassociatedtwo- wayANOVAP-valuesummaryinTable2.At8weeksofage,there wasa significant(P<0.001)reduction in vocalization rate with mazeattempt,namelya12% reductionfromthefirsttosecond attemptfollowedbyanegligibledecrease(1%)betweenthesec- ondandthirdmazeattempt.Atboth27and41weeksofage,no significantchangeswereseeninvocalizationrateoverthreemaze attempts.

Treatmentsignificantly(P<0.001)affectedvocalizationrateat 27weeksofage,andthiseffectwasdependentonmazeattempt (P<0.05).Thesestatisticaldifferencesreflectedthefactthatvocal- izationratewasalwayshighestintheGnRHaramsandonaverage increased(+14%),whereasitwasalwayslowestanddecreased(- 37%)intheGnRHa+Tgroup,andremainedrelativelyconstantin theControls,overthethreemazeattempts.At41weeksofage, significant(P<0.001)effectsoftreatmentwereagainapparent,the GnRHaramsvocalizingthemostandtheGnRHa+Tramstheleast, regardlessofmazeattempt.

At8weeksofage,escapeattemptratedecreasedsigniﬁcantly (P<0.001) with maze attempts (total of 211 escape attempts), regardlessoftreatmentgroup.At27and41weeksofage,there werenoeffectsoftreatment,mazeattemptsorinteractionbetween thesefactorsonescapeattemptrate(27weeksofage:totalof33 attempts;41weeksofage:totalof36attempts).

3.3. Long-termspatialmemory 3.3.1. Mazetraining

Treatmentsigniﬁcantly(P=0.020)affectedthenumberoftrain- ingattemptsrequiredtolearnhowtocompletethemazewithin 1min. The GnRHa+T group required fewer training attempts (3.5±0.41)thantheGnRHagroup(6.2±1.09),butneitherofthese twogroupsweredifferentfromtheControls(4.7±0.67).

3.3.2. Traversetimes

InFig.5A,theaveragetraversetimesareshown fortheend of training, as well as when long-term memorywas assessed, andwhenthemazetrapswerepresentedtoanimalsina novel order/orientation (‘Trained<1min’, ‘Long-term memory’, ‘Novel maze’,respectively).Allthree groupshadsimilartraversetimes (P=0.51)attheendoftraining.Whencomparingtraversetimes duringlong-termmemoryassessment withthose attheend of training,traversetimesweresigniﬁcantly(TimeP<0.001)longer for the GnRHa and GnRHa+T groups. There was also a signiﬁ-

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Fig.5. Summaryofspatialperformanceat45weeksofageforlong-termspatialmemoryandnovelmazeassessments.A:Traversetimesattheendoftraining(41weeksof age‘Trained<1min’),duringlong-termspatialmemoryassessment(‘Long-termmemory’usingmazelayout1),andinanunfamiliarmazedesign(‘Novelmaze’usingmaze layout2).Differentlettersontopofbarsindicatesigniﬁcantdifferencesbetweentreatmentgroupmeans,asdeterminedbyTukeyposthoctests.B:Progressionthrough mazezonesduringlong-termspatialmemoryassessment.*P<0.05fromTukeyposthoctest,whencomparingtheeffectsoftreatmentinthatparticularzonewithaone-way ANOVA.C:Progressionthroughmazezonesduringnovelmazeassessment.GnRHa:GnRHa-treatedrams;GnRHa+T:GnRHa-treatedramsthatalsoreceivedtestosterone replacement.Control:untreatedrams.

cant(P=0.032)effectoftreatment,wherebythesizeoftheeffect tendedtobedifferentbetweenthetwotreatmentgroups(Treat- ment×TimeP=0.085).ComparedtotheControls,theGnRHaand GnRHa+Tramstook1.5fold(TukeyHSDadjustedP=0.090)and 1.9 fold (TukeyHSD adjusted P=0.019) longer to complete the maze,respectively(One-wayANOVAoflong-termmemoryassess- mentonly:P=0.043).Comparingtraversetimeduringlong-term memoryassessment of only theControl and GnRHagroups (t- test),conﬁrmedthattheGnRHagrouptooksigniﬁcantly(P=0.030) longerthantheControlgroup.

Duringnovelmazeassessment(Fig.1,Layout2),traversetimes forControlandGnRHagroupsweresigniﬁcantly(P<0.001)longer thaninthefamiliarmazelayout(Fig.1,Layout1).Treatmentsig- niﬁcantly(P=0.015)affectednovelmazetraversetimes,relativeto long-termmemoryassessment,witha1.2,1.8and2.2-foldincrease intheGnRHa+T,GnRHaandControlsgroups,respectively(Treat- ment×TimeP=0.231).However,traversetimesduringthenovel mazeassessmentwere notdifferentbetweentreatmentgroups (One-wayANOVAofnovelmazeassessmentonly:P=0.435).

Theproportionsoftimeanimalsspentineachmazezone,when long-termmemoryandthenovelmazewereexamined,areshown inFig.5B&C. Overall,theproportionatetimeanimalsspentin eachmazezone,whenlong-termmemorywasassessed,wassim- ilarexceptforzoneE,inwhichtheyspentthemosttime.There wasasigniﬁcant(P<0.05)effectoftreatmentontheproportionof timespentinzoneA,whereControlsspentthegreatest,andthe GnRHa+Tgrouptheleast,proportionoftime.Asimilartrendinthe effectsoftreatmentonproportionatetimespentinazonewasseen inzonesBandC.

Whenramsweretested in thenovel mazelayout,later the sameday,theproportionaltimeallocationpatternchangedsub- stantially,relativetothatofthefamiliarmaze(Fig.5A,Long-term memoryassessment).Inthenovelmaze,animalspassedrelatively quicklythroughthefirsttrap(zoneB),butsloweddownandspent approximatelyequalproportionsoftimeinthesecond(zoneD), third(zoneE),andfinal(zoneC)traps.Thegreatestproportionof timewasspentinthefourthtrap(zoneA).Relativetothelong- termmemoryassessment,theproportionoftimespentinzoneA wassignificantly(P<0.001)increased,andzoneEwassignificantly (P<0.001)reducedduringthenovelmazeassessment.Therewere nostatisticallysignificanteffectsoftreatmentontheproportionof timespentinanyofthezonesduringthenovelmazeassessment, comparedtothelong-termmemoryassessment.

4. Discussion

Thisstudydemonstratedthatthereisanimprovement,formale sheepfromtheearlyadolescenttoyoungadultagerange,inspatial orientationandlearningperformance,aswellasbehavioralmea- suresofemotionalreactivitythataredisplayedwhenperforming spatialtasks.Whiletheresultsdonotsupportasignificantrolefor GnRHaortestosteroneontheoutcome(i.e.traversetime)ofspatial orientationandlearningperformance,theyrevealedthatblockade ofGnRHandtestosteronesignalingsystemsaffectedthemannerin whichanimalsmovedthroughaspatialmaze.Specifically,blockade ofGnRHsignalingincreased,whereasrestorationoftestosterone signalingdecreased,howquicklysheepprogressedbeyondaspe- cificpointinthemazeandhowemotionallyreactivetheywere, i.e.howlikelytheyweretovocalizeorattempttoescape.Assess- mentoflong-termspatialmemorydemonstratedthattheblockade of GnRHsignalingimpaired spatialreferencememory, and this effectwasindependentoftestosteronereplacement.Incontrast, spatialworkingmemorywasprimarilyaffectedbythesuppres- sionofgonadalsteroidsignalingassociatedwiththeblockadeof GnRHsignaling.

4.1. Spatialorientationandlearning

Thefindings,inthepresentstudy,thattheblockadeofGnRH signalingdoesnotaffectmazetraversetimesofmalesheepdur- ingspatialorientationandlearningassessments,areinagreement withthepreviousfindings ofWojniusz et al.(2013).Minimum traversetimesincreasedfrom5to16sinthecurrentstudy’smod- ified5-trapmazecomparedtothepreviousstudy’s3-trapmaze.

ThesefindingsconfirmedthatthelackofaGnRHa-treatmenteffect onspatialorientationreportedbyWojniuszetal.(2013)wasnot duetoaceilingeffectassociatedwithfasttraversetimesinasim- plemaze.Instead,thepresentstudyrevealedthatthemannerin whichsheepmovedthroughthemazewasaffectedbytheblockade ofGnRHandtestosteronesignaling,asconcludedfromthemore detailedanalysisofemotionalreactivityandprogressionwithinthe maze,togetherwiththeinclusionofanadditionalGnRHa-treated groupinwhichtestosteronewasreplaced.Specifically,itwasseen thatramsthathadbothGnRHandtestosteronesignalingblocked (GnRHagroup)exhibitedincreasedemotionallyreactivebehavior, asreflectedbyhighervocalizationfrequenciesat27and41weeks ofage(Fig.4).ThiseffectofperipubertalGnRHa-treatment was lostwhentestosteronesignalingwasreplaced,whichindicatesthat theeffectswereduetosuppressionoftestosterone,secondaryto theblockadeofGnRHasignaling.Testosteronereplacementalso decreasedthefrequencyofescapeattemptsasramsaged,indepen-

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dentofGnRHa-treatment(Fig.4,Attempt1),whichagainsuggests thattestosteronereducedemotionalreactivity.Thiseffectoftestos- terone haspreviously been reported in other species, typically inrelationtoreductioninfearfulbehavior(BoissyandBouissou, 1994; Fryeand Seliga,2001; Aikey et al., 2002)or increase in hypothalamic-pituitary-adrenalaxisresponsestostress(Viauand Meaney,1996;Sealeetal.,2004).

TheloweremotionalreactivityofGnRHa+Tanimalsmayhave indicatedthatthisgroupwaslessanxiousandmotivatedtotra- versethemazetoreunitewiththeirpeers,asreflectedbytheir slowerprogressionthroughthemazeovermultiplemazeattempts at 41 weeks of age (Fig. 3), compared to Control and GnRHa groups.Differencesinemotionalreactivitymayalsoexplainthe trend for GnRHa-treatedanimals to have faster traversetimes, comparedtoGnRHa+Tanimalsduringspatiallearningassessment (Fig.2,Attempt1&2at41weeksofage),astheymayhavebeen moreanxiousandmotivatedtoreunitewithflockmembers.Itis noteworthythatrestorativeeffectsoftestosteroneonspatialper- formancehavebeenreportedpreviouslyinadultmaleratswhere itwasshowntominimizetheinfluenceofnon-mnemonicfactors inahippocampus-dependentversionoftheY-maze(Hawleyetal., 2013).Thatstudyisofparticularnote,asitalsoutilizedaninnate behavior−i.e.novelty-seekingofrats,whereasthecurrentstudy usedflockinginsheep−tostudytheeffectsoftestosteroneonspa- tialability.Itwasarguedthatresultsfromstudiesthatusedfood rewardorwaterescapeasmotivationtocompletetasks,werecom- promisedbytheeffectsoftestosterone(andotherandrogens)on foodintake,bodyweightgain,anxietyandhypothalamic-pituitary- adrenalaxisreactivity.

Suppression of gonadal steroid signaling is therefore likely toincrease fearfulbehavior orelevate stress responses viathe hypothalamic-pituitary-adrenalaxis.Indeed,previouswork(Evans etal.,2012)hasindicatedthattheGnRHa-mediatedblockadeof pubertalhormone production increased anxietyand emotional reactivityinramsduringa stresstest.Thus,thelossofgonadal steroidsignalingmayalterthemannerinwhichsubjectsbehave duringaspatialtask,withoutaffectingtheperformanceoutcome forspatialorientationandlearning(i.e.nochangeintraversetime).

4.2. Long-termspatialmemory

Thesuccessfulretentionandrecollectionofspatialinformation wasreflectedbyControlramsretainingtheabilitytotraversethe mazewithin1min,4weeksaftertraining.Thisagreeswithprevi- ousfindings(Leeetal.,2006)thatsheepareabletorecallspatial information6weeksaftertraining.Furtherevidencethatanimals retainedspatialinformationwasreflectedinthepatternofmaze progressionduringthelong-termmemoryassessment.Familiarity withthemazelayoutwasreflectedbythefactthatramspassed quicklythroughthefirstmazezonebutspentalongerproportion oftimeinthelastzone.Thispatternofmazezoneprogressionalso becamemoreprominentacrossthethreeattemptsat8weeksof ageandtoalesserextentacrossthethreeagesassessed,asanimals becamemorefamiliarwiththemaze.Furthermore,this pattern ofmazeprogressionwaslostwhenanimalsencounteredanunfa- miliarmazelayoutat45weeksofage.Finally,thesimilarityinthe patternofmazeprogressionforthethreegroupswouldsuggestthat theyallretainedspatialinformation,eventhoughtraversetimes indicateddifferencesinlong-termspatialrecall.

AspeciﬁceffectofGnRHsignalingonlong-termspatialmemory wasevidentfromtheimpairedabilityofGnRHatreatedanimalsto recallspatialinformationandthatthiseffectcouldnotbecoun- teractedwiththerestoration oftestosteronesignaling.Thelack ofarestorativeeffectoftestosteroneonlong-termspatialmem- oryisinteresting,becausetheGnRHa+Tramsweretheeasiestto traintocompletethemazewithin1min,butweretheslowestto

traversethemazeduringthelong-termmemoryassessmentand exhibitedslowmazeprogressionduringtheassessmentofspatial learning.However,itshouldbenotedthattheconditionsduring trainingfortheassessmentoflong-termspatialmemorydiffered fromthoseofthespatiallearningassessment.Specifically,maze attemptsduringlong-termmemorytrainingfollowedimmediately afteroneanotherandanimalswereshownhowtocompletethe mazeafter1min,whereasspatiallearningassessmentconstituted threeattemptsthatwereseparatedbyapproximately2handrams thatdidnot completethemazewithin5minwereguided back totheaudiencepenviatheentrance.Thus theapparently con- flictingresultscouldreflectdifferencesinthelearningpatternof theGnRHa+Tgroupacrossthesetwocomponentsofthestudy,i.e.

effectsonrapidrepetitivelearningvs.thelongertimescaleused inthespatiallearning assessment.Furthermore,it couldreﬂect theeffectsoftestosteroneindecreasingemotionalreactivityand motivation tocomplete themaze,as discussed above.We can, therefore,notentirelyruleoutthepossibilitythattheeffectsof long-termperipubertalGnRHa-treatmentonspatialmemorywere not,inpart,mediatedviadifferencesinmotivationoremotional reactivityinthisstudy.

Comparisonofperformancein long-termmemoryand novel mazeassessmentsisofinterestasitprovidesameanstoassess whetherblockade ofGnRH/testosterone signalinghad aneffect on spatial reference or spatial working memory. Spatial refer- encememoryisdefinedasthecategorizationofinformationthat remainsthesameamongtrials,whichiscomparabletohavingthe sametrapsinmultipleassessments(OltonandPapas,1979).Spatial workingmemoryreferstothecategorizationofinformationabouta particularsequenceofspatialcues,whichiscomparabletochang- ingtheorderoftrapsin thenovelmazeassessment(Oltonand Papas,1979).Performanceduringlong-termspatialmemoryand novelmazeassessmentswassimilarforbothgroupsinwhichGnRH signalingwasblocked(GnRHa&GnRHa+T),irrespectiveoftestos- teronereplacement.However,testosteronereversedtheincrease intraversetimesfromfamiliartonovellayoutswhenGnRHsig- nalingwasblocked(GnRHagroup).Therefore,itisconcludedthat spatialworkingmemorywasinfluenced bytestosteronesignal- ing,whereasspatialreferencememorywasexplicitlyinfluencedby GnRHsignaling.Thisconclusionissupportedbyworkinadultmale rats,whichalsonotedthatspatialworkingmemorywasmoresen- sitivetotestosteronethanspatialreferencememory(Sandstrom etal.,2006;Spritzeretal.,2008;Hawleyetal.,2013).

4.3. Potentialmechanismsinvolved

While endogenous GnRH secretion is pulsatile, the contin- uous GnRHa administration in this study suppressedsignaling downstreamoftheGnRHreceptor.Theexactchangesinducedby GnRHa-treatmentwithinthecentralnervoussystemarelikelytobe complexandinvolveanarrayofalteredpathways.Herewediscuss thepotentialmechanismsinvolvedwiththesuppressionofGnRH andtestosteronesignaling,fromevidenceintheliterature,with- outhavingdirectmeasuresofGnRHortestosteroneincirculation, cerebrospinalﬂuid,ortissuehomogenatesinthisstudy.

Thehippocampusiscentraltospatial learningand memory, andAlbertsonetal.(2009)hasdemonstratedthatthereareGnRHR typeIimmunoreactivecellsintheCA1to4regions,aswellasthe dentategyrus,oftheovinehippocampus.Nuruddinetal.(2013b) reportedthatperipubertalblockadewithGnRHa,resultedinsex speciﬁc changes in hippocampal (CA1 to 3) mRNAexpression.

Speciﬁcally,whiletherewerenochangesinthelevelsofexpres- sionof mRNAfor GnRH receptor (TypesI and II) and estrogen receptor(alphaandbeta),inrams,mRNAexpressionofendocrine variables(androgenreceptor,aromataseandgrowthhormone),as wellasneuroplasticitymarkers(neuralcelladhesionmolecule1,

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VGFnervegrowthfactorinducibleandLIMhomeobox5),decreased andmRNAexpressionfortheAMPA1glutamatereceptor,anaddi- tionalneuroplasticitymarker,increased,relativetotheControls.

Alterations in LIM homeobox5 are associated with hippocam- paldevelopment,whereitisvitalinregulationofprecursorcell proliferation, as well as the control of neuronal differentiation andmigration (Zhaoet al.,1999).TheAMPA1glutamaterecep- torplaysanimportantroleinlong-termpotentiation,whichis− togetherwithitscounterpartlong-termdepression−anactivity- dependentsynapticplasticitymechanisminvolvedinlearningand memory(Benkeetal.,1998).Anincreaseinthenumberofpost- synapticAMPA1glutamatereceptors,theirprobabilitytobeopen, theirkineticsoranincreaseintheirsingle-channelconductance wouldgenerallyindicateimprovedlearningandmemory(Benke etal.,1998).Inthepresentstudy,GnRHa-treatedanimalsshowed impaired spatial learning and memory, despite the increase in AMPA1glutamatereceptormRNAthatwasreportedbyNuruddin etal.(2013b)usingthesameovinemodel.However,mRNAlev- els do not necessarily reﬂect protein expression, or any other alterationsinAMPA1glutamatereceptorfunction,andwouldbe valuabletostudytheseinthepresentcohortofanimals.

Androgenreceptorsmediatealterationsinsynapticplasticityin thehippocampus(MacLuskyetal.,2006)andithasbeensuggested thatimpaired spatialmemoryin adultrats,followinggonadec- tomy,occursduetoreducedexpressionofhippocampalandrogen receptors(particularly in areaCA1) (Hawley etal., 2013).Such areduction in mRNAexpressionof androgenreceptor andaro- matase reported by Nuruddin et al. (2013b) in GnRHa-treated rams,providessupportthatlong-termGnRHa-treatmentmayalter thewayin whichsubjectsrespondtotestosterone.Ward etal.

(1999)alsoreportedthatalteredreceptorexpressioninthebrain, ratherthanaltered gonadalsteroidproduction, wasresponsible forunderperformanceincopulatorybehaviorthatresultedfrom prenatalexposuretostressandalcohol. Furthermore,thestudy bySealeetal.(2004)providedevidencethatrestorativeeffectsof testosteronereplacementonhypothalamic-pituitary-adrenalaxis stressresponses,ingonadectomizedmalerats,aremediatedvia theandrogenreceptor.PeripubertalGnRHa-mediateddifferences inhippocampalandrogenreceptormRNAmaythereforealsorelate totheobserveddifferencesinemotionalreactivityinthepresent study.However,furtherphysiologicalevidenceofsteroidreceptor expression and circulating steroidlevels is required to investi- gatewhateffectswouldbeassociatedwithGnRHa-treatmentand testosteronereplacementandhowthisrelatestothedifferences observedinspatialmemory.

Finally,thesuppressionofLHsignalingthatisassociatedwith GnRHa-treatment,mayalsoplayarole intheobservedchanges in learning and memory, as LH receptors are expressedin the CNS,includingthehippocampus(Blair etal.,2015).Inaddition, it hasbeen shown in human and rodent studies that theage- relatedincreaseincirculatingLHanddeclineingonadalsteroid production,correlateswithimpairedcognitivefunction(Blairetal., 2015).However, theroute through which circulating LH could reachreceptorsinthecentralnervoussystemisnotyetcharac- terized.

5. Conclusion

Theresultsofthis studydemonstratethatspatialorientation and learning were indirectly affected by peripubertal GnRHa- treatment,wherebytheblockadeoftestosteronesignaling,rather than theblockadeof GnRH signalingonly, increasedemotional reactivityand motivationtoreunitewithﬂockmembers. Long- termretentionofspatialinformationwasimpairedbythespeciﬁc blockadeofGnRHsignaling,anddidnotimprovewhentestosterone

signalingwasrestored.Itisthereforeconcludedthatblockageof thepubertaltransitionwithGnRHa-therapygreatlyaffectedlong- termspatialmemory,particularlywithregardtospatialreference memory,butminimallyaffectedspatialorientationandlearning in males.Furthermore,therestorationof testosterone signaling exaggeratedchangesinspatialreferencememoryandcounteracted thechangesinspatialorientationandspatialworkingmemory.It isnotknownwhetherperipubertalGnRHa-treatmentinfemales willalsoresultinareductioninlong-termspatialmemory.Fur- therinvestigationisrequiredtoestablishthemechanismsthrough whichGnRHaalterspatialmemoryandlearning−whetheritis directlyviaGnRHRinthehippocampusorindirectlyviachanges inemotionalreactivity−andiftheseeffectsarereversiblewhen GnRHa-treatmentisterminatedafterthenormalpost-pubertalage.

Acknowledgements

This work was funded by the BBSRC (Project reference:

BB/K002821/1) and forms part of the Sex On Brain European Research(SOBER)Group.Theauthorswouldliketothankthestaff atCochnoFarmandResearchFacilityforcareandmaintenanceof animals:MorvenCampbell,CarolChestnut,StephenCrozier,Ken- nethDrummond,RobertKnottandMalcolmMcColl.Theauthors wouldalsoliketothankalltheUniversityofGlasgowstudentsthat assistedinthebehavioraltrials.

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