A reduction in long-term spatial memory persists after discontinuation of peripubertal GnRH agonist treatment 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

A reduction in long-term spatial memory persists after

discontinuation of peripubertal GnRH agonist treatment in sheep

D. Hough

^a

, M. Bellingham

^a

, I.R. Haraldsen

^b

, M. McLaughlin

^c

, J.E. Robinson

^a

, A.K. Solbakk

^d,e,f

, N.P. Evans

^a,∗

aInstituteofBiodiversityAnimalHealthandComparativeMedicine,CollegeofMedicalVeterinaryandLifeSciences,UniversityofGlasgow,GlasgowG61 1QH,UK

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

cDivisionofVeterinaryBioscienceandEducation,SchoolofVeterinaryMedicine,CollegeofMedicalVeterinaryandLifeSciences,UniversityofGlasgow, GlasgowG611QH,UK

dDepartmentofPsychology,UniversityofOslo,Pb1094Blindern,0317Oslo,Norway

eDepartmentofNeurosurgery,DivisionofClincialNeuroscience,OsloUniversityHospital–Rikshospitalet,0027Oslo,Norway

fDepartmentofNeuropsychology,HelgelandHospital,8651Mosjøen,Norway

a r t i c l e i n f o

Articlehistory:

Received12October2016 Receivedinrevisedform 23November2016 Accepted23November2016

Keywords:

Spatialmemory Hippocampus GnRH Puberty

Precociouspuberty Genderidentitydisorder

a b s t r a c t

Chronicgonadotropin-releasinghormoneagonist(GnRHa)administrationisusedwheresuppressionof hypothalamic-pituitary-gonadalaxisactivityisbeneﬁcial,suchassteroid-dependentcancers,earlyonset genderdysphoria,centralprecociouspubertyandasareversiblecontraceptiveinveterinarymedicine.

GnRHreceptors,however,areexpressedoutsidethereproductiveaxis,e.g.brainareassuchasthehip- pocampuswhichiscrucialforlearningandmemoryprocesses.Previouswork,usinganovinemodel, hasdemonstratedthatlong-termspatialmemoryisreducedinadultrams(45weeksofage),follow- ingperipubertalblockadeofGnRHsignaling(GnRHa:goserelinacetate),andthiswasindependentofthe associatedlossofgonadalsteroidsignaling.Thecurrentstudyinvestigatedwhetherthiseffectisreversed afterdiscontinuationofGnRHa-treatment.TheresultsdemonstratethatperipubertalGnRHa-treatment suppressedreproductivefunctioninrams,whichwasrestoredaftercessationofGnRHa-treatmentat44 weeksofage,asindicatedbysimilartestessize(relativetobodyweight)inbothGnRHa-Recoveryand Controlramsat81weeksofage.RamsinwhichGnRHa-treatmentwasdiscontinued(GnRHa-Recovery) hadcomparablespatialmazetraversetimestoControls,duringspatialorientationandlearningassess- mentsat85and99weeksofage.FormerGnRHa-treatmentalteredhowquicklytheramsprogressed beyondaspeciﬁcpointinthespatialmazeat83and99weeksofage,andthedirectionofthiseffect dependedongonadalsteroidexposure,i.e.GnRHa-Recoveryramsprogressedquickerduringbreeding seasonandslowerduringnon-breedingseason,comparedtoControls.Thelong-termspatialmemory performanceofGnRHa-Recoveryramsremainedreduced(P<0.05,1.5-foldslower)afterdiscontinuation ofGnRHa,comparedtoControls.Thisresultsuggeststhatthetimeatwhichpubertynormallyoccurs mayrepresentacriticalperiodofhippocampalplasticity.Perturbingnormalhippocampalformationin thisperipubertalperiodmayalsohavelonglastingeffectsonotherbrainareasandaspectsofcognitive function.

1. Introduction

Gonadotropin-releasing hormone (GnRH) is a hypothalamic decapeptidethat,followingitsreleasefromaxonterminalsatthe medianeminence,stimulatesthereleaseofluteinizinghormone

∗Correspondingauthor.

E-mailaddress:Neil.Evans@glasgow.ac.uk(N.P.Evans).

(LH) and folliclestimulating hormone (FSH) from thepituitary gland. GnRH can also reach thecentral nervous system (CNS), as GnRH neurones in the hypothalamus can have axons that extendintootherregionsoftheCNSincludingthelimbicsystem (Silvermanetal.,1987).In addition,GnRHcancrosstheblood- brainbarrier,fromthemedianeminence,intothethirdventricle cerebrospinalﬂuid,albeitwithlowefﬁciency(CaratyandSkinner, 2008).GnRHreceptorexpressionhasbeendemonstratedatsites withintheCNS(Jennesetal.,1997;Albertsonetal.,2009;Schang

http://dx.doi.org/10.1016/j.psyneuen.2016.11.029

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2 D.Houghetal./Psychoneuroendocrinology77(2017)1–8

etal.,2011)andarangeofperipheraltissues(Hapgoodetal.,2005;

Skinneretal.,2009).Thus,whenGnRHanalogsareusedtherapeu- ticallyinhumanandveterinarymedicine,itisalsoimportantto considertheeffectsatthesenon-reproductivesites.

AsGnRHagonists(GnRHa)resultincontinuedreceptorstimu- lation,asopposedtoultradiancyclicchanges,theiradministration initiallyresultsinanincreaseinLHandFSHsecretion(‘ﬂare-effect’), followedbythedown-regulationofGnRHreceptorexpressionin thepituitaryglandandsuppressionofreproductiveaxisfunction (Garner,1994;ChenandEugster,2015).GnRHaistypicallypre- scribedwhenthesuppressionofthereproductiveaxisisrequired, suchassteroid-sensitiveconditionslikeprostatecancer,uterine ﬁbroidsandendometriosis(Garner,1994).Inchildrenandadoles- cents,GnRHacanbeprescribedfortreatmentofcentralprecocious puberty(CPP)(ChenandEugster,2015)andgenderdysphoria(GD) (Hembreeetal.,2009)totemporarilyhaltreproductivedevelop- ment.

Carelet al. (2009)emphasized theneed for investigationof the potential psychological effects associated with peripubertal GnRHa-treatment in CPP. Similarly, the potential effects of GnRHa-treatment on cognition during this important develop- mentalperiodarenotwellcharacterized.Wojniuszetal.(2016) recentlydemonstrated thatperipubertal GnRHaincreases emo- tionalreactivity(i.e.emotionalandbehavioralresponsestoafearful situation)ingirlswithCPP,whereasrestingheartratedecreased and this effectwas more pronounced withlongerdurations of GnRHa-treatment.Studies,usinganovinemodel,havealsodemon- stratedthat peripubertalGnRHa-treated ramsdisplayincreased risk-takingbehavior(Wojniuszetal.,2011),alteredemotionalreac- tivity(Evansetal.,2012)andreducedlong-termspatialreference memory(Hough et al.,2016).Physiological changes withinthe limbicsystem have alsobeen reportedin this ovinemodel, as peripubertalGnRHa-treatmentaltersamygdalavolume(Nuruddin etal.,2013a)andtheexpressionofhippocampalgenesthatare involvedinendocrinesignalingandsynapticplasticity(Nuruddin etal.,2013b).Withthisgrowingbodyofevidencethatperipuber- talGnRHa-treatmentmayaffectdevelopmentofcognitivefunction, thereisnowarequirementtoinvestigatewhethertheseeffectsare reversiblewhenGnRHa-treatmentisdiscontinued.

In the current study, we investigated whether effects from peripubertalGnRHa-treatment,persistedinramsfollowingthedis- continuationoftreatment.Speciﬁcally,weinvestigatedwhether thepreviouslyreportedreduction in long-termspatialmemory persists,orifeffectsonspatialorientationand learningemerge laterinlife,followingthediscontinuationofperipubertalGnRHa- treatment.

2. Materialsandmethods

2.1. Animals

AllanimalprocedureswereconductedattheUniversityofGlas- gowCochnoFarm andResearch Centre(55^◦ 55N)under Home OfﬁceRegulations (ProjectLicense: 60/4422). Theexperimental animalsusedinthisstudywereScottishMule,Texelcrossmales, bornfromsamesexlittersbetween23Marchand12April2013.At birth,lambswereassignedtooneofthetreatmentgroupsdescribed below.Lambsfromtwinandtripletpregnancieswereallocatedto differentgroups, sothatonly1 siblingwasrepresentedineach treatmentgroup. Puberty was delayed in the GnRHa-Recovery (GnRHa-Rec,n=25)lambs bysubcutaneousimplantation ofthe GnRHa,goserelinacetate(Zoladex3.6mg,kindlydonatedbyAstra Zeneca,Macclesﬁeld,UK),everyfourweeksfrom8to44weeksof age(averageageofpubertalonsetinmalesheepis10weeksofage (WoodandFoster,1998)andareexpectedtobesexuallycompetent

withintheﬁrstyearoflife).Control(n=30)andGnRHa-Recrams weregrazedonpasture,exceptduringbehavioraltrials,whenthey werehousedindoorswithadlibitumaccesstohayorsilage,with supplementsasdeemednecessarybystandardmanagementprac- tices.Allanimalswereeuthanizedattheendofthestudyperiod, whentheywereapproximately2yearsofage.

2.2. Testesdevelopment

Approximately every four weeks, morphometric data were collectedfromallanimals,includingtestes size,tomonitorthe effectivenessofGnRHatosuppressthereproductiveaxis.Scrotal lengthandcircumferenceweremeasuredwithatailortapemea- surewhilesheepwereheldinasitting-position.Testessizewas calculated fromthe scrotal length×circumference and normal- izedtobodyweight.Testessizedataat28(ﬁrstbreedingseason), 44(ﬁrstanestrus),81 (secondbreedingseason) and99 (second anestrus)weeksofagearepresented,asthesemeasurementswere nearesttothedatesofspatialmazeperformanceassessment.

2.3. Assessmentofspatialorientationandlearning

Thisstudyusedmodiﬁcationsoftheassessmenttechniquesand spatialmazesdescribedpreviously(Houghetal.,2016),asitfollows onfromthisstudyontheeffectsofchronicperipubertalGnRHa- treatment(with/withouttestosteronesupplementation)onspatial orientation,learningandmemoryinramsfrom8to45weeksof age.Thecurrent study reportsanalysesof spatial mazeperfor- mancedatafollowingthediscontinuationofGnRHa-treatmentat 44weeksofage.Speciﬁcally,performancewasevaluatedat83and 95weeksofage,andcomparedtothatobservedat41weeksofage (Houghetal.,2016)whentheGnRH-Recoverygroupwasstillbeing treatedwithGnRHa.

2.3.1. Spatialorientationandlearning

SheepwereindividuallyassessedinspatialmazeLayout1of Fig.1at41weeksofage,andinLayout2ofFig.1at83and95 weeksofage.Achangein mazelayout wasnecessary,as some ofthesheepwerealreadyfamiliarwiththeformerlayout(used inlong-termspatial memoryassessment at45 weeksofageas reportedbyHoughetal.,2016).Eachsheepwasgiventhreemaze attemptswithinthesameday(eachattemptseparatedby∼2h)to traversethemazeandreunitewithﬂockmembersintheaudience pen.Approximately30sheepwereassessedperdayandkeptinthe audiencepenthroughoutthedaywithadlibitumaccesstowater andhay.Duringeachmazeattempt,asheepwascalmlyushered fromtheaudiencepentothestartofthemaze.Sheepthatfailedto completethemazewithina5mintimelimit,wereusheredbackto theaudiencepenviathemazeentrancesothatthecorrectroute remainedunknown.Onthelastattemptoftheday,unsuccessful sheepproceededtotheaudiencepenviathequickestrouteSpatial orientationwasassessedastheperformanceofsheepintheﬁrst spatialmazeattemptoftheday,ateachage.Spatiallearningwas assessedastheperformanceofsheepover threemazeattempts withinthesameday,ateachage.

2.3.2. Recordedobservations

Spatialperformancewasindividuallyassessedbyrecordingtra- versetimes(min:s: ms),i.e. thetimetakentomovefromthe entrancetotheﬁnishline(5min=incomplete),aswellasrecod- ingtheprogressthroughthemazeasthetimedifferencetomove betweenlinesAtoE,judgedontheplacementofafrontlegacross theline.Emotionalreactivitywasnotassessedinthisstudy,asonly afewvocalizations,escapeattempts,urinationsanddefecations wereobservedat83and95weeksofage.

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Fig.1.Spatialmazelayoutsthatwereusedinthisstudy,withlettersindicatingzonesthatcontainthesametrapsacrossalllayouts,butpresentedinadifferentorderor orientation.SpatialorientationandlearningwasassessedinLayout1and2at41and83weeksofage,respectively.Layout2wasusedfortrainingandtheassessmentof long-termspatialmemoryat99weeksofage.Layout3wasusedforthenovelmazeassessmentat99weeksofage.

2.4. Assessmentoflong-termspatialmemory

Long-termspatialmemorywasassessedasdescribedpreviously (Houghetal.,2016).

2.4.1. Training

Education and conﬁrmation runswere performed, over two days,at95weeksofage,inthemazelayoutusedintheassess- ment of spatialorientation and learning (Layout 2,Fig. 5).The educationrunswerecompletedwhen a sheepwasabletotra- verse themaze within 1min ontwo successive attempts. The retentionofthisabilitywastestedintheconﬁrmationrun,which consistedof two attempts to complete themaze within1min.

Ifunsuccessful,sheeprepeatedthecycle ofeducationand con- ﬁrmation runs, with a maximum of 3 cycles within the same day.Thetotalnumberofattemptsduringtheeducationandcon- ﬁrmation runs was recorded for each ram, together with the quickesttraversetime,toserveasameasureoftheeaseoftrain- ing.

2.4.2. Long-termspatialmemory

Retentionoflong-termspatialmemorywasassessed4weeks aftertrainingwascompleted(99weeksofage),inthesamemaze design. Each sheep wasgiven one mazeattempt, and traverse times(incomplete=5min)andprogressthroughmazezoneswas recorded.Theproportionoftimespentineachzonewascalculated foreachanimalasapercentageoftotaltimespentinthemaze.

2.4.3. Familiarityinanovelmazedesign

Immediatelyafterassessmentoflong-termmemory,eachsheep wasgivenoneattempttotraverseanewspatialmazelayout(Lay- out3,Fig.1),whichcontainedthesame‘traps’butinadifferent orderororientation.Mazetraversetimes(incomplete=5min)and progressthroughmazezoneswererecorded.

2.5. Statisticalanalysis

Data were excluded from analysis where performance was judged to have been compromised because of temporary

incapacity,i.e.health concerns.Inaddition,datawereexcluded fromanalysiswhereanimalsescapedfromthemazeareaorjumped overinternalmazewalls.Exclusionofdatawasdonebyspecifying amissingvaluefortherelevantresponsevariable(s)inthatpartic- ularmazeattempt(numberofobservationsisspeciﬁedinFig.3).

AllstatisticalanalyseswereperformedwithRsoftware(Version 3.2.1, © 2015The RFoundation for Statistical ComputingPlat- form)usingtheRStudiointerface(Version0.99.467,©2009–2015 RStudioInc.).Responsevariableswereanalyzedusingthegener- alizedlinearmodel(glm)function;ramidentitywasincludedas anexplanatoryvariable,toaccountforindividualvariationacross timeorrespectivemazeattempts.

TheeffectofGnRHaonbodyweightandnormalizedtestessize, wereanalyzed using a:(1) Two-wayANOVA(Treatment×Age) in year 1 and 2, (2) Two-way ANOVA during breeding and non-breedingseason.Effectsofageandtreatmentonspatialori- entationwereassessed withdatafromtheﬁrstattemptof the maze,acrossallages(41,83and95weeksofage),usingatwo-way ANOVA(Treatment×Age).Effectsoftreatmentonspatiallearning, overthreeconsecutivemazeattempts,wereassessedwithtwo- wayANOVA(Treatment×Mazeattempt)ateachrespectiveage.

One-wayANOVAwasusedtoassesstheeffectsoftreatmentonthe easeofmazetraining(numberoftrainingattemptsat95weeksof age),aswellastraversetimesuponcompletionoftraining.Effects oftreatmentonlong-termspatialmemoryweretestedbycom- parisonoftraversetimesat: (1)99weeksofageonly(one-way ANOVA);(2)95(lasttrainingattempt)versus99(theassessment attempt)weeksofage(two-wayANOVA:Treatment×Time);and 99(GnRHa-Recovery)versus45(duringGnRHa-treatment)weeks ofage(two-wayANOVA:Treatment×Time).Theeffectofmaze designfamiliaritywasexaminedbycomparisonofthetraversetime ofmazelayouts1and2at83weeksofage,orlayouts2and3at 95weeksofage(two-wayANOVA:Treatment×MazeLayout).The effectsoftreatmentandageontestessizewasevaluatedwitha two-wayANOVA.AllstatisticaltestswerefollowedbyaTukeyHon- estSignificantDifferenceposthoctest,toassesswheresignificant differencesexistedbetweentreatmentgroups.Allgraphsrepresent meansandstandarderrorsofthemean.StatisticalP-values≤0.05 wereconsideredsignificant.

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Fig.2.Testicularsizemean±s.e.m.,asafactorofbodyweight,duringGnRHa-treatmentintheﬁrstyear,andafterdiscontinuationofGnRHa-treatmentinthesecondyear, ofliferelativetoControlrams.Measurementsweretakenduringthebreedingseasonsat28and81weeksofage.Non-breedingseasonmeasurementsoccurredat44and 99weeksofage.Differentlettersontopofbarsindicatesigniﬁcantdifferencesinthemeansacrossbothyears.Control:notreatment;GnRHa:peripubertalGnRHa-treated from8to44weeksofage;GnRHa-Rec:GnRHa-treatmentdiscontinuedat44weeksofage.

Fig.3. Mean±s.e.m.traversetimeoframsat41,83and95weeksofageduringspatialorientationandlearningassessments,overthreemazeattemptswithinthesame day.Differentlettersontopofbarsindicatesigniﬁcantdifferencesbetweenmeansatthatparticularage.Control:notreatment;GnRHa:peripubertalGnRHa-treatedfrom8 to44weeksofage;GnRHa-Rec:GnRHa-treatmentdiscontinuedat44weeksofage.

3. Results

3.1. Bodyweightandtestesdevelopment

Therewasnodifference(P>0.05)inbodyweightbetweenthe ControlandGnRHa-treatedramsatanyage.PeripubertalGnRHa- treatment signiﬁcantly (P<0.01) suppressed normalized testes size,comparedtoControlrams,atboth28and41weeksofage (Fig.2).IntheGnRH-Recgroup,normalizedtestessizeincreased signiﬁcantly(P<0.001)by25%overthe37weeksfollowingdis- continuationofGnRHa-treatment.Normalizedtestessizeduring thebreedingseasoninthesecondyearoflife,being47%higher comparedtothebreedingseasonofthepreviousyear.At81and 99weeksofage,therewasnodifference(P>0.05)innormalized testessizebetweenControlandGnRHa-Recgroups.

3.2. Spatialorientation–completionofmazeattheﬁrstattempt 3.2.1. Traversetimes

Theaveragetimetakenforsheeptotraversethemaze(Fig.3) wassigniﬁcantly(P<0.001)differentoverthethreeagestested.

Comparedto41weeksofage(Layout1),averagetraversetimes at83and95weeksofagewere84and62%slower,respectively.

Adifferentmazelayoutwasusedat83weeksofage(Layout2)to thatusedat41weeksofage,andcomparisonofthesetwoages only,indicatedatendency(P=0.082)foraveragetraversetimesto belongerat83weeksofage;aneffectthatwasseenequallyin bothgroups(Controls:2.92±0.29vs.3.47±0.30min;GnRHa-Rec:

2.96±0.38vs.3.49±0.26min).

Therewerenosigniﬁcanteffects(P>0.05)oftreatmentorthe interactionbetweentheeffectsoftreatmentandage,onmeantra- versetimes.

3.2.2. Progressthroughthemaze

Therewerenosignificanteffects oftreatment,orinteraction betweentheeffectsofageandtreatment,ontheproportionoftime spentinanyofthemazezones(Fig.4,Attempt1).Regardlessof age(P>0.05),ramsspentthegreatestproportionoftimeinzones CandE.However,ramsspentsignificantly(P<0.001)moretimein zonesAandB,andsignificantly(P<0.01)lesstimeinzoneD,at95 comparedto83weeksofage.

3.3. Spatiallearning–completionofmazewithsame-day repeatedattempts

3.3.1. Traversetimes

Themeantimestakentocompletethemazeacrossallthree attemptswithinthesameday,ateach age,areshown inFig.3 and theP-value summary isreportedin Table1. Therewasno significant(P>0.05)differenceinspatiallearningbetweentheCon- trolandGnRHa-Recgroups,atanyageandnosignificant(P>0.05) interactionbetweentreatmentandage.Significantimprovement intraversetimesoverthethreeattemptswereseenat41(P<0.01), 83(P<0.001)and95(P=0.050)weeksofage,butat83weeksof agetheimprovement(P<0.001)wasgreaterinthenewmazelay- outwitha29.7%decreaseintraversetimesfromthefirsttosecond attempt,and10.7%fromthesecondtothirdattempt.

Themeanproportionoftimespentineachmazezone,forthe twogroupsofanimals,areshowninFig.4withtheassociatedP- valuesummaryinTable1.At41weeksofage,therewasnoeffect oftreatmentonthetimespentinanymazezone.At83weeksof age,ontheﬁrstattempt,animalsspentmosttimeinzonesB,C andE.Onthesecondattempt,theproportionoftimespentineach

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Fig.4.Mean±s.e.m.proportionatetimespentineachzoneofthemaze,expressedhereasapercentageofthetotaltimespentinthemazeduringspatialorientationand learningassessmentat83and95weeksofageoverthreemazeattempts.Control:notreatment;GnRHa-Rec:GnRHa-treatmentdiscontinuedat44weeksofage.*P<0.05.

Table1

Spatiallearning.Summaryoftwo-wayANOVAP-valuestoassesstheeffectsoftreatmentonspatialperformanceacrossallthreemazeattemptswithinthesamedayatages 41,83and95weeks.P-valuesinboldarestatisticallysigniﬁcant(P<0.05)thoseinitalicsarewherethereisatrend(P<0.1).

ResponseVariable 41wks 83wks 95wks

Treatment Attempt Treatment×Attempt Treatment Attempt Treatment×Attempt Treatment Attempt Treatment×Attempt

Traversetime 0.226 0.001 0.566 0.552 <0.001 0.790 0.394 0.050 0.440

Proportionoftime

ZoneA 0.508 0.076 0.827 0.368 <0.001 0.734 0.450 0.018 0.417

ZoneB 0.853 0.010 0.915 0.021 0.860 0.647 0.403 0.486 0.895

ZoneC 0.273 <0.001 0.373 0.610 <0.001 0.126 0.128 0.065 0.057

ZoneD 0.597 <0.001 0.783 0.441 0.006 0.286 0.726 0.005 0.812

ZoneE 0.180 0.380 0.743 0.905 0.145 0.080 0.207 0.280 0.879

zonewasmoreequal,withtheexceptionofzoneE,whereanimals spentthegreatestproportionoftime. Bythethird attempt,the timespentineachzonewasapproximatelyequal.Thesechanges inthepatternofmazeprogressionwerereflectedbyasignificant (P<0.01)increaseintheproportionoftimespentinzonesA(1st trap)andD(4thtrap),andasignificant(P<0.001)decreaseinthe proportionoftimespentinzoneC(3rdtrap)overthecourseofthe threeattempts.TheGnRHa-Recgroupspentsignificantly(P<0.05) lesstimeinzoneBthantheControlgroup,particularlyinattempt 2.TheGnRHa-Recgroupalsotended(P=0.080)tospendlesstime inzoneEinattempt1,butmoretimeinthiszoneinattempt3, comparedtoControls.

At95weeksofage,progressthroughthemazeduringtheﬁrst attemptfollowedasimilarpatterntothatseenat83weeksofage, withthemosttimebeingspentinzonesCandE.Onthesecond attempt,therewasareduction intheproportionatetimespent in thesezones, togetherwitha statistically signiﬁcantincrease (P<0.05)intheproportionoftimespentinzonesAandD,butthis patternchangewasnotasmarkedasseenat83weeksofage.The proportionoftimespentinzoneCtended(P=0.057)todecrease witheachmazeattemptintheControls,sothattheyspentnearly equalproportionsoftimeacrossallzonesbyattempt3,butthe GnRHa-RecgroupstillspentmostoftheirtimeinzonesCandE, eveninattempt3.

3.4. Long-termspatialmemory 3.4.1. Traversetimes

Fig.5+Adepictsthemeantraversetimesattheendoftrain- ing(‘Trained<1min’),and4weekslaterwhenperformancewas assessedinthesamemaze(‘Long-termmemory’)andinanovel mazedesign(‘Novelmaze’).Therewerenoeffectsoftreatmenton thenumberofattemptsrequiredtocompletethetraining(Con- trols 6.6±0.8; GnRHa-Rec7.1±0.9 attempts)or traversetimes at the end of training. Compared to traversetimes at the end of training, all animals took signiﬁcantly (P<0.001) longer to traverse the maze during long-term memory assessment, and GnRHa-treatmentsigniﬁcantlyexaggeratedthiseffect(Treatment P=0.041,Treatment×TimeP=0.041), asGnRH-Recand Control animalswere2-foldand1.3-foldslower,respectively.

Whenanimalswere tested ina novelspatial maze,thetra- verse times were similar (P=0.564) for GnRH-Recand Control animals. Comparison of individuals’ performances in the long- term spatial memory and novel maze assessments, indicated thatallanimalstooksigniﬁcantly(FamiliarityP<0.001)longerto completethenovelmaze,irrespectiveoftreatmentgroup(Treat- ment×FamiliarityP=0.146). Theincrease in traverse time was signiﬁcantlyaffectedbytreatment(P=0.008),wherebyControlani- malstook1.6-foldlonger,andGnRH-Recanimalsonlytook1.1-fold

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Fig.5.Spatialperformanceduringlong-termspatialmemoryandnovelmazeassessmentsat99weeksofage.(A)Mean±s.e.m.traversetimesaftertrainingwascompleted (‘Trained<1min’),and4weekslaterinthesamefamiliarmazedesign(‘Long-termmemory’)oranunfamiliarmazedesign(‘Novelmaze’).(B)Progressionthroughmaze zonesduringlong-termspatialmemoryassessment;and(C)duringnovelmazeassessment.DifferentlettersabovebarsindicatesigniﬁcantdifferencesfromTukeyposthoc test.Control:notreatment;GnRHa-Rec:GnRHa-treatmentdiscontinuedat44weeksofage.

longer,tocomplete thenovelmazecomparedtothelong-term memoryassessment.

Themeanproportionoftimespentineachmazezoneduring thelong-termspatialmemoryandnovelmazeassessmentsare showninFig.5BandC.Duringthelong-termmemoryassessment, theproportionoftimespentinthemazezoneswasnotdifferent betweentheGnRHa-RecandControlgroups,althoughtherewas atrend(P=0.079)fortheControlgrouptospendaproportionally greatertimeinzoneBcomparedtotheGnRHa-Recgroup.

Whenanimalsweretestedinthenovelmaze,progressthrough themazezoneswasnotaffectedbytreatment,butanimalsspent signiﬁcantly(P<0.05)lesstimeinthelastthreezonesofthenovel mazecomparedtothe‘familiar’mazelayoutusedinthelong-term spatialmemoryassessment.

4. Discussion

FollowingthediscontinuationofGnRHa-treatmentat44weeks ofage,thereproductiveaxiswasnolongersuppressed,astestes sizeincreasedtoa similarsize asuntreated ramsby83 weeks ofage and waslargerat thetime ofthe breedingversus non- breedingseason.Thisprovidesevidencethatendogenous GnRH andgonadal steroid signalingwasrestored.This delayedexpo- suretogonadalsteroidsignalingdidnotalterthespeedatwhich animalscompletedthespatialtasksduringassessmentsofspatial orientation(i.e.ﬁrst mazeattempt) andlearning (progressover 3same-daymazeattempts)afterperipubertalGnRHa-treatment hadceased,butaffectedthemannerinhowquicklyramsmoved beyonda speciﬁc point withinthe maze, over three same-day attempts.Onassessmentoflong-termspatialmemoryat99weeks ofage,GnRHa-Recramstooklongertotraverseafamiliarspatial task,4weeksaftertraining,thanage-matchedControls,whereas theirperformanceduringanunfamiliarspatialtaskwasthesame astheControls. Thisreduction in long-termspatial memoryin GnRHa-Recanimals wasalsoobserved prior tothe withdrawal ofGnRHa-treatment(Houghet al.,2016)and thecurrentstudy therefore reports that this reduction persisted into adulthood and wasnot reversed afterthe discontinuation of peripubertal GnRHa-treatment.Adetaileddiscussiononthesemainobserva- tionsfollowsbelow.

4.1. Spatialorientationandlearning 4.1.1. PriortoGnRHawithdrawal

AlthoughperipubertalGnRHa-treatmentdidnothaveprofound effectsonspatialorientation(i.e.ﬁrstmazeattempt)andlearning (i.e.changeover3same-daymazeattempts)duringtheﬁrstyearof

life(Wojniuszetal.,2011;Houghetal.,2016),peripubertalGnRHa- treatmentwasassociatedwithalterationstothemannerinwhich ramsmovedwithinthemaze.Thiswasevidencedbyadecreased motivationtocompletethemazeandincreasedemotionalreac- tivitywithinthemaze,butdidnothaveasigniﬁcanteffectonthe patternofprogressionthroughthemazezones.Supplementation ofperipubertalGnRHa-treatmentwithexogenousgonadalsteroids counteractedtheeffectsofGnRHaonmotivationandemotional reactivity.

4.1.2. AfterGnRHawithdrawal

Giventheobservationsintheﬁrstyearoflife,itisnotsurprising thatafterdiscontinuationofGnRHa-treatmentinthesecondyear oflife,traversetimesduringassessmentsofspatialorientationand learningwereunaffectedintheGnRHa-Recgroup.Interestingly, mazeprogressionoverthreesame-dayattemptsintheGnRHa-Rec groupwasquickerat83,butslowerat95weeksofage,compared toControls.Asbreedingandnon-breedingseasonsarerepresented at83and95weeksofage,respectively,itcouldbeindicativethat, intheGnRHa-Recrams,highgonadalsteroidlevelshadagreater impacttoimprove theability oframstolearnhowtoprogress beyondaspeciﬁcpointinthespatialmaze,comparedtotheCon- trols.Themanifestationofthesedifferencesinmazeprogression patternsduringthesecondyearoflife,suggeststhattheprogram- mingofmotivationalbehaviorand/oremotionalreactivitymight bedependentonexposuretogonadalsteroidsduringacriticalwin- dowofdevelopmentwhichcoincideswiththeperipubertalperiod.

4.2. Long-termspatialmemory 4.2.1. PriortoGnRHawithdrawal

Asreportedpreviously(Houghetal.,2016),duringtheﬁrstyear oflife,peripubertalGnRHa-treated ramsrequired1.3-foldmore trainingattemptstolearnhowtocompletethespatialmazethan untreated rams.Thiseffectwascounteracted withtestosterone supplementation,indicatingthat theeaseoftraining wasinﬂu- encedbytestosterone,ratherthanGnRHsignaling.

Long-termspatialmemoryperformance wasalsoreduced in peripubertalGnRHa-treatedrams,comparedtoControlrams(1.5- fold).AssupplementationoftheGnRHa-treatmentwithexogenous testosteronedidnot counteractthis reduction, itindicated that long-termspatialmemoryisaffectedbythelossofGnRH,rather thantestosterone,signaling.

Lastly,theassessmentoframsinaspatialmazewithanovel layoutdemonstratedthattestosteronecounteractedtheeffectsof peripubertalGnRHa-treatmenttoreducethespatialperformance oframs.Thisindicatedthattestosterone,ratherthanGnRH,sig- nalinginﬂuencedtheabilityoframstosolveaspatialtaskwhere familiarcueswerepresentedinanovelsequence.

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4.2.2. AfterGnRHawithdrawal

Thepresentstudyreportsthat,afterGnRHawithdrawaltheease withwhichanimalscouldbetrainedtocompleteaspatialmaze inthesecondyearoflifewasnotdifferentfromControls.Thus thedeﬁcitintrainingabilityobservedduringperipubertalGnRHa- treatmentwasnotpermanentandadelayedexposuretogonadal steroidswassufﬁcienttorestorethespeedofspatialmazetraining.

Duringtheassessmentoflong-termspatialmemoryat99weeks ofage,GnRHa-Recramswerefoundtobe1.5-foldslowerthanCon- trolsintraversingthespatialmaze.Interestinglythis difference relativetotheControlsisofthesamemagnitudeasthosereported when animals were receiving GnRHa-treatment (Hough et al., 2016).Themaintenanceofthisdifferenceinspatialperformance, followingthediscontinuation ofperipubertal GnRHa-treatment, demonstratedthattheeffectsofperipubertalGnRHaonlong-term spatialmemorywerenotreversedandpersistedintoadulthood, despitetherestorationofnormalGnRHandgonadalsteroidsig- naling. This result indicates that long-term spatial memory is dependentonchangesthatoccurredduringacriticalwindowof developmentthat is sensitive toalterations in GnRH signaling.

Thesechangesmightberelatedtoneuralplasticityandendocrine signalingassuggestedbyNuruddinetal.(2013b).Spatialmemory canbesubdividedintolong-termspatialreferencememory,which categorizesspatialinformationaccordingtocuesthatremainthe samebetweenspatialtasks (Oltonand Papas,1979)andwork- ingspatialmemory,whichcategorizesinformationbasedonthe sequenceofspatialcues(OltonandPapas,1979).Astheperfor- manceoftheControlandGnRHa-Recramswerecomparableat 99weeksofageinthenovelmaze,whicheffectivelytestsspatial workingmemory,itcanbeconcludedthattheobservedperma- nenteffectsofperipubertalGnRHa-treatmentonlong-termspatial memoryisspeciﬁctospatialreferencememory.Itisalsoreason- abletoarguethattheincreaseincirculatingtestosterone,which shouldhaveaccompaniedgonadaldevelopmentintheGnRHa-Rec group,musthave beensufﬁcienttoeliminateanyeffectsofthe peripubertalGnRHa-treatmentonspatialworkingmemory.

4.3. Implicationsfordiscontinuingperipubertal GnRHa-treatment

Toourknowledge, this is theﬁrst reportthat theeffects of peripubertal GnRHa-treatment toreduce long-term spatial reference memory will persists following GnRHa withdrawal and suggeststhattheseeffectsarepermanent.Studiesontheuseof GnRHainadulthumans(Freedlandetal.,2009;Greenetal.,2000) havereportedimpairedcognitionandmemoryloss,whichwere ascribedtotheassociatedlossofestrogenand/ortestosterone.Beer etal.(2006)reportedthatlong-termmemorywasreduced,interms ofcompromisedimmediateanddelayedverbalmemory,inmen withprostatecancerandandrogendeprivation(primarilyGnRHa- mediated)comparedtountreatedhealthyControls.Anotherstudy, onelderlymenwithprostatecancerandGnRHa-mediatedandro- gendeprivation(Salminenetal.,2004),reportedthatadeclinein testosteronewasassociatedwithslowervisuo-motorspeed,slower reactiontimesrelatingtoworkingmemory,reducedrecognition speed and delayed recall of letters,but improvedobject recall.

Theseobservations supporttheconclusionof thecurrent ovine studythatspatialworkingmemoryisreducedbythelossoftestos- terone,andthatrestorationoftestosterone,eitherviareplacement therapy (Hough etal.,2016)orgonadal developmentfollowing GnRHa-withdrawal,resultsinnormalfunction.However,deﬁcits inlong-termspatialreferencememorydidnotimprovewithexpo- suretoendogenoustestosteroneandarelikelypermanentlyaltered bythechangesinGnRHsignalingthatoccurredduringtheperipu- bertalperiod.Insodoing,itidentiﬁestheperipubertalperiodas acriticalwindowofdevelopmentwithregardtospatialmemory,

inwhichGnRHsignalingisinvolved.Theobservationthatperipu- bertalGnRHa-treatmentisassociatedwithpermanentchangesin braindevelopmentraisesparticularconcernsaboutthecognitive changesassociatedwiththeprolongeduseofGnRHa-treatmentin childrenandadolescents.

Limitedstudieshavelookedatthelong-termeffectsofGnRHa- treatmenton cognition in children and adolescents. One study reportedthat3-yearGnRHa-treatmentofgirlswithearlypubertal onsetwasassociatedwitha7%reductioninIQ(Muletal.,2001).

Thiseffectwashypothesizedtobeattributedtothesuppressionof sexsteroidsandtheireffectonbraindevelopmentwhichresultedin amoreage-appropriateIQ(Muletal.,2001).Arecentstudyofgirls treatedwithGnRHaforCPP,reportedthattheyexhibitedincreased emotionalreactivityandadecreasedrestingheartrate(Wojniusz etal.,2016).Interestingly,thechangesinheartrateobservedin thatstudyweredependentuponthedurationofGnRHa-treatment.

Takenwiththeresultsofthisstudy–inwhichitisindicatedthat peripubertalGnRHa-treatmentaffectsaspectsofcognitivefunction –itmaybeworthconsideringthedurationoftheGnRHa-treatment inchildrenandadolescents,andlimitingitwherepossible(Hayes, 2016).

TheresultsofthisstudywhenconsideredwiththoseofHough etal.,2016suggestthatthereisacriticalperiodofbraindevel- opmentassociatedwiththeperipubertal period.For earlyonset GD,GnRHaisprescribedfromchildhood,throughouttheadoles- centperiodandintoearlyadulthood,whenaninformeddecision canbemadeaboutgenderreassignment.Thisprolongedtreatment thatmayencompasssuchcriticaldevelopmentalperiods,however, maybejustiﬁedbytheincreasedriskoflife-threateningbehav- iors (e.g.risktakingand suicideattempts),theeffects ofwhich couldoutweigh anyminor cognitiveand psychologicalimpacts ofGnRHa-treatment(GrossmanandD’Augelli,2007;Vanceetal., 2014).For CPP, GnRHa-treatmentcommences fromthetime of diagnosisofearlypubertalonset(8or9yearsofageforgirlsorboys, respectively)andcontinuesuntilapproximately11years ofage (Careletal.,2009),withthemaingoaltoincreasepredictedadult heightbyallowingmoretimeforgrowthpriortothefusionofbones duringpuberty.However,thisgoalhasbeenprimarilyachieved whenGnRHa-treatmentcommencedwithpubertalonset<6years ofage(Careletal.,2009;Hayes,2016).Thequestionthenremains whetherGnRHaexposurecanbelimitedinchildrenwithpubertal onsetbetween6to8or9yearsofage,orifGnRHa-treatmentcan bediscontinuedearlierthan11yearsofage.

While continuous GnRHa-treatment may limit psychological problems in CPP, the decision to commence GnRHa-treatment needsfurtherexploration(Careletal.,2009)asproperparental andphysiciansupportmightbeanalternativeintervention(Hayes, 2016).Ithasbeensuggested bySteinberg(2004)thata special windowofsusceptibilitytopsychosocialandpsychopathological conditionsexistsduringadolescence,whichiscreatedbythetem- poralgapbetweentheonsetofpubertyandlateadolescence.At theonsetofpubertytherearechangesinthelimbicsystemand increasingrewardsensitivity,whereasthematurationofthepre- frontalcortexisslowerandage-dependentsothatself-regulatory systems are onlydeveloped in late adolescence. In this regard, early-maturingchildren would experience a greatervulnerabil- itytowardsrisk-takingandnovelty-seekingbehavior,aswellas psychopathologicalconditions,thanchildrenthatenterpubertyat anolderage(Steinberg,2004).Thisisevidencedbycorrelations betweenearlypubertalonsetandarelativelyyoungageofﬁrstsex- ualintercourse,increasedincidenceofdrugandalcoholabuse,as wellasincreasedriskofsexualabuse(MulandHughes,2008;Kim andLee,2012;Hayes,2016).Againstthisbackground,thereductioninlong-termspatialmemoryfollowingperipubertalGnRHa, asobservedintheramsinthisstudy,mightnotoutweightherisks involvedinwithholdingthistreatment,butprovidesevidencethat

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somecognitivefunctionsmaybeirreversiblyalteredbyperipu- bertalGnRHa-treatment.Furtherinvestigationonotherpotential cognitiveandpsychosocialeffectsarerequired–includingwhether theseeffectsarereversibleorsexuallydimorphic–toassistinmak- inginformeddecisionsaboutthetimingofthecommencementand discontinuationofperipubertalGnRHa-treatment.

5. Conclusion

Spatial orientation and learning performance (i.e. traverse times)werenotdifferentfromuntreatedrams,whenassessedat 83and95weeksofage,followingthediscontinuationofperipu- bertalGnRHa-treatmentat44weeksofage.However,theeffects ofperipubertalGnRHa-treatment toincreaseemotional reactivity,persistedintothesecondyearoflifeafterGnRHa-treatment hadbeendiscontinued,becausethemannerinwhichramsmoved throughthemaze(i.e.mazeprogresspattern)overmultiplesame- daymazeattemptsdifferedfromtheControls.Interestingly,these aspectsofhowtheramsprogressedthroughamazeappearedto bedependentonthelevelofgonadalsteroidexposure(i.e.quicker mazeprogressionduringbreedingseasonvs.slowermazeprogres- sionduringnon-breedingseason).

Thereductioninlong-termspatialmemoryinducedbyperipu- bertalGnRHa-treatment persisted in rams intoadulthood even afterGnRHa-treatmentwasdiscontinued.Developmentofthiscog- nitivefunctionis,therefore,likelytooccurduringacriticalwindow ofdevelopment,whichmayreﬂectatime-limitedperiodofhip- pocampalplasticity.PerturbationsinGnRHsignalingduringthis peripubertalperiod mayalsohavelong lastingeffects onother brainareasand/oraspectsofcognitivefunction.

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.

References

Albertson,A.J.,Navratil,A.,Mignot,M.,Dufourny,L.,Cherrington,B.,Skinner,D.C., 2009.ImmunoreactiveGnRHtypeIreceptorsinthemouseandsheepbrain.J.

Chem.Neuroanat.35,326–333.

Beer,T.M.,Bland,L.B.,Bussiere,J.R.,Neiss,M.B.,Wersinger,E.M.,Garzotto,M., Ryan,C.W.,Janowsky,J.S.,2006.Testosteronelossandestradioladministration modifymemoryinMen.J.Urol.175,130–135.

Caraty,A.,Skinner,D.C.,2008.Gonadotroping-releasinghormoneinthird ventricularcerebrospinalﬂuid:endogenousdistributionandexogenous uptake.Endocrinology149,5227–5234.

Carel,J.C.,Eugster,E.A.,Rogol,A.,Ghizzoni,L.,Palmert,M.R.,Antoniazzi,F., Berenbaum,S.,Bourguignon,J.P.,Chrousos,G.P.,Coste,J.,Deal,S.,deVries,L., Foster,C.,Heger,S.,Holland,J.,Jahnukainen,K.,Juul,A.,Kaplowitz,P.,Lahlou, N.,Lee,M.M.,Lee,P.,Merke,D.P.,Neely,E.K.,Oostdijk,W.,Phillip,M., Rosen-ﬁeld,R.L.,Shulman,D.,Styne,D.,Tauber,M.,Wit,J.M.,2009.Consensus statementontheuseofgonadotropin-releasinghormoneanalogsinchildren.

Pediatrics123,e752–e762.

Chen,M.,Eugster,E.A.,2015.Centralprecociouspuberty:updateondiagnosisand treatment.Pediatr.Drugs17,273–281.

Evans,N.P.,Robinson,J.E.,Erhard,H.W.,Ropstad,E.,Fleming,L.M.,Haraldsen,I.R.H., 2012.Developmentofpsychophysiologicalmotoricreactivityisinﬂuencedby peripubertalpharmacologicalinhibitionofgonadotropinreleasinghormone action-resultsofanovinemodel.Psychoneuroendocrinology37,1876–1884.

Freedland,S.J.,Eastham,J.,Shore,N.,2009.Androgendeprivationtherapyand estrogendeﬁciencyinducedadverseeffectsinthetreatmentofprostate cancer.ProstateCancerProstaticDis.12,333–338.

Garner,C.,1994.UsesofGnRHagonists.J.Obstet.Gynecol.NeonatalNurs.23, 563–570.

Green,H.J.,Pakenham,K.I.,Gardiner,R.A.,2000.Effectsofluteinizinghormone releasinghormoneanalogsoncognitioninwomenandmen:areview.Psychol.

HealthMed.5,407–418.

Grossman,A.H.,D’Augelli,A.R.,2007.Transgenderyouthandlife-threatening behaviors.SuicideLifeThreat.Behav.37,527–537.

Hapgood,J.P.,Sadie,H.,vanBiljon,W.,Ronacher,K.,2005.Regulationofexpression ofmammaliangonadotrophin-releasinghormonereceptorgenes.J.

Neuroendocrin.17,619–638.

Hayes,P.,2016.Earlypuberty,medicalisationandtheideologyofnormality.

Women’sStud.Int.Forum56,9–18.

Hembree,W.C.,Cohen-Kettenis,P.,Delemarre-vandeWaal,H.A.,Gooren,L.J., Meyer,W.J.,Spack,N.P.,Tangpricha,V.,Montori,V.M.,2009.Endocrine treatmentoftranssexualpersons:anendocrinesocietyclinicalpractice guideline.J.Clin.Endocrinol.Metab.94,3132–3154.

Hough,D.,Bellingham,M.,Haraldsen,I.R.,McLaughlin,M.,Rennie,M.T.,Robinson, J.E.,Solbakk,A.K.,Evans,N.P.,2016.Spatialmemoryisimpairedby

peripubertalGnRHagonisttreatmentandtestosteronereplacementinsheep.

Psychoneuroendocrinology75,173–182.

Jennes,L.,Eyigor,O.,Janovick,J.A.,Conn,P.M.,1997.Braingonadotropinreleasing hormonereceptors:localizationandregulation.RecentProg.Horm.Res.52, 475–491.

Kim,E.Y.,Lee,M.I.,2012.Psychosocialaspectsingirlswithidiopathicprecocious puberty.PsychiatryInvest.9,25–28.

Mul,D.,Hughes,I.A.,2008.TheuseofGnRHagonistsinprecociouspuberty.Eur.J.

Endocrinol.159,S3–S8.

Mul,D.,Versluis-denBieman,H.J.M.,Slijper,F.M.,Oostdijk,W.,Waelkens,J.J.,Drop, S.L.,2001.Psychologicalassessmentsbeforeandaftertreatmentofearly pubertyinadoptedchildren.ActaPaediatr.90,965–971.

Nuruddin,S.,Bruchhage,M.,Ropstad,E.,Krogenæs,A.,Evans,N.P.,Robinson,J.E., Endestad,T.,Westlye,L.T.,Madison,C.,HeboldHaraldsen,I.R.,2013a.Effectsof peripubertalgonadotropin-releasinghormoneagonistonbraindevelopment insheep—amagneticresonanceimagingstudy.Psychoneuroendocrinology38, 1994–2002.

Nuruddin,S.,Wojniusz,S.,Ropstad,E.,Krogenæs,A.,Evans,N.P.,Robinson,J.E., Solbakk,A.-K.,Amiry-Moghaddam,M.,HeboldHaraldsen,I.R.,2013b.

Peri-pubertalgonadotropin-releasinghormoneanalogtreatmentaffects hippocampusgeneexpressionwithoutchangingspatialorientationinyoung sheep.Behav.BrainRes.242,9–16.

Olton,D.S.,Papas,B.C.,1979.Spatialmemoryandhippocampalfunction.

Neuropsychologia17,669–682.

Salminen,E.K.,Portin,R.I.,Koskinen,A.,Helenius,H.,Nurmi,M.,2004.Associations betweenserumtestosteronefallandcognitivefunctioninprostatecancer patients.Clin.CancerRes.10,7575–7582.

Schang,A.L.,Ngo-Muller,V.,Bleux,C.,Granger,A.,Chenut,M.C.,Loudes,C.,Magre, S.,Counis,R.,Cohen-Tannoudj,J.,Laverriere,J.N.,2011.GnRHreceptorgene expressioninthedevelopingrathippocampus:transcriptionalregulationand potentialrolesinneuronalplasticity.Endocrinology152,568–580.

Silverman,A.J.,Jhamandas,J.,Renaud,L.P.,1987.Localizationofluteinizing hormone-releasinghormone(LHRH)neuronsthatprojecttothemedian eminence.J.Neurosci.7,2312–2319.

Skinner,D.C.,Albertson,A.J.,Navratil,A.,Smith,A.,Mignot,M.,Talbott,H., Scanlan-Blake,N.,2009.GnRHeffectsoutsidethe

hypothalamo–pituitary–reproductiveaxis.J.Neuroendocrinol.21,282–292.

Steinberg,L.,2004.Risktakinginadolescence:whatchanges,andwhy?Ann.N.Y.

Acad.Sci.1021,51–58.

Vance,S.R.,Ehrensaft,D.,Rosenthal,S.M.,2014.Psychologicalandmedicalcareof gendernonconformingyouth.Pediatrics134,1184–1192.

Wojniusz,S.,Vögele,C.,Ropstad,E.,Evans,N.P.,Robinson,J.E.,Sütterlin,S.,Erhard, H.W.,Solbakka,A.K.,Endestadg,T.,Olbergh,D.E.,HeboldHaraldsen,I.R.,2011.

Prepubertalgonadotropin-releasinghormoneanalogleadstoexaggerated behavioralandemotionalsexdifferencesinsheep.Horm.Behav.59,22–27.

Wojniusz,S.,Callens,N.,Sütterlin,S.,Andersson,S.,DeSchepper,J.,Gies,I., Vanbesien,J.,DeWaele,K.,VanAken,S.,Craen,M.,Vögele,C.,Cools,M., Haraldsen,I.R.,2016.Cognitive,emotional,andpsychosocialfunctioningof girlstreatedwithpharmacologicalpubertyblockageforidiopathiccentral precociouspuberty.Front.Psychol.7,1–12.

Wood,R.I.,Foster,D.L.,1998.Sexualdifferentiationofreproductive neuroendocrinefunctioninsheep.Rev.Reprod.3,130–140.