ABSTRACT

Introduction:Brainregionsimplicatedinsexualbehaviorbegintodifferentiateinthelast

trimesterofgestation.Antenataltherapywithcorticosteroidsisoftenusedinclinicalpractice

duringthisperiodtoacceleratelungmaturationinpre‐termriskpregnancies.Clinicaland

animalstudieshighlightedmajorbehavioralimpairmentsinducedlaterinlifebythese

treatments,especiallywhensyntheticcorticosteroidsareused.

Aim:Toevaluatetheimplicationsofacuteprenataltreatmentwithnaturalversussynthetic

corticosteroidsonadultmaleratsexualbehavioranditsneurochemicalcorrelates.

Methods:TwelvepregnantWistarratswereinjectedwithdexamethasone(DEX‐1mg/kg),

corticosterone(CORT‐25mg/kg)orsalineonlategestation(pregnancydays18and19).

Followingthisbriefexposuretocorticosteroids,weassessedthesexualbehavioroftheadult

maleprogenyandsubsequentlycorrelatedthesebehaviorswiththelevelsofcathecolamines

andmRNAofdopamineandandrogenreceptors(AR)inbrainregionsrelevantforsexual

behavior.

MainOutcomeMeasures:Sexualbehaviorofadultmaleoffspringwasassessedbyexposureto

receptivefemales.Thiswascorrelatedwithserumtestosteronelevelsandlevelsof

cathecolamines(determinedbyHPLC)anddopamineandandrogenreceptorsmRNAexpression

(real‐timePCR)inbrainregionsimplicatedinsexualbehavior.

Results:PrenatalDEXexposureresultedinadecreasednumberandincreasedlatencytimeto

mountsandintromissionsinadulthood.Thesefindingscorrelatedwithdecreasedlevelsof

2

serumtestosteroneandincreasedhypothalamicexpressionofARmRNA.DEXanimalsalso

displayedlowerdopaminelevelsandhigherdopaminereceptormRNAexpressionbothin

hypothalamusandnucleusaccumbens(NAcc).ThemilderphenotypeofCORTanimalswas

correlatedonlywithdecreaseddopaminelevelsinNAcc.

Conclusion:Antenatalcorticotherapyprogramsadultmalesexualbehaviorthroughchangesin

specificneuronalandendocrinemediators.Importantly,equipotentdosesofcorticosterone

triggerlessdetrimentalconsequencesthandexamethasone,emphasizingthedifferentialimpact

ofactivationofthedifferentcorticosteroidreceptors.

Keywords:Antenatalcorticotherapy;Corticosteroids;Dopamine;Neurodevelopment;Sexual

behavior

Wordcountoftext(includingabstract):2494

3

INTRODUCTION

Thelasttrimesterofgestationandearlypostnatalperiodarecriticalforbrainsexual

differentiation(Segarraetal.,1991).Insultsatthisperiod,includingstressandprolonged

exposuretocorticosteroids,havebeenshowntodisruptseveralbehaviorsinadulthood,namely

malesexualbehavior(Holsonetal.,1995,Gerardinetal.,2005,Pifferetal.,2009).Interestingly,

exposuretocorticosteroidsduringlategestationhasbeencorrelatedwithasustained

perturbationinmalesteroidogenesis(Pageetal.,2001)andanimpoverisheddopaminergic

innervationofthenucleusaccumbens(NAcc)(Leaoetal.,2007),whichisofparticularrelevance

whenconsideringthefacilitatoryroleofdopamineinthedifferentaspectsofsexualbehavior

(GiulianoandAllard,2001).

Inclinicalpractice,glucocorticoidsareprescribedinabout10%ofpregnanciesatriskofpreterm

deliveryinordertopromotefetallungmaturation(Crowley,1995,NIH,1995,Craneetal.,

2003).Dexamethasone(DEX)andbetamethasone,thepreferreddrugs(Jobeetal.,2003),are

syntheticcorticosteroidsthatcrosstheplacentawith100%efficacyandhavebeenshownto

reducethemorbidityandmortalityofthepreterminfantafterdelivery(NIH,1995).Despite

this,thesafetyoftheexposureofthedevelopingfetalbraintoglucocorticoidshasbeen

questionedasitmighthavelife‐longeffectsonadultbehaviorandneuroendocrinefunction

(Matthews,2000,Welbergetal.,2001,Oliveiraetal.,2006).Availabledatasuggeststhatthe

activityofthehypothalamic‐pituitary‐adrenal(HPA)axis,whichisvitaltostressresponse,might

4

bereprogrammedbymanipulationsinthecorticosteroidmilieuduringlategestation;this

alteredpatternoftheHPAisbelievedtobe,atleastinpart,responsibleforthebehavioraland

neuroendocrinechanges(WelbergandSeckl,2001),aswellasforincreasedriskfor

hypertension,type2diabetes(Levittetal.,1996,Lindsayetal.,1996,Sapolskyetal.,2000)and

neuropsychiatricdisorders(Welbergetal.,2001).Ofparticularinterestistheevidence

suggestingalessdeleteriouseffectonadultemotionalbehavioroftheacuteadministrationof

endogenouscorticosteroids(Oliveiraetal.,2006),especiallyinlightofevidenceshowingthat

cortisoldisplayssimilartherapeuticefficacytoDEXduringpregnancyandneonatallife(Crowley,

1995).

Inlightofthisevidence,wedecidedtoassesstheimpactofshort‐termantenatalcorticosteroid

exposureinmalesexualbehaviorandsearchforitsneurochemicalandendocrinecorrelates.

Furthermore,wealsowantedtocomparenaturalandsyntheticcorticosteroidsintermsoflong‐

termadverseeffects.

5

METHODS

Animalsandtreatments

Experimentswereconductedinaccordancewithlocalregulations(EuropeanUnionDirective

86⁄609⁄EEC)andNIHguidelinesonanimalcareandexperimentation.

TwelveadulttimedpregnantWistarHanrats(Charles‐RiverLaboratories,Barcelona,Spain)

receivedatday14ofgestationwereindividuallyhousedunderstandardlaboratoryconditions

(12/12hlight/darkcycle,withlightsonat8a.m.;foodandwateradlibitum).

SubcutaneousinjectionsofDEX(1mg/kg,Sigma‐Aldrich;n=4),corticosterone(CORT,25mg/kg,

Sigma‐Aldrich;n=4),orsaline(controls,1mL/kg;n=4)wereadministeredonembryonicdays

(ED)18and19ofpregnancy(Oliveiraetal.,2006).Drugdosageswerechosentoachieve

comparabletransrepressivepotenciesattheglucocorticoidreceptors(GR)(SchimmerBP,2005).

Weaningwasperformedatpostnatalday21andpupswerepair‐housedaccordingtogender

andprenatalexposure.Maleoffspring(2siblingsperdam;n=4dams/group),weretestedat3

monthsforsexualbehavior.

Preparationofsexuallyreceptivefemales

Adult3monthsfemaleratswereindividuallyhousedandovariectomized,aspreviously

described(Agmo,1997).Sexualreceptivitywasinducedbysubcutaneousestradiolbenzoate(20

µg/rat,SigmaAldrich)andprogesterone(1mg/rat,Sigma‐Aldrich)52and4hoursbeforemale

exposure,respectively.

6

Malesexualbehavior

ThetestarenaconsistedofarectangularPlexiglasbox(40x60x40cm)withatransparenttopand

avideocameraoverit.Exposurewasconductedtwohoursaftertheonsetofthedarkphase,in

aquietroom,withadimredlight.Sexuallyexperiencedmaleswereplacedinthearenaten

minutesbeforeareceptivefemalewaspresentedandactivitywasrecordedfor20minutes;

latencytimeandnumberofmountsandintromissions(vaginalpenetration)wereregistered

andintromissionratiocalculatedasintromissions/[intromissions+mounts].

Biometricandtestosteronemeasurements

Animalsweresacrificedoneweekafterbehaviorassessmentandbloodcollectedfor

determinationofserumtotaltestosteronelevelsbyelectrochemiluminescenseimmunoassay

(ElecsysTestosteroneIIreagentkit,RocheDiagnostics;measuringrange2.5‐1500ng/dL).Testis

wetweightwasassessed.

Braincathecolamines

Afterbrainsnapfreezing,theregionsofinterestwererapidlydissectedunderthescopeusing

macrodissectionofspecificbrainareas.Thehypothalamuswasisolatedbyplacingwholebrains

upsidedownandusingdelicateforceps(Dumont#7forceps,FineScienceToolsUSAInc.,Foster

City,CA,USA)todetachitfromtherest.Thehypothalamuswasidentifiedastheroundshaped

areainthecenterofthebrain.NAccwasisolatedusingpunchdissectionin2mmsectionsof

brains(AltoTMbrainmatrix,StoetlingCo.,WoodDale,IL,USA)andidentifiedundera

stereomicroscope(ModelSZX7,OlympusAmericaInc.,CenterValley,PA,USA).NAccwas

7

identifiedasthetissueinthevicinityoftheanteriorbranchoftheanteriorcommissure,

accordingtoPaxinosstereologicalcoordinates(PaxinosandWatson,2005).Sampleswere

frozeninliquidnitrogen(overnightat‐20ºC)afteraddingpercloricacid0.2M.Sampleswere

brieflysonicated,centrifugedand50µlaliquotsofthesupernatantinjectedonahigh

performanceliquidchromatography(HPLC)combinedwithelectrochemicaldetectionsystem.A

mobilephaseof0.7Maqueouspotassiumphosphate(monobasic)(pH3.0)in10%methanol,1‐

heptanesulfonicacid(222mg/l)andNa‐EDTA(40mg/l)wasused.

Levelsof5‐HT,5‐hydroxyindoleaceticacid(5‐HIAA),dopamine,3,4‐dihydroxyphenylaceticacid

(DOPAC)and4‐hydroxy‐3‐methoxyphenylaceticacid(homovanillicacid,HVA)weredetermined

usingaGilsoninstrument(GilsonInc.,Middleton,WI,USA),fittedwithananalyticalcolumn

(SupelcoSupelcosilLC‐183M;7.5cmx4.6mm;flowrate:1.0–1.5ml/min;Supelco,Bellefonte,

PA,USA).Astandardcurvewasthenobtainedanddatapresentedasconcentration(nanogram

permilligramoftissueprotein).

Molecularanalysis

ForReal‐TimePCRanalysis,totalRNAwasisolatedfromfrozenareasusingTrizol(Invitrogen)

andDNasetreatment(Fermentas),accordingtomanufacturer.TwoµgofRNAwereconverted

intocDNAusingtheiSCRIPTkit(Biorad).RT‐PCRwasperformedusingSyberGreen(Qiagen)and

theBioradq‐PCRCFX96apparatus.HPRTwasusedasahousekeepinggene.Weusedrelative

quantificationtodeterminethefoldchangedifferencebetweencontrol,CORTandDEXanimals,

usingtheΔΔCTmethodasdescribedbefore(Pfaffl,2001).Primersequenceswere

AR_F:GGGTGACTTCTCTGCCTCTG,AR_R:CCACAGATCAGGCAGGTCTT(androgenreceptor);

8

ESR1_F:CAGGTGCCCTACTACCTGGA,ESR1_R:GGTAGCCAGAGGCATAGTCG(estrogenreceptor1);

ESR2_F:AACCGCCATGAGTATTCAGC,ESR2_R:GTAACAGGGCTGGCACAACT(estrogenreceptor2);

nNOS_F:GACAACGTTCCTGTGGTCCT,nNOS_R:GAAGAGCTGGTCCTTTGTGC(neuronalnitricoxide

synthase);D1R_F:TCCTTCAAGAGGGAGACGAA,D1R_R:CCACACAAACACATCGAAGG(dopamine

D1receptor);D2R_F:CATTGTCTGGGTCCTGTCCT,D2R_R:GACCAGCAGAGTGACGATGA(dopamine

D2receptor);HPRT1_F:GCAGACTTTGCTTTCCTTGG,HPRT1_R:TCCACTTTCGCTGATGACAC.

Statisticalanalysis

Forstatisticalanalysis,the“n”ofeachexperimentgroupwasconsideredthenumberoflitters

fromwhichindividualswerederived.Resultsarepresentedasaverage±SE.Datawasanalyzed

byPASWStatistics18.0(SPSSInc,Chicago,IL,USA),usingANOVA.Wheneverappropriate,post

hoccomparisonswereperformedusingTukeytest;statisticalsignificancewasconsideredwhen

p<.05.

9

RESULTS

AcuteantenatalDEXexposureaffectsadultmalesexualbehavior

Treatmentsignificantlyaffectedsexualmotivationandthevolitiveaspectsofcopulatory

behavior(F=20.057,p<.001),asrevealedbyanincreasedlatencytomountinDEX‐exposed

animalscomparedtocontrols(p<.001)andCORTsubjects(p=.029),respectively(Figure1).

CORTanimalswerealsodifferentfromcontrols(p=.027).

Treatmentalsoaffectedthenumberofmounts(F=5.233,p=.031),anothermeasureofsexual

motivation(Agmo,1997),whichwassignificantlydecreasedinDEX‐exposedsubjectswhen

comparedtocontrols(p=.027).Interestingly,CORTratswerenotdifferentfromtheother

groups.

Incontrasttomounts,intromissionsarenotexclusivelydependentonsexualmotivation.

Treatmentaffectedthisparameter(F=23.081,p<.001)asrevealedbyanincreasedlatencyof

DEXprogenyincomparisontocontrols(p<.001)andCORTsubjects(p=.016).Again,CORT

animalsalsotookmoretimetointromissionthancontrols(p=.024).

Moreover,thenumberofintromissionswasaffectedbytreatment(F=8.083,p=.010).This

indicatoroftheeasinessintheactivationofejaculatoryreflexes(Agmo,1997)wasfoundtobe

decreasedinDEX‐exposed(p=.008),butnotonCORT‐exposedsubjects(p=.103).

10

Intromissionratio(Figure2),anindicatorofefficiencyofpenileerection,wasalsoaffectedby

treatment(F=22.972,p<.001).DEX‐subjectsdisplayedsignificantlylowerratioscomparedto

controls(p<.001)andCORT‐exposedanimals(p=.003).

AdulttestosteronelevelsareaffectedbyantenatalDEX

SerumtestosteronelevelsweresignificantlyreducedbyantenatalDEXexposure(F=15.815,

p=.001;vsCORTp=.001;vscontrolsp=.004)butnotbyCORT(Figure3).Testiswetweightwas

similarbetweengroups(datanotshown).

Antenatalcorticosteroidsinfluencebraindopaminelevels

AntenatalcorticosteroidsexposureinfluencedthelevelsofdopamineinNAcc(F=39.911,

p<.001;Table1).BothprogenyofCORTandDEXdamsdisplayeddecreasedlevelsofdopamine

inNAccincomparisontocontrols(p<.001andp<.001,respectively).Treatmentalsoaffected

dopamineturnover(F=6.162,p=.021),withanincreaseinDEXsubjectswhencomparedtoCORT

(p=.026)andcontrols(p=.047).

Hypothalamiclevelsofdopaminewerealsosignificantlyaffectedbyantenataltreatment

(F=5.817,p=.024),withadecreaseonlyinDEX‐treatedsubjects(p=.022)whencomparedto

controls.Dopamineturnoverinthisareawasalsoaffected(F=10.286,p=.005),withCORTand

DEXanimalsdisplayingalowerratiowhencomparedtocontrols(p=.010and.008,respectively).

Treatmentdidnotaffectserotoninlevels(F=3.564,p=.072)noritsturnover(F=1.076,p=.381)in

theNAcc.However,hypothalamiclevelsofserotoninwerereduced(F=14.050,p=.002)whileits

11

turnoverwasincreased(F=7.552,p=.012)inbothDEX(p=.003;p=.029)andCORT(p=.004;

p=.016)groups.

Molecularcorrelates

IntheNAcc,dopamineD1(D1R)andD2(D2R)receptorsmRNAlevelsweresignificantlyaffected

byprenatalexposuretocorticosteroids(F=111,471,p<.001andF=76.383,p<.001,respectively;

Table2),withanincreaseinDEXgroupwhencomparedtocontrols(p<.001;p<.001,

respectively)andCORT(p<.001;p<.001,respectively).Asimilareffectwasalsoobservedinthe

hypothalamus,butonlyforD1R(F=7.868,p=.011;vscontrolsp=.009);CORTanimalswerenot

differentfromanyothergroup.

LevelsofARandneuronalnitricoxidesynthase(nNOS)mRNAwerenotdifferentbetween

groupsintheNAcc.However,inhypothalamus,therewasasignificanteffectofexposureonAR

(F=13.049,p=.002)andnNOS(F=27.056,p<.001)mRNAlevels,withanincreaseinDEXprogeny

whencomparedtocontrols(p=..020;p<.001,respectively)andCORTsubjects(p=.002;p=.002,

respectively).

Levelsofestrogenreceptors1and2werenotaffectedbytreatmentinneitherarea.

12

DISCUSSION

Inrats,brainsexualdifferentiationoccursmainlyduringlategestation(ED14–21)andthefirst2

weeksofpostnatallife(Segarraetal.,1991).Thevulnerabilityofthistime‐windowtoseveral

insults,includingstressandcorticosteroidexposure,maythusleadtolong‐termconsequences

onadultmalesexualbehavior(Gerardinetal.,2005,Pifferetal.,2009).Infact,previousdata

suggeststhatprenatalstressdisruptsthenormalmaternalhormonalmilieuandsuppressesthe

fetaltestosteronepeakonED18and19,requiredforlaterexpressionandmaintenanceofmale

sexualbehavior(WardandWeisz,1984,Lalauetal.,1990).Suchdatasustainthatinterferences

inthematurationofthehypothalamic‐pituitary‐adrenal(HPA)axis,affectthehypothalamic‐

pituitary‐gonadal(HPG)axis,sincebothareregulatedbycommonplayersbothcentrallyandat

theperiphery(Pageetal.,2001).Followingtheinitialexperimentalevidenceshowingthatlate

gestationwhole‐bodyrestraintunderbrightlightsresultsindelayedinitiationofcopulation

(WARD1972),morerecentdatacorrelatedprolongedprenatalstress/corticosteroidswith

impairedadultmalesexualbehaviorandreducedserumtestosteronelevels(Gerardinetal.,

2005,Pifferetal.,2009).However,nopreviousstudiesfocusedonthepotentialeffectsofa

short‐termexposurewhichbettermimicstheeverydayclinicalpractice,norinthecomparison

betweendifferenttypesofcorticosteroids.

Theanalysisofseveralbehavioralparameterspermittedustodistinguishbetweenappetitive

andconsummatorycomponentsofmaleratsexualbehavior.Maleratsexualbehavioris

characterizedbyaseriesofmounts,eitherwithorwithoutvaginalpenetration(intromission),

13

ultimatelyleadingtoejaculation(HullandDominguez,2007).Whilelatencyuntilthefirstmount

reflectssomeoftheappetitiveaspectsandsexualmotivation,intromissionandejaculation

latenciesbutalsomountandintromissionfrequenciesreproduceconsummatorycomponents

ofcopulatorybehavior(Pfausetal.,1990b,Agmo,1999).Interestingly,theimpairmentofmale

sexualbehaviorobservedinthisstudywasmainlycharacterizedbyalterationsinsexual

appetite(increaseinmountandintromissionlatencies);moreover,thereducednumberof

mountsmightreflectdecreasedsexualmotivation(Agmo,1997).Importantly,thesedifferences

aremorestrikinginanimalsexposedtoDEXthantoCORT.

Regardingtheneurobiologyofsexualbehavior,threemajorintegrativesystemsregulatesexual

motivationandgenitalandmotorresponses(Hulletal.,2004).Whereasthemesolimbicsystem

iscriticalforappetitivebehaviorandreinforcement,themedialpreopticsystemcontributesto

genitalreflexes,sexualmotivationandmotorpatternsofcopulation.Finally,thenigrostriatal

systemenhancesthemotoricreadinesstorespondtostimuli.Dopamineisthecommonkey

playerinallthreesystems,easingsexualmotivation,copulatoryproficiency,andgenitalreflexes

(GiulianoandAllard,2001).Thepathwaysforsexualexcitationinvolvetheactivationof

incertohypothalamicandmesolimbicdopaminetransmissionthattargetsthehypothalamic

medialpreopticarea(MPOA)andNAcc,respectively(Pfaus,2009).Asaresult,inthemalerat

thereisaslightincreaseindopaminereleaseinNAccfollowingpresentationtoareceptive

femalethatisfollowedbyasharpincreaseindopaminetransmissionduringcopulation,that

graduallydeclinesaftertheremovalofthefemale(Pfausetal.,1990a).

14

Apreviousstudyfocusingontheeffectsofprolongedprenatalimmobilizationstressonthe

adultmaleratcorrelatedtheabsenceofcopulatorybehaviorswithunchangedNAcc

extracellularlevelsofdopamine,DOPACandHVAduringexposuretoreceptivefemales.Such

data,obtainedthroughsimultaneoussexualbehaviortestingandconcomitantmicrodialysis

samplig,suggestedthatintenseenvironmentalstressorsmightimpairNAccdopaminerelease

(WANG1995).

Inourexperiment,aninterestingneurochemical‐behaviorcorrelatewasestablished,aswe

founddecreaseddopamineinhypothalamusandNAccofcorticosteroidbrieflyexposed

animals.Interestingly,wehadpreviouslyreportedareduceddopaminergicinnervationofthe

NAccfollowingprenatalshort‐termexposuretoDEX,revealedbyareduceddensityoftyrosine

hydroxylase‐positivefibersinthesesubjects(Leaoetal.,2007).Inaddition,thedecreasein

dopaminelevelsinNAcchereinreportedislikelytobeofrelevanceforthechangesinsexual

behaviorifonetakesintoaccountdescriptionscorrelatingadelayedonsetofcopulationand

ejaculationwithadiminishedreleaseofthisneurotransmitterinthemesolimbictract(Hullet

al.,2004).Furthermore,theincreaseindopamineD1andD2receptorsmRNAintheNAcc

followingDEXexposurehereinshownfurthersupportstheexistenceofahypodopaminergic

statusintheseanimals,andmayappearasacompensatorymechanismduetothelow

dopaminelevelsobserved.

AlthoughweobservedsubstantialdifferencesinthedopaminelevelsandreceptorsintheNAcc,

onedrawbackofthisworkisthefactthatwedidnotdiscriminatebetweenitstwofunctionally

distinctregions:thecoreandtheshell.C‐fosexpressionisincreasedinthecorebutnottheshell

duringsexualbehavior(BradleyandMeisel,2001).Onthecontrary,administrationofdrugsof

15

abuseresultsinincreaseddopaminelevelsintheshelloftheNAcc(Pontierietal.,1995,Nisell

etal.,1997,DiChiaraetal.,1999)(DICHIARA2002).Thissuggeststhatshellandcoremightbe

activateddifferentlyinresponsetonaturalreinforcersanddrugsofabuse.Indeed,NAcc

neuronsexhibitsimilarneuronalactivityduringrespondingtotwonaturalrewards‐foodand

water,butdifferentfiringpatternsduringrespondingforanaturalrewardversuscocaine(Carelli

etal.,2000).Therefore,consideringthedifferentfunctional/activationalrolesofcoreandshell,

itwouldbeinterestingtoanalyzedopaminemetabolismandreceptorsineachsubareainorder

todissectwhatisthemostaffectedarea.

ThehypodopaminergicstatusofDEX‐exposedanimalsintheNAccmighthaveotherbehavioral

consequencesbesidesalteredsexualbehavior,consideringtheimportanceofcorrectdopamine

inputforfeeding,rewardandaddiction,amongothers.DopamineisreleasedintheNAccin

responsetodrugsofabusebutalsootherconsumatorybehaviorssuchassexandfoodandthus

theVTA‐NAccpathwayisalsoknownasthe“rewardpathway”(PiazzaandLeMoal,1996).

Interestingly,somestudieshavereportedcross‐sensitizationbetweenrepeatedexposuresto

pharmacologicalagentsandnaturalmotivatedbehaviorssuchassex(MitchellandStewart,

1990a,b,FiorinoandPhillips,1999).Forexample,sexualexperiencecancross‐sensitize

neuronalresponsestoamphetamineandthisseemstodependondopaminereleaseinthe

NAcc(BradleyandMeisel,2001).

Theintricately‐regulatedbalancebetweenhypo‐andhyper‐dopaminergicstatesinthe

mesolimbiccircuit,speciallyintheNAccarea,underliesanindividual’scyclesofdrug‐seeking

behavior/abuseandresponsetonaturalrewards.Whileahyperdopaminergicstateseemsto

enhancethemotivationalorrewardingpropertiesofdrugsofabuse,hypodopaminergicstates

16

appeartoenhancedrug‐seekingbehaviorinparallelwithreductionsintheperceived

motivationalimpactof‘natural’rewardssuchasfoodandsex(Dianaetal.,1993,Dianaetal.,

1998,Melisetal.,2005).Thistheoryisinagreementwithourbehaviouralandneurochemical

results,giventhefactthatDEX‐animalshavelowdopaminelevelsintheNAccand,

concomitantly,impairedappetitivesexualbehaviour.Additionally,itsuggeststhatthese

animalsmightalsodisplaydifferentialsusceptibilitytoaddiction,aphenomenonalsoobserved

inothermodelsofearlylifestress(Kippinetal.,2008).

Dopamineinthehypothalamus,particularlyintheMPOA,isessentialforgenitalreflexes,motor

patternsofcopulation,andprobablysexualmotivation(HullandDominguez,2006);several

studiesdescribedthefacilitativeroleofincreasedlevelsintheMPOAonsexualbehavior,

suggestingthattestosteronemightmediatethiseffect(DOMINGUEZ2005).Inthepresent

study,conclusionsontheimpactofprenatalexposuretonaturalversussynthetic

corticosteroidsarelimitedbythefactthatdissectionofthewholehypothalamicareawas

performedinsteadofisolatingtheMPOA.Nonetheless,thedecreasedhypothalamiclevelsof

dopaminehereinreportedintheDEXgroup,butnotintheCORTgroup,isassociatedwitha

significanteffectonD1receptorsmRNA.Thisfactislikelytobeofsignificancetoexplainthe

differentialneuroendocrineandbehavioraleffectsofCORTfromDEX.

Inaddition,anincreaseintheandrogenreceptormRNAwasobservedinthehypothalamusof

DEXprogeny,possiblyreflectingareductioninthecirculatingandrogens.Interestingly,previous

17

studiesshowedthatalthoughnormalbasallevelsofdopamineintheMPOAareadequateto

allowsomecopulatorybehavior,efficientmatingrequiresanandrogen‐dependentfemale‐

stimulatedincrease(Putnametal.,2005).Also,byup‐regulatingnNOSintheMPOA,

testosteroneenhancesnitricoxideproduction,whichcontrolsdopaminerelease(Sandersonet

al.,2008).Inthepresentstudy,wefoundincreasedlevelsofnNOSmRNAinthehypothalamus

ofDEXsubjects.ItwouldbeofaddedvaluetoassessiftheseschangespersistinMPOAsamples,

whichwouldbeinaccordancetopreviousdescriptionsintheMPOAofgonadectomizedrats

(Singhetal.,2000).However,technicalissuesintheisolationoftheMPOAandthefactthat

neighborhypothalamicsubareasmightdisplaydifferentsusceptibilitiestocirculatingandrogens

couldjustifywhyotherstudiesdidnotconfirmtheoriginalfindings(Satoetal.,2005).

Thus,inordertodrawfurtherconclusionsontheimpactoftheinuterocorticosteroids

exposureontheadultmaleMPOA,itwouldbeofinteresttospecificallyanalyzethis

hypothalamicareainfuturestudies.

18

CONCLUSIONS

Earlylifeexposuretoshort‐termglucocorticoidligandstriggerslifelongprogrammingeffectsin

brainregionsimplicatedindistinctaspectsofmalesexualbehavior.Thebehavioralchanges

correlatewithaltereddopaminergicsystemsandneuroendocrinemarkers.Thesefindingsareof

clinicalrelevance,astheyprovidesupporttotherapeuticinterventionsforsexualdysfunction

thatmodulatebraindopaminergiclevels(Montorsietal.,2003a,Montorsietal.,2003b,Padma‐

Nathanetal.,2004,MinerandSeftel,2007)andperipherallevelsoftestosterone

(HatzimouratidisandHatzichristou,2007,Traishetal.,2007,Hatzimouratidisetal.,2010).

Noticeably,equipotentCORTadministrationtriggersalessdetrimentalimpairmentthanDEX,

highlightingtheroleofthedifferentcorticosteroidreceptorsonthesystemsregulatingsexual

behavior.

19

FIGURELEGENDS

Figure1–Latencytimestomountandintromissionwereincreasedindexamethasone(DEX)

andcorticosterone(CORT)exposedanimalswhencomparedtocontrols(left);thenumberof

mountsandintromissionswassignificantlyreducedinDEX‐exposedrats(right).*p<.05.

Figure2–Intromissionratio,calculatedasintromissions/[intromissions+mounts],was

diminishedinDEX‐exposedratswhencomparedtocontrolsandCORTanimals.*p<.05.

Figure3–AntenatalDEXadministrationledtodiminishedserumtestosteronelevels(ng/dL)in

adultmalerats,whencomparedtoCORTandcontrols.*p<.05.

20

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