Effect of prenatal stress on memory nicotine withdrawal and 5HT1A expression in raphe nuclei of adult rats

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ContentslistsavailableatScienceDirect

International Journal of Developmental Neuroscience

jo u r n al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / i j d e v n e u

Effect of prenatal stress on memory, nicotine withdrawal and 5HT1A expression in raphe nuclei of adult rats

N. Said

^a,∗

, S. Lakehayli

^a

, M. El Khachibi

^b

, M. El Ouahli

^c

, S. Nadiﬁ

^b

, F. Hakkou

^a

, A. Tazi

^a

Q2

aLaboratoryofPharmacology,FacultyofMedicineandPharmacyofCasablanca,19RueTarikBnouZiad,Casablanca,Morocco

bGeneticsandMolecularPathologyLaboratory,FacultyofMedicineandPharmacyofCasablanca,19RueTarikBnouZiad,Casablanca,Morocco

cSultanMySlimaneUniversity,FacSciences&TecniquesBeni-Mellal,LifeSciences,Morocco

a r t i c l e i n f o

Articlehistory:

Received11February2015

Receivedinrevisedform28March2015 Accepted16April2015

Availableonlinexxx

Keywords:

Prenatalstress Nicotine Withdrawal Anxiety Memory 5HT1A

RealtimePCR

a b s t r a c t

Maternaldistresshasoftenbeenassociatedwithcognitivedeﬁcienciesanddrugabusedependencein rats.Thisstudyexaminedthesebehavioraleffectsinoffspringofmothersstressedduringgestation.To thisend,pregnantdamsweresubjectedtodailyelectricfootshocksduringthelast10daysofpregnancy.

Wemeasuredlitterparametersandbodyweightsofthedescendantsafterweaning(21days)andat adulthood(80days).Afterwards,prenatallystressedandcontrolrats’performancesinthenovelobject recognitiontestwerecomparedinordertoevaluatetheirmemorywhileothersunderwenttheWater consumptiontesttoassessthenicotinewithdrawalintensityafterperinatalmanipulations.Meanwhile, anothersetofratsweresacrificedand5HT1Areceptors’mRNAexpressionwasmeasuredintheraphe nucleibyquantitativeRealTimePCR.Wenoticednosignificantinfluenceofmaternalstressonlitter sizeandbodyweightrightafterweaning.However,controlratswereheavierthanthestressedratsin adulthood.Theresultsalsoshowedasignificantdecreaseintherecognitionprocessinratsstressedin uterocomparedtothecontrols.Moreover,aheightenedanxietysymptomwasobservedintheprenatally stressedoffspringfollowingnicotinewithdrawal.Additionally,theRealTimePCRmethodrevealedthat prenatalstressinducedasignificantdecreasein5HT1Areceptors’levelsintheraphenucleiwhilethe nicotinehadasimilareffectthesereceptors’expressioninbothnicotine-treatedcontrolandprenatally stressedgroups.Takentogether,thesefindingssuggestthatthecognitivefunctionsanddrugdependence canbetriggeredbyearlyadverseeventsinrats.

1. Introduction

Prenatalexposuretostresscangeneratepermanentphysiolog- Q3

icaland developmentalimpairmentsand raise theincidenceof stress-relatedpsychiatricdisordersinoffspring.Thesealterations oftenextendtoadulthood,affectingawiderangeofvitalfunctions inducinganxiety-relatedsyndromes(Pallarésetal.,2007;Laloux etal.,2012),depression(Smithetal.,2004)andsocialinteraction impairments(Patinetal.,2005).Prenatalstressisalsoknownto producelong-lastingmemorydeﬁciencies inrodents(Markham etal.,2010;Modiretal.,2014).Theycanbecausedbyasevere reductioninneurogenesis(Lemaireetal.,2000;Fujiokaetal.,2006), volume(Martinez-Tellezetal.,2009)andgranulecellnumberinthe hippocampus(Schmitzetal.,2002).

∗Correspondingauthorat:PharmacologyDepartmentintheFacultyofMedicine andPharmacyofCasablanca,19TarekIbnZyad,Casablanca,Morocco.

Tel.:+212620729222.

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

Thepronouncedeffectsofmaternalstressonbraindevelopment hasbeenassociatedwiththehyperactivationofthehypothalamic pituitary-adrenal(HPA)axis(Valléeetal.,1997;Morley-Fletcher etal.,2003),thatproduceshighlevelsofcorticosterone(Takahashi etal.,1998;Williamsetal.,1999)andcatecholamines(Rohdeetal., 1989),capableofreachingthefetalbrainstructuresviamaternal circulationduringpregnancy.Moreover,thedensityoftheminer- alocorticoid(MR)andglucocorticoid(GR)corticosteroidreceptors arealarminglylowinhippocampiofprenatallystresseddescen- dants(Maccarietal.,1995;VanWaesetal.,2006),suggestinga molecularbackgroundtothebehavioralabnormalitiesreported.

In these developmental studies,prenatal stress was applied topregnantdamseither byrestraint stress(Zuena etal.,2008;

Morley-Fletcheretal.,2011;Lalouxetal.,2012),electricfootshocks (EstanislauandMorato,2005,2006),chronicvariablestress(Koenig etal.,2005;Leeetal.,2007)orimmobilizationstress(Abrahamyan etal.,2011;Chigretal.,2014).Thestressparadigmusedinthis studyhavebeenproposedasavalidatedanimalmodeltoassess theeffectsofgestationalstressontheoffspring.Sincetheelectric

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Fig.1.Schematicrepresentationofthestressprocedureofcontrolandprenatalstressdams.

shockswerenotphysicallywounding(0.5mA)somuchasirritat- ingforthepregnantfemalerats,thisanimalmodelmimicsstressful circumstancespregnantwomencanfaceonadailybasis.

Maternal stress alters the dopamine transmission in limbic structuressuchasthenucleusaccumbens (Thierryet al,.1976;

Rouge-Pontetal.,1998).Therefore,itissafetospeculatethatperi- natalmanipulationsmightheightentheresponsetodrugsofabuse andincreasethedrug-seekingbehaviorinrodents.Indeed,mater- naldistresswasfoundtoincreasevulnerabilitytobenzodiazepines (Lakehaylietal.,2015),amphetamine(Deminiereetal.,1992),alco- hol(Van Waesetal.,2011)andcocaine(Kippinetal.,2008)by elicitinganintensiﬁedaddictiontothesesubstance.Inthiswork, weevaluatedtheintensityofnicotinewithdrawalin prenatally stressedratsthroughanxietyassessmentsafternicotinecessation.

Theserotoninergicsystemisknowntoplayamajorroleinmood regulation,depression, impulsivenessand anxiety(Murphy and Pigott,1990;Miyagawaetal.,2011).Manyresearchershavealso reportedtheimportantrole of5HT_1A auto-receptorsin mediat- ingthisanxietysyndromeintheraphenuclei(Cheetaetal.,2000;

Kennyetal.,2001;Franklinetal.,2012).Consideringtheprevi- ouslyreportedeffectofmaternalstresson5HT_1Areceptorsinthe prefrontalcortexandhippocampus(Morley-Fletcheretal.,2004;

ZoharandWeinstock,2011;Zoharetal.,2014),wehypothesized thattheimpactofprenatalontheoffspringmightinvolvethese serotoninergicauto-receptors.

Thus,thepurposeofthisstudyisto(i)evaluatetheimpactof prenatalstressonrecognitionmemoryinadultratsinthenovel objecttest,(ii)assesstheintensityofthewithdrawalsymptoms inprenatallystressedratsincomparisonwithcontrolratsbythe waterconsumptionparadigmand(iii)measurethe5HT_1AmRNA expressionintheraphenucleiofadultoffspring.

2. Materialandmethods 2.1. Animals

Weusedeightyexperimentallynaïve adultmale andfemale Wistarrats(LaboratoryofPharmacologyCasablanca)inthebehav- ioral assessments and twenty-four adult rats for the qRTPCR technique.Allanimals(250–350g)werehousedfourpercagewith foodandwateradlibituminahousingroomkeptunderastabilized temperature:22±1^◦C(normal12hdark/lightphotoperiod,light:

8h00–20h00).

All procedures conducted were reviewed and approved by theEthicalCommitteeforbiomedical researchof theFaculty of MedicineandPharmacyofCasablanca(Comitéd’Éthiquepourla RechercheBiomédicaledeCasablanca:CERBC).Differentmeasures weretakentominimizepainanddiscomfortinaccordancewiththe

NationalInstitutesofHealthGuideforCareandUseofLaboratory Animals.

2.2. Drugtreatments

Nicotinehydrogentartrate(SigmaAldrich,Paris,France) was usedinthewaterconsumptiontestanddissolvedinphysiologi- calsaline(0.9%sodiumchloride).BothSalineandNicotinewere administeredsubcutaneously(SC)ataninjectionvolumeof1ml/kg ofbodyweight.

Thedoseofnicotineused(0.5mg/kg)waschosenaccordingto previousstudiesthatreporteditseffectivereinforcingproperties (Brielmaieretal.,2008;Thieletal.,2009).Thedrugdoserefersto thesaltformofnicotine.

2.3. Prenatalstressprocedure

Thestressprocedurewasperformedas previouslydescribed inourlaboratory(Lakehaylietal.,2015).Fourteenvirginfemale Wistarratsweretime-matedwithsexuallyexperiencedmalerats andisolatedinindividualcagesoncethepregnancywasconﬁrmed.

Duringthegestationperiod,thecageswereregularlycleanedand additionalbeddingwasprovidedforthedamstopreparefortheir delivery.Thepregnantdamswererandomlyassignedtotwogroups (n=7each)atday10ofpregnancy.Halfofthedamswerestressed dailywithelectricfootshocksduringtheirlast10gestationaldays whereastheotherhalfremainedunstressedintheircageswithin thehousingfacility.We chosetoapplythestressprocedureon damsontheirlast10daysofpregnancyinwhichtheneuraldevel- opmentofthefetusoccurinrats(Clancyetal.,2001).

ThepregnantdamsunderwentthefootshockstressinaPlex- iglasboxequippedwithagridﬂoor.Theyweresubjectedto80 electricshocksof0.5mAfor5seach,witha1–2minintervalon arandombasis.Eachstresssessionlasted100minandwascar- riedoutbetween8:00and16:00daily.AShockModulegenerator Q4 (modelLE2706)deliveredtheelectricshocksFig.1.

Thetwogroupsofdamswereleftundisturbedduringtheirdeliv- ery.Rightafterbirth,alllitterswereadjustedtothesamelittersize (8pupsperlitter).Attheageof21days,theoffspringwerehoused Q5 ingroupsoffourpercageandsortedaccordingtotheirlitterand gender.Toavoidlittereffects(HolsonandPearce,1992),nomore thantwoanimalspereachlitterwereusedineachtest.

2.4. Bodyweightsandlitterparameters

WecomparedthetwoControl(C:N=10)andPrenatallyStressed (PS:N=10)groups in litter size, male and female number and ratioandbodyweightsafterweaningattheageof21daysandin

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Fig.2.Schematicrepresentationoftheexperimentalprocedureinthewaterconsumptiontest.

adulthood(80days).Wedidn’tmeasurethemortalityratedueto itsrarity.

2.5. Behavioralassessments

Toavoidcontact-relatedstress,theratswereproperlyhandled (1–2minperrat)everydayfor7daysstraight.Then,theywere allocatedtospeciﬁcgroupsbasedontheirbodyweight.Theexper- imenterswereblindtotheorderandgroupassignmentsofthe rats.

Thedayoftheexperiment,theanimalswerebroughttoaprivate roomdesignedforbehavioralexperiments30minbeforetesting.

Whitenoisewasplayedinthebackgroundinorder tocoverall potentialsurroundingnoises.Thetestswereperformedduringthe lightphaseofthelight/darkcycle(from9:00amto4:00pm).All theoffspringweresubmittedtothebehavioralexperimentswhen theyreached80days.

2.6. Novelobjectrecognitiontest

Thistestwasperformedasdescribedelsewhere(Markhametal., 2010).Twentyadultratswereassignedtotwogroups(N=10each) inthisexperiment.Controland prenatallystressedgroupswere testedwhentheyreachedtheageof80days.Theaimofthisexper- imentwastoassesstheobjectrecognitionmemoryinadultrats.

Thetestwascarriedoutintheopenﬁeldapparatus(asquare 100×100cmblackwoodenarenasurroundedby40cmhighwalls) andlastedthreedayswiththreemainphases.Betweentrials,the objectswerecleanedwithanethanolsolution.

2.6.1. Thehabituationphase

Duringtheﬁrstday,theanimalwasgentlyplacedatthecenter oftheopenﬁeldapparatusandallowedtoexplorethetestingarea freely.Thisphaseiscrucialtofamiliarizetheanimalwiththearena and improveitsinteraction withtheobjectsduringthesecond phase.

2.6.2. Theacquisitionphase

Twenty-fourhours afterthe habituation, theanimal is once againplacedintheopenﬁeldarenawithtwoidenticalobjects(A1 andA2)for3min.TheLegotoyswerepositioned10cmfromthe walls.

Duringthisphase,thenumberofinvestigatoryboutswasmea- suredtogetherwiththeexplorationtimeoftheobject.

2.6.3. ThetrialPhase

Duringthethirddayoftheexperiment,prenatallystressed(PS) andcontrol(C)ratsunderwentthelast3-mintrial.Inthearena,

thesecondobjectA2wasexchangedwithanewobjectB.Ifthe ratexhibitsunalteredmemoryskills,itwillrecognizethefamiliar objectandwillspendmoretimeexploringthenewone.Here,we measuredtheRecognitionpercentage[(NovelObjectExploration time/Totaltime)×100].

ThenewobjectBisalsoalegotoydifferentinshapefromthe twootheridenticalobjectsA1andA2.

2.7. Waterconsumptiontest

Toassesstheeffectofprenatalstressonwithdrawalintensity inrats,weusedtheWaterconsumptiontestasdescribedinour Q6 laboratory(Tazietal.,1992;ElGanounietal.,2004).Anothersetof sixtymaleandfemaleratswereusedinthistest.

ThetestwascarriedoutinaPlexiglasYMazewiththreeidentical arms(50cmlong,15cmwideand35cmhigh).Twoofthosearms (Arm1and2)wereblackwhereasthethirdonewaswhiteand separatedfromtheothersbyaremovableguillotinedoor.Alight bulbwassuspended40cmabovethethirdarm.Duringtrials,a bottlewasplacedinthearm2or3dependingonthetestingphase.

Theratswerewaterdeprivedfor36hpriortotheexperiment.

2.7.1. Trainingphase

Theprenatallystressed(PS)andcontrol(C)treatedgroupswere dividedintotwogroupseachasrepresentedinFig.2.Inthepre- natallystressedrats,aPSgroupwastreatedwithsaline(PSVeh:

N=10)whileanotherreceivednicotine(PSNIC:N=20).Thesame treatmentswereadministratedtotheCgroupsaswell,yielding twoothergroups:CVeh(N=10)andCNIC(N=20).

For7consecutivedays,those4groupsweredailysubjectedto 10-minsessionsintheYMazerightaftertheirs.cinjectionofNico- tineorsaline.Accesstothethirdwhitearmwasblockedbythe guillotinedoorandthewaterbottlewasplacedinthesecondblack arm.Theratswereallowedtodrinkwaterinthisarmduringthe trials.Afterwards,wemeasuredthewaterintakebyweighingthe bottlebeforeandafterthesessions.

2.7.2. Testsession

Onthetestday(day8),ratswererandomizedbytheirwater intakescoresandassignedtosixgroupsaccordingly.ThetwoNIC groups(PSNICandCNIC)weredividedtotwogroupseachduring thissession:PSNICreceivedeitherasalineinjectionorcontinuedto betreatedwithNICwhileCNICwerealsodividedintotwogroups.

Thisresultedinthefollowingfourgroups:PSNIC/VehPSNIC/NIC, CNIC/NICandCNIC/Veh.

Duringthetesttrial,theguillotinedoorwasremovedtoallow freeaccesstothewhitebrightlylitthirdarminwhichthewater bottlewasﬁxed.Normally,theanimalwouldhesitatebetweenhis

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naturalwater-seekingbehaviorandhisfearofunfamiliarwell-lit spaces.Thepurposewastohighlightthereboundofanxietyasso- ciatedwithnicotinewithdrawalsyndromeasreportedpreviously (Irvineetal.,2001).Theanxiety-likebehaviorherewasexpressed bythedecreaseinwaterconsumption.

2.8. mRNAstudies

Anothersetofratswereusedforthismethod.Control(C)and prenatallystressed(PS)offspringweredistributedintofourmain groups:

-CVeh:acontrolgroupthatreceived4dailys.c.injectionsofNaCl 9%

-CNIC:acontrolgroupthatreceived4dailys.c.injectionsofnico- tine(0.5mg/kg)

-PSVeh:aprenatallystressedgroupthatreceived4dailys.c.injec- tionsofNaCl9%

-PSNIC:aprenatallystressedgroupthatreceived4dailys.c.injec- tionsofnicotine(0.5mg/kg)

2.9. Tissuecollection

Animalswereeuthanizedatadulthood(80days).Brainswere quicklyremovedandraphenucleiwereisolated,frozenondryice andkeptat−80^◦CuntilthemRNAextractionprocedure.

2.10. TotalRNAisolationandreversetranscriptionPCR(RTPCR)

Using Trizol (Invitrogen),total RNA wasextracted from the frozentissuesamplesasdescribedbythemanufacturer.Then,RNA concentrationandqualityweremeasuredusingtheNanoVue^TM PlusSpectrophotometer(GEHealthcare,UK).

ThecDNAwereobtainedbyreversetranscriptionfrom2␮gof RNAusing1␮lrandomExamer(Invitrogen),1␮lofRTSuperscript at200U/␮l(Invitrogen),10nMdNTP(Invitrogen),and4␮lofM- MLVreversetranscriptase(Invitrogen).

2.11. QuantitativeRealtimePCR(qRTPCR)

Inaﬁnalvolumeof25␮l,2␮lperwellofcDNA(50ng)was usedalongwith:5×reactionbuffer,10␮Mprimers1.5mMMgCl2, 0.25UTaqpolymerase(Bioline,London,UK).Thethermalcycling wasperformed:35 cyclesofDNAdenaturation(5minat95^◦C), primerhybridization(30sat95^◦C),andelongation(30sat72^◦C) followed bya ﬁnal elongation step(7min at72^◦C). Separation byelectrophoresison2%agarosegelwasperformedandvisual- izedusingEthidiumBromidestraining.Sampleswereanalyzedin triplicatestomakesureproductsfromallPCRrunswerecross- comparable.

Real time PCR Applied Biosystem FAST 7500 apparatus and SYBR Green were used according to the manufacturer’s pro- cedure. To determine fold changes, we usethe DCT method (Livak and Schmittgen, 2001).The primers sequences were as

Table1

Litterparameters(mean±S.E.M.)analyzedincontrolandprenatallystressedlitters.

Parameter Controlrats Prenatalstressrats Statistics^a

Littersize 10.4±0.2 9.6±1.3 p=0.62

Numberofmalesperlitter 4.6±0.6 4.4±0.2 p=0.41 Numberoffemalesperlitter 5.4±0.5 4.8±0.7 p=0.85 Male:femaleratio 1.19±0.3 1.38±0.2 p=0.40 Bodyweight(atWeaning) 54.490±3.2 38.490±5.1 p=0.83 Bodyweight(atAdulthood) 358.340±15263.970±18**(p<0.01)p=0.002

aStudentt-test,non-pairedsamples(pvalues).

Fig.3.NovelObjetRecognitionTest.(A)Mean(±SEM)numberofinvestigatory bouts:Intheacquisitionphase,therewasanosignificantdifferencebetweentheC andthePSgroup.(B)Time(±SEM)spentinvestigating:TheCgroupspentlesstime investigatingtheobjectsduringtheacquisitionphase,butnostatisticallysignificant changewasnoted.(C)Percentage(±SEM)ofRecognition:Asignificantdifference wasnoticedbetweenthePSandCgroupat*p<0.05.

followed:5-HT1Areceptorforward,5-CGTGCACCATCAGCAAGGA- 3; 5-HT1Areceptor reverse,5-CTGAAGATGCGCCCGTAGAGA-3; Hprt forward, 5-GACCGGTTCTGTCATGTCG-3; Hprt reverse, 5- ACCTGGTTCATCATCACTAATCAC-3.

2.12. Statisticalanalysis

Whenexaminingthestatisticalsigniﬁcanceofthebodyweights, litterparametersanddiscriminationmeasuresinthenovelobject recognitiontest,weusedtheStudentt-testfornon-pairedsamples tocomparethegroups.

Inthewaterconsumptiontest,resultswereexploredbytwo- wayANOVAs.Theposthoccomparisonwasperformedusingthe Newman–Keulstestwiththelevelofsigniﬁcancesetatp<0.05.All statisticalanalyseswereperformedusingtheSPSS15.0software package.

TheAnalysisofvarianceANOVAwasalsousedtoexaminethe resultsof theqRTPCR technique.Thevariableswerethegroups

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(ControlvsPS)anddrugtreatments(salinevsnicotine).Thestu- dent’st-testwasperformedafterwardsforpairwisecomparisons.

Thesigniﬁcancelevelwassetatp<0.05.TheSPSS15.0software packagewasusedtoperformthestatisticalanalysis.

3. Results

3.1. Bodyweightsandlitterparameters

AsshowninTable1,nostatisticallysigniﬁcantdifferencewas foundinweightsatweaningbetweentheprenatallystressedrats andthecontrolgroup.Incontrast,PSratswerelighterthantheC groupwhentheyreachedadulthood(**p<0.01).

TheStudentt-test(non-pairedsamples)failedtoﬁndasignif- icantdifferenceinlittersize,numberofmalesandfemales and Male:Femaleratiointhetwogroups.

3.2. Novelobjectrecognition

Nosigniﬁcantdifferencewasfoundintheinvestigatorybouts and time spentinvestigating theobjectsduring theacquisition test.However,therecognitionpercentagewassigniﬁcantlylower intheprenatallystressedgroupswhencomparedtothecontrol rats(Fig.3C:T₍₁₈₎=16.147;p<0.05).

3.3. Waterconsumptiontest

TheFig.4depictstheanxietylevelsofratsintheYMazetest.The ANOVAtestshowedsigniﬁcantdifferencebetweentheCVeh/Veh and theCNIC/NIC groups (F_(5,54)=17.948; p<0.001; posthocC NIC/NICp<0.01).Similarly,theCNIC/Vehgroupexhibitedalower waterintakethantheC Veh/Vehgroupaswell(F_(5,54)=17.948;

p<0.001;posthocCNIC/Vehp<0.001).

Intheprenatallystressedgroups,therewasastatisticallysigniﬁ- cantreducedwaterdrinkinginthePSNIC/Vehgroupincomparison withthePS Veh/Vehrats (F_(5,54)=17.948; p<0.001;post hocC NIC/Veh p<0.001). Similarly, the PS NIC/NIC group drank less thanthePSVeh/Veh(F_(5,54)=17.948;p<0.001;posthocCNIC/NIC p<0.01).

A signiﬁcant difference was also found between the two Veh/Vehgroups (F_(5,54)=17.948; p<0.001; posthocPSVeh/Veh p<0.05). Moreover, the PS NIC/Veh presented a pronounced decrease of water consumption than the C NIC/Veh groups (F_(5,54)=17.948;p<0.001;posthocPSNIC/Vehp<0.01).

Fig.4.WaterConsumptionTest.Mean(±SEM)waterintakeinoffspring.CVeh/Veh groupexhibitedahigherwaterconsumptioncomparedtoCNIC/NIC(**p<0.01),C NIC/Veh(***p<0.001)andPSVeh/Vehgroups(*p<0.05).ThePSNIC/Vehpresented lesswaterintakevaluescomparedtothePSNIC/NIC(=/ =/p<0.01),PSVeh/Veh (=/ =/ =/p<0.001)andCNIC/Veh(++p<0.01).

.0 .5 1.0 1.5 2.0 2.5 3.0 3.5

C Veh PS Veh C NIC PS NIC

5HT1A Relave Fold Change

+

≠

***

Fig.5.QuantitativeRealTimePCR.Dataareexpressedasmean±SEMof5HT1A mRNAexpressionintheraphenuclei.Thevehicletreatedprenatallystressedgroup (SPPLB)exhibitedasigniﬁcantlylowerlevelof5HT1Areceptorswhencompared withthevehicletreatedControlat***p<0.001.Nicotinedecreased5HT1Aexpres- sionintheraphenucleiofthecontroloffspringincomparisonwiththevehicle treatedcontrolgroupat+p<0.05,andasigniﬁcantlyhigherlevelsofthissametype ofreceptorswhencomparedwiththenicotine-treatedprenatallystressedgroupat

=/ p<0.05.

3.4. QuantitativerealtimePCR

Asseen in Fig. 5, prenatalstress signiﬁcantly decreased the 5HT1A mRNA expression in the raphe nuclei (F_(3,20)=8.514;

p=0.001;posthocPSVehp<0.001).Nicotinehasalsoinﬂuenced thesereceptors’levelsincontrolandprenatallystressedoffspring (F_(3,20)=8.514;p=0.001;PosthocCNICp<0.05;PSNICp<0.05).

4. Discussion

Inthisstudy,weevaluatedtheimpactofprenatalexposureon recognitionmemoryinadultratsusingtheNovelObjectrecog- nitionparadigm.Theresultsshowednosignificantdifferencein investigatoryboutsandtimeinprenatallystressedratscompared totheircontrols.Yettherecognitionpercentageshowedthatthe PSgroupdidnotidentifythefamiliarobjectduringthetrialsession sincetheytookmoretimeexploringitcomparedtothecontrol group. These data corroborate withearlier findings stating the strongnegativeinfluenceofearlyadverseeventsonmemoryin rodents(Szuranetal.,1994;Kapooretal.,2009;Markhametal., 2010).Tothis dayhowever,nostudyhasassessed theeffectof electricfootshockonmemoryinrats.

Giventheinvolvementofthehippocampusinthemechanism underlyingdiscriminationmemory(Steckleret al.,1998), these deficienciesmightbecausedbytheoffspring’searlycumulative exposuretoglucocorticoidsinutero(Williamsetal.,1999),induc- ingseriousdamagestothatspecificstructure,asfirstdescribedin the‘glucocorticoidcascadehypothesis’bySapolskyetal.(1986).

Besides,prenatally stressedoffspring displayeda reduced sero- tonin, noradrenaline and dopamine levels and turnover during adulthood (Peters, 1982; Takahashiet al., 1992; Hayashiet al., 1998).Thismaysuggestthattheneurotransmittersystemsmediate theinﬂuenceofperinatalmanipulationsoncognitivefunctions.

Furthermore,thecurrentstudyshowedforthefirsttimethat prenatalstressinducesaninterestinganxietyreboundfollowing nicotinewithdrawalbythewater consumptiontest.Theresults confirmedtheanxiogeniceffectofnicotinechronicexposure(File etal.,1998;Irvineetal.,1999)sincethenicotinegroups(CNIC/NIC andSPNIC/NIC)dranklesswatercomparedtotheVehiclegroups (CVeh/VehandSPVeh/Veh).Moreover,wenoticedthatthewith- drawnrats(CNIC/VehandPSNIC/Veh)weresignificantlyreluctant tovisitthebrightlylitthirdarmintheYMazewhencompared totheirrespectiveVehiclegroups (CVeh/VehandPSVeh/Veh), reflecting a withdrawal-induced anxietyoutburst in these animals.Thesedataarein agreementwithpreviousstudies(Irvine

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etal.,2001;Jonkmanetal.,2005)reportingthesameanxiety-like behaviorafternicotinecessation.Asinalldrugsofabuse,nicotine abruptinterruptiondrasticallyreducesthedopaminereleaseand increasesitsuptake,triggeringaseverehypodopaminergicstate (Hildebrand etal.,1998;Ducheminetal.,2009).Thisraisesthe dopamineoutputintheprefrontalcortex (Carbonietal.,2000), leadingtotheanxiogeniceffectobservedintheYMazeapparatus (Broersenetal.,2000).

However,themoststrikingresult inthewaterconsumption testwastheeffectoftheprenatalonthenicotinewithdrawalpro- cessinrats,greatlyconveyedbythesigniﬁcantdecreaseofwater intakeinthePSNIC/VehgroupincomparisonwiththeCNIC/Veh rats.Indeed,theprenatallystressedwithdrawnanimalsexhibited aheightenedanxiogenicresponsetonicotinewithdrawalthanthe controlwithdrawngroup.Thecauseofthisintensiﬁedwithdrawal symptomisstillunknown.Itcanbeexplainedbythedepletionof dopamineinducedbyprenatalstressinrats(Alonsoetal.,1994), renderingthePSoffspring vulnerabletoaddiction,andhelpless againstthewithdrawalprocess.

Additionalnon-behavioralobservationswerereportedinthis study.Weassessedthelittersizeandbodyweightofthedescen- dantsinthe21stand80thpostnatalday.Nodifferencewasfound betweentheCandPSlittersinsize,numberofmales,femalesand male:femaleratio.However,theAdultprenatallystressedoffspring (80days)werelighterthantheircontrolcounterpartsasreportedin pastresearches(Dragoetal.,1999;EstanislauandMorato,2006) whileotherauthorsfoundconﬂictingresults(Wardetal.,2000;

Hougaardetal.,2005).Thiscanbeduetothedifferencesindietor theperiodschosenforbodyweightmeasurements.

Onamolecularlevel,ourresultsshowedthatprenatalstress alteredthe5HT1Areceptors’mRNAexpressionintheraphenuclei oftheadultoffspring.Indeed,theratsstressedinuteroexhibited signiﬁcantlylowerlevelsofthesereceptorsincomparisonwiththe controlgroups.

Previousstudiesassociatingprenatalstresswith5HT_1Afound anupregulationofthistypeofreceptorsintheprefrontalcortex (Morley-Fletcheret al.,2004)whileothersreporteda reduction in5HT1Alevelsinthehippocampus(VandenHoveetal.,2006) andintheGABAergicneuronsoftheprefrontalcortex(Zoharand Weinstock,2011;Zoharetal.,2014).Thesediscrepanciesmaybe attributedtodifferenttypesofprenatalstressapplied,thelocation ofthe5HT_1Areceptorsevaluated andwhethertheywerepost- Q7

synapticorauto-receptors(Fletcheretal.,2008;Mülleretal.,2007).

InthesamemRNAmeasurements,weobservedasigniﬁcant nicotine-induceddownregulationofthe5HT_1Aauto-receptorsin theraphenucleiinthecontrolgroup(CNIC)andtheprenatally stressedgroup(PSNIC)whencomparedwiththeirvehicle-treated counterparts.Kennyetal.(2001)reportedthatchronicnicotinehad thesameeffecton5HT_1Areceptorsintheprefrontalcortexatthe samedose(0.5mg/kg),andahighersensitivityinthedorsalraphe nucleuswasstatedelsewhere(Rasmussenetal.,1997).Besides, theenhancementofauditorystartleresponseelicitedbynicotine withdrawalwasinhibitedby5HT_1Aantagonists(Rasmussenetal., 1997,2000).

Takenaswhole,thisstudydemonstratedthattheprenatalstress affectsthecognitivefunctionsespeciallytherecognitionanddis- criminationmemoryin ratsand induceda heightenednicotine withdrawalsyndromeinprenatallystressedrats.Besides,theqPCR showedthatperinatalmanipulationselicitedmolecularpermanent changesinthe5HT1AmRNAexpressionintheraphenucleiofthe prenatallystressedanimals.Thatreﬂectstheneurologicalabnor- malitiesinkeybrainstructuressuchastheraphenuclei.

Thesefindingsareverycrucialtofurtherunderstandthepre- natalstressandpossiblyfindsuccessfultherapies.Nevertheless, furthermolecularstudiesoughttobecarriedouttoconfirmour

resultsatamolecularor/andcellularlevelandopennewinteresting researchperspectives.

Uncitedreferences Q8

Citóetal.(2012),Jonkmanetal.(2008),Kabbajetal.(1994), Tzavaraetal.(2002)andWeinstocketal.(1992).

Acknowledgment

WedeeplyacknowledgetheparticipationofPrChigrFatiha,Pr NajimiMohamed,PrBattasOmar,PrTahiriJoutiNadiaandtheir preciouscollaborationinthis study.Theexperiments described herein werepartially funded by the Association for the Devel- Q9 opment of Scientiﬁc Research (Association Mostaqbal pour le DéveloppementdelaRechercheScientiﬁque:AMDRS).

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