Neonatal handling alters the structure of maternal behavior and affects mother-pup bonding

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ContentslistsavailableatScienceDirect

Behavioural

Brain

Research

j o ur na l h o me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / b b r

Research

report

Neonatal

handling

alters

the

structure

of

maternal

behavior

and

affects

mother–pup

bonding

A.R.

Reis

a,∗

_,

_M.S.

_de

_Azevedo

a

_,

_M.A.

_de

_Souza

a

_,

_M.L.

_Lutz

a

_,

_M.B.

_Alves

a

_,

_I.

_Izquierdo

b

_,

M. Cammarota

c

_,

_P.P.

_Silveira

d

_,

_A.B.

_Lucion

a

a_Departamento_de_Fisiologia,_Instituto_de_Ciências_Básicas_da_Saúde,_Programa_de_{Pós-graduac¸}_ão_em_{Neurociências,}_Universidade_Federal_do_Rio_Grande_do

Sul(UFRGS),SarmentoLeite,500,PortoAlegre,RS,CEP90050-170,Brazil

b_Centro_de_Memória,_Instituto_do_Cérebro,_Pontifícia_Universidade_Católica_do_Rio_Grande_do_Sul_(PUCRS),_Porto_Alegre,_RS,_CEP_90610-000,_Brazil c_Instituto_do_Cérebro,_Universidade_Federal_do_Rio_Grande_do_Norte_(UFRN),_Avenida_Nascimento_de_Castro,_2155,_Natal,_RN,_CEP_59056-450,_Brazil d_Faculdade_de_Medicina,_Universidade_Federal_do_Rio_Grande_do_Sul_(UFRGS),_Ramiro_Barcelos,_2350,_Porto_Alegre,_RS,_CEP_90035-003,_Brazil

h

i

g

h

l

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g

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t

s

•Neonatalhandlingaffectsmaternalcareandaltersmother–pupsrelationship.

•Handlingdesynchronizesmother–pupinteractionsbychangingmaternalbehaviorsequence. •Neonatalhandlinginducessex-dependentchangesinthenestodorpreference.

•HandlingaffectsCREBandBDNFlevelsinpup’solfactorybulb,inasex-speciﬁcmanner. •Resultssuggestadifferentialolfactorylearningandpreferencefornestodorinpups.

a

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t

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c

l

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n

f

o

Articlehistory: Received2October2013

Receivedinrevisedform17February2014 Accepted21February2014

Availableonline2March2014 Keywords: Neonatalintervention Neurotrophicsignaling CREB Attachment Olfactorylearning

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Duringearlylife,amotherandherpupsestablishaverycloserelationship,andtheolfactorylearning ofthenestodorisveryimportantforthebondformation.Theolfactorybulb(OB)isastructurethat playsafundamentalroleintheolfactorylearning(OL)mechanismthatalsoinvolvesmaternalbehavior (lickingandcontact).Wehypothesizedthathandlingthepupswouldalterthestructureofthematernal behavior,affectOL,andaltermother–puprelationships.Moreover,changesinthecyclicAMP-response elementbindingproteinphosphorylation(CREB)andneurotrophicfactorscouldbeapartofthe mecha-nismofthesechanges.Thisstudyaimedtoanalyzetheeffectsofneonatalhandling,1minperdayfrom postpartumday1to10(PPD1toPPD10),onthematernalbehaviorandpups’preferenceforthenest odorinaYmaze(PPD11).WealsotestedCREB’sphosphorylationandBDNFsignalingintheOBofthe pups(PPD7)byWesternblotanalysis.Theresultsshowedthathandlingaltersmother–pupsinteraction bydecreasingmother–pupscontactandchangingthetemporalpatternofallcomponentsofthe mater-nalbehaviorespeciallythedailylickingandnest-building.Wefoundsex-dependentchangesinthenest odorpreference,CREBandBDNFlevelsinpupsOB.Malepupsweremoreaffectedbyalterationsinthe lickingpattern,andfemalepupsweremoreaffectedbychangesinthemother–pupcontact(thetime spentoutsidethenestandnursing).

1. Introduction

Inmammals,theimpactofleaving thesafeuterine environ-mentandfacingmanyunfamiliarenvironmentalstimuliandrisks requirestheprotectionofacaregiver.Duringtheneonatalperiod,

∗ Correspondingauthor.Tel.:+5505133083578;fax:+5505133083092. E-mailaddress:bioadolfo@gmail.com(A.R.Reis).

themotherisaninterfacebetweenthenewlybornmammaland theenvironmentandcanpowerfullyshapeinfantdevelopment[1]. Earlylifeisacriticalphaseforthenervoussystem,whenthebrain undergoesfunctionalorganization,neuronalproliferation, migra-tionanddifferentiation,gliogenesisandmyelination[2].Morethan 50 yearsof studyhave exploredthe implicationsofchanges in maternalbehavior onneonatal programmingand its persistent consequencesonbehavioralandneurochemicaloutcomeslaterin life[3–12].

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A.R.Reisetal./BehaviouralBrainResearch265(2014)216–228 217 During early life, mother and infant establish a very close

relationship.Auditoryandvisualsensorysystemsareheavily impli-cated in this process but during theneonatal period, olfactory learningisa keyfactorfor theattachmentestablishment, espe-ciallyinsmall-brainmammalssuchasrodents[13–19].Inrats,the pupslearnhowtoidentifythemotherthroughaprocesssimilar totheclassicparadigmofconditioning,involvingtactile stimula-tionfromthedam(unconditionedstimulus)andthedam’sodor (conditionedstimulus)[16,17,19–21].Theolfactorybulb(OB)and thelocuscoeruleus(LC)areimportantstructuresintheolfactory learningmechanism[14,22–24],enablingtheratpup,borndeaf andblind,todirectitsbehavior towardthemother[23].Tactile stimulationcanactivatepups’LC,whichincreasesNoradrenaline (NA)intheolfactorybulbmitralcells[22].Thisassociationactivates achainofeventsinthemitralneuronsofthepup’sOB increas-ingthephosphorylation ofCREB(cyclicAMP-response element bindingprotein),whichisresponsibleforthetranscriptionofa vari-etyoffactors(includingbrainderivedneurotrophicfactor[BDNF]) thatleadtobiochemicaland morphologicalchanges inmemory formation[14,25–29].BDNFappearstobeakeyfactor in olfac-toryassociationlearning[30].BDNFgeneexpressionincreasesin responsetoseveralstimuli,includingneurotransmitterssignaling andCREBphosphorylation[31];forareview,see[32]andiscritical intheOBmorphologicdevelopment[33–38].

Neonatalhandlingisanexperimentalprocedurethatinvolves briefmaternalseparationandtactilestimulation,whichis exten-sivelyusedtoinvestigatetheeffectsofearlylifeinterventionson behavioralandendocrinealterations.Thisrepeateddisruptionin themother–pup relationship reducesfear [39], altersHPA axis (Hypothalamic-Pituitary-Adrenal)responsetoavarietyof stress-ors[40–44]andmayalsoaffectsocialbehaviorsandfertilityinboth maleandfemalerats[45–48].Inadditiontothebehavioraland neu-roendocrineaspectsofthesechanges,neonatalhandlingaltersthe brainplasticityandneurotrophicsignaling,thusproducing long-lastingstructuralchanges[49–51].

Previousstudiesfromourlaboratoryhaveshownthatthe han-dlingprocedurereducesthepup’spreferenceforthenestodorina sexdependway[45,48].Thislackofpreferencemaybeduetoan alterationintheolfactorylearningmechanisms;changesintheNA activityandCREB’sphosphorylationintheOBof7-day-oldratpups suggestthatthishypothesiscouldberight[48].BDNFistheperfect candidatetotestthishypothesissinceitisimplicatedinplasticity, dendriticbranching,neuronalsurvival,migrationand differentia-tionandaxonalcompetitioninthisareaduringtheneonatalperiod [33–38].

Otherstudiesusingearlyhandlingshowthatneurotrophic fac-torslikeBDNFplayakeyroleintheestablishmentofthesechanges andalsopointoutsexdependentchanges[52–54].However,isstill notclearifthehandlingprocedurecouldaffecttheneurotrophic signalingintheolfactorybulbofratpupsandifthesechangescould alsopresentasexdependentpatternlikeinotherbrainareas.

Therefore,ourhypothesisisthatneonatalhandlingaltersthe dailypatternofmaternalbehaviorcomponentsbeyondthe lick-ingcomponent,andthatthesechangesarepartofthemechanism thataltersBDNFgeneexpressionthroughsex-dependent modiﬁca-tionsinCREBphosphorylationandproductioninthepup’sOB.For that,weanalyzedtheeffectsofhandlingonCREBphosphorylation andBDNFlevelsintheolfactorybulbof7-day-oldratpupsto ver-ifywhetherthealterationsinCREBphosphorylationaretransient oraretranslatedintoproteinalterations(changesinBDNFlevels), whichwouldindicateamoreprolongedeffect.Finally,expecting toassociatethechangesinmaternalbehaviorwiththepups’social behavior,wealsoanalyzedthepups’socialbehaviorwiththenest odorpreferencetestonPPD11toconﬁrmwhetherthe biochem-icalalterationsintheolfactorybulbwouldaffectsocialbehavior alreadyinearlylife.

2. Experimentalprocedures

2.1. Animals

PregnantfemaleWistarratswerebroughtfromthecolonyof theFederalUniversityofRioGrandedoSul(PortoAlegre,Brazil) totheanimalroominourlaboratory.Approximately7daysbefore delivery,thefemaleswerehousedindividually,andthepresenceof thepupswascheckedtwicedaily.Birthwasconsideredtobeday0, andonpostpartumday1(PPD1),thenumberofpupswasculledto 8perdambyrandomlyremovingafewpupswhileensuring min-imalcontactwiththeremainingrats,thesexofthepupswerenot consideredinthisprocedure.Alloftheanimalsweremaintained ona12-hlight/darkcyclewiththelightsonat6a.m.Theroom tem-peraturewas22±1◦C,andwaterandfood(Rodentchow,Nutrilab, Colombo,Brazil)wereavailableatalltimes.Cagebeddingwasnot changedfromPPD0to10.Theexperimentswereperformedin accordancewiththeNationalInstitutesofHealth(NIH)and Colé-gioBrasileirodeExperimentac¸ãoAnimal(COBEA)guidelines.These guidelinesweredesignedtominimizethediscomfortofanimals andwereapprovedbytheEthicsinResearchCommitteeofFederal UniversityofRioGrandedoSul(ProcessCEP/UFRGSnos.2007937 and19759)andfollowedBrazilianlegislation.

2.2. Neonatalhandling

Pupswerehandledfor1minperdayfromPPD1toPPD10for behaviouralstudieswhileforWesternblotanalysisthisprocedure lasteduntilPPD7.First,thehomecagecontainingthemotherand thelitterwasmovedtoaquietroomnexttotheanimalfacilityand weregiventhesamelightperiodandtemperatureasdescribed above.Then,themotherwasremovedfromthehomecageand placedintoanothercage.Theexperimentergentlyhandledallof thepupsatthesametimeusingbothhands,coveredwithﬁnelatex gloves,for60s.Noapparentharmwasinﬂictedtothepups;they weresimplytouched.Afterhandling,allofthepupsweretakento thenestatthesametime,andthemotherwasplacedbackinside thehomecage.Thehomecagewasthenreturnedtotheanimal facilityroomandleftundisturbeduntilthesametimethenextday. Thepupswerehandledduringthelightperiodofthedaily photope-riod cycle(10:00–12:00)at adistanceofapproximately100cm fromthemother.Thetotaltimeofthemother–infantseparation wasapproximately90s[39,45–48,50].

2.3. Experimentsandgroups

Intheﬁrstexperiment,weanalyzedtheeffectofneonatal han-dling onmaternal behavior. Litters weredivided into2 groups basedonthehandlingprocedure:thenon-handledgroupor con-trolgroup(NH,n=9),inwhichthepupswereleftundisturbedwith theirmotherduringtheﬁrst10postnatal days,andthe repeat-edlyhandledgroup(H,n=9),inwhichthepupswerehandledas describedabovefromPPD1to10.

Inthesecondexperiment,thelittersthatoriginatedfromthe experimentone(NH,n=9;H,n=9)wereusedfortheodorofthe nestpreferencetesttoanalyzethesocialbehaviorofthepupson thePPD11.

Inthethirdexperiment,themolecularmechanism intheOB related to maternal behavior wasanalyzed onPPD 7. Previous studiesshowedchanges inthemonoaminergic systemafterthe handlingprocedureonthatday[48].Atotalof48pups(24males and24females)fromthetwoexperimentalgroupsdescribedabove (NH;n=12fromeachsexandH;n=12fromeachsex)weredivided into4subgroupsbasedonthetimeoftissuecollectionforWestern blotanalysis(n=6ofeachsexinallgroups).Twosamplesoftissue

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wereobtainedinbothgroups,30and120minafterthehandling procedure.

2.4. Experiment1:maternalbehavior

Fromthe1sttothe10thPPD,thematernalbehaviorwasscored dailyusingaprocedureadapted frompreviousstudies[55–59]. Four72-minrecordingsessionswereconducted.Therecording ses-sionsoccurredatregulartimeswith3periodsduringthelightphase (9:00,12:00and15:00)and1periodduringthedarkphase(18:00) ofthelight-darkcycle.Withineachobservationsession,the behav-iorofthemotheratthatspeciﬁcmomentwasscoredevery1min and30s.Thus,wehad50observationspersessionin4periodsper day,renderingatotalof200observationspermotherperday. Usu-ally,onlyonebehaviorwasrecordedineachobservationsample; however,2simultaneousbehaviors(forexample,nursingand lick-ing)werealsorecorded.Thedatawereexpressedasthenumberof observationsinwhichthepupsreceivedthetargetmaternal behav-iorduringthe10postpartumdays(thetotalnumberofeventswas 2000).

Thetargetmaternalbehaviorswereasfollows:(1)lickingany pup(thebodysurfaceand/oranogenitalregion),(2)nestbuilding, (3)goingoutsideofthenest,(4)carryingpups(retrievingthepups andplacingtheminthenest),and(5)nursingpups(ineithera higharched-backpostureoralowarched-backposture,inwhich themotherlaysoverthepups,orapassiveposture,inwhichthe motherislyingeitheronherbackorsidewhilethepupssuckle).A detaileddescriptionofthebehaviorsisprovidedinMyers,Brunelli [60]andChampagne,Francis[56].

2.5. Experiment2:nestodorpreferencetest

Thesocialbehaviorofthepupswastestedinthe11thPPDwith aprotocoladaptedfrom[45,48].Briefly,thematernalodor pref-erencetestwasa two-odorchoicebetweenareaswiththenest orfreshbedding.AY-maze(neutralarm15cm×10cm,testarms 20cm×10cm)wasusedtoperformthistest.Ineacharea,300mL offreshornestbeddingwereplacedinadjacentarms.AtPPD11, apupwasremovedfromthenestandplacedintheneutralarmof theY-maze.Allofthesessionswerevideotapedandfurther ana-lyzedbyaresearcherwhowasblindtotheexperimentalgroups. Duringthe1-mintrial,theamountoftimethatthepup(whole body)spentover each of thetwo armswas recorded.We also recordedthetimethatwasspentinreachingthenestarea. Ani-malsweretestedfor5trials,withaninter-trialintervalof2min, duringwhichthemazewascleanedwith70%ethanoltoremove traceodors.Weusedonemaleandonefemalepupfromeach lit-ter,andtheorderofthetestwascounterbalancedacrosslittersto preventanyeffectfromthetestingorder.Ineachtrial,thenestor freshbeddingwasswitchedbetweenthedifferentarmsofthe Y-maze.ThevideorecordingswerelateranalyzedusingtheNoldus Observersoftware(NoldusInformationTechnology,Wageningen, Netherlands).Weanalyzedthetimeineachareaineachtrial,the percentageoftotaltimeovertheareasandthelatencytoreachthe nestarea.Thetotaltimeineachareawasobtainedbycalculating thesumofthefivetrials,andthedatawereexpressedasthe per-centageoftimethattheanimalspentoverthebeddingareas(fresh orthenest).Thelatencytoreachthenestareawasobtainedby calculatingthemeanofthefivetrials,andthedatawereexpressed inseconds.

2.6. Experiment3:Westernblotanalysis

ThelevelsofBDNF,CREBandpCREBwereanalyzedonPND7 (postnatalday7),which istheoptimaldayfor expressingodor preferencelearningforthematernalodor[25,26,61]byWestern

blotanalysis,asdescribedbyRaineki,DeSouza[48].Brieﬂy,pups wererapidlydecapitatedimmediatelyaftertakingthemfromthe homecage.TheOBswereremovedandimmediatelyfrozenina containerwithisopentaneindryiceandstoredat−70◦_C._They

werehomogenizedusing500␮Lofhomogenizationbuffer(20mM Tris–HCl, pH 7.4 containing 1mM sodium orthovandate, 1mM EDTA,1mMEGTA,50mMNaFand1mMPMSF).Proteincontent wasdetermined using the Bradford (1976) method.Equivalent amountsofprotein(20_␮gforeachsample)wereloadedintoeach lane.Proteinswereseparatedonthebasisofgelmobilityusing SDS-PAGE; theywere thenelectrotransferred topolyvinylidene diﬂuoridemembrane(PVDFmembrane;Immobilon-P,Millipore, Billerica,USA).ThemembraneswereimmersedinPonceauS solu-tiontodeterminewhetherequalamountsofproteinwereloaded; theywerethenwashedwithwater.Themembraneswere incu-bated in Tween–Tris buffersaline(TTBS; 100mM Tris–HCl,pH 7.5, containing 0.9% NaCl and 0.1% Tween 20) that contained 5% albumin for 2h at room temperature to block nonspeciﬁc binding.Afterward, the membraneswere rinsed fourtimes for 15min in TTBS, followed by incubation overnight ona shaker at 4◦C in the presence of primary antibodies that recognize the following antigens: BDNF (1:5000, Santa Cruz Biotechnol-ogy,SantaCruz,USA),CREB(1:5000,NewEnglandBioLabs,USA) andpCREB(1:4000,Sigma–Aldrich,USA).Theblotswererinsed four times for 15min in TTBS, and they were then incubated for2hwiththesecondaryantibody,goatanti-rabbitIgG conju-gatedwithhorseradishperoxidase(SantaCruzBiotechnology),at a1:80,000(BDNF)or1:50,000(CREBandpCREB)dilutioninTTBS. Themembraneswerethenrinsedfourtimesfor15mininTTBS. Immunoreactivity was detected using the West-Pico enhanced chemiluminescencekit (Pierce, IL,USA). Densitometric analysis wasconductedusinganImageQuantRT-ECLsystem(GE, Piscat-away,NJ,USA).

2.7. Statisticalanalysis

Weusedtimeseriesdecompositiontoaccesstrendsand sea-sonal patterns in thecomponents of the maternal behavior of the two groups (NH and H) in all observation sessions in the ﬁrst10PPDs.Toaccesstrendinthecomponentsofthematernal behaviorweusedDickey–FullerTestandwereconsidered signif-icantwhen p>0.05. ToaccessseasonalpatternsweusedFisher GTestandwereconsideredsigniﬁcantwhenp<0.05 (Fig.1A–D ).

Thenumberofeventsofeachcomponentofthematernal behav-iorwasexpressedasthemean(±SEM).Weanalyzedthematernal behavioracrossthe4daily observationsessions(sumofthe10 days in each daily observation sessions,with 500observations persession)foreachcomponentofthematernalbehaviorinthe ﬁrst10postpartumdays.WeusedaGeneralizedEstimating Equa-tionsanalysis(GEE)tocomparethematernalbehaviorinthetwo groups (NH and H) acrossthe 4 daily observationsessions. To assessdifferencesbetweenthegroupsforeachobservationpoint, weusedBonferronimultiplecomparisonstestwhenappropriate. Inallcases,differenceswereconsideredsigniﬁcantwhenp<0.05 (Fig.2A–D).

Weanalyzedthecomponentsofthenursingbehavior(Active Nursing–HighandLowArched-backNursingandPassive Nurs-ing)acrossthe4dailyobservationsessionsusinga Generalized EstimatingEquationsanalysis (GEE)comparing thetwo groups (NH and H) across the 4 daily observation sessions. To assess differences betweenthegroups for each observationpoint, we usedBonferronimultiplecomparisonstestwhenappropriate.In all cases, differences wereconsidered signiﬁcant when p<0.05 (Fig.3A–C).

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A.R.Reisetal./BehaviouralBrainResearch265(2014)216–228 219

Fig.1.Timeseriesdecompositionanalysisofthenursingbehaviorofnon-handled(A)andhandleddams(C)andLicking(BodyandAnogenital)ofnon-handled(B)and handleddams(D)intheﬁrst10PPDs.Datawereanalyzedusingdecompositionofadditivetimeseries,verifyingtrendandseasonalpatternsintheﬁrst10dayspostpartum acrossthe4dailyobservationsessions(before,after,3hafterand5hafterhandling).*Representspresenceoftrendorseasonalpattern,n=9inallgroups.

Toanalyzetheperformanceinthenestodorpreferencetest, wecompared thepercentage oftime spentin thenest bedding areaandinthefreshbeddingareausingananalysisofvariance (ANOVA)forhandlingprocedure,sexandbeddingarea(Freshor Nest). Toassessdifferences betweenthegroupsfor each point, weusedBonferronimultiplecomparisonstestwhenappropriate (Fig.4).

Toanalyzetheperformanceineachtrialofthenestodor pref-erencetest,wecomparedthetime(seconds)spentinthebedding areas(FreshandNest)usingusedaGeneralizedEstimating Equa-tionsanalysis(GEE)fortime(timeineach ofthe5trialsofthe nestodorpreferencetest),handlingprocedure,sexandbedding area(FreshorNest).Toassessdifferencesbetweenthegroupsfor eachpoint,weusedBonferronimultiplecomparisonstestwhen appropriate(Fig.5A–D).

To analyze the latency to reach the nest bedding area and thetotalofmovementduringtheodorpreferencetest,weused an analysis of variance (ANOVA) for handling procedure and sex.Toaccessdifferencesbetweenthegroups,weused Bonfer-roni multiple comparisons test when appropriate. In allof the cases,differenceswereconsideredtobesigniﬁcantwhenp<0.05 (Fig.6).

ThelevelsofCREBandpCREBintheOBwereanalyzedusing Studentt-test,andtheBDNFlevelswereanalyzedusinga two-wayanalysisofvariance(ANOVA)followedbyBonferronimultiple comparisonstest(factorstreatment×timeoftissuecollection).In allcases,differenceswereconsideredtobesigniﬁcantwhenp<0.05 (Figs.7A–Cand8A–C).

3. Results

3.1. Timeseriesanalysisofmaternalbehavioracrossthefrist10 postpartumdays

3.1.1. Nursingbehavior

Weobservedatrendinthenursingbehaviorinthenon-handled group(Dickey–Fuller=−1.56,Lagorder=3,p=0.74)andalsointhe handledgroup(Dickey–Fuller=−2.51,Lagorder=3,p=0.36).Dams showedanaturalreductioninthepatternofthenursingbehavior acrosstheﬁrst10PPDs,butthisreductionwasmorepronounced in thedamsfromthehandledgroup(Fig.1C)compared tothe non-handledgroup(Fig.1A).Therewasalsoa seasonalpattern acrossthe4dailyobservationsessionsin bothgroups(FisherG test,NH=p<0.01andH=p<0.01)butthehandledgroup(Fig.1C) exhibitsadifferentdailypatterncomparedtonon-handledgroup (Fig.1A).

3.1.2. Motheroffthenest

Weobservedatrendinthetimethatthemotherspentoutside the nest in the non-handled group (Dickey–Fuller=−2.07, Lag order=3, p=0.54) and also in the handled group (Dickey–Fuller=−1.92, Lag order=3, p=0.60). Dams showed anaturalincreaseinthetimespentoutsidethenestacrosstheﬁrst 10PPDs,butthisincreasewasmorepronouncedinthedamsfrom thehandledgroup(datanotshow)comparedtothenon-handled group(datanotshow).Therewasalsoaseasonalpatternacross the 4 daily observation sessions in both groups (Fisher G test,

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Fig.2.Componentsofthematernalbehaviorinthe4dailyobservationsessions:nursingbehavior(A),motheroffthenest(B),licking(BodyandAnogenital)(C)andmother buildingnest(D).Datawereexpressedmean(±SEM)andanalyzedusingGEEforhandlingprocedureand4dailyobservationsessions(before,after,3hafterand5hafter handling)foreachcomponentofmaternalbehavior(nursing,offthenest,lickingandbuildingnest)followedbyBonferroni’smultiplecomparisonstesttoaccessdifferences ineachpoint.*Representsasigniﬁcantdifference(p<0.05)forinteractionhandling×observationsessionintheallgraphics,n=9inallgroups.

NH=p<0.05 and H=p<0.01) but the handled group (data not show)exhibitsadifferentdailypatterncomparedtonon-handled group(datanotshow).

3.1.3. Lickingpup(BodyandAnogenital)

Weobservedatrendinthelickingbehaviorinthenon-handled group(Dickey–Fuller=−2.29, Lag order=3, p=0.45)and also in thehandledgroup(Dickey–Fuller=−2.33,Lagorder=3,p=0.44). Althoughtherewasobservedatrendinbothgroups,thedamsof thenon-handledgroup(Fig.1B)showmorecomplextrend (poly-nomial)themthehandledgroupthatexhibitsalinearreduction oflickingacrosstheﬁrst10PPDs(Fig.1D).Therewasnoseasonal pattern detectedacrossthe 4 dailyobservation sessionsin the non-handledgroup(Fig.1B)thereisnosigniﬁcantchangeacross theday(Fisher Gtest,p>0.05)but thehandledgroup(Fig.1D) exhibitsadifferentdailypatternwithanabruptincreasein lick-inginthesessionimmediatelyafterhandlinggroup(FisherGtest, p<0.01).

3.1.4. Nest-buildingbehavior

Weobservedatrendinthetimenest-buildingbehaviorinthe non-handledgroup(Dickey–Fuller=−3.29,Lagorder=3,p=0.08) andalsointhehandledgroup(Dickey–Fuller=−3.03,Lagorder=3, p=0.16). Dams showed a natural decrease in the time spent buildingthenestacrosstheﬁrst10PPDs,butthisdecreasewas lesspronouncedinthedamsfromthehandledgroup(datanot show) compared to the non-handled group (data not show). Therewas also a seasonal pattern across the4 daily observa-tion sessions in both groups (Fisher G test, NH=p<0.01 and

H=p<0.001)but thehandledgroup(datanot show) exhibitsa differentdailypatterncomparedtonon-handledgroupwithan abruptincreaseinthesessionimmediatelyafterhandling(datanot show).

3.2. Maternalbehavioracross4dailyobservationsessions 3.2.1. Nursingbehavior

Damsshowedanaturalreductioninthepatternofthe nurs-ingbehavioracrossthe4dailyobservationsessions(maineffect observationsessionWaldchi-square(3,14)=132.05p<0.001),but

thisreductionwasmorepronouncedinthedamsfromthehandled group(maineffecthandlingWaldchi-square(1,16)=6.81,p<0.01)

especiallyintheobservationsessionsimmediatelyafter (Bonfer-ronimultiplecomparisonstestp<0.05)and3hafterthehandling procedure(Bonferronimultiplecomparisonstestp<0.01) (Inter-actionhandling×observationsessionWaldchi-square(3,14)=2.68

p<0.05)(Fig.2A). 3.2.2. Motheroffthenest

Damsshowedanaturalincreaseinthetimespentoutsidethe nestacrossthe4daily observationsessions (maineffect obser-vationsessionWaldchi-square(3,14)=230.99,p<0.001),butthis

increase was more pronounced in the dams from the handled group(maineffecthandlingWaldchi-square(1,16)=7.53,p<0.01).

The results showed signiﬁcant main effects for handling and observationsessions,and therewasaninteractionbetweenthe twofactors(Interactionhandling_×observationsessionWald chi-square(3,14)=23.39,p<0.001)thatshowedthattheincreaseinthe

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Fig.3. Componentsofthenursingbehaviorinthe4dailyobservationsessions:activenursing–higharched-backnursing(A),passivenursing(B)andactivenursing– lowarched-backnursing(C).Datawereexpressedmean(±SEM)andeachcomponentofthenursingbehaviorwasanalyzedusingGEEforhandlingprocedureand4daily observationsessions(before,after,3hafterand5hafterhandling)followedbyBonferronimultiplecomparisonsteststoaccessdifferencesineachpoint.*Representsa signiﬁcantdifference(p<0.05)forinteractionhandling×observationsession,n=9inallgroups.

the handling procedure (Bonferroni multiple comparisons test, p<0.05)and3hafterthehandlingprocedure(Bonferronimultiple comparisonstest,p<0.001)(Fig.2B).

3.2.3. Lickingpup(BodyandAnogenital)

Whenthe distributionof thelicking behavior wasanalyzed acrossthefourdailyobservationsessions,theresultsshowedno signiﬁcantmaineffectforthetreatmentand(maineffecthandling Wald chi-square (1,16)=0.73, p>0.05). Dams of handled pups

showedanincreaseinlickingbehavior(maineffectobservation session, Wald chi-square (3,14)=11.96, p<0.05 and interaction

handling×observation session Wald chi-square (3,14)=24.09,

p<0.001),butthisincreasewaslimitedtotherecordingsession immediatelyafter the handlingprocedure (Bonferronimultiple comparisonstest,p<0.01)(Fig.2C).

3.2.4. Nest-buildingbehavior

Dams showed a natural increase in nest-building behavior acrossthe4dailyobservationsessions(maineffectobservation ses-sions,Waldchi-square(3,14)=32.88p<0.001).Theresultsshowed

nomaineffectofthetreatmentalone(maineffecthandlingWald chi-square(1,16)=0.07,p>0.05),buttherewasasigniﬁcant

inter-actionbetweenthetwofactors(Interactionhandling×observation sessionWaldchi-square(3,14)=23.89p<0.001),inwhichdamsof

thehandledpupsshowedanincreaseinnest-buildingbehaviorthat waslimitedtotherecordingsession immediatelyafterthe han-dlingprocedure,whichissimilartowhatwasfoundforthelicking behavior(Bonferronimultiplecomparisonstest,p<0.05)(Fig.2D).

3.3. Activeandpassivenursingbehavioracross4daily observationsessions

3.3.1. Activenursing–higharched-backnursing

Damsshowedanaturalreductioninthepatternofthe nurs-ingbehavioracrossthe4dailyobservationsessions(maineffect observation session Waldchi-square (3,14)=67.66p<0.001) but

therewerenodifferencesbetweenthegroups(maineffecthandling

Fig.4.Percentageoftimespentinthebeddingareas(FreshandNest)intheodor preferencetestonPPD11.Datawereexpressedmean(±SEM)andanalyzedusing ANOVAforhandlingprocedure,sexandbeddingarea(FreshandNest)followedby Bonferronimultiplecomparisonsteststoaccessdifferencesineachpoint. *Repre-sentssigniﬁcantdifference(p<0.05)forthemaineffecthandlingandformaineffect sex,n=9inallgroups.

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Fig.5. Timeinsecondsspentinthebeddingareas(FreshandNest)ineachsessionoftheodorpreferencetestonPPD11.Datawereexpressedmean(±SEM)andanalyzed usingGEEfortime(sessionsoftheodorpreferencetest),handlingprocedure,sexandbeddingarea(FreshandNest)followedbyBonferronimultiplecomparisonsteststo accessdifferencesineachpoint.*Representssigniﬁcantdifference(p<0.05)fortheinteractiontime×handling×sex×beddingarea,n=9inallgroups.

Waldchi-square(1,16)=1.32,p>0.05)andnointeractionbetween

thetwofactors(Interactionhandling×observationsessionWald chi-square(3,14)=2.79p>0.05)(Fig.3A).

3.3.2. Passivenursing

Therewerenodifferences in thepassive nursingacrossthe 4 daily observation sessions (main effect observation session Wald chi-square (3,14)=2,18 p>0.05), no differences between

the groups (main effect handling Wald chi-square (1,16)=3.17,

p>0.05)andnointeractionbetweenthetwofactors(Interaction

Fig.6. Timespendbythepupstoreachthenestbeddingareainthenestodor preferencetestonPPD11.*Representssigniﬁcantdifference(p<0.05)forthemain effecthandling,n=8inallgroups.

handling×observation session Wald chi-square (3,14)=3.35

p>0.05)(Fig.3B).

3.3.3. Activenursing–lowarched-backnursing

Dams showeda natural reduction in thepattern ofthe low arched-back nursing across the 4 daily observation sessions (main effectobservation session Wald chi-square(3,14)=101.26

p<0.001),butthisreductionwasmorepronouncedinthedams fromthehandledgroup(maineffect handlingWaldchi-square

(1,16)=6.01,p<0.05)especiallyintheobservationsessions

imme-diatelyafter(Bonferronimultiplecomparisonstestp<0.05)and 3hafterthehandlingprocedure(Bonferronimultiplecomparisons testp<0.01)(Interactionhandling×observationsessionWald chi-square(3,14)=7.96p<0.05)(Fig.3C).

3.4. Nestodorpreferencetest

3.4.1. Percentageoftimespentovertheareas

There were no main differences between the pups of both groupsinthenestodorpreferencetestwhenthepercentageofthe totaltime(sumofthe5trials)wasanalyzed.Maleandfemalepups ofbothgroupsappearedtoshowpreferenceforthesideofthenest bedding(MaineffecthandlingF(1,34)=0.01p>0.05;maineffectsex

F(1,34)=3.02p>0.05;maineffectarea–FreshorNestBeddingArea

–F(1,34)=45.25p<0.0001).

There was a difference between male and female pups in thetimespentoverthearea(Interactionsex×areaF(1,34)=6.18

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Fig.7. WesternblotanalysisofmalepupsolfactorybulbafterthehandlingprocedureonPPD7.CREB(A),pCREB(B),andBDNFlevels(C)intheolfactorybulbofmalepups wereexpressedasmean±S.E.M.andanalyzedusingStudentttest(AandB)orTwo-WayANOVAfollowedbytheBonferroniPost-tests(C).*Representssigniﬁcantdifference (p<0.05)comparedtocontrolgroup(non-handled);n=6inallgroups,exceptforpCREBlevelsinthehandledgroup(n=5).

didthefemalepups(Bonferronimultiplecomparisonstest,p<0.05) (Fig.4).

There were no interactions between handling×sex (F(1,34)=0.33, p>0.05) and handling×sex×area (F(1,34)=

0.02,p>0.05).

3.4.2. Timespentovertheareasineachtrialoftheodor preferencetest

Therewerenomaindifferencesbetweenthepupsofbothsex and groups in the nest odorpreference test when the time in each trialwas analyzed (Maineffect handlingWaldchi-square

(1,34)=0.02p>0.05;main effectsexWaldchi-square(1,34)=2.19

p>0.05)butthereweredifferencesbetweenthetimespentinthe beddingareas(maineffectarea–FreshorNestBeddingAreaWald chi-square(1,34)=41.58,p<0.001)andintheperformanceineach

trials(maineffecttrialWaldchi-square(1,31)=43.59,p<0.001).

Thetrialthatthepupshadtheworstperformancewasthe sec-ond(Interactionarea×trialWaldchi-square(1,31)=16.71,p<0.01).

There was a difference between male and female pups in the timespentoverthearea(Interactionsex×areaWaldchi-square

(1,34)=7.79p<0.01).Malepupsspentmoretimeinthenestbedding

areathandidthefemalepups(Bonferronimultiplecomparisons test,p<0.05),andthisresultwasclearlycausedbythelackof pref-erenceforthenestbeddingareaobservedinthefemalepupsthat werehandledintheneonatalperiod(handling×sex×area×trial

Waldchi-square(1,31)=10.59,p<0.05).Malepupsfrombothgroups

preferredthenestareainthetrial3(Bonferronimultiple com-parisonstest,NHp<0.05andHp<0.001),4(Bonferronimultiple comparisonstest,NH p<0.001andHp<0.05)and5(Bonferroni multiplecomparisonstest,NHandHp<0.001),femalepupsthe non-handledgrouppreferredthenestareainthetrials1 (Bonfer-ronimultiplecomparisonstest,p<0.05)and4(Bonferronimultiple comparisonstest,p<0.001),femalepupsofthehandledgroupdid notpreferredanysideofthemazeinthe5trialsoftheOdor Pref-erenceTest.

There were no interactions between handling×sex Wald chi-square(1,34)=1.72,p>0.05),handling×areaWaldchi-square (1,34)=0.03,p>0.05),handling×trialWaldchi-square(1,34)=2.60,

p>0.05), sex×trial Wald chi-square (1,31)=0.98, p>0.05),

han-dling×sex×area Wald chi-square (1,34)=0.11, p>0.05),

han-dling×sex×trial Wald chi-square (1,31)=5.22, p>0.05),

han-dling×area×trialWaldchi-square(1,31)=7.41,p>0.05).

3.4.3. Timetoreachthenestbeddingarea

Theresultsshowedthathandledpupsspendmoretime reach-ing the nest bedding area than the pups of the non-handled group (main effect handling F(1,28)=7.23, p<0.05; main effect

sex F(1,28)=0.31 p>0.05; interaction handling×sex F(1,28)=0.00

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Fig.8. Westernblotanalysisoffemalepupsolfactorybulbafterthehandlingprocedureonpost-natalday7.CREB(A),pCREB(B),andBDNFlevels(C)intheolfactorybulb offemalepupswereexpressedasmean±S.E.M.andanalyzedusingStudentttest(AandB)orTwo-WayANOVAfollowedbytheBonferroniPost-tests(C).*Represents signiﬁcantdifference(p<0.05)comparedtocontrolgroup(non-handled);n=6inallgroups.

3.4.4. Totaltimeofmovementintheodorpreferencetest

Therewere no differences between thegroups in thetotal amount of movement during the 5 trials of the Odor Prefer-enceTest(maineffecthandlingF(1,28)=2.09,p>0.05;maineffect

sex F(1,28)=0.23 p>0.05; interaction handling×sex F(1,28)=3.33

p>0.05)(datanotshow).

3.4.5. Percentageoftimespentovertheareas,timetoreachthe nestbeddingareaandthematernalbehavior

Alterationsinthematernalbehaviorcoulddisrupttheolfactory learning,andourresultsshowedthattheeffectcouldbe differ-entformaleandfemalepups.Althoughmaleandfemalepupsof thehandledgroupusedmoretimetoreachthenestbeddingarea, thisalterationwascorrelatedwiththematernallickingpatternfor malepupsofthehandledgroup(anincreaseinlickinginthe ses-sionimmediatelyafterhandlingr=0.568,p<0.05).Forthefemale pups,therewasanegativecorrelationbetweenthetimethateach pupspenttoreachthenestbeddingareaandthematernal nurs-ingpattern(r=−0.5,p<0.05)andacorrelationtendencybetween thetimespentbythepuptoreachthenestbeddingareaandthe maternaltimespentoutsidethenest(r=0.49,p=0.054).

Anassociationwasalsopresentbetweenthenestodor pref-erenceof femalepupsand thematernal behavior.Therewas a correlationbetweenthepercentageoftimethatthefemalepups spendinthenestareaandthenursingbehavior(r=0.62,p<0.01) andanegativecorrelationwiththematernaltimespentoutside thenest(r=−0.57,p<0.05).

3.5. Westernblotanalysis

3.5.1. CREBandBDNFlevelsintheOBof7-day-oldmales

There were no differences in the CREB level 30min after handling between the group submitted to repeated handling (PND1 to PND7) compared to the non-handled group in male pups (t10=0.722, p>0.05) (Fig. 4A). There were alsono

differ-encesinthepCREBlevelbetweenthegroups(t9=1.219,p=0.25)

(Fig.4B).

TheBDNFlevelwasincreasedintheRHgroupat30(Interaction handling×time F(1,10)=16.71 p<0.01) and 120min (Interaction

handling×timeF(1,10)=7.43,p<0.05)afterhandling(maineffect

handlingF(1,10)=56.99,p<0.0001).Therewasnoeffectofthetime

alone(maineffecttimeoftissuecollectionF(1,10)=0.023,p>0.05)

(Fig.4C).

3.5.2. CREBandBDNFlevelsintheOBof7-day-oldfemales

TherewerenodifferencesintheCREBlevel30minafter han-dlingbetweenthegroups(t10=0.536,p>0.05)(Fig.5A).Therewas

adecreaseinthepCREBlevelinthefemalepupsoftheRHgroup (t10=4.298,p<0.01)(Fig.5B).

TheBDNFleveldidnotdifferinanyofthegroupsat30and 120minafterhandling(maineffecthandlingF(1,10)=0.474,p>0.05;

maineffecttimeoftissuecollectionF(1,10)=0.001,p>0.05;

Inter-action handling×time F(1,10)=0.556 and F(1,10)=0.166,p>0.05)

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4. Discussion

Theresultsshowedthathandlingthepupsduringthe neona-talperiodinducescomplexchangesonmaternalbehaviorthatare not limitedtoan increase ordecrease in licking behaviorafter theintervention.Moreover,wedemonstratedthat thehandling interventionaltersCREBphosphorylationandBDNFlevelsinasex dependentwayinthepups’OB,whichcouldbeassociatedwith differentialolfactorylearningandtheodorofthenestpreference inthepups.

4.1. Neonatalhandlingandmaternalbehavior

Thisstudyshows,fortheﬁrsttime(tothebestofourknowledge) thathandlingaltersthetrendalongtheﬁrst10postpartumdays andtheseasonaldailypatternoflicking,nestbuilding,nursingand timespentoutsidethenest.

Studiesthathadinterventionsinthepostpartumperiodusing slightlydifferenthandlingprotocols,suchasbrief(15min)daily separationfromthepups,showedanenhancementinmother–pup interactions by provoking bursts of maternal sensory stimula-tionofthepupsimmediatelyaftertheirreturntothehomecage [7,9,52–54,56,62,63].

Inthisstudy,weexpectedtoﬁndthesameincreaseinmaternal care,especiallyinthelickingbehavior.Indeed,ourresultsshowed anincreaseinlickingbehaviorimmediatelyafterthehandling pro-cedure,aswehadpreviouslydemonstrated[64],butthisincrease waslimitedtothatspeciﬁcperiod, whichisin agreementwith theresultsdescribedbyClaessens,Daskalakis[9].Increasedlicking behaviorcouldrepresentamaternalresponsetothe environmen-talinterventionbecausemothersofnon-handledpupsshowedan expectedstabledailypatterninthismaternalbehavior.Changesin thestabilityofthelickingpatternthatwereinducedbyhandling couldbeperceivedbytheoffspringasanenvironmentalthreat, whichcouldleadtopersistentalterationsintheirdevelopment.

One important suggestionmade by Pryce, Bettschen [65] is thattheratstraincouldaffecttheresultofthehandlingprocedure in thematernalbehavior. Using Wistar ratherthan LongEvans rats(usedinotherstudies)[6,9],theydidnotﬁndanincreasein lickingafterthehandlingprocedure[51].WealsousedWistarrats, butwefoundanincreaseinlickingafterthehandlingprocedure inagreementwithotherthestudiesthatalsousethisratstrain [52–54,63].Therearetwoimportantdifferencesintheprotocol ofthematernalbehaviorobservationthatcouldexplainthis dis-crepancy.Pryce,Bettschen[65]measuredtheeffectofhandlingon maternalcareduringthedarkperiod,whenratsaremoreactive, and the mothersusually spend less time in contact with their pupsincomparisontothelightperiodinwhichweperformedour observations.Moreover,westartedrecordingmaternalbehavior immediatelyafterthehandlingprocedureandtheirobservations began15minaftertheintervention.

Wealsoobservedareactiveincreaseinthenest-building behav-ior in the session immediately after handling, which was also expectedbecausethehandlingprocedurealtersthenestareaand introducesexternalunfamiliarodors thatcantriggerthemotor activecomponentsofthematernalbehavior[66,67].

In additionto differencesin thefrequency of licking, rather complexchangesinothercomponentsofthematernalbehavior werefound,whichweremorepersistentthanthelickingburst. The total frequency of nursing decreased in dams whose pups were handled compared to the non-handled pups especially in the sessions immediately and 3h after handling procedure. Moreover,damsthathadtheirspupshandledincreasedthetime spentoutsidethenest,andthischangewasnotobservedafterthe procedure,aswouldbeexpected;instead,apersistenteffecton thisbehaviorwasobservedbeforethehandlingandonlyafter3h

aftertheintervention,thisdifferencemaybeduetotheincreasein lickinginthesessionimmediatelyafterhandling,decreasingthe timeoutsidethenestinthisperiod.

Maternalbehaviorsthatinvolvemoreactivemovements(such aslickingandnestbuilding)andthematernalbehaviorsthatare morequiescent(suchasnursing)arebecontrolledbydifferentCNS regionswithoppositerelationstothedopaminergicsystem[68]. Maternalactivebehaviorsarestimulatedbydopaminergic recep-torsintheshelloftheNucleusAccumbens(NCc)[68].Incontrast, quiescentbehaviorsareinhibitedbydopaminergicreceptors,so thattheonsetofquiescentbehaviordemandsa decreaseinthe dopaminergictonuswithintheNCc[69].Thesemechanismscould beinvolvedinthedisparitylicking/nursingbehavioralsequencein handlednestsasdescribedinourstudy.

Handlingpersecanaffectdirectlythepups[12],althoughthe roleoftheoffspringinearlysocialdynamicisdifﬁculttoanalyze, asitisdominatedbythebehaviorofthecaregiver[70].However, pups’behaviormayplayanimportantroleintheeffectofhandling controllinginpartthematernalbehavior[71–73].Theincreasein lickingcouldbeareactionofthedamtoanincreaseinultrasonic vocalizationofthepups.Thebehaviorofthepupscouldalsoexplain thetrendofdecreasingthelickingacrossthepostpartumdays.

Neonatal handling alters the functioning of the HPA axis (Hypothalamic-Pituitary-Adrenal)inresponsetoavarietyof stress-ors[40–43]andmayalsochangethemorphologyandfunctionof brainstructures[49–51].Ifthesealterationsoccuralreadyinthe ﬁrstdaysofthepups’life[43,49,50,74,75]theymayaffectthepups’ earlybehavioralresponsetothehandlingprotocolandleadtoa differentialstimulationofthematernalbehavioracrossthe post-partumdays.Otherpossibilityisthatthemothercouldadapttothe repeatedhandlingandreducethereactivitytotheprotocolacross the10postpartumdays.Futuremeasuresofultrasonicvocalization ofthepupswilladdtothestudyintermsofbeingabletoevaluate thepup’slevelofmotivationandaffectivestatechange.

Theresultsareintriguingandleaveopenthepossibilitythat pup behavior could beplaying a major role in the changes in maternalcare.Recentworkhasemphasizedtherolethatpup moti-vationplaysintriggeringandmaintainingmaternalcareinrodents [70],infactactivenursingbehaviorthatispersistentlyalteredby thehandlingprocedure,isdirectlyaffectedbythepupsbehavior [12,65,76–78].

Basedonthenaturalsequenceofthematernalbehavior[66,67], we expectedtoﬁnd anincrease in thenursingbehavior and a decreaseinthetimespentoutsidethenestaftertheincreaseinthe motoractivecomponentsofthematernalbehavior,butwefound theopposite.Theseresultssuggestthathandlingdoesnotonlyalter maternalbehaviorbyincreasingordecreasingitsbehavioral com-ponents,butalsodesynchronizesthemother–pupinteractionsby changingthebehavioralsequence.

Stabilityofthemother–infantrelationshipisimportantforthe developmentofemotionalandcognitivefunctions;forareview, see[3].Indeed,synchronyinmother–infantinteractionsappears to bea crucial variable in humandevelopment [79] and addi-tionally for rats [80,81]. We infer that these alterations in the behavioralsequenceofthematernalcareassociatedwithchanges inthemother–pupinteractionsynchronyleadtoareductioninthe mother–pupcontact(increasedtime spentoutsidethenestand decreasednursing)andaltersthestabilityofthematernalbehavior throughouttheday(especiallylickingandnestbuilding).

Besidesthechangesintheseasonaldailypatternofnursingand time spentoutside thenest,handlingalsoaffectedthetrendof bothcomponentsofthematernalbehavior.Damsthathadtheirs pupshandledshowedareductioninthecontactwiththepupsand increasedtrendtobeoutsidethenestacrosstheﬁrst10postpartum days.Damsofthenon-handledgroupalsoshowatrendof increas-ingthetimeoutsidethenest,butthehandlingprocedureseems

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toacceleratetheseparationbetweenthemotherandthepups,as damsofthehandledgroupshowthesameamountoftimeoutside thenestinthe4PPDthandamsofthenon-handledgroupinPPD8. Thesamepatternoccurswiththenursingbehaviorbutinthe oppo-sitedirection,inwhichdamsofhandledgroupshowpracticallythe samescoreofnursingin4PPDthandamsofthenon-handledgroup inPPD8.Thesechangescouldaffecttheolfactorylearningprocess duringacriticalperiodforthebondingformationandcouldbeakey causeoflong-lastingeffectsofthisinterventiononsocialbehaviors [45,50].

4.2. Maternalbehaviorandolfactorylearningofthepups

Infant rats rapidly and naturally learn to identify, orient, approachandpreferthenestodor[22,25,27].Thenestodor pref-erencedepends onthematernalbehavior, especiallythetactile stimulationoflickingandmother–pupcontact[14,19,20,82], con-sideringthatthehandlingprocedureincreasesthelickingbehavior andthenaturaltactilestimulationofthepupsbythemother, acti-vatestheLC[82],increasesnoradrenaline(NA)levelsintheOB ofthepups[14,22]andinducesthephosphorylationofthecyclic AMPresponseelementbindingprotein(CREB)[14,27,29,83],we expectedtoﬁndanincreaseinthelevelsofCREBphosphorylation inthepups’olfactorybulbs;however,theresultsshowednosuch increase.Infact,inhandledfemalepups,CREBphosphorylationwas evenreduced,whileinmales,weobservednosigniﬁcanteffecton PPD7.Onepossibleexplanationcouldbeanadaptationprocessto therepeatedincreasedlicking,which,aftersomedaysofthe han-dling,couldcauseareductioninthepup’sresponsetothisincrease. WehavedescribedareductioninNAintheOBonPPD7afterthe handlingprocedure[48],andthisreductioncouldleadtoalackof increaseinCREBphosphorylationafterthehandlingonPPD7.

ThefactthatonlythehandledfemalesshowadecreaseinCREB phosphorylationcouldindicatethat,inayetunknownway,females aremoresensitivetothedecreaseinmother–pupcontact than males.Indeed,thedelayinthetimespentinreachingthenestarea wascorrelatedtothenursingbehavioronlyinthefemalepups.The resultsofthenestodorpreferencetestalsoagreewiththis hypoth-esisbecausefemalepupsthathadlesscontactwiththeirsdams (decreasednursingandincreasedtimeoutsidethenest) demon-stratedlesstimeinthenestarea.Infact,whenweanalyzedthe trialsofthenestodorpreferencetest,femalepupsofthehandled groupdidnotshowanysignoflearningacrossthe5trailsin con-trastwiththemalepupsofthesamegroupandthefemalepupsof thenon-handledgroup.Astherewasanoveralldifferencebetween maleandfemalepups,itispossiblethatthetaskwasmoredifﬁcult tofemalethanmalepups,regardlessoftheexperimentalgroup.

Differencesinthematernallickingdistributionamongthepups ofdifferentsexescanalsocontributetothiseffect.Studieshave shownthat,inrats,motherslickthemalemorethantheydothe femalepupsduetotheirdifferenturineodors[84–87],and we demonstratedthatthelickingpatternwascorrelatedtothesocial behaviorchangesinthemalepups.Alternatively,itispossiblethat earlylifeinterventionscaninducesex-changesaccordingtothe variablemeasuredlaterinlife.

Thesecorrelationalresultssuggestrelationshipsbetweenthe carebehaviorsandpup learningbutitisdifﬁculttoteaseapart theeffectsofhandlingversusmaternalcareontheneurochemical changes.Studiesusinganartiﬁcialmodelofmaternalbehaviorare abletodisentangletheseeffectsandareveryusefultoevaluatethe degreeofimportanceofeachvariableinthismatter[88–90].

Thebiochemicalalterations foundintherepeatedlyhandled pupsaremostlikelyassociatedwithchangesinthe monoaminer-gicsystem[48,63,91,92].Raineki,DeSouza[48]showedthatmale andfemalerepeatedlyhandledpups(PPD1–PPD7)demonstrate adecreaseinthenoradrenergictonusintheOBafterhandlingon

PPD7,butonlymaleshaveanincreaseinserotoninactivity,which couldactasacompensatorymechanisminmales.However,itis stillpoorlyunderstoodwhymaleandfemaleratpupsonPPD7 showthesedifferencesinthepCREBlevelsintheOB.

Itispossiblethat,alreadyatthis earlyage,maleandfemale pupshavedifferentresponsestoenvironmentaloradverse stim-uli[93–95].Stamatakis,Mantelas[63]showedthataprotocolof neonatalhandlinginducessexuallydimorphicchangesinthe lev-elsofhippocampal5-HT1Areceptorsinadultanimals,withmales havinganincreaseandfemalesadecrease.Inaddition,repeated neonatalhandlingincreasesBDNFlevelsintheCA4areainthe hippocampusofmaleratpups,whilefemalesshownoalteration [52,96].OurresultsintheOBareinlinewiththatstudy.Handled malepupsdidnot demonstratedifferencesinCREB phosphory-lation,buttheyhadincreasedlevelsofBDNFat30and120min aftertheinterventioncomparedtocontrolgroups.Becausethere wasnochangeintheCREBphosphorylation,itispossiblethatthe increaseinBDNFlevelsat30and120minafterthehandling proce-durecouldbecausedbyacumulativeeffectoftheincreasedlicking overthepreviousdays.Thus,theincreasedBDNFsignalinginthe OBinmalepupscouldcharacterizeapersistenteffectofthe han-dlingprocedure.AnincreaseinhippocampalBDNFisassociated withanincreaseinspatialmemoryinadultratsthatwerehandled intheneonatalperiod[52,96],andthereforeitispossiblethatthe sameeffectexistsfortheolfactorylearning.

IntheOB,nosigniﬁcantchangeintheBDNFlevelswasdetected intherepeatedlyhandledfemalepups;however,thisresultdoes notnecessarilymeanthattherewerenochangesintheBDNF lev-elsindifferentregionsofthatstructure.WeusedaWesternblot protocolwiththehomogenizationoftheentireOB,butthis struc-turehasseverallayerswithdifferentpopulationsofneurons.The useofmorphologicalanalysisthatcouldallowdifferentiationofthe layersintheOBwouldbeagoodexperimentalapproachto detect-ingmorespeciﬁcandpreciseeffectsofneonatalhandlingonthe functioningandmorphologyoftheOB.

5. Conclusions

Inconclusion, wedemonstratedthatthehandlingprocedure inducescomplexchangesincriticalcomponentsofmaternalcare. Thisstudyshows,forthefirsttime(tothebestofourknowledge), thathandlingcanchangethemother–pupinteractionsynchrony, thusalteringthebehavioralsequenceofmaternalcare,reducing themother–pups contactduringthefirst10 PPDsandchanging thedailypatternof nursing,licking behaviorandnest building. Handlinginducesaburstinlickingbehaviorimmediatelyafterthe interventionandcausesbiochemicalchangesonthepup’sbrain ina sex-specificway. Thesematernalbehavioral changes could explaintheincreaseinBDNFintheolfactorybulbofmalepups andthedecreaseinCREBphosphorylationinfemalepupsbecause differencesinthebehaviorofthepupswerecorrelatedwith differ-encesinthematernalbehaviorcomponents(alsoinasex-specific way).Thesebiochemicalchangesinthenervoussystemofthepups couldbethecauseofthesexualdifferencesshowninthenestodor preferencetest.

Althoughthefemalepupsappeartobemoreaffectedbythe handlingprocedure, malesalso have alterations in thetime to reachthenestarea,andtheincreasedlevelsofBDNFinthe olfac-torybulbcouldcontributetothelong-lastingeffectsofearlylife environmentalinterventiononthedevelopmentofthebehavioral strategiesinmales.

Conﬂictofinterest

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Acknowledgements

This study was supported by grants from Coordination of Improvement of Higher Education Personnel (CAPES), National Research Council of Brazil (CNPq) and FAPERGS-PRONEX (10/0018.3).

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