Addendum. Environmental and tactile stimulation modulates the neonatal handling effect on adult rat spatial memory.

Transcription

1 ddendum Environmentl nd tctile stimultion modultes the neontl hndling effect on dult rt sptil memory. Nikolos P. Dsklkis 1,2, Mri Kperoni 1, Christos Koros 1, E. Ronld de Kloet 2, nd Efthimi Kitrki 1,3 1 Lbortory of Histology & Embryology, University School of Medicine, University of thens 2 Division of Medicl Phrmcology, Leiden/ msterdm Center for Drug Reserch, Leiden University Medicl Center, Leiden University 3 Deprtment of Bsic Sciences, School of Dentistry, University of thens Int J Dev Neurosci. 29 Dec;27(8):

2 186 bstrct Hndling of rt pups promotes their dult cognitive performnce. However, new dt suggest tht individul components of the hndling procedure, like exposure to novelty or tctile stimultion, hve distinct lsting effects on behviour. In this study we exmined the interction of erly novelty exposure with vrying mount of tctile stimultion on sptil recognition memory nd corticosterone secretion of dult mle nd femle rts. split litter design ws used nd the experimentl nimls were lso compred to niml fcility rered controls. The experiment ws conducted in two phses. In the first phse, we exmined the effect of novel or home environment during the 15-min of neontl hndling, following 1 bck-strokes. Tctile stimultion of 1 bck-strokes combined with novelty exposure, enhnced novel rm discrimintion in Y-mze tsk in dult rts of both sexes compred to their siblings tht styed t home, s well s to the niml fcility rered controls. In the second phse, dditionl bck-stroking (totl of 2 bck-strokes) reduced the Y-mze performnce of mles neontlly exposed to novelty, while the sme tretment enhnced the performnce of their siblings tht styed t home. Bsl corticosterone levels, determined one week post Y-mze, were significntly incresed only in the novelty exposed/1 bck-stroked femles compred to sme sex non-hndled controls. In contrst, 1 bckstrokes combined with the home cge environment incresed corticosterone in mles. Increse to 2 bckstrokes reversed the impct of neontl environment on corticosterone levels. These dt suggest tht the nture nd intensity of the individul components of mild erly-life mnipultion, like hndling, re criticl in modifying spects of dult memory performnce nd bsl drenocorticl function. 1. Introduction There is ccumulting evidence tht dult rts exposed s pups to erly-life experience during the perintl period show ltered behviour nd neuroendocrine function [1, 2]. For instnce, n dverse experience, like prolonged mternl seprtion of 24 hours, impirs lter life cognitive performnce nd enhnces drenocorticl nd emotionl rectivity. On the contrry, mild mnipultions, like neontl hndling, cn led, vi developmentl progrmming, to nimls with better coping with stressful situtions lter in life [1, 3-7]. Such hndled nimls, which re seprted from their mother dily for 15 minutes over the first three weeks of life, exhibit s dults reduced nxiety. In response to mild stressor they hve lower output of corticosterone, prolctin nd drenline [7-1]. On the other hnd, hndled nimls hve incresed levels of glucocorticoid receptors in the hippocmpus nd frontl cortex [11-13]. ccordingly, their limbic - hypothlmic pituitry drenl (LHP) xis feedbck sensitivity to corticosterone is enhnced, providing more efficient termintion of the stress response. The forementioned fetures of hndled nimls hve been linked to

3 vritions of neontl hndling nd sptil memory 187 their generlly improved performnce in cognitive tsks [14, 15]. Neontl hndling consists of t lest three different mnipultions: brief mternl seprtion (up to 15min), touching (tctile stimultion) by the experimenter, nd exposure to novelty. Firstly, the short term mternl seprtion elimintes olfctory cues s well s the tctile stimultion by the mother, for which the ltter compenstes t reunion [16, 17]. Secondly, touching by the experimenter during the seprtion cn, to some extent, mimic the mternl tctile stimultion, but the vrious protocols re different in this respect. Finlly, the dditionl exposure to novelty offers new olfctory nd visul stimultion (from 2nd week on). lthough the effect of neontl hndling on the dult niml s physiology nd behvior hs been extensively studied since the mid 5s [6], only few studies hve exmined the impct of its seprte components. The estblished hndling prdigm [7] ws modified by introducing split litter design, for studying the impct of exposure to novelty seprtely [18]. In this model, ll pups re experiencing dily short term seprtion from their mother s well s the experimenter s touching. However, only hlf of the pups in ech litter re exposed individully to novelty, while the rest re left in their home cge. The dult mle rts, exposed to novelty exposure s prt of this hndling protocol, exhibited s dults enhnced sptil nd non-sptil memory [18, 19] nd showed incresed hippocmpl LTP [2]. More recently, by using the sme prdigm [21], it ws shown tht brief exposure of pups to n unfmilir environment produced in dult mles n ttenuted fer nd stress response, s well s diminished sexul behvior. Femles hve been scrcely studied in this prdigm. Juvenile nd dult femle rts, tht hd experienced s pups prolonged bsence of the mother while being plced in novel environment, were less rective to stressors s compred to femle pups tht styed in their nest during mternl bsence [22]. However, others did not observe this difference using the sme protocol [23]. During prolonged (24h) mternl seprtion, rtificil tctile stimultion of mle pups, especilly in the nogenitl region, hs been shown to reverse the centrl effects of seprtion in rts, while dditionl feeding restored corticosterone secretion [24-26]. Tctile stimultion cn lso enhnce the mturtion of corticl neurons [27], improve pssive voidnce response [28] nd sptil working memory [29]. In prdigms of one hour mternl seprtion, tctile stimultion of the dorsl re hs been shown to reverse the effect of seprtion on nxiety-like behvior nd pin sensitivity in dult rts of both sexes [3]. The lsting behviourl effects of tctile stimultion during the hndling prdigm hve not been studied. The im of this study ws to exmine the impct nd the interction of erly novelty exposure with vrying mount of tctile stimultion on sptil recognition memory nd bsl corticosterone secretion of dult mle nd femle nimls (Fig. 1 for n overview).therefore the experiment ws conducted in two phses. We first exmined the effect of novel or home environment, following 1 bck strokes, on the bove prmeters. The outcome ws further investigted with n dditionl mount of tctile

4 188 stimultion (totl of 2 bck-strokes) preceding the dily novelty or home exposure. In both phses, split litter design ws used. The experimentl nimls were lso compred to non-hndled, niml fcility rered (FR) controls. 2. Mterils nd Methods 2.1 Subjects 18 femle nd 6 mle Wistr rts (obtined t 6 weeks of ge from Hellenic Psteur Institute, thens, Greece) were used for breeding. fter hbitution period of two weeks, 3 femles were put in cge with one mle rt Type IV (mcrolon-polycrbonte cges with wire lid; 6 x 38 x 2 cm; contining swdust bedding). Pregnncy ws determined by vginl smers obtined every morning nd pregnnt femles were trnsferred in individul cges (home cge: mcrolonpolycrbonte type III cges with wire lid; 42.5 x 26.6 x 18.5 cm) contining swdust. The dy of birth ws defined s postntl dy (= PND ). We ccepted litter with mximl excess of one mle or femle nd our litter size ws 7-9 pups. The pups of experimentl groups were exposed to neontl mnipultions from PND1 to PND21. In the control litters, the pups remined undisturbed with their dms in the housing room until the time of behviorl testing, receiving only the niml fcility rering (chnge of cge once per week; strting t PND1). In totl, offspring of 13 litters ws used in this study. ll nimls were wened t PND22, group housed with the sme sex littermtes nd received the norml niml fcility rering fterwrds. t PND9 they were tested in the Y-mze tsk nd one week lter they were scrificed under bsl conditions. Estrus phse ws detected in scrificed femles by vginl smers. Rts were housed under 12:12 h light/drk cycle (lights on t 7. h, illumintion inside the cge: 2-3 lux, temperture: 22 ± 2 C, reltive humidity 6 ± 1%). Food nd tp wter were provided d libitum. ll niml tretments were pproved by the locl ethicl committee nd hve been crried out in greement with ethicl recommendtion of the Europen Communities Council Directive of 24 November 1986 (86/69/EEC). ll efforts were mde to minimize the number of nimls used nd their suffering. 2.2 Erly-life mnipultions Erly-life mnipultions consisted of 4 consecutive steps (see in Fig. 1B detiled description): i. Dms trnsfer out of the litter. Between 14: nd 15:h, dms were removed from their cge nd plced in sme type cge, in the sme room, t distnce from the home cge. ii. Bck-pinting (tctile stimultion): In Experimentl Phse I, bck-pinting, lsted 1min per pup with 1 pssges of the mrker (1 strokes, 1S). In Experimentl Phse II, bck- pinting lsted 1min per pup with 2 pssges of the mrker (2 strokes, 2S). iii. Novelty exposure (environmentl stimultion) of 3min dily. Exposure to novelty ws performed ccording to Tng [18]. Home nd Novel pups within ech experimentl phse (see following section) were dily experiencing the brief mternl seprtion nd the sme mount of tctile stimultion. Novel pups were dditionlly exposed to different context, compred to home pups, nmely the novel environment in isoltion from their littermtes. iv. Dms reunion. ll nimls with the exception of FR were bck-pinted. From dy 2 to 21, the nimls were

5 vritions of neontl hndling nd sptil memory 189 identified (on step 2) by the experimenter by the different colour in the bck tht ws renewed dily. The two mrkers used hd the sme odour. 2.3 Experimentl design To study the impct of erly novelty exposure on sptil recognition memory nd bsl corticosterone secretion of dult mle nd femle nimls we used (tretment) x (sex) design nd the tretment ws comprised of three levels with control group (niml fcility rering), home group (bck-stroked in the home cge), nd novel group (bck-stroked nd exposed to novelty). To study, dditionlly, the interction of erly novelty exposure with vrying mount of tctile stimultion we conducted the experiment in two phses: - In Phse I, 21 mle nd 21 femle pups from 6 different litters received, on their bck, 1 strokes (1S) during bck-pinting, with or without novelty exposure ( 1S-Novel nd 1S-Home groups, respectively). Mternl seprtion lsted in totl 15min. - In Phse II, 16 mle nd 13 femle pups from 4 different litters received on their bck 2 strokes (2S) during bck-pinting, with or without novelty exposure ( 2S-Novel nd 2S-Home groups, respectively). Mternl seprtion lsted in totl 15min. PND1-21 PND22 PND9 PND97 Erly life mnipultions Wening Y- mze test Decpittion in bsl conditions B N N N N N N N N N N N N N N N N N N N N H H H H H H H H H H H H H H H H H H DM H H DM i. Dm out ii. 1-min Bck-pinting iii. 3-min Novelty exposure iv. Dm bck DM TIME min Figure 1. Experimentl methods. () Time line of longitudinl study. (B) Sequentil steps of erly-life mnipultions. (i) Dm out. The dms were trnsferred out of the litter. This step took pproximtely 1min (s indicted in the clock). (ii) Bck-pinting (tctile stimultion): Bck-stroking, consisting of pssges of mrker tip over the pups bck, ws used s dily tctile stimultion; strted 1min fter the dm ws removed nd lsted 1min per niml. For ll the pups of litter, this step took pproximtely 9min (s indicted in the clock). (iii) Novelty exposure (environmentl stimultion): Exposure to novelty ws performed ccording to Tng [18] nd strted 1min fter the dm ws removed. split-litter design ws used so tht hlf of the nimls in ech litter 1 min 1 min 14 min (pinted with certin color during stroking) were put individully in novel cges (mcrolonpolycrbonte 18 x 2 x 14 cm; contining fresh swdust bedding), under heting lmps for 3min (Novel nimls). The other hlf of the litter (pinted with nother color during stroking) remined for 3min in the home cge under heting lmps (Home nimls). fter 3min the Novel pups were trnsferred bck to their home cge. This step took pproximtely 4min (s indicted in the clock). During the trnsfer of Novel (bck nd forth), the home pups were lso eqully touched. (iv) Dms bck (dms reunion): Finlly, the dms were returned in the home cge. This step took pproximtely 1min (s indicted in the clock). 15 min

6 19 In both phses of the experiment, the experimentl nimls were lso compred to FR controls. Control litters were the sme group of nimls in both phses nd consisted of 1 mle nd 13 femle pups tht derived from 3 different litters. For synopsis of the different experimentl groups ccording to the erly-life mnipultions see Tble 1. Tble 1 Synopsis of the experimentl groups ccording to the different erly-life mnipultions (ELM) ELM Tctile Stimultion niml Fcility PND 2-21 Rering Novelty exposure PND 2-21 Number of nimls per group Note: PND, postntl dy Group nme Experimentl Phse I Experimentl Phse II CON 1S-HOME 1S-NOVEL 1S-HOME 1S-NOVEL 1 bck-strokes dily 1 bck-strokes dily 2 bck-strokes dily bck-strokes dily 2 (1 13 ) 21 (1 11 ) 2 (11 9 ) 15 (8 7 ) 14 (8 6 ) 2.4 Behviorl Testing Y-mze test The nimls bility for sptil recognition memory ws ssessed t PND 9 by using the Y-mze test [31, 32]. In brief, blck, wooden Y-mze pprtus, hving three identicl rms (5 x 16 x 32 cm) ws used. Extr-mze only, visul cues (posters nd lbortory glsswre equipment) were plced round the mze nd bove the top of the blck wooden sides. No visul cues were plced inside the mze, nd the extr-mze cues were not eqully visible from ll points within the mze, thus enbling sptil orienttion. The investigtor ws not visible by the rt. Rts were tested during the light phse of their cycle (between 9:-13: h). The testing room ws next to nimls room. The floor of the mze ws covered with swdust bedding tht ws renewed for ech niml using mixture of swdust tht differed for mles nd femles. The tsk consisted of two trils hving 4h intertril intervl. The sme rm ws lwys designted s strt rm, where the niml ws introduced fcing the wll of the rm. The other two rms were lternting s Novel nd Other rm between rts to reduce rm-bis effects. In Tril 1, the novel rm ws blocked with blck wooden guillotine. t the strt of testing, rt ws plced in the strt rm nd the rt ws llowed to explore the strt nd other rms for 15min. t the end of Tril 1, the rt ws returned to its home cge nd the bedding inside the mze ws mixed, to reduce the possibility of using odors s cue. In Tril 2, ll rms were ccessible nd the rt ws llowed to explore them for 5min. The nimls were recorded with video-cmer djusted on the ceiling of the room. The videos were nlyzed blindly by two independent observers by using computer softwre [33]. The dependent vribles mesured in the 1 st nd the whole 5min period of Tril 2 were: ) number of totl entries b) % entries in ech rm, c) % durtion in ech rm nd in the center of the pprtus. Entry into n rm ws counted when the hed nd two front pws were inside the rm, nd the durtion of n rm visit ended when the hed nd two front pws were outside of the rm gin. 2.5 Corticosterone mesurement One week following Y-mze testing, the nimls were decpitted under bsl conditions between h. Trunk blood ws collected in heprinized tubes on ice nd centrifuged t 14. rpm, for 5min, t 4 C. Plsm ws collected nd kept t -8 o C until use. Corticosterone levels were

7 vritions of neontl hndling nd sptil memory 191 determined by using stndrd rdioimmunossy kit for smll rodents (MP Biomedicls, LLC, NY, US; sensitivity 1.25 ng/ml, intr-ssy vrition, 4.4%, interssy vrition 6.5%). 2.6 Dt nlysis nimls behviour in Y-mze ws nlyzed in the 1 st min nd in the whole-5min- period of Tril 2, s it hs been reported tht nimls might grdully loose their interest during testing period [32]. The following behviourl mesures were nlysed: - Generl locomotion nd explortion behviour during Y-mze test ssessed by the totl number of entries in ll rms, during Tril 2. - Novel rm discrimintion during Y-mze test. To mesure novel rm discrimintion in the Y-mze test, Difference score ws clculted by subtrcting the % entries in Other rm from the % entries in Novel rm for ech niml. dditionlly the % entries in Other nd Novel rms were compred. - Strtegy in Y-mze performnce. Differences in the strtegy of explortion were exmined by nlyzing the % of entries in the strt rm nd the % durtion of sty in the center of the mze during the 1 st min of Tril 2. - First choice. s first choice ws considered the first rm visited. ll the bove vribles, long with corticosterone mesurements, were sttisticlly evluted by two-wy nlysis of vrince (NOV) with tretment nd sex s the independent vribles. When pproprite, Bonferroni or Lest Significnt Difference (LSD) posthoc nlysis ws pplied (s specified in the legends). The preference for Novel versus Other rm ws lso nlyzed by repeted two-wy NOV with rm s the within-subject fctor. In this cse, pired t-tests within the groups were pplied for further comprisons of % entries between these two rms. Experimentl Phses I nd II were nlyzed seprtely. Choice of first rm entered fter leving the strt ws compred to chnce (5%) by binomil 2-tiled test [32, 34]. ccording to the vginl smers obtined t scrifice, femles comprised mixed popultion s to the estrus cycle in ll groups. Since they hd comprble distribution of the phses mong groups, we hve pooled the dt within ech femle group for ll the prmeters mesured. The sme non-treted nimls were used s the control group in sttisticl nlysis of Experimentl Phses I nd II. The level of significnce ws set t p<.5. The sttisticl nlysis ws djusted for non-equivlent groups. 3. Results 3.1 Effect of neontl exposure to novelty following 1 bck-strokes on Y-mze performnce (Experimentl Phse I) Generl locomotion nd explortion behviour during Y-mze test ll nimls showed the sme motivtion nd bility to explore the Y-mze pprtus during Tril 2, s ssessed by the totl number of entries in ll rms tht did not differ significntly mong groups (Fig. 2).

8 192 8 MLE Totl Entries (Tril 2) 1 min 6 ENTRIES 4 2 CON 1S-HOME 1S-NOVEL CON 1S-HOME 1S-NOVEL B 25 2 MLE 5 min ENTRIES CON 1S-HOME 1S-NOVEL CON 1S-HOME 1S-NOVEL Figure 2. Generl locomotion nd explortion behviour during Y-mze test in Experimentl Phse I. Men ± SEM of entries in ll rms of the Y-mze during the 1 st nd whole 5min period of Tril 2. CON, niml fcility rered controls; 1S-HOME, nimls neontlly 1 times bckstroked in the home cge; 1S-NOVEL, nimls neontlly 1 times bck-stroked nd exposed to novelty. No significnt differences were observed (2-wy NOV; significnce ccepted for p <.5) Novel rm discrimintion during Y-mze test Erly-life exposure to novelty following 1 bck-strokes significntly enhnced the dult nimls performnce in the Y-mze memory test (Fig. 3). nlysis of the difference score for the 1 st min nd 5min of Tril 2 (Fig. 3,B), gve n overll effect of tretment [1st min: F(2,53) = 5., p =.11; 5 min: F(2,54) = 9.33, p =.1], sex [1st min: F(1,53) = 4.59, p =.37; 5min: non significnt] nd tretment-sex interction[ F(2,53) = 3.62, p =.34; 5min: non significnt]. In generl, Novel nimls (1S-NOVEL) showed incresed bility to discriminte the novel rm in the Y-mze, s depicted by their higher difference scores. Novel mles hd higher score in the 1st nd 5min of Tril 2, compred to controls (p =.4 nd p =.29 respectively), but they did not differ significntly from the Home (1S-HOME) mles (p =.84 nd p =.22 for the 1st nd 5min respectively). On the other hnd, Novel femles were superior to both Home femles (p =.14 nd p =.8 for the 1 st nd 5min of Tril 2, respectively; Fig. 3, B) nd control femles in the 5min period (p =.45; Fig. 3B). Home femles were impired, s compred to control femles (p =.17) nd Home mles (p =.11) in the 1st min of Tril 2 (Fig. 3). Novel nimls of both sexes entered more often the novel thn the other rm, compred to Home nimls nd controls (Fig. 3C, D). More specificlly, during the 1 st

9 vritions of neontl hndling nd sptil memory 193 min (Fig. 3C) of testing Novel nd control nimls entered more often the novel thn the other rm (p =.6 for Novel mles; p =.45 for control mles; p =.36 for Novel femles nd p =.49 for control femles). In the whole 5min period (Fig. 3D), only Novel nimls still showed preference for the novel rm (p =.19 for Novel mles nd p =.1 for Novel femles). Home nimls did not show ny rm preference for novel or other rm during the test. Novel rm discrimintion B Difference score Difference score min MLE b b c CON 1S-HOME 1S-NOVEL 5 min MLE b b b c$ CON 1S-HOME 1S-NOVEL b C % ENTRIES ,3 2 CON 1S-HOME 1S-NOVEL MLE 1 min CON 1S-HOME 1S-NOVEL OTHER NOVEL D % ENTRIES ,3 2 CON 1S-HOME 1S-NOVEL CON 1S-HOME 1S-NOVEL MLE 5 min OTHER NOVEL CON 1S-HOME1S-NOVEL CON 1S-HOME 1S-NOVEL Figure 3. Novel rm discrimintion during Y-mze test in Experimentl Phse I. Men ± SEM of (, B) Difference score (% entries in Novel rm - % entries in Other rm) nd (C, D) of % entries in the Other nd Novel rm during the 1 st nd whole 5min period of Tril 2. CON, niml fcility rered controls, 1S-HOME, nimls neontlly 1 times bck-stroked in the home cge; 1S-NOVEL, nimls neontlly 1 times bck-stroked nd exposed to novelty. In (, B), different letters (one or combintion of two) over the brs denote significntly different groups within the sme sex (2-wy NOV nd LSD posthoc test). In (C, D), strs over horizontl lines denote significnt differences between rms for ech niml group (repeted 2-wy NOV nd pired t-tests). Doted lines define chnce level; significnce ccepted for p < Strtegy in Y-mze performnce Differences in exploring the strt rm nd the center of the pprtus were detected

10 194 mong groups in the 1 st min [NOV tretment effect: F(2,51):4.23, p=.21, sex: F(1,51):4.44, p=.41, tretment - sex F(2,51):7.71, p=.1 ; Fig. 4 ], but not the 5min period of Tril 2. Home mles visited the strt rm significntly less thn the Novel (p =.24) nd control (p =.1) mle groups nd the femle Home group (p =.1). No significnt differences were detected mong femle groups. Concerning the time spent in the center during the 1 st min [NOV tretment effect: F(2,55):3.357, p=.43 ; Fig. 4B], both Home nd Novel mles spent less time in the center thn controls (p =.1 nd p =.29, respectively) (Fig. 4B). No difference in this prmeter ws detected in femle groups. 6 MLE STRT RM (1 min) % ENTRIES 4 33,3 2 b $ b CON 1S-HOME 1S-NOVEL CON 1S-HOME 1S-NOVEL B % DURTION MLE b b CENTER (1 min) CON 1S-HOME 1S-NOVEL CON 1S-HOME 1S-NOVEL Figure 4. Strtegy in Y-mze performnce for nimls of Experimentl Phse I. Men ± SEM of () % entries in the Strt rm nd (B) % durtion in the Center of the pprtus during the 1st min of Tril 2. CON, niml fcility rered controls, 1S-HOME, nimls neontlly 1 times bck-stroked in the home cge; 1S-NOVEL, nimls neontlly 1 times bck-stroked nd exposed to novelty. Dotted line in () defines chnce level. Different letters over the brs denote significntly different groups within the sme sex (2-wy NOV nd Bonferroni posthoc test). Significnce ccepted for p < First choice Only mle rts of the Novel group showed trend (p=.65) in entering first the novel thn the other rm (Tble 2), but the sme trend ws found towrds the right rm compred to the left one (p=.65; Tble 2). Control mles entered first more the right rm (p=.21) compred to the left one, independently of its novelty (Tble 2). In the femles first choice there ws not specific novel-other or right-left preference.

11 vritions of neontl hndling nd sptil memory 195 Tble 2 First choice in Experimentl Phse I Experimentl group Novel rm s 1st choice Right rm s 1st choice CON mle 1S-HOME mle 1S-NOVEL mle CON femle 1S-HOME femle 1S-NOVEL femle 6.% (6/1) 8.% (8/1) 9.9% (1/11) b 46.2% (6/13) 66.7% (7/11) 44.4% (4/9) Note. Given re percentges of rts entering the novel rm or the right rm first fter leving the strt rm. First number in prentheses indictes the number of rts tht entered the novel or right rm first nd the second number indictes the totl number of rts tested. P<.5, compred 9.% (9/1) 2.% (2/1) 81.8% (9/11) b 61.5% (8/13) 33.3% (4/11) 77.8% (7/9) to chnce (5%); b P=.65, compred to chnce (5%). CON, niml fcility rered controls; 2S-HOME, nimls neontlly 2 times bckstroked in the home cge; 2S-NOVEL, nimls neontlly 2 times bck-stroked nd exposed to novelty. 3.2 Effect of neontl exposure to novelty following 2 bck-strokes on Y-mze performnce (Experimentl Phse II) Generl locomotion nd explortion behviour during Y-mze test ll nimls showed the sme motivtion nd bility to explore the Y-mze pprtus during the 1st min of Tril 2 (Fig. 5). n overll effect of tretment on the totl number of entries ws obtined for the whole 5min period of Tril 2 [F(2,43) = 5.65, p =.7]. Further nlysis per sex showed tht the effect of tretment ws mrginlly nonsignificnt in mles [F(2,21) = 3.47, p =.52], with 2 bck-strokes Novel (2S-NOVEL) mles showing trend for less totl entries versus controls (p =.55). No significnt differences were observed between Novel nd Home mles. In femles, n nlogous trend ws observed towrds reduced explortion versus control, which did not rech significnce Novel rm discrimintion during Y-mze test Erly novelty or home exposure in combintion with 2 bck-strokes significntly modified the nimls bility for sptil recognition memory in dulthood (Fig. 6). nlysis of the difference score for the 1 st min nd 5min of Tril 2, gve n overll effect of tretment [1st min: F(2,47) = 3.29, p =.47; 5min: F(2,45) = 6.22, p =.4 ]. In the 1st min, there ws trend of 2 bck-stroked Home (2S-HOME) nimls to outperform Novel (2S-NOVEL) nimls, which however did not rech significnce when further nlyzed per sex (Fig. 6). In the 5min period (Fig. 6B), mles 2S-Home significntly outperformed 2S-Novel (p =.21) nd control (p =.19) nimls of the sme sex (F(2,24) = 4.52, p =.23). n nlogous trend in 2S-Home femles did not rech significnce (p =.79). Home nimls of both sexes, s well s controls entered more often the novel

12 196 8 MLE Totl Entries (Tril 2) 1 min 6 ENTRIES 4 2 B 25 2 CON 2S-HOME 2S-NOVEL CON 2S-HOME 2S-NOVEL 5 min MLE ENTRIES CON 2S-HOME 2S-NOVEL CON 2S-HOME 2S-NOVEL Figure 5. Generl locomotion nd explortion behviour during Y-mze test in Experimentl Phse II. Men ± SEM of entries in ll rms of the Y-mze during the 1st nd whole 5 min period of Tril 2. CON, niml fcility rered controls, 2S-HOME, nimls neontlly 2 times bckstroked in the home cge; 2S-NOVEL, nimls neontlly 2 times bck-stroked nd exposed to novelty. No significnt differences were observed (2-wy NOV nd Bonferroni posthoc test; significnce ccepted for p <.5). thn the other rm during the 1st min of Tril 2 (p =.42,.45,.3 nd.49 for the comprisons between Other nd Novel rm in 2S-HOME mles, control mles, 2S-HOME femles nd control femles, respectively) (Fig. 6C). t the end of 5min, 2S-Home mles were still showing preference for visiting novel versus other rm (p =.44, Fig. 6D), while this preference ws lost over time in the femle 2S-Home nd control groups. 2S-Novel nimls of both sexes showed no preference for novel versus other rm in either time point Strtegy in Y-mze performnce No differences in exploring the strt rm were observed mong the groups of Phse II (dt not shown). Tretment [F(2,43) = 4.19, p =.23] nd sex [F(1,43) = 1.2, p =.3] significntly ffected the time spent in the center of the mze during the 1 st but not the whole 5min period of Tril 2 (Fig. 7). Further nlysis showed tht 2S-Home femles spent more time in the center compred to 2S-Novel femles (p =.46). In ddition, 2S-Novel femles spent less time in the center compred to 2S-Novel mles (p =.33).

13 vritions of neontl hndling nd sptil memory 197 Novel rm discrimintion Difference score MLE 1 min B C Difference score CON 1S-HOME 1S-NOVEL CON 1S-HOME 1S-NOVEL 5 min MLE b CON 2S-HOME 2S-NOVEL CON 2S-HOME 2S-NOVEL % ENTRIES 1 min MLE 6 OTHER NOVEL 4 33,3 2 D CON 2S-HOME 2S-NOVEL CON 2S-HOME2S-NOVEL % ENTRIES 5 min 6 MLE OTHER NOVEL 4 33,3 2 CON 2S-HOME 2S-NOVEL CON 2S-HOME2S-NOVEL Figure 6. Novel rm discrimintion during Y-mze test in Experimentl Phse II. Men ± SEM of (,B) Difference score (% entries in Novel rm - % entries in Other rm) nd (C,D) of % entries in the Other nd Novel rm during the 1st nd whole 5 min period of Tril 2. CON, niml fcility rered controls; 2S-HOME, nimls neontlly 2 times bck-stroked in the home cge; 2S-NOVEL, nimls neontlly 2 times bck-stroked nd exposed to novelty. In (,B), different letters (one or combintion of two) over the brs denote significntly different groups within the sme sex (2-wy NOV nd LSD posthoc test). No significnt differences were observed in the 1st min period for both sexes nd in the 5min period for femles. In (C,D), strs over horizontl lines denote significnt differences between rms for ech niml group (repeted 2-wy NOV nd pired t-tests). Doted lines define chnce level; Significnce ccepted for p < First choice No specific novel-other or right-left preference ws observed for the 2 bck- stroked mle groups (Tble 3). Control mles, s mentioned in Experimentl Phse I, entered first the right rm (p=.21) compred to the left one, independently of its novelty. In femle rts, the 2 bck-stroked Novel nimls showed preference for entering first

14 198 the right rm (p=.31) compred to the left (Tble 3), while no specific preference ws observed for the other femle groups. 4 MLE CENTER (1 min) % DURTION c bc$ CON 2S-HOME 2S-NOVEL CON 2S-HOME 2S-NOVEL Figure 7. Strtegy in Y-mze performnce for nimls of Experimentl Phse II. Men ± SEM of % durtion in the Center of the pprtus during the 1 st min of Tril 2. CON, niml fcility rered controls; 2S-HOME, nimls neontlly 2 times bck-stroked in the home cge; 2S-NOVEL, nimls neontlly 2 times bck-stroked nd exposed to novelty. Different letters (one or combintion of two) omit over the brs denote significntly different groups within the sme sex; $: denotes sex difference between sme tretment groups (2-wy NOV nd Bonferroni posthoc test; significnce ccepted for p <.5). Tble 3 First choice in Experimentl Phse II Experimentl group Novel rm s 1st choice Right rm s 1st choice CON mle 2S-HOME mle 2S-NOVEL mle CON femle 2S-HOME femle 2S-NOVEL femle 6.% (6/1) 75.% (6/8) 62.5% (5/8) 46.2% (6/13) 85.7% (6/7) 66.7% (4/6) 9.% (9/1) 75.% (6/8) 62.5% (5/8) 61.5% (8/13) 71.4% (5/7) 1.% (6/6) Note. Given re percentges of rts entering the novel rm or the right rm first fter leving the strt rm. First number in prentheses indictes the number of rts tht entered the novel or right rm first nd the second number indictes the totl number of rts tested. P<.5, compred to chnce (5%); b P=.65, compred to chnce (5%). CON, niml fcility rered controls; 2S-HOME, nimls neontlly 2 times bckstroked in the home cge; 2S-NOVEL, nimls neontlly 2 times bck-stroked nd exposed to novelty. 3.3 Bsl corticosterone levels (Experimentl Phse I & II) Erly-life exposure to novelty following 1 bck-strokes (Phse I) significntly ffected the nimls bsl corticosterone levels in dulthood in sexully dimorphic mnner (NOV tretment effect: F(2,53):6.15, p=.1, sex effect: F(1,53):32.44, p=.1; Fig. 8). Home (1S-HOME) mles nd Novel (1S-NOVEL) femles hd elevted corticosterone levels compred to sme sex controls (p =.43 nd.7, respectively).

15 vritions of neontl hndling nd sptil memory 199 No significnt differences were observed between Home nd Novel nimls of either sex. ll femle groups hd higher corticosterone levels from the respective mle ones (p =.1,.29,.1 for control, 1S-HOME nd 1S-NOVEL, respectively). Bsl corticosterone levels B Corticosterone (ng/ml) 6 MLE b$ 4 b$ b $ b 2 CON 1S-HOME 1S-NOVEL CON 1S-HOME 1S-NOVEL Corticosterone (ng/ml) MLE b$ b b $ CON 2S-HOME 2S-NOVEL CON 2S-HOME 2S-NOVEL Figure 8. Bsl corticosterone levels. Men ± SEM of plsm corticosterone levels in nimls from Experimentl Phse I () nd II (B) determined one week fter Y-mze test. CON, niml fcility rered controls; 1S-HOME, nimls neontlly 1 times bck-stroked in the home cge; 1S-NOVEL, nimls neontlly 1 times bck-stroked nd exposed to novelty; 2S-HOME, nimls neontlly 2 times bck-stroked in the home cge; 2S-NOVEL, nimls neontlly 2 times bckstroked nd exposed to novelty. Different letters (one or combintion of two) omit over the brs denote significntly different groups within the sme sex; $: denotes sex differences between sme tretment groups (2-wy NOV nd Bonferroni posthoc test; Significnce ccepted for p <.5). Exposure to novelty following 2 bck-strokes (Phse II) reversed the corticosterone pttern observed in experimentl groups of Phse I (NOV tretment effect: F(2,39):4.1, p=.26, sex effect: F(1,39):31.38, p=.1, tretment - sex interction: : F(2,39):4.22, p=.23 ; Fig. 8B). Novel (2S-NOVEL) mles hd elevted corticosterone levels, s compred both to control nd Home (2S-HOME) mles (p <.1 for both). Corticosterone levels of 2S-Novel mles did not differ from 2S-Novel femles. 2S-Home femles hd higher corticosterone levels thn femle control (p =.49), s well s, thn 2S-HOME mles (p <.1). Finlly, control femles hd higher corticosterone levels thn control mles (p =.1).

16 2 4. Discussion The beneficil effects of neontl hndling, comprised of brief mternl seprtion, rtificil tctile stimultion of the pup nd exposure to novelty [6], on dult brin function, hve extensively been studied in rodents. However, the effects of the individul components of hndling nd their possible interction hve not been exmined in detil. This study imed to exmine whether the mount of novelty exposure nd tctile stimultion, during the hndling procedure, is reflected in lter behviour nd circulting corticosterone levels. To diminish the effects of mternl cre we dopted split litter design, previously used to study the effects of novelty exposure in dult behviour nd physiology: hlf of the pups styed in the home cge nd the other hlf in novel cge [18]. 4.1 Sptil memory: inverted U-shped curve effect of mild erly-life mnipultions The min finding of the present study is tht novelty in the hndling prdigm enhnces dult sptil recognition memory, nd tht the mount of tctile stimultion in the form of bck-stroking cn modify this effect. 1 times bck-stroking llows the beneficil effect of novelty on memory performnce, while 2 times bck-stroking countercts this effect. Interestingly, 2 times bck-stroking hd n enhncing effect on memory of dult rts tht styed during neontl hndling in their home cge, denoting tht the overll mount of stimultion (novelty nd stroking), received by the pups during the hndling procedure, ppers to be criticl for the dult outcome. miniml mount of neontl stimultion, s well s over-stimultion, were disdvntgeous for sptil recognition memory in dulthood. The bove dt suggest the existence of n inverted U-shped curve effect of mild erly mnipultions, such s novelty nd tctile stimultion, on dult sptil performnce. In previous studies it ws shown tht novelty exposure enhnces the wter mze performnce of mle rts, s compred to their home-cge siblings receiving the sme mount of hndling [18, 19]. The Y-mze dt of Phse I re in complince with this finding. We hve further shown tht the erly novelty experience hs the sme beneficil effect on femle sptil recognition memory, nd is superior in comprison to miniml neontl stimultion (niml fcility rering). The superiority of 1S-Novel nimls in Y-mze tsk is not due to chnges in their generl locomotion ctivity, s the number of totl entries ws similr in ll niml groups. Erly exposure to novelty hs been shown to enhnce LTP in the C1 re of the hippocmpus in dulthood [2] nd to reduce emotionl rectivity in the open field [18]. These findings could ccount for the better performnce of 1S-Novel nimls in the Y mze test. Furthermore, it ws reported [19] tht mle rts exposed to novelty cn mount n dditionl corticosterone response to n unexpected event. These nimls correspond to the 1S-Novel mles in our study, nd their presumed more effective HP-xis coping could hve helped them

17 vritions of neontl hndling nd sptil memory 21 to outperform 1S-Home in the Y-mze test. 4.2 Endocrine correltes The HP-xis of nimls exposed to novelty hs been suggested [19, 35] to be similr to tht of the hndled rts. However, study compring directly these two mnipultions is missing. The only neuroendocrine evidence so fr concerns corticosterone levels. In our study, 3 month old mle 1S-Home nd 1S-Novel rts did not differ in circulting corticosterone levels, in greement with previous reports [19]. Compred to non-hndled controls, only 1S-Home mles hd higher corticosterone levels, in complince with the previously reported enhnced emotionl rectivity of 1S-Home mles versus 1S-Novel (Tng, 21). Notbly, dditionl stimultion reversed the sitution, with 2S-Novel mles hving higher corticosterone levels from both 2S-Home nd non-hndled controls. This increse in bsl hormone levels suggests higher impct of erly-life mnipultions for this group t the level of HPxis s well. n opposite effect of novelty ws observed in femles, which ws gin reversed by dditionl bck-stroking. The pttern in femles implies more sensitive HP-xis response in the first plce (1S-Novel femles versus 1S-Home), which is not further responding fter dditionl stroking (2S-Home nd 2S-Novel did not differ in hormone levels). 4.3 Gender differences on corticosterone nd sptil performnce Bsl corticosterone levels of dult controls were higher in femles, s expected [36]. In mles, higher hormone levels coincided with poor memory performnce (groups 1S-HOME nd 2S-NOVEL), while in femles incresed corticosterone levels were in prllel with enhnced sptil memory (groups 1S-NOVEL nd 2S-HOME). lthough in our study no direct correltion cn be mde between corticosterone levels nd nimls performnce, s we hve determined the hormone one week following Y-mze, previous studies hve shown tht stress levels of corticosterone impir sptil recognition memory in mle rts nd fcilitte it in femles. [37, 38]. The reson for this dimorphism is not cler. This my be due to counterction exerted by the higher estrogen levels of femles to the impiring effects of corticosterone in sptil memory. The effect of erly novelty exposure on dult sptil recognition memory ws generlly the sme for both sexes under the sme tretment. Sex differences were observed in the behviorl strtegy used during the memory test (s those concerning the visits of the strt rm or the center of the pprtus). Studies on the influence of estrogens on memory give equivocl results, with some studies showing improvement, impirment or no effect [for review, see [39, 4]]. The estrus phse of femle rts cn contribute to their cognitive performnce depending on the tsk used nd the context of lerning. For exmple, lthough it hs been reported tht stressed femles t proestrus differ in their performnce in the Y-mze tsk from those in estrus [41], this hs not been observed for non-stressed femle nimls [37]. In the Y-mze tsk, control

18 22 mles nd femles (t different estrus phses) do not show significnt differences in performnce [38]. In the current study, we used mixed popultion of intct femles, rndomly ssigned s to the estrus cycle nd we found no significnt differences in memory performnce of the control nimls, in greement with the previous findings. 4.4 Mternl cre lthough we used split litter design, our results do not completely exclude the contribution of mternl cre. In the clssicl hndling prdigm, hndled nimls receive greter mounts of mternl cre t the reunion thn the non-hndled [17, 42]. In the novelty prdigm, it ws proposed tht the mothers were retrieving more the home-stying pups [19]. However, this mternl cre difference ws not found by others [43] nd ws not confirmed in following study [44]. dditionlly, Tng s experimentl litters did not hve n equl sex distribution nd this could be confounding fctor, s it hs been shown tht dms differentilly cre for their mle or femle pups [45, 46]. 5. Conclusion In conclusion, this study provided evidence tht the nture nd intensity of the individul components of mild erly-life mnipultion, like hndling, ffect outcome in spects of sptil memory nd bsl drenocorticl function of dult rts. Trnsient vritions in erly-life experience could thus led to behviourl vrition or behviourl plsticity of the response towrds lter-life event. 6. cknowledgment This reserch ws supported by grnt 7/3/8372 of the Greek Generl Secretrit of Reserch & Technology. 7. References [1] Chmpgne, D. L., Ronld de Kloet, E., Joels, M. Fundmentl spects of the impct of glucocorticoids on the (immture) brin. Seminrs in fetl & neontl medicine. 29,14: [2] de Kloet, E. R., Sibug, R. M., Helmerhorst, F. M., Schmidt, M. V. Stress, genes nd the mechnism of progrmming the brin for lter life. Neuroscience nd biobehviorl reviews. 25,29: [3] Lehmnn, J., Pryce, C. R., Jongen-Relo,. L., Stohr, T., Pothuizen, H. H., Feldon, J. Comprison of mternl seprtion nd erly hndling in terms of their neurobehviorl effects in ged rts. Neurobiology of ging. 22,23: [4] Plotsky, P. M., Thrivikrmn, K. V., Nemeroff, C. B., Cldji, C., Shrm, S., Meney, M. J. Long-term consequences of neontl rering on centrl corticotropin-relesing fctor systems in dult mle rt offspring. Neuropsychophrmcology. 25,3: [5] DeNelsky, G. Y., Denenberg, V. H. Infntile stimultion nd dult explortory behviour in the rt: effects of hndling upon visul vritionseeking. niml behviour. 1967,15: [6] Levine, S. Infntile experience nd resistnce to physiologicl stress. Science (New York, N.Y. 1957,126:45. [7] Meney, M. J., itken, D. H., Bodnoff, S. R., Iny, L. J., Spolsky, R. M. The effects of postntl hndling on the development of the glucocorticoid receptor systems nd stress recovery in the rt. Progress in neuro-psychophrmcology & biologicl psychitry. 1985,9:731-4.

19 vritions of neontl hndling nd sptil memory 23 [8] Nunez, J. F., Ferre, P., Escorihuel, R. M., Toben,., Fernndez-Teruel,. Effects of postntl hndling of rts on emotionl, HP-xis, nd prolctin rectivity to novelty nd conflict. Physiology & behvior. 1996,6: [9] Levine, S., Hltmeyer, G. C., Krs, G. G., Denenberg, V. H. Physiologicl nd behviorl effects of infntile stimultion. Physiology & behvior :5. [1] Meerlo, P., Horvth, K. M., Ngy, G. M., Bohus, B., Koolhs, J. M. The influence of postntl hndling on dult neuroendocrine nd behviourl stress rectivity. Journl of neuroendocrinology. 1999,11: [11] Meney, M. J., itken, D. H. The effects of erly postntl hndling on hippocmpl glucocorticoid receptor concentrtions: temporl prmeters. Brin reserch. 1985,354:31-4. [12] Meney, M. J., Diorio, J., Frncis, D., LRocque, S., O'Donnell, D., Smythe, J. W., et l. Environmentl regultion of the development of glucocorticoid receptor systems in the rt forebrin. The role of serotonin. nnls of the New York cdemy of Sciences. 1994,746:26-73; discussion 74, [13] Viu, V., Shrm, S., Plotsky, P. M., Meney, M. J. Incresed plsm CTH responses to stress in nonhndled compred with hndled rts require bsl levels of corticosterone nd re ssocited with incresed levels of CTH secretgogues in the medin eminence. J Neurosci. 1993,13: [14] Kosten, T.., Lee, H. J., Kim, J. J. Neontl hndling lters lerning in dult mle nd femle rts in tsk-specific mnner. Brin reserch. 27,1154: [15] Stmtkis,., Pondiki, S., Kitrki, E., Dimntopoulou,., Pngiotropoulos, T., Rftoginni,., et l. Effect of neontl hndling on dult rt sptil lerning nd memory following cute stress. Stress (msterdm, Netherlnds). 28,11: [16] Villescs, R., Bell, R. W., Wright, L., Kufner, M. Effect of hndling on mternl behvior following return of pups to the nest. Developmentl psychobiology. 1977,1: [17] Pryce, C. R., Bettschen, D., Feldon, J. Comprison of the effects of erly hndling nd erly deprivtion on mternl cre in the rt. Developmentl psychobiology. 21,38: [18] Tng,. C. Neontl exposure to novel environment enhnces hippocmpl-dependent memory function during infncy nd dulthood. Lerning & memory (Cold Spring Hrbor, N.Y. 21,8: [19] Tng,. C., kers, K. G., Reeb, B. C., Romeo, R. D., McEwen, B. S. Progrmming socil, cognitive, nd neuroendocrine development by erly exposure to novelty. Proceedings of the Ntionl cdemy of Sciences of the United Sttes of meric. 26,13: [2] Tng,. C., Zou, B. Neontl exposure to novelty enhnces long-term potentition in C1 of the rt hippocmpus. Hippocmpus. 22,12: [21] Benetti, F., ndrde de rujo, P., Snvitto, G. L., Lucion,. B. Effects of neontl novelty exposure on sexul behvior, fer, nd stress-response in dult rts. Developmentl psychobiology. 27,49: [22] Rees, S. L., Steiner, M., Fleming,. S. Erly deprivtion, but not mternl seprtion, ttenutes rise in corticosterone levels fter exposure to novel environment in both juvenile nd dult femle rts. Behviourl brin reserch. 26,175: [23] Pryce, C. R., Bettschen, D., Bhr, N. I., Feldon, J. Comprison of the effects of infnt hndling, isoltion, nd nonhndling on coustic strtle, prepulse inhibition, locomotion, nd HP ctivity in the dult rt. Behviorl neuroscience. 21,115: [24] Puk, J., Kuhn, C. M., Field, T. M., Schnberg, S. M. Positive effects of tctile versus kinesthetic or vestibulr stimultion on neuroendocrine nd ODC ctivity in mternlly-deprived rt pups. Life sciences. 1986,39: [25] vn Oers, H. J., de Kloet, E. R., Wheln, T., Levine, S. Mternl deprivtion effect on the infnt's neurl stress mrkers is reversed by tctile stimultion nd feeding but not by suppressing corticosterone. J Neurosci. 1998,18: [26] Suchecki, D., Rosenfeld, P., Levine, S. Mternl regultion of the hypothlmic-pituitry-drenl xis in the infnt rt: the roles of feeding nd stroking. Brin Res Dev Brin Res. 1993,75: [27] Schpiro, S., Vukovich, K. R. Erly experience effects upon corticl dendrites: proposed model for development. Science (New York, N.Y. 197,167: [28] Zhng, M., Ci, J. X. Effects of neontl tctile stimultion nd mternl seprtion on the nxiety nd the emotionl memory in dult femle rts. Zoologicl Reserch. 26,27:7. [29] Zhng, M., Ci, J. X. Neontl tctile stimultion enhnces sptil working memory, prefrontl longterm potentition, nd D1 receptor ctivtion in dult rts. Neurobiology of lerning nd memory. 28,89: [3] Imnk,., Morinobu, S., Toki, S., Ymmoto, S., Mtsuki,., Kozuru, T., et l. Neontl tctile stimultion reverses the effect of neontl isoltion on open-field nd nxiety-like behvior, nd pin sensitivity in mle nd femle dult Sprgue-Dwley rts. Behviourl brin reserch. 28,186:91-7. [31] Conrd, C. D., Gle, L.., Kurod, Y., McEwen, B. S. Chronic stress impirs rt sptil memory on the Y mze, nd this effect is blocked by tineptine pretretment. Behviorl neuroscience. 1996,11: [32] Dellu, F., Myo, W., Cherkoui, J., Le Mol, M., Simon, H. two-tril memory tsk with utomted recording: study in young nd ged rts. Brin reserch. 1992,588: [33] Spruijt, B. M., Gispen, W. H. Behviorl sequences s n esily quntifible prmeter in experimentl studies. Physiology & behvior.

20 ,32:77-1. [34] Conrd, C. D., Lupien, S. J., Thnsoulis, L. C., McEwen, B. S. The effects of type I nd type II corticosteroid receptor gonists on explortory behvior nd sptil memory in the Y-mze. Brin reserch. 1997,759: [35] Tng,. C., Reeb, B. C., Romeo, R. D., McEwen, B. S. Modifiction of socil memory, hypothlmicpituitry-drenl xis, nd brin symmetry by neontl novelty exposure. J Neurosci. 23,23: [36] tkinson, H. C., Wddell, B. J. Circdin vrition in bsl plsm corticosterone nd drenocorticotropin in the rt: sexul dimorphism nd chnges cross the estrous cycle. Endocrinology. 1997,138: [37] Conrd, C. D., Jckson, J. L., Wieczorek, L., Brn, S. E., Hrmn, J. S., Wright, R. L., et l. cute stress impirs sptil memory in mle but not femle rts: influence of estrous cycle. Phrmcology, biochemistry, nd behvior. 24,78: [38] Conrd, C. D., Grote, K.., Hobbs, R. J., Feryorni,. Sex differences in sptil nd nonsptil Y-mze performnce fter chronic stress. Neurobiology of lerning nd memory. 23,79:32-4. [39] Pckrd, M. G. Posttrining estrogen nd memory modultion. Hormones nd behvior. 1998,34: [4] Willims, C. L., Meck, W. H. The orgniztionl effects of gondl steroids on sexully dimorphic sptil bility. Psychoneuroendocrinology. 1991,16: [41] Korol, D. L., Kolo, L. L. Estrogen-induced chnges in plce nd response lerning in young dult femle rts. Behviorl neuroscience. 22,116: [42] Liu, D., Diorio, J., Tnnenbum, B., Cldji, C., Frncis, D., Freedmn,., et l. Mternl cre, hippocmpl glucocorticoid receptors, nd hypothlmic-pituitry-drenl responses to stress. Science (New York, N.Y. 1997,277: [43] Mcri, S., Mson, G. J., Wurbel, H. Dissocition in the effects of neontl mternl seprtions on mternl cre nd the offspring's HP nd fer responses in rts. The Europen journl of neuroscience. 24,2: [44] kers, K. G., Yng, Z., DelVecchio, D. P., Reeb, B. C., Romeo, R. D., McEwen, B. S., et l. Socil competitiveness nd plsticity of neuroendocrine function in old ge: influence of neontl novelty exposure nd mternl cre relibility. PLoS ONE. 28,3:e284. [45] Moore, C. L., Morelli, G.. Mother rts interct differently with mle nd femle offspring. Journl of comprtive nd physiologicl psychology. 1979,93: [46] Clessens, S., Dsklkis, N., Oitzl, M. S., De Kloet, E. R., Chmpgne, D. Development of individul differences: Impct of erly-life experience. FENS bstr. 28,4:59.6.