Anxiety-Like, Novelty-Seeking and Memory/Learning Behavioral Traits in Male Wistar Rats Submitted to Early Weaning

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Physiology & Behavior 124 (2014) 100–106

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Physiology & Behavior

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Anxiety-like, novelty-seeking and memory/learning behavioral traits in male Wistar rats submitted to early weaning

Mabel Carneiro Fraga a,b, Egberto Gaspar de Moura b, Natália da Silva Lima b, Patrícia C. Lisboa b, Elaine de Oliveira b, Juliana Oliveira Silva a, Sylvio Claudio-Neto a, Cláudio C. Filgueiras a, Yael Abreu-Villaça a,AlexC.Manhãesa,⁎

a Laboratório de Neuroﬁsiologia, Departamento de Ciências Fisiológicas, Instituto de Biologia Roberto Alcantara Gomes, Centro Biomédico, Universidade do Estado do Rio de Janeiro, Brazil b Laboratório de Fisiologia Endócrina, Departamento de Ciências Fisiológicas, Instituto de Biologia Roberto Alcantara Gomes, Centro Biomédico, Universidade do Estado do Rio de Janeiro, Brazil

HIGHLIGHTS

• The breastfeeding period has been shortening progressively. • Models of early weaning show that adult rats present several endocrine alterations. • Maternal deprivation and pharmacological early weaning affect behavior in adult rats. • Early weaning by wrapping dams with a bandage does not affect behavior at adulthood. • Hypercorticosteronemia at early weaning may be a relevant imprinting factor.

article info abstract

Article history: The most frequently used animal models of early weaning (EW) in rodents, maternal deprivation and pharma- Received 1 July 2013 cological inhibition of lactation, present confounding factors, such as high stress or drug side effects, that can Received in revised form 25 October 2013 mask or interact with the effects of milk deprivation per se. Given these limitations, the development of new Accepted 6 November 2013 models of EW may provide useful information regarding the impact of a shortened period of breastfeeding on the endocrine and nervous systems, both during development and at adulthood. Using a model of EW in Keywords: which lactating Wistar rat dams are wrapped with a bandage to block access to milk during the last three days Early weaning Anxiety of lactation, we have recently shown that the adult offspring presented higher body mass, hyperphagia, Novelty-seeking hyperleptinemia, leptin as well as insulin resistance, and higher adrenal catecholamine content at adulthood. Memory Here, we used this EW model, which involves no pharmacological treatment or maternal separation, to analyze Learning anxiety-like, novelty-seeking and memory/learning behavioral traits in the adult male offspring. To that end, an- Motor activity imals were tested in the elevated plus maze, in the hole board arena and in the radial arm water maze. Except for an increased number of rearing events (a measure of vertical activity), no other behavioral differences were observed between EW and control animals. The contrasting behavioral results between the three EW models may be associated with differences in HPA axis function in the offspring at weaning, since it has been observed that bandaging does not affect corticosteronemia while maternal separation and pharmacological EW increase it. © 2013 Elsevier Inc. All rights reserved.

1. Introduction long-term chronic diseases, such as obesity and diabetes [33].Because breast milk mediates unequaled beneficial effects regarding nutritional, According to the World Health Organization (WHO), “exclusive con- immunological and cognitive outcomes in infants, a global public health sumption of breast milk and no other food or drink, not even water for recommendation is that infants should be solely breastfed for the first 6 - 6 months of life” is defined as exclusive breastfeeding [88].Exclusive months of life [45,46]. On the other hand, women are currently becoming and prolonged breastfeeding has been associated with protection against more active in the labor market and, for many new mothers, the breastfeeding period has been shortening progressively [31].Infact,no more than 35% of infants worldwide are exclusively breastfed during ⁎ Corresponding author at: Laboratório de Neurofisiologia, Departamento de Ciências the first four months of life [88]. Fisiológicas, Instituto de Biologia Roberto Alcantara Gomes, Centro Biomédico, Universidade Epidemiological and experimental studies have shown that nutri- do Estado do Rio de Janeiro, Av. Prof. Manuel de Abreu 444, 5 andar, Vila Isabel, Rio de Janeiro, RJ 20550-170, Brazil. Tel.: +55 21 2868 8195; fax: +55 21 2868 8029. tional alterations during early periods of life can imprint epigenetic E-mail addresses: [email protected], [email protected] (A.C. Manhães). changes, which lead to disorders such as obesity, diabetes, hypertension

[3,19] and vulnerability to psychopathological disorders such as anxiety Helsinki and with the Guide for the Care and Use of Laboratory Animals and depression at adulthood [13]. This phenomenon, which was initially as adopted and promulgated by the National Institutes of Health, and denominated programming [3] and now is also known as developmen- followed the principles described in the guide for the care and use of tal plasticity [32],hasbeendefined as a biological phenomenon that de- laboratory animals [4]. All animals (Wistar rats) used in the present termines the relationship between physical and chemical stimuli during study were kept in a temperature-controlled (23 ± 1 °C) vivarium on critical periods of early life (gestation and lactation) with the future a 12 h light/dark cycle (lights on at 7:00 a.m.). functional status of the adult offspring. Experimental models that address the immediate, early and late- 2.1. Experimental model of programming by early weaning emerging repercussions of shortened lactation may be useful to assess its long-lasting effects on the nutritional, metabolic, endocrine and behav- Three-month-old, nulliparous female rats were caged with male rats ioralstatusoftheprogeny.DosSantosetal.[23] observed, for example, (3:1). After mating, each female was placed in an individual cage with that animals weaned at postnatal day (PN) 15 by physical separation free access to food (standard chow for rodents, Nuvilab CR1 — São from the progenitors are more likely to prefer high-fat diets when com- Paulo, SP, Brazil) and water until delivery. Pregnant rats in our vivarium pared to animals weaned at PN30, showing a behavioral programming usually have litters consisting of 10 to 12 pups. In order to avoid the caused by early weaning. Other behavioral changes associated with influence of litter size on the programming effect, we only used litters early weaning through maternal deprivation (MD) include higher anx- consisting of 10 pups and, at birth, litters were adjusted to 6 male iety [42,61], higher motor activity [43] and reduced cognitive perfor- pups per dam to maximize lactation performance [62]. mance [68]. In the 1st postnatal (PN) day, 20 lactating rats were randomly Since maternal deprivation can, by itself, be an imprinting factor, it is assigned to the two following groups: EW (early weaning, n = 10) – conceivable that its effects on the development of the offspring can dams were lightly anesthetized with thiopental (0.06 mg/ml/100 g) mask or interact with the effects of milk deprivation. In addition, it is a and wrapped with a breast bandage (physical barrier) to interrupt suck- less representative model of early weaning in humans since mothers ling during the last 3 days of the lactation period – from PN19 to PN21 do not usually separate completely from their babies, and when separa- [50,51]; and C (control, n = 10) — no barrier was used to avoid suck- tion is inevitable, a caregiver is frequently present. Thus, our laboratory ling. After weaning (PN21), all EW and C animals had free access to developed two different models of early weaning (EW) without mater- food and water. Siblings were kept together throughout the experiment nal deprivation. In the first EW model, lactation is interrupted during since single housing could influence behavior (cage size: 39 cm the last three days (PN19–PN21) of the lactation period through maternal long × 32 cm wide × 14 cm high) [81]. treatment with bromocriptine, a prolactin (PRL) inhibitor [6–8,18,28]. We have shown that, at weaning (PN21), the offspring presented 2.2. Behavioral studies hyperleptinemia [8], hypothyroidism and hypercorticosteronemia [28]. At adulthood, in addition to the development of alterations as- From PN160 to PN178, 32 EW and 38 C offspring were submitted to sociated with the metabolic syndrome (MS), such as obesity and in- the behavioral tests (pups per litter — EW: 3.2 ± 0.1; C: 3.8 ± 0.2). The sulin and leptin resistance [7], adult rats programmed by maternal following tests were used: 1) elevated plus-maze (EPM); 2) hole board hypoprolactinemia presented lower levels of serum TSH, T3 and T4, arena (HB); 3) radial arm water maze (RAWM). A full description of characterizing a central hypothyroidism [6], hypoadiponectinemia, these testing equipments and protocols can be found elsewhere hypoprolactinemia, hypercorticosteronemia and higher total adre- [27–30]. All testing sessions were performed between 1:00 p.m. and nal catecholamine content [18]. Moreover, we observed that mater- 6:00 p.m. in a sound attenuated room. All tests were video recorded nal hypoprolactinemia resulted in long lasting effects on behavioral and all behavioral analyses were carried out using the video images of parameters: The adult EW offspring displayed higher anxiety-like the tests. behavior and poorer memory/learning performance. No differences Anxiety-like behavior was assessed at PN160 in the EPM. Rats were were observed regarding novelty-seeking behavior [28].Inthesec- allowed 10 min to explore the maze. The number of entries and the ond model, EW is caused by a physical barrier — abreastbandage total time spent in open (respectively: Time OA and Entries OA) and that prevented pups from suckling from PN19 to PN21 [50,51].In closed (respectively: Time CA and Entries CA) arms and in the central previous reports, we have shown that, at weaning (PN21), the offspring area (Time CN and Entries CN) of the EPM were recorded. The percentage presented hypoleptinemia [51], hypothyroidism, but normal adrenal of open arms entries [%Entries OA: Entries OA / (Entries OA + Entries hormones [50]. At adulthood, these EW animals presented obesity, CA)] and the percentage of time spent in the open arms [%Time OA: hyperphagia, hypoadiponectinemia, hyperglycemia, leptin and insu- Time OA / (Time OA + Time CA)] were used as anxiety measures. In ad- lin resistance, hypoprolactinemia [51] and higher total adrenal cate- dition, Entries CA was used a measure of locomotor activity and the per- cholamine content [50]. centage of time spent in the center of the maze (%Time CN: time spent in Given our previous behavioral results in the experimental model in center divided by total time) was used as an independent measure of which EW was caused by maternal treatment with bromocriptine decision-making [55,69]. In addition, ethological measures such as [28], the aim of the present study was to analyze behavior in the adult head-dipping, grooming and return CA were analyzed [12,15,38]. offspring of dams in which breastfeeding was interrupted by a physical All animals were returned to their home cages after the EPM test, barrier, that is, lactation interruption that does not involve the use of where they remained until they were tested in the HB. The EPM pharmacological substances or maternal deprivation. Here we focused was cleaned with paper towels soaked in 50% ethanol and dried be- on anxiety-like and novelty-seeking behaviors, as well as on memory/ fore each trial. learning performance. Since we have already published the hormonal Novelty-seeking behavior was assessed at PN163 in the HB. Rats profile of the animals used in the current study [50,51], we will discuss were allowed 10 min to explore the arena. The total number of holes our behavioral results in light of the previously reported hormonal and explored (nose-pokes) by each animal in the center and in the periph- metabolic alterations. ery of the equipment (respectively CH and PH) was noted and used as a measure of exploratory activity [27– 30]. The %CH [CH / (CH + PH)] 2. Materials and methods was also calculated. By using a 4 × 4 grid of same-sized rectangles drawn on a transparent overlay positioned on top of the video-images This study was conducted under the institutional approval of the of the tests, it was possible to measure locomotor activity and anxiety- Universidade do Estado do Rio de Janeiro (protocol: CEUA/017/2009). like behavior [1]. The total number of rectangles crossed was used as a All experiments were carried out in accordance with the declaration of measure of locomotor activity. The number of rectangles crossed in 102 M.C. Fraga et al. / Physiology & Behavior 124 (2014) 100–106 the center of the arena and the time spent in this area were considered 4. Discussion measures of anxiety-like behavior [1]. Ethologically derived measures were also evaluated and included rearing, stretching, grooming and It is well known that events in early life can result in permanent fecal boli [1,12,28]. The HB was cleaned with paper towels soaked in reorganization or imprinting of physiological systems and that these 50% ethanol and dried before each test. All animals were returned to events could lead to long-lasting or late-emerging effects, including be- their home cages after the HB tests, where they remained until they havioral ones, both in humans [60,71] and in animal models [10,86,90]. were tested in the RAWM. In particular, experimental studies have suggested that the lactation pe- Memory and learning were assessed at PN167 in the RAWM. Rats riod is the most crucial phase during development for nutritional im- were tested for 5 consecutive days, 4 trials per day (inter-trial interval: printing in mammals [20,21,62,65,83]. Weaning is one of the most 15 min). Animals were allowed 2 min per trial to explore the maze and important events in the early mammalian life, and it corresponds to a find the hidden platform that was positioned at the end of one of the period during which the offspring gradually becomes nutritionally arms. If a given animal failed to find the platform during these 2 min, and behaviorally independent from their progenitors under natural it was gently guided to it, where it could stay for 20 s. For the first conditions [63]. 4 days, the escape platform was not moved from its initial position. As previously mentioned, the increased participation of women in the On the 5th day, the platform was moved to the arm located in the oppo- labor force has been associated with a reduced period of breastfeeding site side of the central arena. These probe trials on the last day of testing [31], a fact that makes the study of the consequences of early weaning were used to assess working memory when testing parameters were in animal models relevant. As a result, several experimental models of changed [27–30,37,80]. The following variables were used to assess per- early weaning have been developed. These models can be classified into formance in the RAWM [25,33]:1)Latencytofind the hidden platform three major groups depending on the strategy used to induce early on each trial of each testing day (LAT); 2) Latency to first arm entry weaning: 1) Physical separation from the progenitors [34,35,38,68,79]; (LATARM); 3) Number of first entries into any arm which didn't have 2) Interruption of lactation by the use of pharmacological agents the platform (reference memory errors — RM); 4) Number of re-entries [2,6–9,18,28,87]; 3) Interruption of suckling by a physical barrier [50,51]. into the arm that had the platform (working memory correct errors — All three strategies are known to cause significant metabolic and WMC); 5) Number of re-entries into any arm that did not have the plat- endocrine alterations in the young as well as in the adult offspring form (working memory incorrect errors — WMI). The pool was cleaned [6–8,50,51,58,76]. However, behavioral alterations were only present (bedding residue and fecal boli were removed) and the water thoroughly when early weaning was caused by maternal deprivation (MD) or mixed between tests. The water was completely changed every day. use of pharmacological agents [28,40,41,54,74].Inrodents,MDre- sults in higher anxiety levels both during adolescence [61] and at adulthood [40,41,74] associated with enhanced corticosterone se- 2.3. Statistical analysis cretion [76] and tachycardia [35]. This association led to the sugges- tion that this model of EW could be in fact useful in assessing the Kolmogorov–Smirnov one-sample tests (K–S) were used to assess psychoneuroimmunoendocrine interactions underlying the long- the normality of the distributions of each of the variables. Significance lasting consequences of early life stress [70,78,85,89]. In addition, was assumed at the level of P b 0.05. Differences between EW and C Lomanowska et al. [54] have shown that MD increases motor activity, groups regarding anxiety, novelty-seeking and ethological measures while other studies demonstrated that it reduces the number of rearing were analyzed by means of multivariate analyses of variance (mANOVA). events during childhood as well as during adolescence [25,85]. Regard- GROUP (EW or C) was used as the between-subjects factor. Differences ing the pharmacological approach, we have recently shown that EW by between the EW and C groups regarding latency to find the hidden plat- bromocriptine treatment resulted in poorer memory/learning perfor- form (LAT) in the RAWM were analyzed by means of a repeated measures mance and higher levels of anxiety in the adult offspring [28]. analysis of variance (rANOVA). DAY and TRIAL were considered the Prior to the present study, two different hypotheses were proposed within-subjects factors. TREATMENT (EW or C) was used as the to explain the altered behaviors at adulthood in early-weaned rats. between-subjects factor. The RAWM variables RM and WMI were The first one suggested that behavior was affected as a direct conse- analyzed using a multivariate ANOVA (mANOVA), while the LATARM quence of malnutrition on brain development and function [49,66,77]. and WMC variables were analyzed using the Mann–Whitney test The second hypothesis suggested that behavior was altered due to en- (M–W). docrine changes – either involving the hypothalamic–pituitary–adrenal (HPA)axisorthyroidhormones– that were observed at adulthood [30,67,73,82]. This latter possibility implies that the effects of early 3. Results weaning on brain function at adulthood are indirect, via the endocrine system. As indicated in Fig. 1A and B, EW had no long-term effect on %Time The bandaging procedure [50,51] used in the current study completely OA and on %Entries OA. Ethological measures in the EPM were also not blocks access to the milk produced by the lactating dams. Therefore, the affected (Table 1). No significant difference was observed regarding the pups must rely on their internal stores and must also eat the same number of nose-pokes in the HB (Fig. 1C). Conversely, the number of chow that is available to their progenitors in order to survive early rearing was increased in the EW group (F = 6.9, df = 1, P = 0.011) weaning. Although pups are already transitioning from milk to chow by (Fig. 1D). The other variables in the HB were also not affected by the PN19, their reliance on milk for nutrition is still significant, as indicated EW (Table 1). As for the RAWM, both groups showed a clear and similar by the reduction in body mass gain from PN19 onwards [51].Irrespective improvement in performance from the 1st to the 4th day (DAY effect: of the considerable somatic effects of EW by physically blocking suckling, F = 46.3, df = 2.1, P b 0.001) that is indicated by a progressive reduc- cognition and emotion at adulthood – as indicated by anxiety levels, tion in the time needed to find the hidden platform upon repeated test- novelty-seeking behavior and visuospatial memory/learning – were unaf- ing (Fig. 1E). As expected, both groups presented a marked increase in fected. Considering the behavioral results obtained with the three EW latency in the probe trial when compared to the previous (fourth) strategies, the present study seems to disprove the first hypothesis, indi- day. A significant TRIAL effect (F = 58.7, df = 2.5, P b 0.001) was cating that the altered behaviors that were observed in models involving present, indicating that animals in both groups were able to learn either maternal deprivation or pharmacological approaches are not the the new position of the platform by the end of the probe trial. No dif- result of the shortened lactation period per se. ferences were observed between groups regarding the other RAWM As for the second hypothesis, it has been consistently shown that all variables (Table 1). three models result in significant changes to endocrine and metabolic M.C. Fraga et al. / Physiology & Behavior 124 (2014) 100–106 103

A %TIME OA B %ENTRIES OA 10 20 9 18 8 16 7 14 6 12 5 10 4 8 3 %Time OA 6

2 %Entries OA 4 1 2 0 0 C EW C EW Group Group

C NOSE POKES D REARING 12 16 14 * 10 12 8 10 6 8 6 4 4 2 Number of events Number of events 2 0 0 C W C W

E LATENCY TO PLATFORM 250 200 150 100 Time (s) 50 0 1 2 3 4 5 Day C EW

Fig. 1. Anxiety-like behavior tested in the elevated plus maze (EPM). No differences were observed between early weaning (EW, n = 32) and control (CON n = 38) rats regarding %Time OA (A) and %Entries OA (B), indicating that anxiety levels were not affected by the EW. Novelty-seeking behavior, as measured by the number of nose pokes in the hole board (HB) arena. No difference was observed between early weaning (EW, n = 32) and control (CON n = 38) rats regarding the total number of nose pokes (C). The number of rearings was increased in the EW group when compared to CON rats, indicative of higher motor vertical activity (D). Memory/learning performance as measured by the latency to ﬁnd the hidden platform in the radial arm water maze (RAWM). No differences were observed between early weaning (EW, n = 32) and control (CON n = 38) rats regarding the latency to ﬁnd the hidden platform (E). Values are means ± SEM. *P b 0.05. parameters at adulthood. For instance, MD studies have indicated that content and hypoprolactinemia [18]. As for the physical barrier ap- the adult offspring presents lower leptin, adiponectin and testosterone proach, we have shown that adult rats present obesity, hyperphagia, levels [84], hypercorticosteronemia [76] and tachycardia [35].Regard- hyperleptinemia and leptin resistance, hypoadiponectinemia, hyper- ing the pharmacological approach to EW, we have shown that adult glycemia and insulin resistance and higher adrenal catecholamine con- rats present obesity, hyperleptinemia, leptin resistance [7], hypothy- tent [50,51]. Therefore, all three approaches have long-lasting or late- roidism [6], hypoadiponectinemia, dyslipidemia, hyperglycemia and in- emerging effects on HPA axis and thyroid activity, endocrine systems sulin resistance, hypercorticosteronemia, higher adrenal catecholamine that are known to be involved in cognitive function and emotional

Table 1 Additional behavioral data.

Elevated plus maze

Treatment Time OA Entries OA Entries CA %Time CN Head-dipping Grooming Return CA

Control 29.4 ± 6.3 s 2.5 ± 0.5 11.2 ± 0.8 27.3 ± 2.1 2.3 ± 0.3 4.6 ± 0.5 4.5 ± 0.4 Early weaning 26.6 ± 6.4 s 2.1 ± 0.4 11.8 ± 1.0 24.6 ± 2.4 1.9 ± 0.4 4.6 ± 0.4 4.5 ± 0.5

Hole board

Treatment Entries TT Entries CN@ %Entries CN@ Time CN@ Grooming Bolus Stretching

Control 88.8 ± 6.1 0 (2) 0 (1.8) 0 (2.7) s 8.0 ± 0.7 7.4 ± 0.8 1.7 ± 0.3 Early weaning 95.3 ± 5.5 0 (2) 0 (1.6) 0 (3.8) s 9.1 ± 1.0 8.2 ± 0.7 1.9 ± 0.3

Radial arm water maze

Treatment LATARM 1–4@ LATARM probe@ RM 1–4RMprobeWMC1–4@ WMC probe@ WMI 1–4@ WMI probe@

Control 2.0 (2) 0 (0) 34.1 ± 1.5 12.8 ± 0.7 1 (1) 0 (1) 9 (9) 7 (10) Early weaning 2.5 (2) 0 (0) 33.6 ± 2.4 13.9 ± 1.0 1 (3) 0 (1) 9 (13) 8 (9)

Radial arm water maze: 1–4 — indicates the data from the first 4 testing days (summated); Probe — refers to the 5th day of testing (probe trial); @ — indicates non-parametric data [median (interquartile range)]. 104 M.C. Fraga et al. / Physiology & Behavior 124 (2014) 100–106 response [30,67,73,82]. The absence of significant behavioral effects in preclude the possibility that such differences could be observed in either the current study indicates that the changes in endocrine function at younger or older animals. For instance, we have previously shown that adulthood are not sufficient to explain the altered behaviors observed EW animals present hypothyroidism at P21 while euthyroidism is ob- in the MD and pharmacological approaches. served by P180 [50]. Given the association between thyroid function There is one aspect in which the physical barrier approach markedly and behavior, a significant effect of EW on the behavior of infant or ado- differs from the MD and pharmacological ones that might help explain lescent animals is conceivable [82]. the differences in behavioral outcomes. While the MD and pharmaco- Of the three EW procedures discussed above, the physical barrier logical weaning procedures result in hypercorticosteronemia at PN21 one seems to be the procedure that has less problems modeling early [28,58], the offspring of bandaged dams have normal corticosterone weaning in humans. Even when the mother is not present or is not ca- levels [50]. Many researchers have shown that maternal care associated pable of nursing her child as a result of a medical condition, most infants or not with exclusive breastfeeding is essential for physiologic stability, have caregivers readily available to provide not only food (be it bottled neurobehavioral maturation, social–emotional development and cogni- breast milk, formula or other sources of nutrition), but also emotional tive growth [26]. Specifically, studies carried out in animal models sug- support. Our current data would seem to indicate that maternal care is gest that the quality of the mother–pup interactions, such as increased more relevant, by the end of the lactation period, than constant access maternal licking, grooming, and arched-back nursing, is an important to breast milk for the proper development of cognitive abilities and factor in maintaining a normal HPA-axis response in the offspring emotional responses. However, a considerable body of literature, both [48,53]. Thus, it is conceivable that poorer or absent maternal care in humans and in animal models — including the one used in the in early life causes significant stress, affecting HPA axis function dur- present study, demonstrates that an adequate breastfeeding period ing a critical period of development and, in turn, resulting in long- has both short- and long-term beneficial health effects, particularly lasting changes in the formation and function of brain circuits [5,13]. when considering the metabolic and endocrine profiles of the devel- For instance, it has been shown that hypercorticosteronemia at weaning oping and adult offspring. leads to reduced hippocampal plasticity and induces neuronal apoptosis [17]. 5. Conclusion While it is obvious that the MD approach implies in absent maternal care, and all its associated problems, it is not immediately apparent why In conclusion, our results indicate that EW with a physical barrier should the pharmacological approach result in poorer maternal care has modest, long lasting and selective behavioral effects, demonstrated since the dams are left with the offspring up to PN21. This last approach by increased vertical motor activity without changes in anxiety-like and requires maternal bromocriptine treatment during the last three days of novelty-seeking behavior or in memory/learning performance at adult- lactation. Bromocriptine is an agonist of the type 2 dopaminergic receptor hood. We have shown that precocious weaning induced by physical that, when administered to the dams, inhibits prolactin (PRL) synthesis in blockage of milk ejection does not have the same long lasting effects fi the pituitary gland [59,72]. This treatment signi cantly reduces prolactin on behavioral traits that early weaning caused by pharmacological levels in the dams, which cease to produce milk as a result [8,18].Both agents and maternal deprivation have. Taken together, the results ob- dopaminergic function and prolactin levels have been shown to affect tained with the three EW models suggest that hypercorticosteronemia maternal care [44,57]. For example, low maternal prolactin levels during must be present at weaning to significantly affect behavior. pregnancy are associated with impaired maternal behavior and increased anxiety-levels in the adult offspring [47]. In addition, altered dopami- Declarations of interest nergic function reduces the expression of maternal behavior such as approach, retrieving, grouping and crouching over pups [11,22], which The authors declare no conflict of interest. were shown to affect, for example, spatial learning/memory performance [36,52]. An additional explanation for the altered behavioral results associated with the pharmacological approach resides on the fact Funding that bromocriptine is a lipophilic molecule, which raises the possibility that this prolactin inhibitor can be transferred through the milk to the This work was supported by grants from: Fundação Carlos Chagas pups and directly affect their behavior [14]. However, this possibility Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), is rendered unlikely due to the fact that prolactin levels in P21 pups Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) are normal, which seem to indicate that the amount of bromocriptine and Sub-reitoria de Pós-graduação e Pesquisa da Universidade do Estado that reaches the pups is not sufficient to induce physiological changes do Rio de Janeiro (SR2-UERJ). Authors ACM, CCF, EGM, PCB and YAV re- [18]. ceived fellowships from CNPq. Author MCF received a fellowship from Finally, we observed an increased number of rearing events in the FAPERJ. Authors NSL, JOS and SCN received scholarships from CNPq. Au- HB as a result of EW in the current study. This behavior is considered thor SCN received a scholarship from Coordenação de Aperfeiçoamento a measure of motor activity [64] and it was not present in the pharma- de Pessoal de Nível Superior (CAPES). cological EW model [28]. At this point, it is difficult to reconcile this ethological result with the absence of differences regarding the number Acknowledgments of nose-pokes in the HB, which is considered a measure of novelty- seeking behavior. However, in agreement with our findings, Koike Authors are grateful to Miss Monica Moura for technical assistance et al. [43] showed that higher motor activity could be induced by EW. and Ulisses Risso Siqueira for animal care. 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