Physiology & Behavior 93 (2008) 395 407

Variations in the postnatal maternal environment in mice: Effects on maternal behaviour and behavioural and endocrine responses in the adult offspring
Laurence Coutellier a , Anne-Christin Friedrich a , Klaus Failing b , Hanno Wrbel a,
a

Division of Animal Welfare and Ethology, Institute of Veterinary-Physiology, University of Giessen, Frankfurter Strasse 104, D-35392 Giessen, Germany b Division of Biomathematics and Data Processing, University of Giessen, Frankfurter Strasse 95, D-35392 Giessen, Germany Received 1 May 2007; received in revised form 30 August 2007; accepted 19 September 2007

Abstract According to the maternal mediation hypothesis, brain and behavioural development in rodents is affected by environment-dependent variations in maternal care. Thus, it has been shown that early handling results in reduced behavioural and neuroendocrine responses to stressors and that these effects are associated with increased maternal care received during infancy. To investigate this further in mice, we chose a less artificial paradigm that is not confounded by human manipulation and reflects a more natural form of early environmental variation. We housed lactating C57BL/6 dams and their litters in cage systems composed of a nest cage (NC) and a foraging cage (FC) connected by a tunnel, and varied the dams' access to food by providing food either in the NC (NC dams) or FC (FC dams) until postnatal day 14. FC dams were more frequently observed in the FC than NC dams, and although the frequency of the dams being in physical contact with the pups did not differ between the two treatments, FC dams showed lower levels of active nursing than NC dams during the first week of lactation. These environment-dependent variations in maternal behaviour had sex-specific effects on the adult offspring's behavioural and HPA responses to stressors and altered their social behaviour in the home cage, with NC offspring showing higher levels of socio-positive behaviours than FC offspring. These results provide further independent evidence for the maternal mediation hypothesis and demonstrate that even subtle variations of the maternal environment can affect maternal care and induce persistent changes in offspring phenotype. 2007 Elsevier Inc. All rights reserved.
Keywords: Postnatal manipulations; Maternal care; Stress reactivity; HPA-axis; Social behaviour; Fearfulness; Rodents; Mice; C57BL/6

1. Introduction During the early postnatal development, the brains of both human and non-human mammals are still highly plastic. Consequently, many physiological and behavioural traits are strongly modulated by early postnatal environmental effects on brain development. For example, the capacity to cope with environmental challenge critically depends on stress reactivity and behavioural and social skills that have all been shown to be affected by early experience [15]. Since the earliest account by Levine et al. [1] exactly 50 years ago, the effects of early experience on brain development and the capacity to cope with environmental challenge later in life have been extensively studied in rats.
Corresponding author. Tel.: +49 641 99 38 750; fax: +49 641 99 38 759. E-mail address: hanno.wuerbel@vetmed.uni-giessen.de (H. Wrbel). 0031-9384/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2007.09.008

In rats, the early environment is essentially determined by the mother. The dam represents not only a source of nutrition, care and warmth, but is also a primary source of information about the offspring's future environment. Considering that infancy is characterised by high plasticity of the central nervous system and intense motherinfant interaction, it has been postulated that the pups adaptively adjust their neural systems in response to environment-dependent variations in maternal behaviour [6 8]. Although studies in the wild are lacking, indirect evidence for this hypothesis stems from research showing that postnatal manipulations of motheroffspring contact can persistently alter e.g. hypothalamuspituitaryadrenal (HPA) stress reactivity [9,10], behavioural fearfulness [7,1012] and social behaviour [4,5,13]. More recently, studies in rats indicated that variations in the maternal environment can induce variations in maternal behaviour [14,15] that are associated with changes in the offspring's later behavioural and neuroendocrine responses

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to environmental and social challenges [7,13,16]. Thus, early handling, i.e. brief (315 min) daily separations of dam and pups during the first one or two weeks of life, stimulates active maternal care (licking/grooming and arched-back nursing) [6,12], resulting in a downregulation of the HPA-stress and fear systems in the adult offspring [17,18]. Similar findings were obtained when motheroffspring contact was varied by environmental variation rather than direct manipulation. Thus, even though dams with access to food away from the nest cage spent more time away from their pups than dams with access to food inside the nest cage, they displayed a more active maternal style as indicated by longer nursing bouts, and their offspring showed reduced fearfulness and HPA-reactivity when adult [16]. In contrast, more adverse maternal environments such as maternal separation, i.e. long (34 h) daily separations of dam and pups, or restricted feeding have been shown to induce higher fearfulness and HPA-reactivity in the offspring, but these effects were independent of variations in maternal care [10,16]. Taken together, present evidence suggest that a moderately challenging maternal environment may downregulate behavioural fearfulness and HPA-reactivity in rats, and that this downregulation may be mediated by environment-dependent variations in maternal behaviour, whereas more adverse maternal environments may upregulate behavioural fearfulness and HPA-reactivity through the direct action of environmental adversity on brain development [16]. Due to the availability of genetically modified mouse strains, there is growing interest in using the mouse as a model organism in research, even in the behavioural neurosciences that have traditionally preferred the rat. Especially in view of dissecting the geneenvironment interactions that govern brain development, the mouse has become the favoured species [9]. Compared to rats, however, research in mice on the effects of early postnatal experience on brain and behavioural development is still in its infancy and has produced much more varied results. Consistent with the findings from studies in rats, recent studies in mice showed that maternal separation induced a prolonged corticosterone response to a stressor [9,19] and higher levels of anxiety and fear responses in the adult offspring in open-field and elevated-plus-maze tests [20], whereas early handling decreased anxiety-like behaviour in the elevated-plusmaze test [21] and attenuated learning impairments when tested in the Morris water-maze [22]. However, several other studies failed to replicate similar effects [e.g. [23,24]]. As already noted with respect to rats [25], the artificial and non-specific nature of postnatal manipulations and the confounding effect of the human manipulations are highly unsettling from a scientific point of view. Based on recent studies in rats [16], we therefore chose an experimental approach that allowed us to manipulate the postnatal maternal environment in a more natural and ecologically more relevant way, without direct human manipulation, to study environment-dependent maternal effects on early developmental plasticity of HPA-stress reactivity and behaviour in mice. We used a cage system composed of a nest cage and a foraging cage connected by a tunnel similar to the one used in rats [16]. To induce variations in nest attendance (similar to early handling),

dams were given access to food either in the nest cage or in the foraging cage. The objectives of the present experiment were to study how a moderately challenging maternal environment that forces the dams to leave the nest cage for feeding affects nest attendance and maternal care, and how such environmentdependent variation in maternal behaviour affects HPA-stress responses, behavioural fearfulness and home cage behaviour in the adult offspring. 2. Materials and methods 2.1. Subjects and housing conditions Nineteen adult multiparous female C57BL/6 mice derived from animals obtained from Harlan-Winkelmann (Germany) and their offspring (n = 32 in total) were used. Pregnant females were housed individually in standard polycarbonate cages (Makrolon type III 37.5 22 15 cm) with sawdust as bedding and food (Ssniff Rat/Mouse diet, Spezialditen GmBH, Germany) and water provided ad libitum. The animals were maintained on a 12:12 hour light/dark schedule (lights on at 0800 h) in a temperature- and humidity-controlled animal room. Few days before the expected date of delivery, dams were randomly allocated to two treatment groups (n = 910 per group) and placed in the experimental cage systems (see below) until postnatal day (PND) 14. They were inspected once daily (at 1630 h) for delivery, and day of birth was designated as PND 0. Litters were left intact and dams and their litters were left undisturbed until PND 14, when they were housed in standard Makrolon type III cages. At PND 21, offspring were weaned and housed in same-sex groups of 23 littermates in Makrolon type III cages with sawdust as bedding and a red polycarbonate house (Mouse House, Techniplast, Italy) as shelter. Offspring were left undisturbed until testing except for a weekly cage change. The experiment was run in six replicates. All replicates were conducted under the same conditions and the numbers of dams of the two experimental groups were balanced across replicates. 2.2. Experimental cage system The experimental cage system consisted of two Makrolon type III cages, a nest cage (NC) and a foraging cage (FC), connected by a tunnel (30 cm long, 4 cm of diameter, Fig. 1). Grid floors were used in the FC and in the tunnel to prevent dams from nesting outside the NC, and the attractiveness of the NC for nesting was further

Fig. 1. Schematic representation of the system used to modify the maternal environment. Lactating dams had access to a foraging cage (FC) from their nest cage (NC) via a tunnel from PND 114. The solid floor of the NC was covered with sawdust and the walls were darkened to increase attractiveness for nesting.

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increased by opaque walls and 80 g of sawdust as bedding. This relatively low amount of sawdust was used to facilitate behavioural observations of maternal behaviour from video recordings. The dam had free access to the entire cage system from PND 1 until PND 14, when the tunnel was closed. Thereafter, the NC was replaced by a standard Makrolon type III cage until weaning. 2.3. Treatment groups NC group: the food was provided ad libitum in the nest cage (NC) and the dam was free to move about the entire system (N = 9). FC group: the food was provided ad libitum in the foraging cage (FC) and the dam was free to move about the entire system (N = 10). 2.4. Maternal parameters 2.4.1. Maternal behaviour The behaviour of each dam was recorded using two videocameras (S/W-CCD Minikamera AVC 308, ELV Elektronik A6, Germany), one being placed in front of the NC and the other in front of the FC. Dams were observed during seven 32-min observation periods every day from PND 1 to PND 13. Observations were made at fixed times throughout the day, with four periods during the light phase (starting at 0800, 1100, 1400 and 1700 h) and three periods during the dark phase (starting at 2000, 0000 and 0400 h). Within each observation period, the behaviour of each mother was scored every 4 min (8 observations/period 7 periods per day = 56 observations/ mother/day). On each scan, the position of the dam in the cage system (NC, FC or tunnel) and her behaviour were scored. Maternal behaviour was scored according to a detailed ethogram (Table 1) and grouped into the following categories for further analysis: (1) non-nursing behaviour, including eating, drinking, exploring, self-grooming, resting without body contact with the pups; (2) active nursing, including high kyphosis, partial kyphosis, dog-partial kyphosis, low kyphosis, bear nursing, sit-split nursing; (3) passive nursing, including dog nursing, prone nursing, pig nursing; (4) licking/grooming the pups; (5) undetectable (due to poor visibility). The nursing postures were defined on the basis of the definitions by Macri et al. [10] for rats and Shoji and Kato [26] for mice, and based on our own observation. As already noted for rats [27], behaviours from different categories can sometimes occur at the same time. For example, licking and grooming often occurred while the mother was nursing the pups. In that case, both behaviours were scored. 2.4.2. Plasma corticosterone response to stress On the day of weaning (PND 21), dams were removed from the cage and exposed to a standard stressor to determine their HPA-stress reactivity based on corticosterone (CORT) levels measured in blood samples. This was done during the early light phase between 0800 and 0930 h. Starting at 0800 h, the first dam was removed from the home cage by a familiar experimenter, placed in a new type III cage with sawdust and carried to the

Table 1 Definitions of maternal behaviours Chewing food, sawdust or feces Mouth in contact with spout of the water bottle Sniffing towards the air or any part of the cage Licking, scratching or brushing own fur with tongue or paws Resting without body Inactive without being in contact with any pup contact with pup Other All other behaviours such as defecating, urinating, stereotypic behaviours Active nursing High kyphosis The dam is immobile in high upright dorsal arch posture supported by rigid fore- and hind-limbs, the head is depressed, the trunks and limbs are bilaterally symmetrical, and pups are attached to the nipples [10,26] Low kyphosis The dam is over the pups, immobile, bilaterally symmetric, with the head not depressed and in a low dorsal arch posture supported by pronounced rigid fore- and hind-limbs ([10]; part of the blanket nursing according to [26]). Pups are attached to the nipples Partial kyphosis The dam is over the pups, relatively immobile, bilaterally symmetric, with the head not depressed and in a low dorsal arch posture supported by rigid fore- or hind-limbs [10]. Pups are attached to the nipples Dog-partial kyphosis The dam is over the pups, relatively immobile, in general the legs are buckled. The pups are under the belly bulge. The posture of the dam may or may not be bilaterally symmetric. Pups are attached to the nipples Bear nursing The dam is sitting in an upright posture on its hind quarters with the forepaws lifted off the ground. Pups are attached to the nipples Sit-split nursing Similar to partial kyphosis, but with the hind legs being laterally streched out Passive nursing Dog nursing The dam lies on top of the pups with the back flat and the head lifted or on the ground. All four legs are buckled and close to the body. Pups are attached to the nipples Prone nursing The dam lies flat on top of the pups with little or no imb support [10]. Pups are attached to the nipples Pig nursing The dam lies flat on her side with pups attached to her nipples; correspond to supine nursing according to [26]. Licking/grooming The dam touches any part of a pup's body with her tongue or nose or forepaws, or stretches her nose in the direction of the pups. Non-nursing Eating Drinking Exploring Self-grooming

adjacent room where the first blood sample was taken immediately to obtain a basal measure of CORT. Blood samples were taken by tail incision [28]. For each sample, about 50 l of blood was collected into prechilled ethylenediamine tetraacetic acid (EDTA)-coated tubes (Microvette CB300, Sarstedt, Germany). Sampling was finished within 3 min after the dam was removed from her home cage. Following the first sample, the dam was placed in a type II cage (26.5 20.5 14.5 cm) with sawdust for 20 min of isolation/novelty stress. After 20 min (t20), she was bled again either from the same incision or from a new incision to obtain a measure of the peak CORT response to the stressor. After this second sample, she was placed back in the

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Fig. 2. Experimental protocol followed from birth of the litter until the end of the experiment.

type III cage with food and water, and transported back to the colony room. Forty (t40) and sixty minutes (t60) after the first blood sample, she was transported to the test room again for further blood samples to obtain measures of recovery from the stressor. Between these two samples she was returned to the colony room. All samples were cool centrifuged and the plasma was stored at 20 C until assayed. CORT concentrations were determined by a double antibody radioimmunoassay method, specified for rats and mice, using the commercial kit ImmuChem double antibody corticosteron (MP Biomedicals, Germany). Intraassay and interassay variations were 4.4% and 6.5%, respectively. 2.5. Offspring parameters Fig. 2 illustrates the protocol that was followed to obtain the different measures in the offspring. 2.5.1. Body weight at weaning To control for a treatment effect on pup growth, all offspring were weighed at weaning. 2.5.2. Home cage behaviour At 9 weeks old (1 week), offspring were transferred to a separate room for 24 h video recordings to assess their home cage behaviour. One week prior to this, all groups of offspring were reduced to two mice per cage by removing surplus animals at random from those cages that contained three mice. Eight pairs of males and eight pairs of females of each treatment were observed (N = 8 pairs/treatment/sex). Home cage behaviour was recorded using the same video system as used for recording maternal behaviour with one video-camera placed in front of each cage. Animals were not marked individually since the two mice within a cage were not considered to be statistically independent and therefore behaviour per cage was used in the statistical model. Recordings were made during three consecutive days, and of each day eight 24-min periods were observed. Observations were made at fixed times throughout the day with four periods during the light phase (starting at 0800, 1100, 1400 and 1700 h) and four periods during the dark phase (starting at 2000, 2300, 0200 and 0500 h). Within each observation period, the behaviour of each pair was scored every 4 min (6 observations per period). On each scan the behaviour of both animals was scored. Consequently, on

each scan two behaviours were noted (2 6 observations/period 8 periods per day = 96 observations/pair/day). Home cage behaviour was scored according to a detailed ethogram (Table 2) and grouped into the following categories for further analysis: (1) active socio-positive behaviours (including allo-grooming, playing, non-agonistic interactions), (2) passive socio-positive behaviours (including lying in contact, sitting in contact); (3) offensive socio-negative behaviours (including attacking, escalated fight, offensive posture), (4) defensive socio-negative
Table 2 Definitions of offspring home cage behaviours Socio-positive behaviours Active Allo-grooming Playing Non-agonistic interactions Passive Lying in contact Sitting in contact Socio-negative behaviours Offensive Attacking Escalated fight

Wiping, licking or scratching the cagemate's body Sudden, vertical or horizontal jumps All other interactions with the cagemate except those listed below Inactive with contact to the cagemate's body Sitting with contact to the cagemate's body

Offensive posture

Rushing and leaping at cagemate with bite attempt [29] Physical struggle between the cagemates, which is initiated by a bite and usually involves further biting, kicking, wrestling and rolling over and over [29] Standing on the hind legs, bending slightly forward and pushing the cagemate with the forelegs Similar to offensive posture, but the mouse bends slightly backward Running away from cagemate, generally accompanied by leaps and sudden changes in direction [29] Remaining motionless in response to the cagemate's movement Rapid up-and-down vibration of the tail Wiping, licking or scratching own body Chewing food at the food rack or mouth in contact with spout of the water bottle Sniffing towards the air or any part of the cage Inactive without contact with the cagemate's body Sitting without contact with the cagemate's body All other activities such as locomotion, climbing, digging in the sawdust, urinating, defecating

Defensive Defensive posture Fleeing

Freezing Tail rattling Self-grooming Foraging Exploring Lying alone Sitting alone Other activities

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behaviours (including defensive posture, fleeing, freezing, tail rattling); (5) self-grooming; (6) foraging; (7) exploring; (8) lying alone; (9) sitting alone; (10) other activities; (11) undetectable (due to poor visibility). 2.5.3. Behavioural tests At 10 weeks old ( 1 week) one male and/or one female of each litter were tested in a series of behavioural tests commonly used to test anxiety in animals (N = 8 offspring/sex/treatment). On the first day, all animals were tested on an elevated-O-maze. After one day of recovery, the same animals were exposed to two further tests on two consecutive days: an open-field test and a free exploration test. 2.5.3.1. Elevated-O-Maze test. On an elevated-O-maze, the exploratory drive of mice is competing with their natural tendency to avoid heights and open spaces [30]. The elevated-O-maze (EZM) was derived from the elevated-plus-maze. Both tests were initially developed and pharmacologically validated in rats [31,32], but are now widely used in mice. The advantage of the EZM is that it lacks the ambiguous central square of the traditional plus-maze. The EZM consisted of a circular platform (outer diameter 46 cm, width 5.5 cm) made of grey plastic that was elevated 40 cm above the floor, featuring two open and two enclosed (closed) segments. The closed segments were enclosed by walls extending 20 cm above the surface of the maze, while there were no walls on the open segments. The test was conducted in a test room adjacent to the colony room that was illuminated by low light (48 lx). One of the two littermates previously observed during the 24 h observations was removed from the home cage and placed in a type II cage. The cage was transported to the test room by a familiar experimenter. Each test session started by placing the mouse in one of the two closed segments. The test lasted for 5 min and performance was recorded using a video-camera placed above the EZM and the EthoVision video tracking software (Noldus IT, Netherlands). Measurements of total path moved, percent of time spent and percent of path moved in the open and closed segments, number of open segment entries and percent of protected head dips were used for further analysis. In addition, the number of fecal boli was counted at the end of the 5 min test session. After the test, the mouse was transported back to the colony room in a type II cage and returned to its littermate in the home cage. Between trials, the EZM was cleaned with a 30% alcohol solution. 2.5.3.2. Open-field test. The open-field test is the most widely used behavioural test since it was developed in 1934 [33,34]. It has been validated pharmacologically as a test of anxiety [35]. The apparatus consisted of 4 adjacent dark grey plastic arenas (50 50 40 cm). A camera was mounted above the centre of the four arenas and connected to a video-recorder and the video tracking system EthoVision for analysis. The test took place in the same test room as the EZM. During the test, illumination at floor level in the centres of the arena was set to 67 lx. Four subjects were transported in separate type II cages to the test room and each subject placed in one of the four arenas for 30-min. After the test,

the animals were returned to their home cages. EthoVision allowed automatic recording of the total path moved, as well as the time spent and the path moved in a virtual square centre and in a virtual 5-cm wide corridor along the walls of the arena (thigmotactic behaviour). The number of fecal boli was counted at the end of each trial. The arenas were cleaned with a 30% alcohol solution between trials to remove odour cues. 2.5.3.3. Free exploration test. The free exploration paradigm was first described in 1968 for the rat [36] and adapted to mice in 1981 [37]. For the present study, this procedure was slightly modified. The apparatus consisted of the four arenas used for the openfield test. Before the test started, the shelter (MouseHouse) was removed from the home cage and placed in one corner of the arena 5 cm from the nearest wall, with the opening on the side facing the centre of the arena. Subjects were transported to the test room as described above, placed inside the arena near the shelter and left there for 10 min. After the test, they were returned to their home cages. Again using EthoVision, total distance moved, as well as the time spent and the distance run in various virtual zones (the centre of the open-field and in a 5 cm zone around the shelter) and the number of entries into the shelter and the 5 cm zone around the shelter were analysed. The arenas were cleaned with a 30% alcohol solution between trials. 2.5.4. Plasma corticosterone response to stress At 11 weeks-old (1 week), littermates of those animals that were subjected to the behavioural tests were examined for HPAstress reactivity following the same protocol as described above for the mothers. Sample size was N = 8 per sex and treatment. From t20 to t40 and from t40 to t60, the mice were placed back in their home cages together with their littermate in the colony room. 2.6. Statistical analysis Data were analysed using the BMDP statistical software [38]. The NC treatment was compared to the FC treatment to assess the effects of spatial variation of feeding site. Maternal behaviour was analysed using a repeated measures ANCOVA, with treatment, PND and time-window as main factors (PND and time-window being factors with repeated measures), and litter size, litter sexratio, and age and parity of the dam as covariates. When some covariates showed a significant statistical effect, the corresponding regression coefficients were calculated. The general model to analyse maternal behaviour was 13 PND 7 time-windows 2 treatments. Since the proportion of undetectable behaviour varied across dams and time, analysis of maternal behaviour is based on relative frequencies of behaviours. Because of some missing values, we used a generalized estimating equations model (instead of the least square method (F-tests)). As a method of estimation, the maximum likelihood was used. Thus, the adequate statistical analyses were of Wald type based on a Chi-square analysis. For analysis of the CORT response in the dams (area under the curve AUC) the data were log transformed. A onefactorial analysis of covariance was run to compare NC dams with FC dams.

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Fig. 3. Daily frequency (mean/half-hour SEM) of visits to the FC by NC dams (food in the NC) and FC dams (food in the FC). Daily scores are based on 7 daily halfhour sampling sessions (PND effect: p b 0.001; treatment effect: p b 0.001).

For the analyses of the behavioural tests, weaning weight and the CORT responses in the offspring (area under the curve AUC), the general ANCOVA model was 2 sexes 2 treatments with litter size and sex-ratio of the litter as covariates. For the analysis of the home cage behaviour in the offspring, the general model was 2 sexes 3 days 8 time-windows 2 treatments, with sex, days and time-window being factors with repeated measures. Separate analyses on males and females were performed when a significant sex by treatment interaction was found. These analyses were performed using a comparison related significance level. Significance was set at p 0.05 and trend at p 0.1. 3. Results 3.1. Effects on dams 3.1.1. Nest cage attendance The frequency of the dams' visits to the FC increased in both groups throughout the observation period from PND 113 (2 = 159.04, df = 12, p b 0.001, Fig. 3). However, feeding site had a strong effect on the frequency of visits to the FC, with FC

dams leaving the NC to visit the FC significantly more frequently than NC dams (2 = 166.34, df = 1, p b 0.001, Fig. 3). Despite this difference in nest cage attendance, no difference was found between NC dams and FC dams in the frequencies of them being inside the nest (2 = 1.24, df = 1, p N 0.1). The number of scores inside the nest decreased significantly in both groups throughout the observation period (2 = 336.38, df = 12, p b 0.001, Fig. 4a). Furthermore, the number of scores inside the nest varied across the day. Dams were more often observed in the nest during the light phase than during the dark phase (2 = 152.06, df = 1, p b 0.001, Fig. 4b). There was also a significant litter size effect on nest attendance, with dams with smaller litters being more often observed in the nest (regression coefficient = 0.027; p b 0.001 Fig. 5). 3.1.2. Maternal care Similar to nest attendance, total maternal care decreased with increasing age of the pups, and levels of maternal care were higher during the light phase. Total maternal care was unaffected by treatment (p N 0.1). However, as shown in Fig. 6, feeding site had a significant effect on nursing style. Thus, total levels of

Fig. 4. (a) Daily frequency (mean/half-hour SEM) of nest attendance by NC dams and FC dams. Daily scores are based on 7 daily half-hour sampling sessions (PND effect: p b 0.001; treatment effect: NS). (b) Diurnal distribution of nest attendance (frequency per half-hour). Scores from all 7 daily half-hour sessions were averaged across PND 1 to 13.

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Fig. 5. Frequency of nest attendance according to the size of the litter (regression coefficient = 0.027; p = 0.002).

active nursing differed significantly between NC dams and FC dams, with NC dams showing more active nursing than FC dams (2 = 4.49, df = 1, p = 0.034). However, there was also a significant interaction between treatment and PND (2 = 45.44, df = 12, p b 0.001), indicating that NC dams showed more active nursing during the early postnatal period only (Fig. 6a). There was no main treatment effect on licking/grooming (2 = 0.38, df = 1, p = 0.537), but an interaction between treatment and PND was found for PND1 and 2, with NC dams showing more licking/ grooming than FC dams during the first two days (Fig. 6b; p = 0.061 and p = 0.049, respectively). Conversely, FC dams showed significantly higher levels of passive nursing than NC dams throughout the observation period (2 = 9.21, df = 1, p = 0.002, Fig. 6c), whereas NC dams tended to show more

Fig. 7. Treatment effect on the CORT response to 20 min isolation/novelty stress in the dams. CORT levels (mean SEM; ng/ml) before (basal level: t0) and 20 (t20), 40 (t40) and 60 (t60) min after the onset of the stressor shown by NC dams and FC dams.

non-nursing behaviours than FC dams ( 2 = 3.35, df = 1, p = 0.067, Fig. 6d). 3.1.3. Plasma corticosterone response Twenty minutes isolation/novelty stress induced a dramatic increase in the level of plasma corticosterone that persisted

Fig. 6. Relative frequencies (mean SEM) of maternal behaviours across the two first weeks of lactation by NC dams and FC dams. Daily scores are based on 7 daily half-hour sampling sessions. (a) Active nursing (treatment effect: p = 0.034; treatment PND effect: p b 0.001); (b) licking/grooming (treatment effect: NS; treatment PND effect: NS; except for PND 1: p = 0.061; and PND 2: p = 0.049); (c) passive nursing (treatment effect: p = 0.002; treatment PND effect: NS); (d) nonnursing behaviours (treatment effect: p = 0.067; treatment PND effect: NS).

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Table 3 Statistical analysis of the social behaviours displayed by male and female offspring reared by the NC or FC dams (N = 8 pairs/sex/treatment) over the 3 days of observations Treatment Socio-positive Active Socio-positive Passive Socio-negative Offensive Socio-negative Defensive 1 = 0.50 p = 0.478 21 = 1.00 p = 0.317 21 = 0.078 P = 0.779 21 = 0.449 p = 0.503
2

Day 22 = 7.734 p = 0.021 22 = 5.287 p = 0.071 22 = 7.521 p = 0.023 22 = 0.039 p = 0.98

Time-window 7 = 24.178 p = 0.001 27 = 97.631 p b 0.001 27 = 103.924 p b 0.001 27 = 15.268 p = 0.033

2

Treatment day 2 = 5.580 p = 0.064 22 = 0.467 p = 0.792 22 = 3.291 p = 0.193 22 = 1.473 p = 0.479
2

Treatment time-window 27 = 4.996 p = 0.66 27 = 2.205 p = 0.948 27 = 8.232 p = 0.313 27 = 17.02 P = 0.017

On each day, the mice were observed during 8 time-windows of 24 min each.

beyond t60 (time point effect: p b 0.001). However, neither basal levels (p = 0.446) nor response measures to the stressor differed between NC and FC dams (AUC: p N 0.1 Fig. 7). 3.2. Effects on offspring 3.2.1. Body weight at weaning Body weight at weaning did not differ between the two treatments and there was no significant difference between male and female offspring (NC males: 7.48 0.30 g, females: 7.52 0.32 g; FC males: 8.08 0.41 g, females: 8.25 0.42 g treatment and sex effects p N 0.1). However, there was a significant effect of litter size on weaning weight, with smaller litters consisting of heavier pups (regression coefficient=0.419; p b 0.001). 3.2.2. Home cage behaviour Analysis of home cage behaviour in the adult offspring showed a time-window effect (p b 0.001) reflecting the circadian rhythm of the mice. Thus, they spent more time inactive at 1400, 1700 and 0500 h, whereas foraging and exploration were increased at 0200, 0800, 2000 and 2300 h (p b 0.001). There was also a significant time-window sex interaction (p b 0.05), indicating that some behaviours were more frequently performed by males than by females, such as foraging and exploration (2 = 19.59, df = 1, pb 0.001 and 2 =4.53, df= 1, p = 0.033, respectively). There was no treatment effect on foraging, exploration, inactivity, self-grooming and other behaviours. However, there was a significant treatment by day interaction (2 = 6.18, df = 2, p = 0.045) with respect to social behaviours, indicating that NC offspring showed more social behaviours than FC offspring on day 2 (data not shown). When the different categories of social behaviour were analysed, it was found that males performed more socio-positive (active and passive) behaviours than females (2 = 206.98, df = 1, p b 0.001), whereas females performed more socio-negative (offensive and defensive) behaviours than males (2 = 607.49, df =1, pb 0.001). The significant main effect of time-window (2 = 32.18, df= 7, p b 0.05) reflects the circadian rhythm of social behaviours that occurred mainly during the dark phase. There was no main effect of treatment on any category of social behaviour (Table 3). However, for socio-negative defensive behaviours a significant treatment by time-windows interaction (2 = 17.02, df =7, p = 0.017; Table 3) was found. Furthermore, there was also a

non-significant trend for a treatment by day interaction (2 = 5.58, df= 2, p = 0.06; Table 3) with respect to socio-positive active behaviours. This was due to both male and female NC offspring displaying more socio-positive active behaviours than FC offspring on day two of the observation (p= 0.027 Fig. 8). 3.2.3. Anxiety 3.2.3.1. Elevated-O-Maze test. Males and females tended to behave differently on the EZM depending on treatment when considering total locomotion and percent of time spent on the open sectors (sex treatment: 2 = 3.45, df = 1, p = 0.063 and, 2 = 3.70, df = 1, p = 0.054 respectively). Furthermore, there was a main effect of sex on the number of fecal boli and total locomotion, with males dropping less fecal boli (2 = 4.72, df = 1, p = 0.029) and tending to move less than females (2 = 3.48, df = 1, p = 0.062). Consequently, both sexes were analysed separately. In males, NC offspring showed less locomotion (Fig. 9a, p = 0.032) and tended to spend less time on the open sectors than FC offspring (Fig. 9b, p = 0.1). In females, there were no significant differences between treatments in total locomotion on the EZM and time spent on the open sectors (Fig. 9a and b, p N 0.1). Furthermore, there were no significant treatment effects on the path travelled in the open sectors, number of open sector entries and number of fecal boli. However, offspring reared by NC dams showed less protected head dips than FC offspring

Fig. 8. Frequency of socio-positive behaviours (mean + SEM) displayed by offspring reared by NC dams and FC dams (N = 8 pairs/sex/treatment 8 daily half-hour sessions were averaged across the 3 days of observation; treatment day effect: day 2: p = 0.027).

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Fig. 9. (a) Total locomotion (mean + SEM) and (b) percent of time (mean + SEM) spent by male and female offspring of the two treatment groups in the open sector of the O-maze during a 5-min session (N = 8 per sex per treatment). p = 0.032, p = 0.1.

(NC: 33.3 3.3%; FC: 43.7 3.9%; 2 = 5.08, df = 1, p = 0.024). This effect was mainly due to FC females showing more protected head dips than NC females (p = 0.001), whereas males did not significantly differ (p N 0.1). 3.2.3.2. Open-field test. No main effects of treatment or sex were found (p N 0.1). Offspring of both groups showed similar levels of locomotion in the entire arena (2 = 0.41, df = 1, p N 0.1, Fig. 10a) and similar amounts of time spent and path travelled in the centre of the arena (2 = 0.04, df = 1, p = 0.83 and 2 = 0.63, df = 1, p = 0.43, respectively, Fig. 10b) as well as near the walls (2 = 0.65, df = 1, p = 0.42). There was also no difference between treatments in the number of fecal boli dropped in the arena (2 = 0.63, df = 1, p = 0.42, Fig. 10c) or the number of entries into the centre of the arena (2 = 0.33, df = 1, p = 0.56). 3.2.3.3. Free exploration test. Again, there were no significant main effects of treatment or sex on any of the parameters that were analysed. However, sex-ratio was found to affect time in the centre of the arena (2 = 8.40, df = 1, p = 0.004) and time near the shelter (2 = 3.85, df = 1, p = 0.05), with males from male-skewed litters spending less time in the centre and more time near the shelter. 3.2.3.4. HPA response. There was a main effect of sex indicating that CORT levels in females were higher than in males (2 = 47.99, df = 1, p b 0.001). Consequently, both sexes were analysed separately. Considering the CORT basal level, NC males showed a significantly lower basal level than FC males (Fig. 11a, p b 0.001), whereas there was no such difference in

females (Fig. 11b, p N 0.1). The 20-min exposure to isolation/ novelty induced a dramatic increase in plasma CORT levels (time point effect, 2 = 848.59, df = 3, p b 0.001). However, offspring of both treatment groups showed similar CORT responses to this stressor (t20t0, 2 = 0.19, df = 1, p N 0.1), although females showed a more pronounced peak response (sex effect, 2 = 52.46, df = 1, p b 0.001) and a slower recovery (t20t60) than males (sex effect, 2 = 5.45, df = 1, p b 0.001). In terms of the integral CORT response (AUC), NC females tended to show a higher total CORT response than FC females (Fig. 11c, p = 0.07), while there was no such difference in males (Fig. 11c, p N 0.1). 4. Discussion Compared to rats, the effects of early postnatal experience on HPA-stress reactivity and behavioural measures of coping with environmental challenge have been poorly studied in mice, and the available studies have produced rather inconsistent results. Most studies have used the classical neonatal manipulations (early handling and maternal separation) that involve human intervention and direct manipulation of dams and/or pups. Consequently, the stress and fear induced by these manipulations represent confounding factors that can account, at least in part, for the effects of these manipulations (cf. direct action hypothesis [39]), which renders interpretation of the results difficult. The aim of the present study was to avoid such confounding and create conditions that would reflect more natural variations of the postnatal maternal environment. Thus, we varied the distance between the dams' feeding site and their nest during the first two weeks of lactation to create an

Fig. 10. (a) Total locomotion (mean + SEM), (b) time spent in the centre (mean + SEM) and (c) number of fecal boli (mean + SEM) during a 30-min session in the openfield shown by male and female offspring of the two treatment groups (N = 8 per sex per treatment).

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Fig. 11. CORT levels (mean SEM; ng/ml) before (basal level: t0) and 20 (t20), 40 (t40) and 60 (t60) min after the onset of 20 min isolation/novelty stress shown by (a) male and (b) female offspring of the two treatment groups. (c) Integrated CORT response over the entire 1-hour test period (AUC: area under the curve; N = 8 per sex per treatment; p b 0.001; p = 0.07). Inset: enlargement of the CORT basal level of male and female offspring.

environment that induces variation in motheroffspring contact, without the need for direct human intervention. Dams with access to food in the foraging cage (FC dams) had to leave the nest cage for feeding, whereas mothers with access to food in the nest cage (NC dams) could stay near their pups while feeding. As expected, FC dams displayed considerably more visits away from the nest cage than NC dams. Although this did not result in lower levels of direct contact with the pups, it was associated with significant alterations in maternal style. Thus, NC dams showed more active maternal care, especially during the early postnatal period, whereas FC dams showed more passive nursing. Despite these effects on maternal behaviour, the effects on the adult offspring's fear and HPA-stress responses were rather subtle, yet complex, indicating that no single aspect of maternal behaviour can account for the observed changes, and that there are significant differences in the effects on male and female offspring. Furthermore, the postnatal treatments had some effects on the offspring's social behaviour in the home cage, with NC offspring displaying more active socio-positive behaviours compared to FC offspring. These findings demonstrate that variations of the maternal environment can have significant effects on the behaviour of mouse dams towards their pups and that even subtle modifications of the maternal environment can induce persistent changes in the offspring's phenotype. The observed effects have implications for the offspring's capacity to cope with social and environmental challenge, but whether or not these effects are mediated by environment-dependent variations in the maternal behaviour needs to be investigated further. 4.1. Effects on the dams In line with a recent study in rats [16], mouse dams with access to food in the FC made significantly more visits to the FC and, consequently, were less frequently observed in the nest cage. As in rats, this difference in nest cage attendance did not result in less contact with the pups, but it affected maternal style. However, the effects on maternal style differed markedly from those in rats [16]. In the rats, the FC treatment induced a more active nursing style as indicated by longer bouts of active nursing [16], whereas in the mice, the NC treatment induced a more active nursing style as indicated by higher levels of active

nursing and more licking/grooming, especially during the early postnatal period. Moreover, in the rats, the effects of the FC compared to the NC treatment closely paralleled those induced by early handling compared to non-handling [10,12,40]. Like early handling, the FC condition appeared to model a moderately challenging environment that stimulated active maternal care. In mice, however, neither early handling in NMRI [21] or NC900 mice [23], nor the FC treatment in the present study stimulated active maternal care. While early handling had no effect on maternal behaviour in NMRI and NC900 mice [21,23], FC dams in the present study showed less active maternal care and more passive nursing compared to NC dams. Two alternative explanations may account for these difference to the rat study. First, feeding dams away from their pups may be physically more challenging for mouse dams than for rat dams, thereby compromising their capacity to maintain high levels of active maternal care. Although we cannot exclude this possibility, it is rather unlikely, given that both NC and FC dams visited the nest equally frequently, and foraging in the FC did not appear to be physically more demanding than foraging in the NC. Second, mouse and rat dams may have differed in the psychological perception of the FC treatment. Mice are highly neophobic [40], and the novel environment attached to the home cage may have been aversive to the dam [41]. However, FC dams may have learned to associate the novel cage with the rewarding effect of food, which may have reduced its aversiveness to FC dams more rapidly than to NC dams. This is further supported by the fact that licking/grooming and active nursing were higher in NC dams especially during the first days of lactation. As a result, one might expect NC dams to show signs of stress compared to FC dams, but their corticosterone levels measured at weaning did not differ. However, the stress induced by the unfamiliar environment might have affected maternal behaviour without inducing changes in the HPA system of the dams. It has been shown that similar psychological stressors (e.g. wet bedding, forced foraging) can affect maternal behaviour in rats without activating corticosterone secretion [15]. Alternatively, the novel environment may have altered HPA activity in the dams temporarily, but this effect may have waned by the time of our assessment at weaning three weeks later. Our data are not suitable for exploring these hypotheses any further. However, since elevated corticosterone levels in

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the dams have been suggested to play a major role in alterations in maternal behaviour [42], further research on this issue is clearly warranted. One way to test this idea would be to collect fecal samples throughout lactation to infer changes in HPA activation from changes in corticosterone metabolites in the feces [43]. Regardless of the exact mechanisms underlying these changes, however, our results demonstrate that minor variations in the maternal environment of mice can have significant effects on maternal style, but also that mice and rats may respond differently to seemingly similar environmental variations. 4.2. Effects on the offspring Spatial variation in the lactating dams' access to food also had persistent effects on HPA-stress and fear responses in the adult offspring. Thus, NC males had lower basal corticosterone level than FC males, but they did not differ in their corticosterone response to isolation/novelty stress. Conversely, female offspring did not differ in basal corticosterone levels, but NC females tended to show a more pronounced corticosterone response to the stressor than FC females. These findings partly parallel those found in rats in that the FC treatment was associated with reduced HPA-reactivity, and that this effect was more pronounced in females [16]. A stronger effect on females could reflect their higher sensitivity towards environmental cues compared to males. In wild rats, females show a higher degree of philopatry [44]. Therefore, early environmental cues may predict the future habitat of the females better than that of the males [16]. The same may be true for mice, although so far this has been demonstrated only for the Striped Mouse (Rhabdomys pumilio) [45]. However, in contrast to the rat study, the effects on HPA-reactivity were partly inconsistent with the effects on behavioural measures of fearfulness, especially in females. Thus, although NC males appeared to be more fearful than FC males in terms of the time spent on the open sectors of the O-maze, NC females appeared to be less fearful than FC females, as indicated by fewer protected head dips. However, since neither of these differences in fearfulness was supported by a significant difference in any other measure of fearfulness from the three behavioural tests, the effects of variation in the dams' feeding site on the offspring's HPA and fear responses remain somewhat ambiguous. Considering the difference in the effects of the FC treatment on maternal behaviour in mice and rats, the different effects on the offspring might be due to the complexity of the modulatory effects of early experience on the development of HPA-reactivity and behavioural fearfulness. Studies in rats have shown that several aspects of maternal behaviour as well as direct environmental effects on the offspring together account for the observed changes in offspring phenotype [10,16]. For example, in rats both increased active maternal care as well as more time spent away from the nest cage have been shown to affect offspring development [10,45,46], and to reduce behavioural fearfulness in the offspring [47]. In mice, Calatayud et al. [48] have shown that maternal care affects offspring phenotype, and Priebe et al. [49] have shown that C57BL/6 mothers display more active nursing than BALB/c mothers, and this difference was

associated with C57BL/6 offspring showing less fearful behaviour than BALB/c offspring. If, in mice, the time spent away from the nest cage also contributes to variation in offspring HPA-stress reactivity and fearfulness, resulting in a downregulation similar to that observed in rats, then the dissociation in the present study in the effects of the FC treatment on active maternal care and time away from the nest could have levelled out any differences in HPA-reactivity and behavioural fearfulness between the offspring of the two treatments. Alternatively, the difference in the effects of the two treatments on maternal behaviour could have been too subtle to induce persistent effects on HPA-reactivity and behavioural fearfulness in the offspring. Parfitt et al. [9] have shown that early handling in C57BL/6 mice resulted in reduced measures of anxiety in juvenile offspring, whereas no differences were found when the offspring was an adult. Moreover, C57BL/6 appear to be relatively robust against variations in early experience compared to other strains of mice. For example, in one study, early handling and maternal separation affected fearfulness in BALB/c, 129S1 and FVB/NJ mice, but not in C57BL/6 mice. Similarly, Zaharia et al. [22] found an effect of early handling on the performances of BALB/c mice, but not C57BL/6 mice, in a Morris water-maze test, and BALB/c mice were less fearful than C57BL/6 mice when reared under the same enriched housing conditions [50]. Thus, the relative robustness of C57BL/6 mice towards variations in early experience together with the relatively subtle effects of the two treatments on maternal behaviour may account for the lack of robust effects on HPAstress reactivity and fearfulness in the adult offspring reared by NC and FC dams, respectively. Finally, although our treatments avoided the confounding effect of human manipulation, they resulted in another confounding factor, namely variation in litter size. Culling to standardize litter size would have involved human manipulation, which in turn could have affected offspring development [51]. However, dams raising larger litters were significantly less frequently observed inside the nest. It has previously been shown that litter size affects maternal care [52], which has been explained by extra nutrient requirement, fatigue and discomfort for females with larger litters. More severe variations of the postnatal maternal environment might therefore be needed in future research to unravel the complex relationships between environmental factors, maternal behaviour and phenotypic plasticity of HPA and fear responses in mice. The two treatments also had specific effects on some aspects of social behaviour in the home cage. Thus, adult offspring reared by NC dams showed higher levels of socio-positive behaviours than offspring reared by FC dams. The fact that this effect was statistically significant on only one of the three observation days suggests that it may be expressed under certain circumstances only, e.g. under conditions of social tension. In previous studies, the effects of early experience on social behaviour in the adult offspring have been assessed using a variety of social interaction tests [4,5,13]. To our knowledge, this is the first study to demonstrate an effect of early experience on social behaviour in the home cage. However, our finding supports previous results in rats and mice showing that higher levels of active maternal care result in higher levels of social behaviour when adult [5], whereas lower levels of

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L. Coutellier et al. / Physiology & Behavior 93 (2008) 395407 [3] Holmes A, le Guisquet AM, Vogel E, Millstein RA, Leman S, Belzung C. Early life genetic, epigenetic and environmental factors shaping emotionality in rodents. Neurosci Biobehav Rev 2005;29:133546. [4] Mintz M, Redi-Bettschen D, Feldon J, Pryce CR. Early social and physical deprivation leads to reduced social motivation in adulthood in Wistar rats. Behav Brain Res 2005;156:31120. [5] Branchi I, D'Andrea I, Fiore M, Di Fausto V, Aloe L, Alleva E. Early social enrichment shapes social behavior and nerve growth factor and brain-derived neurotrophic factor levels in the adult mouse brain. Biol Psychiatry 2006;60:6906. [6] Caldji C, Tannenbaum B, Sharma S, Francis D, Plotsky PM, Meaney MJ. Maternal care during infancy regulates the development of neural systems mediating the expression of fearfulness in the rat. Proc Natl Acad Sci 1998;95:533540. [7] Meaney MJ. Maternal care, gene expression and the transmission of individual differences in stress across generations. Annu Rev Neurosci 2001;24:116192. [8] Cirulli F, Berry A, Alleva E. Early disruption of the motherinfant relationship: effects on brain plasticity and implications for psychopathology. Neurosci Biobehav Rev 2003;27:7382. [9] Parfitt DB, Levin JK, Salstein KP, Klayman AS, Gree LM, Helmreich DL. Differential early rearing environments can accentuate or attenuate the responses to stress in male C57BL/6 mice. Brain Res 2004;1016: 1111118. [10] Macri S, Mason GJ, Wrbel H. Dissociation in the effects of neonatal maternal separations on maternal care and the offspring's HPA and fear responses in rats. Eur J Neurosci 2004;20:101724. [11] Denenberg VH, Ottinger DR, Stephens MW. Effects of maternal factors upon growth and behavior of the rat. Child Dev 1962;33:6571. [12] Liu D, Diorio J, Tannenbaum B, Caldji C, Francis D, Freedman A, et al. Maternal care, hippocampal glucocorticoid receptors and hypothalamic pituitaryadrenal responses to stress. Science 1997;277:165961. [13] Macri S, Laviola G. Single episode of maternal deprivation and adult depressive profile in mice: interaction with cannabinoid exposure during adolescence. Behav Brain Res 2004;154:2318. [14] McLeod J, Sinal CJ, Perrot-Sinal TS. Evidence for non-genomic transmission of ecological information via maternal behavior in female rats. Genes Brain Behav 2007;6:1929. [15] Lonhardt M, Matthews SG, Meaney MJ, Walker CD. Psychological stressors as a model of maternal adversity: diurnal modulation of corticosterone responses and changes in maternal behaviour. Horm Behav 2007;51:7788. [16] Macri S, Wrbel H. Effects of variation in postnatal maternal environment on maternal behaviour and fear and stress responses in rats. Anim Behav 2007;73(1):17184. [17] Francis DD, Meaney MJ. Maternal care and the development of stress response. Curr Opin Neurobiol 1999;9:12834. [18] Pryce CR, Feldon J. Long-term neurobehavioural impact of the postnatal environment in rats: manipulations, effects and mediating mechanisms. Neurosci Biobehav Rev 2003;27:5771. [19] Schmidt M, Oitzl MS, Levine S, de Kloet ER. The HPA system during the postnatal development of CD1 mice and the effects of maternal deprivation. Dev Brain Res 2002;139:3949. [20] Romeo RD, Mueller A, Sisti HM, Ogawa S, McEwen BS, Brake WG. Anxiety and fear in adult male and female C57BL/6 mice are modulated by maternal separation. Horm Behav 2003;43:5617. [21] Moles A, Rizzi R, D'Amato FR. Postnatal stress in mice: does stressing the mother have the same effect as stressing the pups? Dev Psychobiol 2004;44:2307. [22] Zaharia MD, Kulczycki J, Shanks N, Meaney MJ, Anisman H. The effects of early postnatal stimulation on Morris water-maze acquisition in adult mice: genetic and maternal factors. Psychopharmacologia 1996;128: 22739. [23] Garipy JL, Rodriguiz RM, Jones BC. Handling, genetic and housing effects on the mouse stress system, dopamine function, and behaviour. Pharmacol Biochem Behav 2002;73:717. [24] Millstein RA, Holmes A. Effects of repeated maternal separation on anxiety- and depression-related phenotypes in different mouse strains. Neurosci Biobehav Rev 2007;31:317.

maternal care are associated with a reduced social behaviour [13]. Reduced socio-positive behaviour in the FC offspring might reflect reduced social motivation [13] and a more solitary life-style. In rats, it has been demonstrated that maternal deprivation (combined with thermal stress) resulted in a preference for solitary home base occupancy rather than social home base occupancy in an open-field test [4]. Our results support these findings and extend their validity to spontaneous social behaviour in the home cage environment. The increased level of socio-positive behaviours in NC offspring might be due to the positive effects of active maternal care on the later ability to establish dominance relationships. Previous studies in mice have shown that increased parental care facilitates the establishment of social status and is associated with increased NGF (nerve growth factor) levels that are known to affect social stability [5]. Although further research is needed to confirm this finding, the present study suggests that variation in socio-positive behaviour may have been mediated by environment-dependent variation in active maternal care. 5. Conclusion To our knowledge, this is the first study to examine the maternal mediation of developmental plasticity in mice, using variation of the early environment that does not involve direct human manipulations. Variation in the distance between the dams' feeding site and their nest induced changes in active maternal care that were associated with some persistent changes in the phenotype of the adult offspring. While the effects on HPA-stress reactivity and behavioural fearfulness differed between male and female offspring, the effects on social behaviour were consistent across both sexes. Moreover, our results suggest that, besides variation in active maternal care, some other factors (e.g. the time spent away from the nest cage, the perceived aversiveness of the treatments) may have contributed to the effects on HPA-reactivity and behavioural fearfulness. Apart from its contribution to our understanding of brain and behavioural development in general, a better understanding of experience-dependent plasticity of brain and behaviour in rodents has also important implications for the housing of laboratory rodents in term of both their well-being and the validity of animal experiments. An approach based on ecologically relevant treatments such as those described here might prove highly beneficial for future research. Acknowledgements This research was supported by the German Research Foundation (DFG). We are grateful to K. Guckelsberger for technical assistance and to R. Winkler and the staff of the central animal facility (ZTL) for animal care. References
[1] Levine S, Alpert M, Lewis GW. Infantile experience and the maturation of the pituitary adrenal axis. Science 1957;126:1347. [2] Laviola G, Terranova ML. The developmental psychobiology of behavioural plasticity in mice: the role of social experiences in the family unit. Neurosci Biobehav Rev 1998;23:197213.

L. Coutellier et al. / Physiology & Behavior 93 (2008) 395407 [25] Macri S, Wrbel H. Developmental plasticity of HPA and fear responses in rats: a critical review of the maternal mediation hypothesis. Horm Behav 2006;50(5):66780. [26] Shoji H, Kato K. Maternal behavior of primiparous females in inbred strains of mice: a detailed descriptive analysis. Physiol Behav 2006;89:3208. [27] Champagne FA, Francis DD, Mar A, Meaney MJ. Variations in maternal care in the rat as a mediating influence for the effects of environment on development. Physiol Behav 2003;79:35971. [28] Drschlag M, Wrbel H, Stauffacher M, Von Holst D. Repeated blood collection in the laboratory mouse by tail incisionmodification of an old technique. Physiol Behav 1996;60:15658. [29] Marashi V, Barnekow A, Ossendorf E, Sachser N. Effects of different forms of environmental enrichment on behavioral, endocrinological, and immunological parameters in male mice. Horm Behav 2003;43:28192. [30] Madini R, Wolfer DP. Elevated null-maze. EMBO/FENS Course Mouse Transgenics and Behaviour. Protocols for demonstrationZurich; 2001. p. 25.1. [31] Pellow S, Chopin P, File SE, Briley M. Validation of open:close arm entries in an elevated plus-maze as a measure of anxiety in the rat. J Neurosci Methods 1985;14:14967. [32] Sheperd JK, Grewal SS, Fletcher A, Bill DJ, Dourish CT. Behavioural and pharmacological characterisation of the elevated zero-maze as an animal model of anxiety. Psychopharmacologia 1994;116:5664. [33] Hall CS. Emotional behavior in the rat. I. Defaecation and urination as measures of individual differences in emotionality. J Comp Psychol 1934;18:385403. [34] Hall CS. Emotional behavior in the rat. III. The relationship between emotionality and ambulatory behaviour. J Comp Psychol 1936;22:34552. [35] Choleris E, Thomas AW, Kavaliers M, Prato FS. A detailed ethological analysis of the mouse open-field test: effects of diazepam, chlordiazepoxide and an extremely low frequency pulsed magnetic field. Neurosci Biobehav Rev 2001;25:25360. [36] Hughes RN. Behaviour of male and female rats with a free choice of two environments differing in novelty. Anim Behav 1968;16:926. [37] Misslin R, Ropartz P. Effects of metamphetamine on novelty-seeking behaviour by mice. Psychopharmacologia 1981;75:3943. [38] Dixon WJ. BMDP Statistical Software Manual, Volume 1 and 2. Berkeley, Los Angeles: University of California Press; 1993. p. 131152. [39] Denenberg VH. Commentary: is maternal stimulation the mediator of the handling effect in infancy? Dev Psychobiol 1998;34:13.

407

[40] Pryce CR, Bettschen D, Feldon J. Comparison of the effects of early handling and early deprivation on maternal care in the rat. Dev Psychobiol 2001;38:23951. [41] File SE. Factors controlling measures of anxiety and responses to novelty in the mouse. Behav Brain Res 2001;125:1517. [42] Catalani A, Casolini P, Scaccianoce S, Patacchioli FR, Spinozzi P, Angelucci L. Maternal corticosterone during the lactation paremanently affects brain corticosteroid receptors, stress response and behaviour in rat progeny. Neuroscience 2002;100(2):31925. [43] Touma C, Palme R, Sachser N. Analyzing corticosterone metabolites in fecal samples of mice: a noninvasive technique to monitor stress hormones. Horm Behav 2004;45(1):1022. [44] Barnett SA. The rat: a study in behavior. Chicago: University of Chicago Press; 1975. [45] Schradin C, Pillay N. The Striped Mouse (Rhabdomys pumilio) from the Succulent Karoo, South Africa: a territorial group-living solitary forager with communal breeding and helpers at the nest. J Comp Psychol 2004;118:3747. [46] Moriceau S, Sullivan RM. Maternal presence serves as a switch between learning fear and attraction in infancy. Nature Neurosci 2006;9:10046. [47] Cirulli F, Capone F, Bonsignore LT, Aloe L, Alleva E. Early behavioural enrichment in the form of handling renders mouse pups unresponsive to anxiolytic drugs and increases NGF levels in the hippocampus. Behav Brain Res 2007;178:20815. [48] Calatayud F, Coubard S, Belzung C. Emotional reactivity in mice may not be inherited but influenced by parents. Physiol Behav 2004;80:46574. [49] Priebe K, Brake WG, Romeo RD, Sisti HM, Mueller A, McEwen BS, et al. Maternal influences on adult stress and anxiety-like behavior in C57BL/6J and BALB/CJ Mice: a cross-fostering study. Dev Psychobiol 2005;47: 398407. [50] Chapillon P, Mannech C, Belzung C, Caston J. Rearing environmental enrichment in two inbred strains of mice: 1. Effects on emotional reactivity. Behav Genet 1999;29(1):416. [51] Plotsky PM, Thrivikraman KV, Nemeroff CB, Caldji C, Sharma S, Meaney MJ. Long-term consequences of neonatal rearing on central corticotropinreleasing factor systems in adult male rat offspring. Neuropsychopharmacology 2005;30:2192204. [52] Priestnall R. Effects of litter size on the behaviour of lactating female mice (Mus musculus). Anim Behav 1972;20:38694.