European Journal of Neuroscience, Vol. 18, pp.

3367±3374, 2003 ß Federation of European Neuroscience Societies

Environmental enrichment during adolescence reverses

the effects of prenatal stress on play behaviour and HPA
axis reactivity in rats

Sara Morley-Fletcher,1,2 Monica Rea,1 Stefania Maccari2 and Giovanni Laviola1

1
Section of Behavioural Pathophysiology, Department of Cell Biology & Neuroscience, Istituto Superiore di SanitaÁ, Viale Regina
Elena, 299, I-00161 Roma, Italy
2
Laboratory Perinatal Stress, University of Lille 1, Villeneuve d'Ascq 59655, France

Keywords: corticosterone, distributed pregnancy, therapeutic strategy

Abstract
Prenatal stress (PS) can produce profound and long-lasting perturbations of individual adaptive capacities, which in turn can result in an
increased proneness to behavioural disorders. Indeed, in PS rats there is evidence of impaired social play behaviour, disturbances in a
variety of circadian rhythms, enhanced anxiety and increased hypothalamic±pituitary±adrenal (HPA) axis reactivity. This study was
designed to experimentally investigate the degree of reversibility of PS-induced disturbances of social play and HPA reactivity by
assessing the effect of the enrichment of the physical environment on PS rats during periadolescence. PS subjects showed a reduced
expression of social play behaviour and a prolonged corticosterone secretion in response to restraint stress, but both these effects were
markedly reversed following environmental enrichment. Interestingly, the enrichment procedure increased social behaviour but had no
effect on corticosterone secretion in nonstressed animals, indicating a differential impact of the postnatal environment as a function of
prenatal background. As a whole, results clearly indicate that rats prenatally exposed to stress can bene®t during periadolescence from
the modulatory effects of an enriched environment. Moreover, they con®rm that PS may well represent a suitable animal model for the
design and testing of new therapeutic strategies for behavioural disorders produced by early insults.

Introduction
In human beings, exposure to stress early in life can increase vulner-
ability to the development of long-term behavioural abnormalities
(Meijer, 1985; Gitau et al., 2001; Heim & Nemeroff, 2001). It has been
hypothesized that a stress-responsive neural network is permanently
altered by early adverse events. Indeed, there is evidence of an
association between foetal exposure to increased levels of glucocorti-
coids and hypertension, increased anxiety disorders and altered neu-
roendocrine responses later in adult life (for review see Seckl, 2001;
Weinstock, 2001). In animal models, prenatal stress (PS) induces a
number of long-term disturbances such as a reduced expression of
juvenile social play (Ward & Stehm, 1991) and an enhanced beha-
vioural reactivity to emotional challenges (Joffe, 1978; Vallee et al.,
1997), together with a persistent impairment of the feedback inhibition
of the hypothalamic±pituitary±adrenal (HPA) axis (Fride et al., 1986;
Vallee et al., 1997; Maccari et al., 2003).
The environmental conditions under which animals are reared after
birth are known to affect the shaping of their adaptive skills (Laviola,
1996; Laviola & Terranova, 1998). In this regard, there is evidence that
postnatal manipulations which directly affect mother±pups interaction,
such as early fostering (Maccari et al., 1995) or handling during the
®rst two weeks of life (Wakshlak & Weinstock, 1990; Smythe et al.,
Correspondence: Dr Giovanni Laviola, as above.
E-mail: laviola@iss.it
Received 23 October 2002, revised 4 July 2003, accepted 21 August 2003
1996), can reverse the effects of prenatal stress on emotional reactivity
and HPA axis function. Manipulation of the environment can also be
obtained by increasing its physical complexity (e.g. novel objects in
the cage changed weekly), i.e. by using an environmental enrichment
procedure. To date, the simultaneous comparison of the effects and/or
possible mechanisms of perinatal manipulation and enriched environ-
ment has been scarce and often limited to the assessment of memory
and learning abilities (Carughi et al., 1990; Hannigan et al., 1993;
Iuvone et al., 1996; Francis et al., 2002). There is apparent incon-
sistency in the effects of enrichment on other parameters such as play
or emotional behaviour (Renner & Rosenzweig, 1986; Van Waas &
Sof®e, 1996) or the HPA axis hormonal response (Mohammed et al.,
1993; van Praag et al., 2000; Schrijver et al., 2002). However, most of
these studies have been carried out in animals with no history of early
insults.
This study was designed to experimentally investigate the effects of
environmental enrichment on PS rats. Early fostering (Meek et al.,
2001) as well as preweaning enrichment (Dell & Rose, 1987) affect the
relationship between dam and pups and can therefore in¯uence early
postnatal development. In order to dissociate the effects of environ-
mental enrichment and prenatal stress we assessed non-fostered
animals which were transferred into an enriched or standard housing
environment at the moment of weaning. We addressed the question of
reversibility of PS-induced disturbances on social play behaviour and
corticosterone secretion in response to restraint stress. Our results
showed that in PS rats environmental enrichment increases play
behaviour and normalizes the HPA axis reactivity.

doi:10.1046/j.1460-9568.2003.03070.x
3368 S. Morley-Fletcher et al.
Materials and methods
Animals
Sprague-Dawley female rats (250 g), without prior breeding experi-
ence, were purchased from a commercial breeder (Charles River,
Italy). Animals were housed in an air-conditioned room (temperature
21  1 8C, relative humidity 60  10%), with a reversed 12-h light±
dark cycle (lights on at 20.00 h). Water and food (Enriched Standard
Diet purchased from Mucedola, Settimo Milanese, Italy) were avail-
able ad libitum. For a week after arrival, animals were group-housed
(four per cage) to coordinate their oestrus cycle. Females were then
placed with a sexually experienced male and daily inspected for
vaginal smear until the discovery of spermatozoids (designated as
day of gestation 0), after which they were housed individually in
Plexiglas cages (30  20  15 cm). Pregnant females were thus ran-
domly assigned to prenatal stress (PS, n ˆ 12 each) or nonstressed
groups (No Stress, n ˆ 12 each). All experiments were carried out in
accordance with the European Communities Council directive of 24
November 1986 (86/609/EEC).
Prenatal stress procedure
The stress procedure started on day 11 of pregnancy until delivery at
22 days as previously reported (see Maccari et al., 1995): pregnant
females were individually placed in transparent plastic cylinders
(diameter 7 cm, length 19 cm) and exposed to bright halogen light
for 45 min. Animals were submitted daily to three stress sessions
during the dark phase of the light±dark cycle, starting at 09.00, 12.00
and 17.00 h. Control No-stress pregnant females were left undisturbed
in their home cages. Male and female offspring were weaned on day 22
after birth, and only male offspring from litters containing 10±14 pups
with a comparable number of males and females were used in the
present study.
Housing and environmental enrichment
The standard laboratory conditions were de®ned as a pair of siblings
housed in transparent cages (32  20  15 cm). Enrichment was
de®ned in terms of physical environment and not social housing
condition (Renner & Rosenzweig, 1986). Therefore, animals were
housed in pairs of siblings in larger and higher cages (40  25  30 cm)
provided with differently shaped plastic containers, coloured plat-
forms, suspended objects and a wheel (diameter 15 cm). The objects
Fig. 1. Time line for experimental protocol showing age of animals (days) during each test. The enrichment condition consisted of differently shaped plastic
containers, coloured platforms, suspended objects and a wheel. The objects were provided in three different sets which were changed twice a week. When the wheel
was present the rats were provided with either a platform or a container suitable for hiding; the bottle of water was suspended above the ceiling and food pellets were
provided on the ¯oor.
ß 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 18, 3367±3374
were provided in three different sets, changed twice a week, in such a
way that animals had a protected environment suitable for hiding and
an open corner suitable for exploration. When the wheel was present
the rats were provided with either a platform or a container, the bottle
of water was suspended above the ceiling and food pellets provided on
the ¯oor.
For both housing conditions, the sawdust of the cage was changed
once a week in association with measurement of animals' body weight.
Rats were put in the standard or enriched cages at the age of 22 days
and maintained in their housing condition throughout all the experi-
mental assessment.
Experimental design
In the present study, 14 No-stress and 14 PS rats were assigned to the
non- enriched condition (NE groups) and 12 No-stress and 14 PS
animals to the enriched one (E groups). In order to prevent litter effects
(Chapman & Stern, 1979), no more than two male siblings from each
original litter were assigned to each housing environment.
The timeline (Fig. 1) illustrates the temporal sequence of events.
Behavioural assessment began after a week of housing in an E or an NE
condition, and corticosterone response was measured at the end of the
housing period. All animals were assessed for social behaviour and
endocrine response.
Social interaction test
The test was conducted on pairs of siblings from the same housing
condition placed for a single 20-min session in a novel environment
represented by the test arena (40  25  30 cm). In order to increase
social behaviour, the rats in a pair had been separated and individually
housed for 24 h before behavioural assessment (Cirulli et al., 1996;
Terranova et al., 1999). The whole session was video-recorded using a
professional Sony videocassette recorder VO-58OOPS apparatus. The
®rst and last 5 min of each session were manually scored (Observer 20
Noldus, Wageningen, the Netherlands) according to an `all-occur-
rence' sampling method (Martin & Bateson, 1986) by an observer
blind to the assignment of animals to the different groups. Separate
scores were obtained for each individual in a pair but, because the two
values cannot be considered statistically independent, pair means were
used for further analysis. Therefore, data presented refer to a total of
seven pairs of rats from each group and housing condition. The
following social and nonsocial behavioural categories were recorded.
 Prenatal stress and enriched environment 3369

Social behaviours
(i) Rough-and-tumble play:. pouncing (the subject lunges toward the
side or back of the partner with the fore paws extended), wrestling and
pinning (one of the animal lying with its dorsal surface on the ¯oor
with the other animal standing over it) with the partner. (ii) Social
investigation: anogenital and body snif®ng. (iii) Social rest: the subject
is lying ¯at or standing still while maintaining physical contact with
the partner, which may be in turn either inactive or involved in
activities which do not require movements around the cage (i.e
social snif®ng or maintenance activities).
Non-social behaviours
(i) Exploring: snif®ng the air, rearing and exploring the cage. (ii)
Grooming.
Radioimmunoassay for corticosterone
At the end of the housing period (see Fig. 1), animals were assessed for
the HPA axis response to a restraint stress. Brie¯y, blood samples
( 100 mL) were collected via the tail vein three times between 09.30
and 11.30 h. The two members of a cage were assessed at the same time
by two separated experimenters in order to avoid differences in the
sampling procedure. Rats were moved to an adjacent room and
individually placed in a restraint transparent tube. Restraint was
carried out in plastic cylinders identical to those used for the prenatal
stress procedure. Blood was collected quickly (< 1 min) to determine
basal corticosterone levels (t0). A second sampling was performed
20 min after restraint stress was initiated (t20). Rats were then returned
to their home cage with their usual partner, until the last blood
sampling was performed 60 min after the initiation of the stress
procedure (t60). Blood was collected in tubes ®lled with 100 mL of
EDTA. Plasma corticosterone levels were obtained using a RIA Kit
(ICN Biomedicals) with a highly speci®c corticosterone antibody and a
detection threshold of 0.1 mg/100 mL. The intra- and interassay coef-
®cients of variation were 5 and 11%, respectively. Numbers of animals
used were nine No-stress and 14 PS rats for both the NE and the E
conditions.
Statistics
Behavioural data were analysed using parametric analysis of variance
(ANOVA) with two levels of prenatal status (PS vs. No-stress) and two
levels of postweaning housing (enriched vs. nonenriched) as between-
subjects factors (Winer, 1971; Chiarotti et al., 1987). Corticosterone
levels were calculated by ANOVA with times of blood sampling
considered as within-subjects factors and prenatal status and post-
weaning housing as between-subjects factors. The area under the curve
of corticosterone data was calculated by use of the trapezoidal rule.
Planned contrast analysis was used to test the locus of signi®cant main
effects and interactions when present. Signi®cance was set at P < 0.05
for all analysis.
Results
Effects of gestational stress on mothers and offspring
No differences as a consequence of prenatal stress were observed for
the length of gestation (22 days), number of pups ( 13 per litter in all
experimental groups), and sex ratio (M : F ˆ 1). At weaning, ANOVA
revealed a tendency for a reduced increment in body weight in stressed
mothers when compared to nonstressed ones (prenatal condition,
F1,22 ˆ 4.05, P ˆ 0.056, with 312  4.5 g for PS vs. 328  6.7 g for
No-stress).
Animals' body weight gain
Results are shown on Fig. 2. A signi®cant increase in body weight was
observed at weaning in rats born from stressed mothers as compared to
nonstressed animals (see inset Fig. 2A, prenatal condition,
F1,22 ˆ 8.75, P < 0.01).
Body weight gain was measured weekly and expressed either in
grams (Fig. 2A, graph) or as ratio to body weight at weaning (Fig. 2B).
A prenatal condition  post-weaning housing interaction (F1,45 ˆ 7.03,
P < 0.01) revealed that within the NE groups PS animals were con-
sistently heavier than No-stress rats throughout the 5 weeks of housing
(F1,45 ˆ 4.86, P < 0.05 for PS±NE vs. No-stress±NE group) whereas
PS rats raised in an enriched environment showed a reduced body
weight gain similar to No-stress±NE rats and signi®cantly lower than
their NE counterparts (F1,45 ˆ 4.77, P < 0.05, for PS±E vs. PS±NE
group).
Social interaction test
Frequency of rough-and-tumble play (Fig. 3A): a main effect of
prenatal condition (F1,21 ˆ 5.74, P < 0.05) and of postweaning housing
(F1,21 ˆ 13.35, P < 0.001) was found although their interaction was
not signi®cant (F1,21 ˆ 1.26, n.s.) Speci®cally planned comparisons
conducted on each main effect revealed that PS decreased social play
(F1,21 ˆ 5.97, P < 0.05 for PS±NE vs. No-stress±NE group) whereas
enrichment markedly increased it in PS rats (F1,21 ˆ 11.85, P < 0.01)

Fig. 2. Body weight gain. (A) Mean (SEM) body weight gain expressed in grams as measured weekly shown by adolescent No-stress and PS rats reared in standard
(NE) or in enriched (E) conditions (n ˆ 11±14 for each group). The inset shows that a signi®cant increase in body weight was observed at weaning in PS rats when
compared to No-stress animals. (B) The same data displayed in A pooled over the time variable and expressed as the ratio to the body weight at weaning (weekly body
weight: weaning body weight). P < 0.05, PS vs. No-stress group within the NE condition; #P < 0.05 E vs. NE condition within the PS group.

ß 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 18, 3367±3374
3370 S. Morley-Fletcher et al.
Fig. 3. Social interaction test. Mean ‡ SEM (A) frequency and (B) duration of
rough-and-tumble play, (C) duration of social rest and (D) duration of environ-
mental exploration shown by adolescent No-stress and PS rats at 36 days of age
reared in standard (NE) or in enriched (E) conditions (n ˆ 6 pairs for each
group). P < 0.05 PS vs. No-stress group within the NE condition; #P < 0.05,
##
P < 0.01 E vs. NE condition within the PS group.
but not in the No-stress group (F1,21 ˆ 3.08, n.s.) Duration of rough-
and-tumble play (Fig. 3B): a main effect of prenatal condition
(F1,21 ˆ 4.11, P < 0.05) and of postweaning housing (F1,21 ˆ 8.26,
P < 0.01) were found although their interaction was not signi®cant
(F1,21 ˆ 1.26, n.s.) Enrichment signi®cantly increased time spent in
social play in PS rats (F1,21 ˆ 4.5, P < 0.05) and slightly in No-stress
subjects (F1,21 ˆ 3.79, P ˆ 0.065).
Duration of social rest: no effects of prenatal stress were observed
(prenatal condition, F1,21 ˆ 1.55, n.s.) whereas a main effect of
enrichment was found (postweaning housing, F1,21 ˆ 6.59
P < 0.01). Speci®cally, levels of social rest were markedly increased
in PS rats (F1,21 ˆ 4.75, P < 0.05) but not in No-stress animals
(F1,21 ˆ 2.15, n.s.) Duration of exploratory behaviour: no effects of
prenatal stress were observed (prenatal condition, F1,21 ˆ 0.01, n.s.)
whereas a strong effect of enrichment was found (postweaning hous-
ing, F1,21 ˆ 10.70, P < 0.01). Time spent exploring the cage was
reduced in all animals.
Basal and stress-induced corticosterone secretion
Plasma corticosterone response, measured immediately before, during
and up to 60 min after the restraint procedure, is shown in Fig. 4A. A
separate analysis performed for each time point of sampling revealed
for basal level (t0) no effect of prenatal stress (prenatal condition,
F1,41 ˆ 0.12, n.s.) or enrichment (postweaning housing, F1,41 ˆ 1.53,
n.s.), whereas at the peak level (t20) enrichment signi®cantly reduced
corticosterone secretion in PS animals (postweaning housing,
F1,31 ˆ 5.28, P < 0.05) but not in No-stress rats. For return to basal
level (t60), ANOVA revealed an effect of prenatal stress (prenatal
condition, F1,31 ˆ 4.04, P < 0.053) and a tendency for an effect of
enrichment (postweaning housing, F1,31 ˆ 3.73, P ˆ 0.06) although
their interaction did not reach signi®cance (n.s.) Speci®cally, a pro-
longed elevation in plasma corticosterone was measured in PS±NE
ß 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 18, 3367±3374
rats compared to the No-stress±NE group (F1,31 ˆ 5.47, P < 0.05 for
PS±NE vs. No-stress±NE, P < 0.05). Interestingly, a more rapid return
to baseline levels similar to non-stressed rats was observed in PS±E
animals (F1,31 ˆ 5.19, P < 0.05).
When considering the area under the curve (AUC, Fig. 4B), ANOVA
revealed a tendency for an effect of PS (prenatal condition,
F1,31 ˆ 3.37, P ˆ 0.06) and enrichment (postweaning housing,
F1,31 ˆ 3.73, P ˆ 0.06) although their interaction was not signi®cant
(F1,31 ˆ 1.05, n.s.) Speci®cally planned comparisons conducted on
each simple effect revealed that the integrated levels of corticosterone
were higher in PS±NE rats than in non-stressed animals (F1,31 ˆ 4.80,
P < 0.05 for PS±NE vs. No-stress±NE) and to the corresponding
enriched group (F1,31 ˆ 5.12, P < 0.05 for PS±NE vs. PS±E). As a
consequence PS±E rats had levels of corticosterone similar to No-
stress±NE animals. Further, enrichment procedure had no effects on
No-stress animals.
Discussion
This study was designed to experimentally investigate the effects of
environmental enrichment during adolescence on PS rats. We found
that, following enrichment, rats prenatally exposed to stress presented
an increased play behaviour and a reduced stress-induced corticoster-
one response. The same environmental procedure increased social
behaviour but did not affect endocrine reactivity in nonstressed
animals.
Previous studies on long-term consequences of prenatal stress have
been mostly carried out on adult subjects. In this regard, some aspects
of the behavioural and physiological pro®le of adolescent PS rat
reported herein need to be described ®rst separately and then in their
interaction with environmental enrichment.
Carry-over effects of prenatal stress on behavioural and
endocrine outcome in the adolescent rat
Male rats were assessed during adolescence, an ontogenetic stage
characterized by high basal levels of behavioural activation (Spear,
2000; Laviola et al., 1999, 2003) and by a prominent expression of the
playful behavioural repertoire (Panksepp, 1981). We found a reduced
amount of rough-and-tumble play in PS subjects when compared to
non-stressed animals. These data con®rm and extend the ®ndings
previously obtained by Ward & Stehm (1991). In that study, however,
the authors assessed basal levels of play behaviour by nonsibling
littermates observed in the home cage. However, in the present work
we assessed the playful interactions of sibling rats placed in a novel
environment and observed in pairs.
Other perinatal manipulations, such as neonatal handling and
prolonged maternal separation, have also been reported to similarly
affect play behaviour in males, with a feminization of their pattern of
responsiveness to playful contacts (Arnold & Siviy, 2002). This is
interesting, because maternal separation permanently increases the
magnitude of neuroendocrine and behavioural responses to stress
(Plotsky & Meaney, 1993) similarly to PS manipulations. On the
other hand, neonatal handling, which involves only a brief period of
mother±pup separation, results in opposite behavioural response to
stress and neuroendocrine effects (Meaney et al., 1988). Early admin-
istration of corticosterone has been reported to decrease play in male
rats while having no effect on females (Meaney et al., 1982). Because
adverse (PS and maternal separation) and positive (neonatal handling)
perinatal manipulations affect corticosterone levels differently, it is
likely that this hormone is not the sole mechanism and that the effects
of early rearing on play are not solely dependent of glucocorticoid
involvement. In this regard, in order to explain the in¯uence of
 Prenatal stress and enriched environment 3371

Fig. 4. Stress-induced corticosterone secretion. (A) Mean ‡ SEM plasma corticosterone secretion (mg/dL) in basal conditions (time 0 min) and in response to a 20-
min period of restraint stress (time 20, and 60 return to baseline) shown by adolescent No-stress and PS rats reared in standard (NE) or in enriched (E) conditions. (B)
Mean (SEM) area under the curve (AUC) analysis for the corticosterone data displayed in A calculated by using the trapezoidal rule (n ˆ 11±14 for each group).

P < 0.05 PS vs. No-stress group within the NE condition; #P < 0.05 E vs. NE condition within the PS group.

perinatal manipulations on the neuroendocrine maturational processes
of the modulatory mechanisms underlying play behaviour, attention
has mostly been focused on gonadal hormones (Thor and Holloway,
1984). Actually, play is a sexually dimorphic behaviour (Meaney,
1989; Hotchkiss et al., 2002) and neonatal testosterone levels are
reduced in PS males (Ward, 1983; Ward & Stehm, 1991; Henry et al.,
1996). However, an involvement of the central opioid system should
also be taken into account to explain the reduction of social play in
male rats subjected to perinatal manipulations and stress (Panksepp,
1981; Ploj et al., 1999; Kalinichev et al., 2001; Terranova et al., 2001;
Weinstock, 2001).
In our experimental conditions, non-stressed animals exhibited the
expected increment in social play behaviour as a coping strategy to
novelty whereas PS animals did not (Terranova et al., 1999; Van der
Berg et al., 1999). The speci®c elevation in levels of social af®liative
interactions (Cirulli et al., 1996), together with the associated intensi-
®cation of exploration and novelty-seeking observed during the ado-
lescent period (Adriani et al., 1998; Laviola et al., 2003), is necessary
for the development of appropriate responses to social situations to be
encountered later in life (Spear, 2000; Von Frijtag et al., 2002). From
an evolutionary perspective, the reduced amount of social play herein
observed in PS rats has important implications for the potential
development of maladaptive behaviours and for the survivability of
individuals in a social setting.
Adolescent PS rats presented higher levels of stress-induced corti-
costerone secretion and a delayed return to the baseline following the
stress extinction when compared to controls. These results are con-
sistent with a previous report by Dugovic et al. (1999). Therefore, at
the adolescent stage we noticed the similar pro®le of impaired feed-
back inhibition of HPA axis activity caused by maternal stress to that
which has been reported in the offspring throughout the different
phases of ontogenesis (Henry et al., 1994).
Our study also indicated that PS rats were consistently heavier than
controls. On this issue, con¯icting results are available in the literature.
Some authors have reported a reduction in body weight in PS compared
to control rats at birth (Pollard, 1984; Cabrera et al., 1999), or at the adult
stage (Vallee et al., 1996), whereas others have found an opposite pro®le
(P®ster & Muir, 1992). A simple explanation may involve an alteration
in feeding behaviour, although the mechanisms involved remain to be
elucidated. A previous work from Vallee et al. (1996) suggested that
prenatal stress may in¯uence metabolic set points through a lower food
intake, associated with increased blood glucose levels. In this regard, the
discrepancies in the effects of prenatal stress on body weight gain should
also be viewed in the light of the differences in developmental stages as
well as in prenatal stress procedures.
Environmental enrichment during adolescence and interplay
with prenatal stress
To date, the effects exerted by environmental manipulations consisting
of increased cage complexity on behavioural parameters other than
learning and memory have been poorly documented. This is surprising,
because there is a large body of evidence on the multifaceted beha-
vioural effects of isolation, which undoubtedly corresponds to an
impoverished environment (Panksepp & Beatty, 1980; Hol et al.,
1999). To our knowledge, there is only one study which has so far
evaluated whether enrichment can affect social play (Renner &
Rosenzweigh, 1986), and it reported negative ®ndings. In general,
studies testing the ability of enrichment to affect emotional behaviour
are lacking or often inconsistent (Van Waas & Sof®e, 1996; Pham et al.,
1999). Furthermore, as for the in¯uence of environmental enrichment
on endocrine responses to stressors, it must be acknowledged that
hormonal ®ndings have not provided additional support for the obser-
vation that enriched animals seem to be less behaviourally stressed
than nonenriched ones. Actually, the HPA axis hormonal response has
not been shown to be differentiated in enriched and non-enriched
subjects (Mohammed et al., 1993; van Praag et al., 2000; Schrijver
et al., 2002; see also Francis et al., 2002).
The experience of a physically enriched environment resulted in a
signi®cant increment in body weight gain in nonstressed animals. An
improved regulation of body temperature in enriched rats could be
accounted as a possible explanation (see Van de Weerd et al., 1997).
The observation of rats resting inside the toy objects was also frequent,
which may have reduced heat loss. Such an effect was less evident in
PS animals housed in enriched conditions, who did not gain weight as
fast as their standard-housed counterparts. In addition to methodolo-
gical differences in time and quality of the enrichment procedure,
among the other factors that may be taken into account to explain
inconsistencies is the fact that usually enrichment studies compare
animals reared in enriched environments in groups with animals reared
in barren environments individually (Renner & Rosenzweigh, 1986;
Mohammed et al., 1993). Most importantly, the great majority of
enrichment studies on parameters other than learning and memory
have been carried out in animals with no history of early adverse
insults (Huck and Price, 1975; see, however, Francis et al., 2002).

ß 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 18, 3367±3374
3372 S. Morley-Fletcher et al.
In our experimental setting, environmental enrichment was found to
be associated with increased levels of play and to a reduction in HPA
axis reactivity in PS rats. Such effects were obtained comparing
enriched animals with animals reared in standard laboratory conditions
but not socially isolated. Moreover, the enrichment procedure involved
the use of cages of laboratory size, thus representing a housing practice
which can easily be standardized. It may be questioned in fact whether
standard housing conditions provide an ideal environmental back-
ground for the study of complex brain functions. In this regard, in the
laboratory, the safety and stability of the rearing environment does not
reliably predict the future challenges involved in life as an experi-
mental animal (Wurbel, 2001).
Play behaviour is associated with a high risk of exposure to
predators, and engaging in vigorous forms of social interaction is
accompanied by lower levels of attention to the environment. If
environmental conditions are such that reduced attention is not advi-
sable, animals will not engage in play (Vanderschuren et al., 1995).
Therefore, the higher amount of play behaviour observed in enriched
animals could also indicate a reduced level of anxiety.
PS rats reared in an enriched environment exhibited higher levels of
play behaviour than No-stress controls. Our study is the ®rst report on
the effects of enrichment on play behaviour following perinatal
manipulation. Although the mechanisms involved in the regulation
of play behaviour were beyond the purpose of this work, some
hypotheses may be offered. A study by Bardo et al. (1997) indicated
reduced sensitivity to morphine administration in enriched rats com-
pared to non-enriched controls. Because the opioid system is over-
expressed in PS rats (Weinstock, 2001), a reduced activation of this
system in PS animals following the experience of enrichment should
be taken into account. Also, the role of corticosterone should be
considered (Meaney et al., 1982). Indeed, we observed that PS±NE
rats showed enhanced levels of corticosterone and reduced play
behaviour, whereas PS±E animals exhibited high levels of play in
association with a reduced HPA reactivity. Actually, the experience of
an enriched environment completely reversed the behavioural and
physiological alterations induced by the prenatal stress.
As for corticosterone, enriched PS animals showed a reduced peak
and a return to baseline levels similar to non-stressed animals, thus
indicating a reduced HPA axis reactivity. Such results are consistent
with those obtained by Francis et al. (2002) in maternally deprived rats
reared in an enriched environment. For future studies, the effects of
enrichment on neurogenesis should also be taken into account. Indeed,
it has been shown that postweaning (but not preweaning) enrichment
(Kohl et al., 2002) enhances hippocampal neurogenesis in adult
animals (Kemperman et al., 1997; van Praag et al., 1999). Prenatal
stress has been found to induce a lifespan reduction of neurogenesis in
the hippocampus (Lemaire et al., 2000). Thus, an improved HPA axis
regulation could have also in¯uenced the rate of neurogenesis in PS
animals. In the non-stressed subjects of the present study, levels of
secreted corticosterone in response to the acute restraint challenge
were unaffected by environmental background. Actually, peak and
recovery levels were very consistent in standard and enriched groups.
This pro®le is in accordance with previous studies which indicate a
weak effect of enrichment on physiological and/or behavioural para-
meters in `naive' animals (van Praag et al., 2000; Schrijver et al.,
2002).
It is important to underline that enrichment exerted a differential
in¯uence as a function of prenatal background. Indeed, it enhanced
play behaviour more markedly in PS rats than in non-stressed animals,
and it did not affect HPA axis reactivity in the latter. It seems therefore
that prenatal stress brought out the behavioural and physiological
consequences of enriched rearing. It is interesting to consider that, in
ß 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 18, 3367±3374
humans, programs of child intervention which serve to offset the risk
associated with family stress for intellectual and emotional develop-
ment have reported that effects are most pronounced in individuals
whose development was compromised by the experience of early
adversities (Ramey & Ramey, 1998). The buffering effects of enriched
environment on PS rats parallel and extend those previously reported,
such as the effects of early adoption on the HPA axis (Maccari et al.,
1995) and of neonatal handling on anxiety-like behaviour (Wakshlak
& Weinstock, 1990; Smythe et al., 1996; see also Francis et al., 2002).
In conclusion, this study provides evidence of a marked reversibility of
PS-induced alterations at both the behavioural and the physiological
level following environmental enrichment. A better understanding of
the environmental factors which are involved in the control mechan-
isms of development might be of great bene®t for a better comprehen-
sion of the long-term effects of early adverse life events.
Acknowledgements
This research was supported as part of the Nervous and Mental Disorders
Research Area Project on `Psychobiological risk or protection factors for
behavioural disorders and vulnerability to recreational substances of abuse
during development' grant SS-1106/RI to G.L., and by the research project
`Hypoxic-Ischemic Brain damage in the Newborn' (OAN/F8), Ministry of
Health; and by the research grant `Pathogenesis and Recovery in Animal and In-
vitro Models of Alzheimer Disease', Italian Ministry of Health, to G.L.
Abbreviations
E, enriched; HPA, hypothalamic±pituitary±adrenal; NE, nonenriched; PS,
prenatal stress (group).
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