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Gestational stress induces post-partum depression-like behaviour and alters maternal care in rats
Psychoneuroendocrinology 29 (2004) 227–244 www.elsevier.com/locate/psyneuen
Gestational stress induces post-partum depression-like behaviour and alters maternal care in rats J.W. Smith a, J.R. Seckl b, A.T. Evans a, B. Costall a, J.W. Smythe a,∗ a
Neuropharmacology Research Group, Department of Pharmacology, School of Pharmacy, University of Bradford, Bradford BD7 1DP, UK b Department of Medical Sciences, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK
Received 28 August 2002; received in revised form 25 October 2002; accepted 14 January 2003
Abstract Gestational stress (GS) produces profound behavioural impairments in the offspring and may permanently programme hypothalamic–pituitary–adrenal (HPA) axis function. We investigated whether or not GS produced changes in the maternal behaviour of rat dams, and measured depression-like behaviour in the dam, which might contribute to effects in the progeny. We used the Porsolt test, which measures immobility in a forced-swim task, and models depression in rodents, while monitoring maternal care (arched-back nursing, licking/grooming, nesting/grouping pups). Pregnant rats underwent daily restraint stress (1 h/day, days 10–20 of gestation), or were left undisturbed (control). On post-parturition days 3 and 4, dams were placed into a swim tank, and time spent immobile was measured. GS significantly elevated immobility scores by approximately 25% above control values on the second test day. Maternal behaviours, in particular arched-back nursing and nesting/grouping pups, were reduced in GS dams over post-natal days 1–10. Adult offspring showed increased immobility in the Porsolt test, and also hypersecreted ACTH and CORT in response to an acute stress challenge. These data show that GS can alter maternal behaviour in mothers, and this might contribute to alterations in the offspring. GS may be an important factor in maternal post-natal depression, which may in turn detrimentally effect the offspring because depressed mothers do not sufficiently care for their offspring.
∗
Corresponding author. Tel.: +44-1274-233-361; fax: +44-1274-234-660. E-mail address: [email protected] (J.W. Smythe).
0306-4530/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0306-4530(03)00025-8
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2003 Elsevier Ltd. All rights reserved. Keywords: Rat; Hypothalamic–pituitary–adrenal; Gestation; Stress; Depression; Porsolt
1. Introduction For many decades it has been recognised that environmental manipulations of infant animals can produce enduring alterations in behavioural and endocrine responses to aversive events. The hypothalamic–pituitary–adrenal (HPA) axis is one of the body’s principal response mechanisms enabling animals to respond appropriately to aversive conditions and maintain homeostasis (Munck et al., 1984; Selye, 1978). Briefly, the paraventricular nucleus of the hypothalamus in the rat contains cells which secrete CRH and AVP into the median eminence (Antoni, 1986; Plotsky, 1987; Rivier and Plotsky, 1986). These secretagogues, in turn, diffuse via the portal vessels to the anterior pituitary corticotrophs and elicit the release of ACTH. The systemic circulatory system transports ACTH to the adrenal gland cortex where it evokes the rapid synthesis of the glucocorticoid, corticosterone (CORT; cortisol in humans). CORT enables a number of catabolic events which promote gluconeogenesis and glycogenolysis, and suppress immune function (Brindley and Rolland, 1989; Munck et al., 1984). All of these responses contribute to the ‘fight/flight’ reflex and together with the release of adrenaline from the adrenal medulla, constitute the endocrine stress response (Selye, 1978). Numerous reports have detailed how the HPA axis is subjected to programming and is permanently altered by manipulations of the mother/pup relationship. Early studies reported on the ‘handling’ phenomenon whereby rat pups are temporarily removed from the nest, isolated for 15–20 min and then returned to the dams. Handled rats, as adults, exhibit a reduced response to an acute stress challenge, secrete less ACTH and CORT, and recover basal levels of these hormones more quickly than non-handled control animals (Ader and Grota, 1969; Hess et al., 1969). The essential difference between handled and non-handled rats is in their sensitivity to CORT-mediated negative-feedback (Dallman et al., 1995; De Kloet, 1991; Feldman and Weidenfeld, 1995; Keller-Wood and Dallman, 1984; Reul and De Kloet, 1985). Handled rats have elevated GC receptor numbers in the hippocampus and frontal cortex compared to non-handled rats; the potency of the negative-feedback signal is augmented by the proliferation of GC receptors in handled animals (Meaney et al., 1989, 1993, 1996). Programming of endocrine function and various stress-related behaviours accompany antenatal manipulations such as gestational stress (GS) and prenatal dexamethasone (DEX) exposure. For instance, prenatal DEX administration leads to suppressed exploration of an open field or elevated-plus maze, while simultaneously increasing CRH mRNA in the PVN (Welberg and Seckl, 2001). Gestationally stressed rats exhibit elevated ACTH and CORT secretion in response to footshock stress, an effect which persists well into adulthood (Weinstock, 1997). We reported that adult female rats who had been gestationally stressed, hypersecreted ACTH and
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CORT in response to brief, restraint stress, and that this was accompanied by fluctuations in GC receptor binding throughout the forebrain (McCormick et al., 1995). Behaviourally, GS is associated with elevated indices of fearfulness in the adult progeny, as revealed by increases in ultrasonic vocalisations, defecation, and reduced ambulation in novel open fields (Braastad, 1998; Lehmann et al., 2000; Suchecki and Neto, 1991; Williams et al., 1998). Moreover, anxiety is also enhanced by GS, as shown by the reluctance of stress-exposed rats to explore the open arms of an elevated-plus maze (Lordi et al., 2000; Vallee et al., 1997). Newport et al. (2002) in a meta-analysis of parenting behaviour suggest that various stressors, including maternal separation and physical insults, ultimately produce highly anxious and depressed affective states in offspring animals, reminiscent of clinical depression in humans. The majority of studies have sought to explain HPA axis programming by identifying the underlying neurochemical and hormonal changes that appear in manipulated rats (Peters, 1982; Takahashi et al., 1992; Weinstock et al., 1998). However, it has been plausibly suggested that the HPA axis may exhibit plasticity as a result of ongoing maternal behaviour, which has been carefully characterised in rats (Rosenblatt, 1993). Lui et al. (1997) showed that rats display varying levels of spontaneous maternal behaviour; some dams are highly nurturant, while other mothers are relatively passive and indifferent towards their litters. What is particularly germane regarding this behavioural dichotomy is that pups from highly nurturant mothers have lower HPA axis responsivity to acute stress challenge, elevated hippocampal mRNA for GC receptors, and lower hypothalamic CRH mRNA compared to progeny from the less nurturant dams; importantly, these changes are linearly related to the magnitude of maternal care. Moreover, Meaney’s group (Meaney et al., 1989, 1993, 1996) have further shown that handling increases maternal care, and that female rats raised by highly nurturant dams exhibit greater maternal care than those raised by less nurturant mothers (Francis et al., 1999). These findings raise the question as to whether or not GS alters maternal behaviour in a manner consistent with the work of Lui et al. (1997) and Francis et al. (1999). That is, given the fact that HPA axis function is programmed by spontaneous maternal behaviour and given that GS has potent influences on HPA axis function, would we observe that stressed dams are less nurturant to their offspring than controls? Furthermore, stress is known to be a major contributory factor in the etiology of depression (Anisman and Zacharko, 1982; Hammen and Gitlin, 1997). Does GS alter maternal behaviour and induce a state of depression in the dam? The following study characterises maternal behaviour in rat dams stressed during gestation, while also measuring depression-like behaviour of the dams and offspring in a modified Porsolt test. We have also included measures of endocrine function as positive controls to ensure the genuine nature of our GS procedure.
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2. Methods 2.1. Subjects A total of 52 pregnant dams were generated in-house from nulliparous, Hooded Lister breeding stock. The procedure entailed placing three females (4 months of age) into a maternity cage (60 × 80 × 20 cm3; Techniplast, UK) along with a single male of the same age. These groupings were left for 5–7 days and if no evidence of mating was obvious, a new male was introduced in place of the original. Mating behaviour was monitored by taking daily vaginal smears and the presence of sperm in the smear was taken to signify day 1 of conception. Dams and their litters were housed singly in polycarbonate maternity cages (60 × 80 × 20 cm3) with wood-chip bedding, and food (Purina rat chow) and water were provided ad libitum. At 21–22 days of age, stressed and non-stressed offspring were weaned to larger, colony cages (80 × 80 × 30 cm3; Techniplast, UK) in groups of five (same sex and same experimental condition). Animals were maintained under normal light cycle conditions (on at 08:00 h; off at 20:00 h), with an ambient temperature of 22 °C, and relative humidity of 45%. 2.2. Gestational stress procedure From days 10–20, 25 randomly selected dams (from alternating cages in the housing room) were subjected to daily, 1 h restraint stress by being placed into plexiglass restrainers (Stoelting, USA), positioned in a brightly lit room. Blood samples were obtained from eight stressed rats via a small incision made in the distal region of the tail to collect plasma for hormone assays (described later). These samples were obtained immediately upon placement into the restrainers on days 10, 15 and 20. Upon removal from the restrainers another blood sample was taken. Twenty-seven, non-stressed control rats were left undisturbed in their home cages, although eight did have blood samples taken on the same days and 1 h apart as per the group of stressed rats. These tests were done for all rats on the same day and for each treatment group. Control rats were not restrained during the sampling procedure, but allowed to wander on a table surface while blood was milked from their tails. Blood sample volume ranged from 200–300 µl per time point. This procedure was carried out between 11:00 and 15:00 h. 2.3. Assessment of mother–pup interactions Starting on the day of birth and then on days 3, 6 and 10, maternal behaviour was observed and recorded using a time-lapse VHS video recorder and camera unit, set to sample activity at 1/3 normal speed. Based on previous findings discussed in the Section 1, we chose to monitor the time dams spent in an arched-back nursing position (active) as opposed to any other position e.g. supine (passive) or prostrate position. Arched-back nursing consisted of periods where the dam held herself erect, over the pups in a canopy posture, with her legs stretched out to provide balance.
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We also measured the time engaged in anogenital licking (of the entire litter, not single pups; this behaviour consisted of the dam holding individual pups between her front paws on the cage floor and visibly licking the hindquarters of the pups) and time spent grouping pups into a nest suitable for permitting suckling (the mothers routinely moved the nest around and carried each pup to a new nest and grouped them). We sampled behaviour over a 2 h span with 1 h from the lights-on and 1 h from the lights-off time periods. We used nine dams from each of the stressed or non-stressed conditions to monitor maternal behaviour, and these had not been subjected to blood sampling, nor any other testing. 2.4. Assessment of depressive behaviour in dams and offspring On post-natal days 3 and 4, selected dams (eight stressed and 10 non-stressed) and offspring (14 stressed males, 10 stressed females, 15 non-stressed males and 10 non-stressed females) between the ages of 3 and 4 months, were randomly selected from each experimental condition and were temporarily removed from their litters for testing in a forced-swim task (Porsolt test). Rats were placed in a 25 cm diameter cylinder, full of water 20 cm deep (25 °C), and monitored for time spent immobile (minimum amount of effort used to float) during a 10 min trial (day 3) or a 5 min trial (day 4). Following testing dams were dried under a fan heater for 2 min before being returned to their home cages. Maternal behaviour (described previously) was not recorded from these mothers. This testing was done between 11:00 and 15:00 h. 2.5. Acute stress challenge in adult progeny In adulthood (ages 3–4 months), progeny were subjected to an acute restraint stress challenge by being placed into plexiglass restrainers for 20 min (as per the dams). Blood samples were obtained from a small incision made in the distal region of the tail. These were taken prior to, post-stress (upon removal from the restrainers), and at 1 and 2 h following removal from the restrainer. This testing was done between 11:00 and 15:00 h. 2.6. Assessment of CORT and ACTH levels Blood samples were collected in ice-cooled, eppendorf tubes rinsed with EDTA, centrifuged at 3000 rpm for 10 min at 4 °C, and the plasma was removed and frozen for later ACTH and CORT determination using commercially available, radioimmunoassay kits. Plasma CORT was measured using a specific CORT antiserum, using [125I] CORT (Immuno Diagnostsics Systems, Ltd, Tyne and Wear, UK) as the tracer. A 10 µl sample was assayed in triplicate, at a minimum detection limit of 1 ng/ml plasma. The antiserum does not cross-react with cortisol, aldosterone or progesterone, but will react slightly with deoxycorticosterone (4%). Plasma ACTH was measured with a specific ACTH antiserum using [125I] ACTH as the tracer. The ACTH antibody cross-reacts 100% with ACTH1-39, but not ACTH1-16, bendorphin, a-MSH, or b-lipoprotein. A 25 µl sample was assayed in triplicate, at a
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minimum detection limit of 15 ng/ml plasma. Radioactivity was counted using a Gamma counter programmed to count for a 2 min period. Intra-and inter-assay reliability is about 4% for both assay procedures. These techniques are similar to those of our previous studies (McCormick et al., 1995; Smythe et al., 1996). 2.7. Statistical analysis All data were assessed by analysis of variance (ANOVA) with or without repeated measures as necessary. For repeated measures design, the degrees of freedom were corrected employing the Greenhouse–Geisser procedure. Post hoc tests where necessary, were done using Bonferonni-corrected t-tests designed to maintain the experimentwise alpha level at 0.05 (Bray and Maxwell, 1982). Integrated CORT and ACTH values were calculated by the trapezoid rule (prestress value × 1 + 20 min value × 20 + 60 min value × 60 + 120 min value × 120/total time 200). 2.8. Ethical statement All procedures carried out in this experiment conform to the requirements of the UK Animals Scientific Procedures Act 1986, under project license number PPL40/1974.
3. Results 3.1. Litter size and infant mortality As shown in Table 1. There were no obvious differences in the number of pups per litter, nor any differences in the survival rates for pups at weaning. 3.2. Maternal Porsolt test On days 3 and 4 following birth, randomly selected control (n = 10) and stressed (n = 8) dams were placed in the Porsolt test. Univariate ANOVAs were performed Table 1 The effects of chronic gestational stress on litter size and infant mortality Measure
Prenatal condition Non-stressed
Number of pups 9.10 (1.1) Number surviving to weaning 7.71 (1.4)
Stressed 7.43 (0.8) 5.90 (1.0)
Values shown are the means and SEM for 12 stressed and 12 non-stressed dams included in the study. The differences are not statistically different.
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for each day’s results because the initial exposure on day 3 is designed to evoke a helpless response on the subsequent test day and is 10 min in length while the day 4 re-exposure time is only 5 min. Analysis on day 3 showed no main effect of prenatal condition on immobility times with F(1,16) = 0.85, ns. On day 4 however, a significant effect of condition emerged with F(1,16) = 5.9, P ⬍ 0.03. Immobility times were elevated in the stressed dams over those of control rats by approximately 25%. Group data are shown in Fig. 1. Thus, during the second exposure, stressed dams quickly ceased struggling to escape the swim tank and spent more time in a motionless posture. 3.3. Assessment of maternal behaviour Three specific behaviours were measured over the first 10 days following birth in groups of control (n = 9) and stressed (n = 9) dams. Arched-back nursing reflects the active form of feeding and ANOVA on these data showed a main effect of days F(3,17) = 7.1, P ⬍ 0.003, and a main effect of prenatal condition F(1,17) = 4.7, P ⬍ 0.05. Means and SEMs are shown in Fig. 2 (top panel). The total amount of arched-back nursing declined over days 1–3 in the control rats and then remained stable; however, arched-back nursing continued to decline over days 3–10 in stressed dams. The overall loss of this active nursing was exaggerated in the stressed dams such that by post-natal day 10 stressed dams spent approximately 40% less time engaged in arched-back nursing than did non-stressed dams. Separate pairwise comparisons failed to isolate any single day effects, however. Fig. 2 (middle panel) shows the results obtained for measures of nesting/grouping pups. Unlike arched-back nursing, there was no main effect of test day on total time spent nesting/grouping the
Fig. 1. Results from the Porsolt forced-swim task assessed in dams on days 3 and 4 following parturition. Stressed (n = 8) and non-stressed (n = 10) dams showed virtually identical immobility times on the initial exposure day. On day 4 however, immobility times were elevated in the stressed dams compared to control rats. Means ± SEM are shown. ∗Significantly different from non-stressed rats on same test day (P ⬍ 0.05).
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Fig. 2. The effects of gestational stress on selected maternal behaviours measured over post-natal days 1–10. Overall, stressed dams (n = 9) spent considerably less time engaged in arched-back nursing and nesting/grouping the pups than did control (n = 9) dams. Values for these variables were approximately 30–40% lower in stressed compared to non-stressed dams. Individual comparisons on a day-by-day basis were not significant. There was no effect of prenatal stress on licking/grouping behaviour. Means ± SEM are shown.
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pups F(3,17) = 2.61, ns. A significant main effect of prenatal condition was obvious with F(1,17) = 4.73, P ⬍ 0.05; as is apparent from Fig. 2, stressed dams spent less time gathering their pups and grouping them in the nest than did control dams. While the total drop was only of the order of 20–25% less time engaged in this behaviour, the fact that it persisted over each of the four assessment ages illustrates the phenomenon was robust. Measures of licking/grooming the pups were surprising, given the first two results. ANOVA on these data failed to reveal any significant results, and as shown statistically, prenatal condition was certainly not a factor in this specific form of maternal care F(1,17) = 0.13, ns. Thus, two out of three measures of maternal behaviour were diminished in the prenatally stressed dams. 3.4. Maternal endocrine responses to daily restraint stress Plasma CORT and ACTH concentrations were measured on days 10, 15 and 20 prior to the stress procedure and immediately upon removal from the restrainers. Control dams (n = 8) had blood samples taken at the same time as the stressed dams (n = 8) were being positioned in the restrainers and having blood taken, and both groups had blood sampled after either 1 h in their home cage (controls) or 1 h in the restrainers (stressed rats). A three-way ANOVA was performed with prenatal condition, pre/post-stress and day of testing as variables. For the CORT data, ANOVA revealed a main effect of pre/post-stress with F(1,15) = 16.7, P ⬍ 0.05, and a significant interaction between prenatal condition and pre/post-stress F(1,15) = 5.2, P ⬍ 0.05. As can be seen in Fig. 3 (top panel), CORT levels remained stable in the control dams, and they were somewhat higher than might be seen in nonpregnant rats; CORT levels rose slightly on gestation day 20 at the post-test time point. Values at this time point were equivalent to those of the stressed dams following 1 h of restraint. Overall, baseline CORT levels were stable in the stressed rats at the pretest measurement (i.e. there was no apparent change in basal CORT over the period of chronic stress from days 10–20), but they did significantly rise following the stress procedure. ACTH data showed considerable variability and statistically no main or interactive effects emerged. These data are depicted in Fig. 3 (bottom panel), where it can be observed that ACTH levels appeared to rise somewhat following the stress procedure, an effect that was most pronounced on gestation day 20. However, these findings were not significant. 3.5. Offspring Porsolt test At 3–4 months of age, randomly selected rats, which were stressed during gestation (males, n = 14; females, n = 10) and non-stressed controls (males, n = 15; females, n = 10), were tested over 2 days in the Porsolt task. There were no significant effects notable on the initial exposure day, with all groups showing immobility time of approximately 380 s. These data are shown graphically in Fig. 4 (top panel). In contrast, ANOVA performed on the data for the re-test on the subsequent day showed an overall main effect of prenatal condition with F(1,45) = 8.11, P ⬍ 0.01. There were neither effects of sex nor any obvious sex x condition interaction
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Fig. 3. Endocrine responses of pregnant dams subjected to daily 1 h restraint stress. The top panel shows basal CORT levels pre- and post-stress in control (n = 8) and stressed (n = 8) rats. Control rats had blood samples taken at the time of the restraint procedure, but were not actually placed into the restrainers. Blood was again sampled after 1 h in the home cage, or following 1 h in the restrainers (stressed group). Overall, CORT levels increased in response to the stress procedure compared to control dams left undisturbed in their home cages. Similar results are shown for the ACTH assay, although the higher degree of intragroup variability precluded identifying any significant effects. The trend was the same however. Means ± SEM are shown.
evident. As can be seen in Fig. 4 (bottom panel), prenatally stressed rats exhibited greater immobility in the swim task than did control rats, an effect that was equally represented in both male and female rats. Thus, like the dams, stress-exposed rats show greater evidence of depression-like behaviour than that of non-stressed rats.
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Fig. 4. Immobility times for the Porsolt task recorded from the offspring of stressed (male, n = 14; female, n = 10) and non-stressed (male, n = 15; female, n = 10) rats in adulthood. Overall there were no differences of note on the first exposure day from either stressed or non-stressed rats, and no obvious sex effects. On subsequent re-exposure, however, a main effect of stress emerged. Prenatally stressed rats exhibited higher immobility times than non-stressed controls, irrespective of sex. Means ± SEM are shown.
3.6. Adult offspring and endocrine responses to acute restraint challenge 3.6.1. Plasma CORT results Groups of randomly selected control (males, n = 7; females, n = 6) and stressed (male, n = 7; females, n = 6) adult offspring were subjected to a 20 min restraint stress challenge. CORT and ACTH determinations were obtained as detailed in Section 2. ANOVA on the CORT data obtained from the female animals showed a significant interaction between prenatal condition and time post-stress with F(3,11) = 4.3, P ⬍ 0.05. As depicted in Fig. 5 (top panel), prenatally stressed female rats initially secreted less CORT than did control animals, but showed continual rise in plasma CORT up to 120 min post-stress whereas the control rats exhibited dimin-
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Fig. 5. Corticosterone levels in response to acute restraint stress in the offspring of prenatally stressed (male, n = 7; female, n = 6) and control (male, n = 7; female, n = 6) rats in adulthood. For both males and females, CORT levels continued to rise in the prenatally stressed rats following the termination of the stressor, although there were no obvious differences in initial response to the stress challenge. Integrated CORT responses are shown in the figures inset to the main ones. Again, both male and female, prenatally stressed rats, showed average, per minute CORT levels that were higher than those of control animals. Means ± SEM are shown in all cases. ∗Significantly different from corresponding non-stressed group (P ⬍ 0.05).
ished levels by 60 min (or at least were returning to baseline while the stressed rats maintained high CORT levels). The net effect of this is shown by the integrated CORT response shown in Fig. 6 as an inset. When the CORT data were analysed using the values of CORT averaged over the duration of the test period, we observed that on a per minute basis, stressed rats have higher plasma CORT levels than do control rats. Similar data were obtained for the male rats. As can be seen in Fig. 5 (bottom panel), stressed and control offspring secreted similar amounts of CORT such that at the time of removal from the restrainers (20 min), each group had virtually the same mean level of plasma CORT. However, while the control rats showed
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Fig. 6. Adrenocorticotrophin levels in response to acute restraint stress in the offspring of prenatally stressed (male, n = 7; female, n = 6) and control (male, n = 7; female, n = 6) rats in adulthood. Overall, in females, ACTH levels were significantly higher in prenatally stressed rats compared to non-stressed controls. In males, the overall effect was that ACTH levels were higher in prenatally stressed animals than compared with control rats, and that prenatally stressed rats showed an initial hypersecretion of ACTH above that of control animals. Integrated ACTH responses are shown in the figures inset to the main ones. Male, prenatally stressed rats, showed average, per minute ACTH levels that were higher than those of control animals. Means ± SEM are shown in all cases. ∗Significantly different from corresponding non-stressed group (P ⬍ 0.05).
decreasing plasma CORT from this point until 120 min post-stress, stressed rats showed increasing plasma levels of CORT at 60 min and values only slightly declined by 120 min post-stress. The ANOVA on these data revealed a significant main effect of prenatal condition F(1,13) = 5.64, P ⬍ 0.05, but surprisingly the prenatal condition x time post-stress was not significant F(3,13) = 2.5, P ⬍ 0.074. The integrated CORT result was significant, as can be seen in the inset bar graph, where the stressed male rats showed higher average CORT values per min of the test duration.
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3.6.2. Plasma ACTH results Measures obtained for the ACTH analysis were in general similar to the CORT results, although there were some qualitative differences. As shown in Fig. 6 (top panel), both stressed and non-stressed female rats showed elevated ACTH secretion in response to acute stress. The ANOVA for these data mirrors this effect with a main effect of prenatal condition F(1,11) = 6.52, P ⬍ 0.05. The initial secretion of ACTH was slightly higher in the prenatally stressed female rats who also showed a slowed decline back to basal levels compared to non-stressed controls. Thus overall, prenatally stressed rats showed higher ACTH levels than did control rats. This finding is generally confirmed by the analysis of the integrated ACTH values (seen in the bar graph inset), where there was a clear tendency for the stressed rats to show higher per minute average plasma ACTH levels. Results for the male rats were even more pronounced where the ANOVA showed a main effect of prenatal condition F(1,13) = 4.81, P ⬍ 0.05, and a significant condition by time post-stress interaction F(3,13) = 4.70, P ⬍ 0.05. Means and SEMs are shown in Fig. 6 (bottom panel). Prenatally stressed rats markedly hypersecreted ACTH in response to acute stress compared to the control rats. At the time of removal from the restrainers, mean ACTH levels were approximately 100% higher in the stressed rats compared with those of the non-stressed animals. Interestingly, the recovery of baseline levels was very quick such that by 120 min post-stress exposure, both groups were almost back to normal basal values. The integrated ACTH response, not unexpectedly, showed that on a per minute basis, ACTH levels were significantly higher in the stressed male rats on average than those from the non-stressed condition. These results are shown in the inset figure on the bottom panel of Fig. 6.
4. Discussion One of the important issues to consider in this study is the mechanism via which GS and post-natal environmental conditions programme changes in the offspring. Considerable speculation has arisen regarding the role of maternal behaviour in the emergence of such programmed changes. Some authors (Maccari et al., 1995; Melniczek and Ward, 1994; Moore and Power, 1986) report that GS impairs maternal behaviour, and this may influence offspring HPA axis function and behaviour. For instance, Moore and Power (1986) reported that stressed dams spent less time licking non-stressed, cross-fostered pups. Interestingly they also showed that stressed pups elicited less licking behaviour from non-stressed, adoptive mothers. This line of evidence would suggest that behavioural patterns of both dam and infant are altered by GS and lead to programmed changes in HPA axis and adult behaviour. Furthermore, the effects of GS on HPA axis activity are significantly reduced if the stress-exposed pups are cross-fostered to an adoptive mother at birth, indicating that some aspect of mother–pup interactions can alter later reactivity to stress (Maccari et al., 1995). Our data presented here show convincingly that GS quantifiably and qualitatively alters some maternal behaviours. For instance, arched-back nursing times are diminished in the stressed dams. Furthermore, stressed dams spend less time gathering
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and grouping their litters under them. Interestingly, we did not observe any overt alteration in the amount of pup licking/grooming that stressed dams did, which shows some dissimilarity with previous reports on handled dams (Francis et al., 1999). Why select maternal behaviours would be specifically influenced by GS, while others remain intact is unclear. It also raises the intriguing question as to the importance of each behaviour, or set of behaviours, in infant development. To our knowledge this has yet to be addressed in any systematic way. The results of the present study are in accordance with those of previous investigations which have reported HPA axis overactivity in gestationally stressed rats, and which have detailed changes in affective behaviour and open field exploration of stressed animals (Braastad, 1998; Weinstock, 1997). We found that gestationally stressed rats exhibited greater immobility in the Porsolt task, suggestive of enhanced depression-like symptoms. These findings are in agreement with those of Alonso et al.’s (1997). Furthermore, gestationally stressed rats displayed exaggerated endocrine responses to an acute stress challenge as demonstrated by the increased ACTH and CORT secretion that stress-exposed rats exhibited compared to control animals. In our study we have presented evidence that the maternal HPA axis is responsive to daily restraint stress (Fig. 3), and that the stress procedure may cause a rise in CORT over the last half of gestation. The same trend was apparent with the ACTH levels, but unfortunately the intragroup variability was quite high and failed to meet criteria for significance. These data are in general agreement with those of Takahashi et al.’s (1998), although our basal CORT values are slightly elevated compared to theirs. However, we can argue that endocrine changes in the dams associated with the stress procedure may contribute to programmed effects in the pups, although intrinsic changes in the pups, such as catecholaminergic alterations cannot be excluded (Alonso et al., 1997; Peters, 1982; Takahashi et al., 1992). One obvious question that arises from these findings is why should stressed dams engage in less frequent and less intense nurturing behaviour? Stress has long been associated with the expression and severity of clinical depression (Gold et al., 1988; Hammen and Gitlin, 1997; Lovallo, 1997). When we examined our dams for signs of stress-related changes in depression-like behaviour, we observed that on postnatal day 4, stressed dams showed greater immobility in the Porsolt task even though they had not been stressed for the previous 6 days. Thus, a chronic stress regimen during pregnancy was sufficient to induce a state of post-natal depression even though the source of the stress had been removed. To our knowledge this is the first report suggesting that post-natal depression in the dam is directly linked with gestational history. Moreover, this state of post-natal depression coincides with reduced maternal behaviours and poorer pup care. It is possible therefore, that a state of depression in the dam promotes less maternal care towards the offspring. It is also conceivable that stressed pups behave in such a way as to alter the maternal behaviour from depressed dams. Hagen (1999) has provocatively posited that post-natal depression may occur in women with unsupportive spouses and who experience difficult labour/childbirth. Because of these difficulties he argues that these women divest themselves from maternal bonding and infant care since the problems of having children may out-
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weigh the benefits of child rearing under these circumstances. Undoubtedly, many women who become pregnant are subject to prepregnancy social and emotional stress, and some will continue to experience problems throughout gestation (Cooper et al., 1988; O’Hara et al., 1983). These are principal risk factors for post-natal depression and puerperal psychosis; importantly, these conditions appear to have a major impact on child cognitive, emotional and physical development (Bhagwanani et al., 1997; Coghill et al., 1986; Glover, 1997; Lovallo, 1997; Stowe and Nemeroff, 1995; Uddenberg and Englesson, 1978). Post-natal depression is a significant and serious illness with a reported incidence of approximately 14%, while a further 30% of women experience adjustment disorder and anxiety following childbirth (Dennerstein et al., 1989). Its impact on family life is pervasive, and its capacity for causing long-term harm in children is disturbing, especially given that some women report depression up to 4 years following childbirth (Buist, 1997; Wisner and Wheeler, 1994; Wrate et al., 1985). In conclusion, GS induces a state of post-natal depression in rat dams and this accompanies less frequent and less intense maternal care. The effects on the offspring include increased endocrine responses to stress and increased levels of intrinsic depression-like behaviour. While maternal care may be suppressed by GS as a consequence of the induction of post-natal depression, it is a possibility that mother/pup signalling is disrupted by prenatal stress, and this is our current focus. Moreover, further testing in a variety of depression models is necessary to corroborate those results obtained here using the solitary Porsolt test.
Acknowledgements This research was supported by BBSRC grant 16/S09519 to James W. Smythe. Jeremy Smith is the recipient of a BBSRC Postgraduate Studentship. The authors thank Osama Al-Khamees and Nickesh Parmar for technical assistance.
References Ader, R., Grota, L.J., 1969. Effects of early experience on adrenocortical reactivity. Physiol. Behav. 4, 303–305. Alonso, S.J., Navarro, E., Santana, C., Rodriguez, M., 1997. Motor lateralization, behavioral despair and dopaminergic brain asymmetry after prenatal stress. Pharmacol. Biochem. Behav. 58, 443–448. Anisman, H., Zacharko, R.M., 1982. Depression—the predisposing influence of stress. Behav. Brain Sci. 5, 89–122. Antoni, F.A., 1986. Hypothalamic control of ACTH secretion: advances since the discovery of 41-residue corticotrophin-releasing factor. Endocr. Rev. 7, 351–370. Bhagwanani, S.G., Seagraves, K., Dierker, L.J., Lax, M., 1997. Relationship between prenatal anxiety and perinatal outcome in nulliparous women: a prospective study. J. Natl Med. Assoc. 89, 93–98. Braastad, B.O., 1998. Effects of prenatal stress on behaviour of offspring of laboratory and farmed mammals. Appl. Anim. Behav. Sci. 61, 159–180. Bray, J.H., Maxwell, S.E., 1982. Analyzing and interpreting significant MANOVAs. J. Educ. Res. 52, 340–367.
J.W. Smith et al. / Psychoneuroendocrinology 29 (2004) 227–244
243
Brindley, D.N., Rolland, Y., 1989. Possible connections between stress, diabetes, obesity, hypertension and altered lipoprotein metabolism that may result in artherosclerosis. Clin. Sci. 77, 453–461. Buist, A., 1997. Postpartum psychiatric disorders. CNS Drugs 8, 113–123. Coghill, S.R., Caplan, H.L., Alexander, H., 1986. Impact of maternal depression in cognitive development of young children. Br. Med. J. 292, 1165–1167. Cooper, P.J., Campbell, E., Day, A., 1988. Nonpsychotic psychiatric disorder after childbirth. Br. J. Psychiatry 152, 799–806. Dallman, M.F., Akana, S.F., Scriber, K.A., Bradbury, M.J., Walker, C.D., Strack, A.M., et al. 1995. Stress, feedback, and facilitation in the hypothalamo–pituitary–adrenal axis. J. Neuroendocrinol. 4, 519–526. De Kloet, E.R., 1991. Brain corticosteroid receptor balance and homeostatic control. Front. Neuroendocrinol. 12, 95–164. Dennerstein, L., Lehert, P., Riphagen, F., 1989. Postpartum depression-risk factors. J. Psychosom. Obstet. Gynaecol. 10, 53–65. Feldman, S., Weidenfeld, J., 1995. Neural mechanisms involved in the corticosteroid feedback effects on the hypothalomo–pituitary–adrenocortical axis. Prog. Neurobiol. 45, 129–141. Francis, D., Diorio, J., Liu, D., Meaney, M.J., 1999. Nongenomic transmission across generations of maternal behavior and stress responses in the rat. Science 286, 1155–1158. Gold, P., Goodwin, P., Chrousos, G., 1988. Clinical and biochemical manifestations of depression: relation to the neurobiology of stress. N. Engl. J. Med. 319, 348–355. Glover, Y., 1997. Maternal stress or anxiety in pregnancy and emotional development of the child. Br. J. Psychiatry 171, 105–106. Hagen, E.G., 1999. The functions of postpartum depression. Evol. Human Behav. 20, 325–359. Hammen, C., Gitlin, M., 1997. Stress reactivity in bipolar patients and its relation to prior history of disorder. Am. J. Psychiatry 154, 856–857. Hess, J.L., Denenberg, V.H., Zarrow, M.X., Pfeifer, W.D., 1969. Modification of the corticosterone response curve as a function of handling in infancy. Physiol. Behav. 4, 109–112. Keller-Wood, M., Dallman, M.F., 1984. Corticosteroid inhibition of ACTH secretion. Endocr. Rev. 5, 1–24. Lehmann, J., Stohr, T., Feldon, J., 2000. Long-term effects of prenatal stress experience and postnatal maternal separation on emotionaility and attentional processes. Behav. Brain Res. 107, 133–144. Lordi, B., Patin, V., Protais, P., Mellier, D., Caston, J., 2000. Chronic stress in pregnanat rats: effects on growth rate, anxiety and memory capabilities of the offspring. Int. J. Psychophysiol. 37, 195–205. Lovallo, W.R., 1997. In: Stress & Health. Sage Publications Inc., New York, USA, pp. 101–121. Lui, D., Sharma, S., Pearson, D., Plotsky, P.M., Meaney, M.J., 1997. Maternal care, hippocampal glucocorticoid receptors, and hypothalamic–pituitary–adrenal responses to stress. Science 277, 1659–1662. Maccari, S., Piazza, P.V., Kabbaj, M., Barbazages, A., Simon, H., LeMoal, M., 1995. Adoption reverses the long-term impairment in glucocorticoid feedback induced by prenatal stress. J. Neurosci. 15, 110–116. McCormick, C.M., Smythe, J.W., Sharma, S., Meaney, M.J., 1995. Gender-specific effects of prenatal stress on hypothalamic-pituitary-adrenal responses to stress and brain glucocorticoid receptor density in adult rats. Dev. Brain Res. 84, 55–61. Meaney, M.J., Aitken, D.H., Viau, V., Sharma, S., Sarrieau, A., 1989. Neonatal handling alters adrenocortical negative feedback sensitivity and hippocampal type II glucocorticoid receptor binding in the rat. Neuroendocrinology 50, 597–604. Meaney, M.J., Bhatnagar, S., LaRoque, S., McCormick, C.M., Shanks, N., Smythe, J.W., et al. 1996. Early environment and the development of individual differences in the hypothalamic-pituitary-adrenal stress response. In: Pfeffer, C. (Ed.), Severe Stress and Mental Disturbance in Children. American Psychiatric Press, Washington, USA, pp. 85–127. Meaney, M.J., O’Donnell, D., Viau, V., Bhatnagar, S., Sarrieau, A., Smythe, J.W., et al. 1993. Corticosteroid receptors in the rat brain and pituitary during development and hypothalamic–pituitary–adrenal (HPA) function. In: McLaughlin, P., Zagon, I. (Eds.), Receptors and the Developing Nervous System. Chapman and Hall, London, pp. 163–201.
244
J.W. Smith et al. / Psychoneuroendocrinology 29 (2004) 227–244
Melniczek, J.R., Ward, I.L., 1994. Patterns of anogenital licking mother rats exhibit towards prenatally stressed neonates. Physiol. Behav. 56, 457–461. Moore, C.L., Power, K.L., 1986. Prenatal stress affects mother infant interaction in Norway rats. Dev. Psychobiol. 19, 235–245. Munck, A., Guyre, P.M., Holbrook, N., 1984. Physiological functions of GCs in stress and their relations to pharmacological actions. Endocr. Rev. 5, 25–44. Newport, D.J., Stowe, Z.N., Nemeroff, C.B., 2002. Parental depression: animal models of an aversive life event. Am. J. Psychiatry 159, 1265–1283. O’Hara, M.W., Reim, L.P., Campbell, S.B., 1983. Postpartum depression: a role for social network and life stress variables. J. Nerv. Ment. Dis. 171, 336–341. Peters, D.A.V., 1982. Prenatal stress: effects on brain biogenic amine and plasma corticosterone levels. Pharm. Biochem. Behav. 17, 721–725. Plotsky, P.M., 1987. Regulation of hypophysiotropic factors mediating ACTH secretion. Ann. NY Acad. Sci. 512, 205–217. Reul, J.M.H.M., De Kloet, E.R., 1985. Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation. Neuroendocrinology 117, 2505–2511. Rivier, C., Plotsky, P.M., 1986. Mediation by corticotrophin-releasing factor of adrenohypophysial hormone secretion. Ann. Rev. Physiol. 48, 475–489. Rosenblatt, J.S., 1993. In: Becker, J.B., Marc Breedlove, S., Crews, D. (Eds.), Behavioral Endocrinology. MIT Press, London, pp. 217–259. Selye, H., 1978. The Stress of Life. McGraw-Hill, New York. Smythe, J.W., McCormick, C.M., Meaney, M.J., 1996. Median eminence corticotrophin-releasing hormone content following prenatal stress and neonatal handling. Brain Res. Bull. 40, 195–199. Stowe, Z.N., Nemeroff, C.B., 1995. Women at risk for postpartum-onset major depression. Am. J. Obstet. Gynaecol. 173, 639–645. Suchecki, D., Neto, J.P., 1991. Prenatal stress and emotional response of adult offspring. Physiol. Behav. 49, 423–426. Takahashi, L.K., Turner, J.G., Kalin, N.H., 1992. Prenatal stress alters brain catecholaminergic activity and potentiates stress-induced behavior in adult rats. Brain Res. 574, 131–137. Takahashi, L.K., Turner, J.G., Kalin, N.H., 1998. Prolonged stress-induced elevation in plasma corticosterone during pregnancy in the rat: implications for prenatal stress studies. Psychoneuroendocrinology 23, 571–581. Uddenberg, N., Englesson, I., 1978. Prognosis of postpartum mental disturbance: a prospective study of postpartum women and their 41/2 year old children. Acta Psychiatr. Scand. 58, 201–212. Vallee, M., Mayo, W., Dellu, F., LeMoal, M., Simon, H., Maccari, S., 1997. Prenatal stress induces high anxiety and postnatal handling induces low anxiety in adult offspring: correlation with stress-induced corticosterone secretion. J. Neurosci. 17, 2626–2636. Weinstock, M., 1997. Does prenatal stress impair coping and regulation of hypothalamic–pituitary–adrenal axis? Neurosci. Biobehav. Rev. 21, 1–10. Weinstock, M., Poltyrev, T., Schorer-Apelbaum, D., Men, D., McCarty, R., 1998. Effects of prenatal stress on plasma corticosterone and catecholamines in response to footshock in rats. Physiol. Behav. 64, 439–444. Welberg, L.A.M., Seckl, J.R., 2001. Prenatal stress, glucocorticoids and the programming of the brain. J. Neuroendocrinol. 13, 113–128. Williams, M.T., Hennessy, M.B., Davis, H.N., 1998. Stress during pregnancy alters rat offspring morphology and ultrasonic vocalizations. Physiol. Behav. 63, 337–343. Wisner, K.L., Wheeler, S.B., 1994. Prevention of recurrent postpartum major depression. Hosp. Comm. Psychiatry 45, 1191–1196. Wrate, R.M., Roony, A.C., Thomas, P.F., 1985. Postnatal depression and child development. Br. J. Psychiatry 146, 622–627.
Gestational stress induces post-partum depression-like behaviour and alters maternal care in rats J.W. Smith a, J.R. Seckl b, A.T. Evans a, B. Costall a, J.W. Smythe a,∗ a
Neuropharmacology Research Group, Department of Pharmacology, School of Pharmacy, University of Bradford, Bradford BD7 1DP, UK b Department of Medical Sciences, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK
Received 28 August 2002; received in revised form 25 October 2002; accepted 14 January 2003
Abstract Gestational stress (GS) produces profound behavioural impairments in the offspring and may permanently programme hypothalamic–pituitary–adrenal (HPA) axis function. We investigated whether or not GS produced changes in the maternal behaviour of rat dams, and measured depression-like behaviour in the dam, which might contribute to effects in the progeny. We used the Porsolt test, which measures immobility in a forced-swim task, and models depression in rodents, while monitoring maternal care (arched-back nursing, licking/grooming, nesting/grouping pups). Pregnant rats underwent daily restraint stress (1 h/day, days 10–20 of gestation), or were left undisturbed (control). On post-parturition days 3 and 4, dams were placed into a swim tank, and time spent immobile was measured. GS significantly elevated immobility scores by approximately 25% above control values on the second test day. Maternal behaviours, in particular arched-back nursing and nesting/grouping pups, were reduced in GS dams over post-natal days 1–10. Adult offspring showed increased immobility in the Porsolt test, and also hypersecreted ACTH and CORT in response to an acute stress challenge. These data show that GS can alter maternal behaviour in mothers, and this might contribute to alterations in the offspring. GS may be an important factor in maternal post-natal depression, which may in turn detrimentally effect the offspring because depressed mothers do not sufficiently care for their offspring.
∗
Corresponding author. Tel.: +44-1274-233-361; fax: +44-1274-234-660. E-mail address: [email protected] (J.W. Smythe).
0306-4530/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0306-4530(03)00025-8
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2003 Elsevier Ltd. All rights reserved. Keywords: Rat; Hypothalamic–pituitary–adrenal; Gestation; Stress; Depression; Porsolt
1. Introduction For many decades it has been recognised that environmental manipulations of infant animals can produce enduring alterations in behavioural and endocrine responses to aversive events. The hypothalamic–pituitary–adrenal (HPA) axis is one of the body’s principal response mechanisms enabling animals to respond appropriately to aversive conditions and maintain homeostasis (Munck et al., 1984; Selye, 1978). Briefly, the paraventricular nucleus of the hypothalamus in the rat contains cells which secrete CRH and AVP into the median eminence (Antoni, 1986; Plotsky, 1987; Rivier and Plotsky, 1986). These secretagogues, in turn, diffuse via the portal vessels to the anterior pituitary corticotrophs and elicit the release of ACTH. The systemic circulatory system transports ACTH to the adrenal gland cortex where it evokes the rapid synthesis of the glucocorticoid, corticosterone (CORT; cortisol in humans). CORT enables a number of catabolic events which promote gluconeogenesis and glycogenolysis, and suppress immune function (Brindley and Rolland, 1989; Munck et al., 1984). All of these responses contribute to the ‘fight/flight’ reflex and together with the release of adrenaline from the adrenal medulla, constitute the endocrine stress response (Selye, 1978). Numerous reports have detailed how the HPA axis is subjected to programming and is permanently altered by manipulations of the mother/pup relationship. Early studies reported on the ‘handling’ phenomenon whereby rat pups are temporarily removed from the nest, isolated for 15–20 min and then returned to the dams. Handled rats, as adults, exhibit a reduced response to an acute stress challenge, secrete less ACTH and CORT, and recover basal levels of these hormones more quickly than non-handled control animals (Ader and Grota, 1969; Hess et al., 1969). The essential difference between handled and non-handled rats is in their sensitivity to CORT-mediated negative-feedback (Dallman et al., 1995; De Kloet, 1991; Feldman and Weidenfeld, 1995; Keller-Wood and Dallman, 1984; Reul and De Kloet, 1985). Handled rats have elevated GC receptor numbers in the hippocampus and frontal cortex compared to non-handled rats; the potency of the negative-feedback signal is augmented by the proliferation of GC receptors in handled animals (Meaney et al., 1989, 1993, 1996). Programming of endocrine function and various stress-related behaviours accompany antenatal manipulations such as gestational stress (GS) and prenatal dexamethasone (DEX) exposure. For instance, prenatal DEX administration leads to suppressed exploration of an open field or elevated-plus maze, while simultaneously increasing CRH mRNA in the PVN (Welberg and Seckl, 2001). Gestationally stressed rats exhibit elevated ACTH and CORT secretion in response to footshock stress, an effect which persists well into adulthood (Weinstock, 1997). We reported that adult female rats who had been gestationally stressed, hypersecreted ACTH and
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CORT in response to brief, restraint stress, and that this was accompanied by fluctuations in GC receptor binding throughout the forebrain (McCormick et al., 1995). Behaviourally, GS is associated with elevated indices of fearfulness in the adult progeny, as revealed by increases in ultrasonic vocalisations, defecation, and reduced ambulation in novel open fields (Braastad, 1998; Lehmann et al., 2000; Suchecki and Neto, 1991; Williams et al., 1998). Moreover, anxiety is also enhanced by GS, as shown by the reluctance of stress-exposed rats to explore the open arms of an elevated-plus maze (Lordi et al., 2000; Vallee et al., 1997). Newport et al. (2002) in a meta-analysis of parenting behaviour suggest that various stressors, including maternal separation and physical insults, ultimately produce highly anxious and depressed affective states in offspring animals, reminiscent of clinical depression in humans. The majority of studies have sought to explain HPA axis programming by identifying the underlying neurochemical and hormonal changes that appear in manipulated rats (Peters, 1982; Takahashi et al., 1992; Weinstock et al., 1998). However, it has been plausibly suggested that the HPA axis may exhibit plasticity as a result of ongoing maternal behaviour, which has been carefully characterised in rats (Rosenblatt, 1993). Lui et al. (1997) showed that rats display varying levels of spontaneous maternal behaviour; some dams are highly nurturant, while other mothers are relatively passive and indifferent towards their litters. What is particularly germane regarding this behavioural dichotomy is that pups from highly nurturant mothers have lower HPA axis responsivity to acute stress challenge, elevated hippocampal mRNA for GC receptors, and lower hypothalamic CRH mRNA compared to progeny from the less nurturant dams; importantly, these changes are linearly related to the magnitude of maternal care. Moreover, Meaney’s group (Meaney et al., 1989, 1993, 1996) have further shown that handling increases maternal care, and that female rats raised by highly nurturant dams exhibit greater maternal care than those raised by less nurturant mothers (Francis et al., 1999). These findings raise the question as to whether or not GS alters maternal behaviour in a manner consistent with the work of Lui et al. (1997) and Francis et al. (1999). That is, given the fact that HPA axis function is programmed by spontaneous maternal behaviour and given that GS has potent influences on HPA axis function, would we observe that stressed dams are less nurturant to their offspring than controls? Furthermore, stress is known to be a major contributory factor in the etiology of depression (Anisman and Zacharko, 1982; Hammen and Gitlin, 1997). Does GS alter maternal behaviour and induce a state of depression in the dam? The following study characterises maternal behaviour in rat dams stressed during gestation, while also measuring depression-like behaviour of the dams and offspring in a modified Porsolt test. We have also included measures of endocrine function as positive controls to ensure the genuine nature of our GS procedure.
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2. Methods 2.1. Subjects A total of 52 pregnant dams were generated in-house from nulliparous, Hooded Lister breeding stock. The procedure entailed placing three females (4 months of age) into a maternity cage (60 × 80 × 20 cm3; Techniplast, UK) along with a single male of the same age. These groupings were left for 5–7 days and if no evidence of mating was obvious, a new male was introduced in place of the original. Mating behaviour was monitored by taking daily vaginal smears and the presence of sperm in the smear was taken to signify day 1 of conception. Dams and their litters were housed singly in polycarbonate maternity cages (60 × 80 × 20 cm3) with wood-chip bedding, and food (Purina rat chow) and water were provided ad libitum. At 21–22 days of age, stressed and non-stressed offspring were weaned to larger, colony cages (80 × 80 × 30 cm3; Techniplast, UK) in groups of five (same sex and same experimental condition). Animals were maintained under normal light cycle conditions (on at 08:00 h; off at 20:00 h), with an ambient temperature of 22 °C, and relative humidity of 45%. 2.2. Gestational stress procedure From days 10–20, 25 randomly selected dams (from alternating cages in the housing room) were subjected to daily, 1 h restraint stress by being placed into plexiglass restrainers (Stoelting, USA), positioned in a brightly lit room. Blood samples were obtained from eight stressed rats via a small incision made in the distal region of the tail to collect plasma for hormone assays (described later). These samples were obtained immediately upon placement into the restrainers on days 10, 15 and 20. Upon removal from the restrainers another blood sample was taken. Twenty-seven, non-stressed control rats were left undisturbed in their home cages, although eight did have blood samples taken on the same days and 1 h apart as per the group of stressed rats. These tests were done for all rats on the same day and for each treatment group. Control rats were not restrained during the sampling procedure, but allowed to wander on a table surface while blood was milked from their tails. Blood sample volume ranged from 200–300 µl per time point. This procedure was carried out between 11:00 and 15:00 h. 2.3. Assessment of mother–pup interactions Starting on the day of birth and then on days 3, 6 and 10, maternal behaviour was observed and recorded using a time-lapse VHS video recorder and camera unit, set to sample activity at 1/3 normal speed. Based on previous findings discussed in the Section 1, we chose to monitor the time dams spent in an arched-back nursing position (active) as opposed to any other position e.g. supine (passive) or prostrate position. Arched-back nursing consisted of periods where the dam held herself erect, over the pups in a canopy posture, with her legs stretched out to provide balance.
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We also measured the time engaged in anogenital licking (of the entire litter, not single pups; this behaviour consisted of the dam holding individual pups between her front paws on the cage floor and visibly licking the hindquarters of the pups) and time spent grouping pups into a nest suitable for permitting suckling (the mothers routinely moved the nest around and carried each pup to a new nest and grouped them). We sampled behaviour over a 2 h span with 1 h from the lights-on and 1 h from the lights-off time periods. We used nine dams from each of the stressed or non-stressed conditions to monitor maternal behaviour, and these had not been subjected to blood sampling, nor any other testing. 2.4. Assessment of depressive behaviour in dams and offspring On post-natal days 3 and 4, selected dams (eight stressed and 10 non-stressed) and offspring (14 stressed males, 10 stressed females, 15 non-stressed males and 10 non-stressed females) between the ages of 3 and 4 months, were randomly selected from each experimental condition and were temporarily removed from their litters for testing in a forced-swim task (Porsolt test). Rats were placed in a 25 cm diameter cylinder, full of water 20 cm deep (25 °C), and monitored for time spent immobile (minimum amount of effort used to float) during a 10 min trial (day 3) or a 5 min trial (day 4). Following testing dams were dried under a fan heater for 2 min before being returned to their home cages. Maternal behaviour (described previously) was not recorded from these mothers. This testing was done between 11:00 and 15:00 h. 2.5. Acute stress challenge in adult progeny In adulthood (ages 3–4 months), progeny were subjected to an acute restraint stress challenge by being placed into plexiglass restrainers for 20 min (as per the dams). Blood samples were obtained from a small incision made in the distal region of the tail. These were taken prior to, post-stress (upon removal from the restrainers), and at 1 and 2 h following removal from the restrainer. This testing was done between 11:00 and 15:00 h. 2.6. Assessment of CORT and ACTH levels Blood samples were collected in ice-cooled, eppendorf tubes rinsed with EDTA, centrifuged at 3000 rpm for 10 min at 4 °C, and the plasma was removed and frozen for later ACTH and CORT determination using commercially available, radioimmunoassay kits. Plasma CORT was measured using a specific CORT antiserum, using [125I] CORT (Immuno Diagnostsics Systems, Ltd, Tyne and Wear, UK) as the tracer. A 10 µl sample was assayed in triplicate, at a minimum detection limit of 1 ng/ml plasma. The antiserum does not cross-react with cortisol, aldosterone or progesterone, but will react slightly with deoxycorticosterone (4%). Plasma ACTH was measured with a specific ACTH antiserum using [125I] ACTH as the tracer. The ACTH antibody cross-reacts 100% with ACTH1-39, but not ACTH1-16, bendorphin, a-MSH, or b-lipoprotein. A 25 µl sample was assayed in triplicate, at a
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minimum detection limit of 15 ng/ml plasma. Radioactivity was counted using a Gamma counter programmed to count for a 2 min period. Intra-and inter-assay reliability is about 4% for both assay procedures. These techniques are similar to those of our previous studies (McCormick et al., 1995; Smythe et al., 1996). 2.7. Statistical analysis All data were assessed by analysis of variance (ANOVA) with or without repeated measures as necessary. For repeated measures design, the degrees of freedom were corrected employing the Greenhouse–Geisser procedure. Post hoc tests where necessary, were done using Bonferonni-corrected t-tests designed to maintain the experimentwise alpha level at 0.05 (Bray and Maxwell, 1982). Integrated CORT and ACTH values were calculated by the trapezoid rule (prestress value × 1 + 20 min value × 20 + 60 min value × 60 + 120 min value × 120/total time 200). 2.8. Ethical statement All procedures carried out in this experiment conform to the requirements of the UK Animals Scientific Procedures Act 1986, under project license number PPL40/1974.
3. Results 3.1. Litter size and infant mortality As shown in Table 1. There were no obvious differences in the number of pups per litter, nor any differences in the survival rates for pups at weaning. 3.2. Maternal Porsolt test On days 3 and 4 following birth, randomly selected control (n = 10) and stressed (n = 8) dams were placed in the Porsolt test. Univariate ANOVAs were performed Table 1 The effects of chronic gestational stress on litter size and infant mortality Measure
Prenatal condition Non-stressed
Number of pups 9.10 (1.1) Number surviving to weaning 7.71 (1.4)
Stressed 7.43 (0.8) 5.90 (1.0)
Values shown are the means and SEM for 12 stressed and 12 non-stressed dams included in the study. The differences are not statistically different.
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for each day’s results because the initial exposure on day 3 is designed to evoke a helpless response on the subsequent test day and is 10 min in length while the day 4 re-exposure time is only 5 min. Analysis on day 3 showed no main effect of prenatal condition on immobility times with F(1,16) = 0.85, ns. On day 4 however, a significant effect of condition emerged with F(1,16) = 5.9, P ⬍ 0.03. Immobility times were elevated in the stressed dams over those of control rats by approximately 25%. Group data are shown in Fig. 1. Thus, during the second exposure, stressed dams quickly ceased struggling to escape the swim tank and spent more time in a motionless posture. 3.3. Assessment of maternal behaviour Three specific behaviours were measured over the first 10 days following birth in groups of control (n = 9) and stressed (n = 9) dams. Arched-back nursing reflects the active form of feeding and ANOVA on these data showed a main effect of days F(3,17) = 7.1, P ⬍ 0.003, and a main effect of prenatal condition F(1,17) = 4.7, P ⬍ 0.05. Means and SEMs are shown in Fig. 2 (top panel). The total amount of arched-back nursing declined over days 1–3 in the control rats and then remained stable; however, arched-back nursing continued to decline over days 3–10 in stressed dams. The overall loss of this active nursing was exaggerated in the stressed dams such that by post-natal day 10 stressed dams spent approximately 40% less time engaged in arched-back nursing than did non-stressed dams. Separate pairwise comparisons failed to isolate any single day effects, however. Fig. 2 (middle panel) shows the results obtained for measures of nesting/grouping pups. Unlike arched-back nursing, there was no main effect of test day on total time spent nesting/grouping the
Fig. 1. Results from the Porsolt forced-swim task assessed in dams on days 3 and 4 following parturition. Stressed (n = 8) and non-stressed (n = 10) dams showed virtually identical immobility times on the initial exposure day. On day 4 however, immobility times were elevated in the stressed dams compared to control rats. Means ± SEM are shown. ∗Significantly different from non-stressed rats on same test day (P ⬍ 0.05).
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Fig. 2. The effects of gestational stress on selected maternal behaviours measured over post-natal days 1–10. Overall, stressed dams (n = 9) spent considerably less time engaged in arched-back nursing and nesting/grouping the pups than did control (n = 9) dams. Values for these variables were approximately 30–40% lower in stressed compared to non-stressed dams. Individual comparisons on a day-by-day basis were not significant. There was no effect of prenatal stress on licking/grouping behaviour. Means ± SEM are shown.
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pups F(3,17) = 2.61, ns. A significant main effect of prenatal condition was obvious with F(1,17) = 4.73, P ⬍ 0.05; as is apparent from Fig. 2, stressed dams spent less time gathering their pups and grouping them in the nest than did control dams. While the total drop was only of the order of 20–25% less time engaged in this behaviour, the fact that it persisted over each of the four assessment ages illustrates the phenomenon was robust. Measures of licking/grooming the pups were surprising, given the first two results. ANOVA on these data failed to reveal any significant results, and as shown statistically, prenatal condition was certainly not a factor in this specific form of maternal care F(1,17) = 0.13, ns. Thus, two out of three measures of maternal behaviour were diminished in the prenatally stressed dams. 3.4. Maternal endocrine responses to daily restraint stress Plasma CORT and ACTH concentrations were measured on days 10, 15 and 20 prior to the stress procedure and immediately upon removal from the restrainers. Control dams (n = 8) had blood samples taken at the same time as the stressed dams (n = 8) were being positioned in the restrainers and having blood taken, and both groups had blood sampled after either 1 h in their home cage (controls) or 1 h in the restrainers (stressed rats). A three-way ANOVA was performed with prenatal condition, pre/post-stress and day of testing as variables. For the CORT data, ANOVA revealed a main effect of pre/post-stress with F(1,15) = 16.7, P ⬍ 0.05, and a significant interaction between prenatal condition and pre/post-stress F(1,15) = 5.2, P ⬍ 0.05. As can be seen in Fig. 3 (top panel), CORT levels remained stable in the control dams, and they were somewhat higher than might be seen in nonpregnant rats; CORT levels rose slightly on gestation day 20 at the post-test time point. Values at this time point were equivalent to those of the stressed dams following 1 h of restraint. Overall, baseline CORT levels were stable in the stressed rats at the pretest measurement (i.e. there was no apparent change in basal CORT over the period of chronic stress from days 10–20), but they did significantly rise following the stress procedure. ACTH data showed considerable variability and statistically no main or interactive effects emerged. These data are depicted in Fig. 3 (bottom panel), where it can be observed that ACTH levels appeared to rise somewhat following the stress procedure, an effect that was most pronounced on gestation day 20. However, these findings were not significant. 3.5. Offspring Porsolt test At 3–4 months of age, randomly selected rats, which were stressed during gestation (males, n = 14; females, n = 10) and non-stressed controls (males, n = 15; females, n = 10), were tested over 2 days in the Porsolt task. There were no significant effects notable on the initial exposure day, with all groups showing immobility time of approximately 380 s. These data are shown graphically in Fig. 4 (top panel). In contrast, ANOVA performed on the data for the re-test on the subsequent day showed an overall main effect of prenatal condition with F(1,45) = 8.11, P ⬍ 0.01. There were neither effects of sex nor any obvious sex x condition interaction
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Fig. 3. Endocrine responses of pregnant dams subjected to daily 1 h restraint stress. The top panel shows basal CORT levels pre- and post-stress in control (n = 8) and stressed (n = 8) rats. Control rats had blood samples taken at the time of the restraint procedure, but were not actually placed into the restrainers. Blood was again sampled after 1 h in the home cage, or following 1 h in the restrainers (stressed group). Overall, CORT levels increased in response to the stress procedure compared to control dams left undisturbed in their home cages. Similar results are shown for the ACTH assay, although the higher degree of intragroup variability precluded identifying any significant effects. The trend was the same however. Means ± SEM are shown.
evident. As can be seen in Fig. 4 (bottom panel), prenatally stressed rats exhibited greater immobility in the swim task than did control rats, an effect that was equally represented in both male and female rats. Thus, like the dams, stress-exposed rats show greater evidence of depression-like behaviour than that of non-stressed rats.
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Fig. 4. Immobility times for the Porsolt task recorded from the offspring of stressed (male, n = 14; female, n = 10) and non-stressed (male, n = 15; female, n = 10) rats in adulthood. Overall there were no differences of note on the first exposure day from either stressed or non-stressed rats, and no obvious sex effects. On subsequent re-exposure, however, a main effect of stress emerged. Prenatally stressed rats exhibited higher immobility times than non-stressed controls, irrespective of sex. Means ± SEM are shown.
3.6. Adult offspring and endocrine responses to acute restraint challenge 3.6.1. Plasma CORT results Groups of randomly selected control (males, n = 7; females, n = 6) and stressed (male, n = 7; females, n = 6) adult offspring were subjected to a 20 min restraint stress challenge. CORT and ACTH determinations were obtained as detailed in Section 2. ANOVA on the CORT data obtained from the female animals showed a significant interaction between prenatal condition and time post-stress with F(3,11) = 4.3, P ⬍ 0.05. As depicted in Fig. 5 (top panel), prenatally stressed female rats initially secreted less CORT than did control animals, but showed continual rise in plasma CORT up to 120 min post-stress whereas the control rats exhibited dimin-
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Fig. 5. Corticosterone levels in response to acute restraint stress in the offspring of prenatally stressed (male, n = 7; female, n = 6) and control (male, n = 7; female, n = 6) rats in adulthood. For both males and females, CORT levels continued to rise in the prenatally stressed rats following the termination of the stressor, although there were no obvious differences in initial response to the stress challenge. Integrated CORT responses are shown in the figures inset to the main ones. Again, both male and female, prenatally stressed rats, showed average, per minute CORT levels that were higher than those of control animals. Means ± SEM are shown in all cases. ∗Significantly different from corresponding non-stressed group (P ⬍ 0.05).
ished levels by 60 min (or at least were returning to baseline while the stressed rats maintained high CORT levels). The net effect of this is shown by the integrated CORT response shown in Fig. 6 as an inset. When the CORT data were analysed using the values of CORT averaged over the duration of the test period, we observed that on a per minute basis, stressed rats have higher plasma CORT levels than do control rats. Similar data were obtained for the male rats. As can be seen in Fig. 5 (bottom panel), stressed and control offspring secreted similar amounts of CORT such that at the time of removal from the restrainers (20 min), each group had virtually the same mean level of plasma CORT. However, while the control rats showed
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Fig. 6. Adrenocorticotrophin levels in response to acute restraint stress in the offspring of prenatally stressed (male, n = 7; female, n = 6) and control (male, n = 7; female, n = 6) rats in adulthood. Overall, in females, ACTH levels were significantly higher in prenatally stressed rats compared to non-stressed controls. In males, the overall effect was that ACTH levels were higher in prenatally stressed animals than compared with control rats, and that prenatally stressed rats showed an initial hypersecretion of ACTH above that of control animals. Integrated ACTH responses are shown in the figures inset to the main ones. Male, prenatally stressed rats, showed average, per minute ACTH levels that were higher than those of control animals. Means ± SEM are shown in all cases. ∗Significantly different from corresponding non-stressed group (P ⬍ 0.05).
decreasing plasma CORT from this point until 120 min post-stress, stressed rats showed increasing plasma levels of CORT at 60 min and values only slightly declined by 120 min post-stress. The ANOVA on these data revealed a significant main effect of prenatal condition F(1,13) = 5.64, P ⬍ 0.05, but surprisingly the prenatal condition x time post-stress was not significant F(3,13) = 2.5, P ⬍ 0.074. The integrated CORT result was significant, as can be seen in the inset bar graph, where the stressed male rats showed higher average CORT values per min of the test duration.
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3.6.2. Plasma ACTH results Measures obtained for the ACTH analysis were in general similar to the CORT results, although there were some qualitative differences. As shown in Fig. 6 (top panel), both stressed and non-stressed female rats showed elevated ACTH secretion in response to acute stress. The ANOVA for these data mirrors this effect with a main effect of prenatal condition F(1,11) = 6.52, P ⬍ 0.05. The initial secretion of ACTH was slightly higher in the prenatally stressed female rats who also showed a slowed decline back to basal levels compared to non-stressed controls. Thus overall, prenatally stressed rats showed higher ACTH levels than did control rats. This finding is generally confirmed by the analysis of the integrated ACTH values (seen in the bar graph inset), where there was a clear tendency for the stressed rats to show higher per minute average plasma ACTH levels. Results for the male rats were even more pronounced where the ANOVA showed a main effect of prenatal condition F(1,13) = 4.81, P ⬍ 0.05, and a significant condition by time post-stress interaction F(3,13) = 4.70, P ⬍ 0.05. Means and SEMs are shown in Fig. 6 (bottom panel). Prenatally stressed rats markedly hypersecreted ACTH in response to acute stress compared to the control rats. At the time of removal from the restrainers, mean ACTH levels were approximately 100% higher in the stressed rats compared with those of the non-stressed animals. Interestingly, the recovery of baseline levels was very quick such that by 120 min post-stress exposure, both groups were almost back to normal basal values. The integrated ACTH response, not unexpectedly, showed that on a per minute basis, ACTH levels were significantly higher in the stressed male rats on average than those from the non-stressed condition. These results are shown in the inset figure on the bottom panel of Fig. 6.
4. Discussion One of the important issues to consider in this study is the mechanism via which GS and post-natal environmental conditions programme changes in the offspring. Considerable speculation has arisen regarding the role of maternal behaviour in the emergence of such programmed changes. Some authors (Maccari et al., 1995; Melniczek and Ward, 1994; Moore and Power, 1986) report that GS impairs maternal behaviour, and this may influence offspring HPA axis function and behaviour. For instance, Moore and Power (1986) reported that stressed dams spent less time licking non-stressed, cross-fostered pups. Interestingly they also showed that stressed pups elicited less licking behaviour from non-stressed, adoptive mothers. This line of evidence would suggest that behavioural patterns of both dam and infant are altered by GS and lead to programmed changes in HPA axis and adult behaviour. Furthermore, the effects of GS on HPA axis activity are significantly reduced if the stress-exposed pups are cross-fostered to an adoptive mother at birth, indicating that some aspect of mother–pup interactions can alter later reactivity to stress (Maccari et al., 1995). Our data presented here show convincingly that GS quantifiably and qualitatively alters some maternal behaviours. For instance, arched-back nursing times are diminished in the stressed dams. Furthermore, stressed dams spend less time gathering
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and grouping their litters under them. Interestingly, we did not observe any overt alteration in the amount of pup licking/grooming that stressed dams did, which shows some dissimilarity with previous reports on handled dams (Francis et al., 1999). Why select maternal behaviours would be specifically influenced by GS, while others remain intact is unclear. It also raises the intriguing question as to the importance of each behaviour, or set of behaviours, in infant development. To our knowledge this has yet to be addressed in any systematic way. The results of the present study are in accordance with those of previous investigations which have reported HPA axis overactivity in gestationally stressed rats, and which have detailed changes in affective behaviour and open field exploration of stressed animals (Braastad, 1998; Weinstock, 1997). We found that gestationally stressed rats exhibited greater immobility in the Porsolt task, suggestive of enhanced depression-like symptoms. These findings are in agreement with those of Alonso et al.’s (1997). Furthermore, gestationally stressed rats displayed exaggerated endocrine responses to an acute stress challenge as demonstrated by the increased ACTH and CORT secretion that stress-exposed rats exhibited compared to control animals. In our study we have presented evidence that the maternal HPA axis is responsive to daily restraint stress (Fig. 3), and that the stress procedure may cause a rise in CORT over the last half of gestation. The same trend was apparent with the ACTH levels, but unfortunately the intragroup variability was quite high and failed to meet criteria for significance. These data are in general agreement with those of Takahashi et al.’s (1998), although our basal CORT values are slightly elevated compared to theirs. However, we can argue that endocrine changes in the dams associated with the stress procedure may contribute to programmed effects in the pups, although intrinsic changes in the pups, such as catecholaminergic alterations cannot be excluded (Alonso et al., 1997; Peters, 1982; Takahashi et al., 1992). One obvious question that arises from these findings is why should stressed dams engage in less frequent and less intense nurturing behaviour? Stress has long been associated with the expression and severity of clinical depression (Gold et al., 1988; Hammen and Gitlin, 1997; Lovallo, 1997). When we examined our dams for signs of stress-related changes in depression-like behaviour, we observed that on postnatal day 4, stressed dams showed greater immobility in the Porsolt task even though they had not been stressed for the previous 6 days. Thus, a chronic stress regimen during pregnancy was sufficient to induce a state of post-natal depression even though the source of the stress had been removed. To our knowledge this is the first report suggesting that post-natal depression in the dam is directly linked with gestational history. Moreover, this state of post-natal depression coincides with reduced maternal behaviours and poorer pup care. It is possible therefore, that a state of depression in the dam promotes less maternal care towards the offspring. It is also conceivable that stressed pups behave in such a way as to alter the maternal behaviour from depressed dams. Hagen (1999) has provocatively posited that post-natal depression may occur in women with unsupportive spouses and who experience difficult labour/childbirth. Because of these difficulties he argues that these women divest themselves from maternal bonding and infant care since the problems of having children may out-
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weigh the benefits of child rearing under these circumstances. Undoubtedly, many women who become pregnant are subject to prepregnancy social and emotional stress, and some will continue to experience problems throughout gestation (Cooper et al., 1988; O’Hara et al., 1983). These are principal risk factors for post-natal depression and puerperal psychosis; importantly, these conditions appear to have a major impact on child cognitive, emotional and physical development (Bhagwanani et al., 1997; Coghill et al., 1986; Glover, 1997; Lovallo, 1997; Stowe and Nemeroff, 1995; Uddenberg and Englesson, 1978). Post-natal depression is a significant and serious illness with a reported incidence of approximately 14%, while a further 30% of women experience adjustment disorder and anxiety following childbirth (Dennerstein et al., 1989). Its impact on family life is pervasive, and its capacity for causing long-term harm in children is disturbing, especially given that some women report depression up to 4 years following childbirth (Buist, 1997; Wisner and Wheeler, 1994; Wrate et al., 1985). In conclusion, GS induces a state of post-natal depression in rat dams and this accompanies less frequent and less intense maternal care. The effects on the offspring include increased endocrine responses to stress and increased levels of intrinsic depression-like behaviour. While maternal care may be suppressed by GS as a consequence of the induction of post-natal depression, it is a possibility that mother/pup signalling is disrupted by prenatal stress, and this is our current focus. Moreover, further testing in a variety of depression models is necessary to corroborate those results obtained here using the solitary Porsolt test.
Acknowledgements This research was supported by BBSRC grant 16/S09519 to James W. Smythe. Jeremy Smith is the recipient of a BBSRC Postgraduate Studentship. The authors thank Osama Al-Khamees and Nickesh Parmar for technical assistance.
References Ader, R., Grota, L.J., 1969. Effects of early experience on adrenocortical reactivity. Physiol. Behav. 4, 303–305. Alonso, S.J., Navarro, E., Santana, C., Rodriguez, M., 1997. Motor lateralization, behavioral despair and dopaminergic brain asymmetry after prenatal stress. Pharmacol. Biochem. Behav. 58, 443–448. Anisman, H., Zacharko, R.M., 1982. Depression—the predisposing influence of stress. Behav. Brain Sci. 5, 89–122. Antoni, F.A., 1986. Hypothalamic control of ACTH secretion: advances since the discovery of 41-residue corticotrophin-releasing factor. Endocr. Rev. 7, 351–370. Bhagwanani, S.G., Seagraves, K., Dierker, L.J., Lax, M., 1997. Relationship between prenatal anxiety and perinatal outcome in nulliparous women: a prospective study. J. Natl Med. Assoc. 89, 93–98. Braastad, B.O., 1998. Effects of prenatal stress on behaviour of offspring of laboratory and farmed mammals. Appl. Anim. Behav. Sci. 61, 159–180. Bray, J.H., Maxwell, S.E., 1982. Analyzing and interpreting significant MANOVAs. J. Educ. Res. 52, 340–367.
J.W. Smith et al. / Psychoneuroendocrinology 29 (2004) 227–244
243
Brindley, D.N., Rolland, Y., 1989. Possible connections between stress, diabetes, obesity, hypertension and altered lipoprotein metabolism that may result in artherosclerosis. Clin. Sci. 77, 453–461. Buist, A., 1997. Postpartum psychiatric disorders. CNS Drugs 8, 113–123. Coghill, S.R., Caplan, H.L., Alexander, H., 1986. Impact of maternal depression in cognitive development of young children. Br. Med. J. 292, 1165–1167. Cooper, P.J., Campbell, E., Day, A., 1988. Nonpsychotic psychiatric disorder after childbirth. Br. J. Psychiatry 152, 799–806. Dallman, M.F., Akana, S.F., Scriber, K.A., Bradbury, M.J., Walker, C.D., Strack, A.M., et al. 1995. Stress, feedback, and facilitation in the hypothalamo–pituitary–adrenal axis. J. Neuroendocrinol. 4, 519–526. De Kloet, E.R., 1991. Brain corticosteroid receptor balance and homeostatic control. Front. Neuroendocrinol. 12, 95–164. Dennerstein, L., Lehert, P., Riphagen, F., 1989. Postpartum depression-risk factors. J. Psychosom. Obstet. Gynaecol. 10, 53–65. Feldman, S., Weidenfeld, J., 1995. Neural mechanisms involved in the corticosteroid feedback effects on the hypothalomo–pituitary–adrenocortical axis. Prog. Neurobiol. 45, 129–141. Francis, D., Diorio, J., Liu, D., Meaney, M.J., 1999. Nongenomic transmission across generations of maternal behavior and stress responses in the rat. Science 286, 1155–1158. Gold, P., Goodwin, P., Chrousos, G., 1988. Clinical and biochemical manifestations of depression: relation to the neurobiology of stress. N. Engl. J. Med. 319, 348–355. Glover, Y., 1997. Maternal stress or anxiety in pregnancy and emotional development of the child. Br. J. Psychiatry 171, 105–106. Hagen, E.G., 1999. The functions of postpartum depression. Evol. Human Behav. 20, 325–359. Hammen, C., Gitlin, M., 1997. Stress reactivity in bipolar patients and its relation to prior history of disorder. Am. J. Psychiatry 154, 856–857. Hess, J.L., Denenberg, V.H., Zarrow, M.X., Pfeifer, W.D., 1969. Modification of the corticosterone response curve as a function of handling in infancy. Physiol. Behav. 4, 109–112. Keller-Wood, M., Dallman, M.F., 1984. Corticosteroid inhibition of ACTH secretion. Endocr. Rev. 5, 1–24. Lehmann, J., Stohr, T., Feldon, J., 2000. Long-term effects of prenatal stress experience and postnatal maternal separation on emotionaility and attentional processes. Behav. Brain Res. 107, 133–144. Lordi, B., Patin, V., Protais, P., Mellier, D., Caston, J., 2000. Chronic stress in pregnanat rats: effects on growth rate, anxiety and memory capabilities of the offspring. Int. J. Psychophysiol. 37, 195–205. Lovallo, W.R., 1997. In: Stress & Health. Sage Publications Inc., New York, USA, pp. 101–121. Lui, D., Sharma, S., Pearson, D., Plotsky, P.M., Meaney, M.J., 1997. Maternal care, hippocampal glucocorticoid receptors, and hypothalamic–pituitary–adrenal responses to stress. Science 277, 1659–1662. Maccari, S., Piazza, P.V., Kabbaj, M., Barbazages, A., Simon, H., LeMoal, M., 1995. Adoption reverses the long-term impairment in glucocorticoid feedback induced by prenatal stress. J. Neurosci. 15, 110–116. McCormick, C.M., Smythe, J.W., Sharma, S., Meaney, M.J., 1995. Gender-specific effects of prenatal stress on hypothalamic-pituitary-adrenal responses to stress and brain glucocorticoid receptor density in adult rats. Dev. Brain Res. 84, 55–61. Meaney, M.J., Aitken, D.H., Viau, V., Sharma, S., Sarrieau, A., 1989. Neonatal handling alters adrenocortical negative feedback sensitivity and hippocampal type II glucocorticoid receptor binding in the rat. Neuroendocrinology 50, 597–604. Meaney, M.J., Bhatnagar, S., LaRoque, S., McCormick, C.M., Shanks, N., Smythe, J.W., et al. 1996. Early environment and the development of individual differences in the hypothalamic-pituitary-adrenal stress response. In: Pfeffer, C. (Ed.), Severe Stress and Mental Disturbance in Children. American Psychiatric Press, Washington, USA, pp. 85–127. Meaney, M.J., O’Donnell, D., Viau, V., Bhatnagar, S., Sarrieau, A., Smythe, J.W., et al. 1993. Corticosteroid receptors in the rat brain and pituitary during development and hypothalamic–pituitary–adrenal (HPA) function. In: McLaughlin, P., Zagon, I. (Eds.), Receptors and the Developing Nervous System. Chapman and Hall, London, pp. 163–201.
244
J.W. Smith et al. / Psychoneuroendocrinology 29 (2004) 227–244
Melniczek, J.R., Ward, I.L., 1994. Patterns of anogenital licking mother rats exhibit towards prenatally stressed neonates. Physiol. Behav. 56, 457–461. Moore, C.L., Power, K.L., 1986. Prenatal stress affects mother infant interaction in Norway rats. Dev. Psychobiol. 19, 235–245. Munck, A., Guyre, P.M., Holbrook, N., 1984. Physiological functions of GCs in stress and their relations to pharmacological actions. Endocr. Rev. 5, 25–44. Newport, D.J., Stowe, Z.N., Nemeroff, C.B., 2002. Parental depression: animal models of an aversive life event. Am. J. Psychiatry 159, 1265–1283. O’Hara, M.W., Reim, L.P., Campbell, S.B., 1983. Postpartum depression: a role for social network and life stress variables. J. Nerv. Ment. Dis. 171, 336–341. Peters, D.A.V., 1982. Prenatal stress: effects on brain biogenic amine and plasma corticosterone levels. Pharm. Biochem. Behav. 17, 721–725. Plotsky, P.M., 1987. Regulation of hypophysiotropic factors mediating ACTH secretion. Ann. NY Acad. Sci. 512, 205–217. Reul, J.M.H.M., De Kloet, E.R., 1985. Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation. Neuroendocrinology 117, 2505–2511. Rivier, C., Plotsky, P.M., 1986. Mediation by corticotrophin-releasing factor of adrenohypophysial hormone secretion. Ann. Rev. Physiol. 48, 475–489. Rosenblatt, J.S., 1993. In: Becker, J.B., Marc Breedlove, S., Crews, D. (Eds.), Behavioral Endocrinology. MIT Press, London, pp. 217–259. Selye, H., 1978. The Stress of Life. McGraw-Hill, New York. Smythe, J.W., McCormick, C.M., Meaney, M.J., 1996. Median eminence corticotrophin-releasing hormone content following prenatal stress and neonatal handling. Brain Res. Bull. 40, 195–199. Stowe, Z.N., Nemeroff, C.B., 1995. Women at risk for postpartum-onset major depression. Am. J. Obstet. Gynaecol. 173, 639–645. Suchecki, D., Neto, J.P., 1991. Prenatal stress and emotional response of adult offspring. Physiol. Behav. 49, 423–426. Takahashi, L.K., Turner, J.G., Kalin, N.H., 1992. Prenatal stress alters brain catecholaminergic activity and potentiates stress-induced behavior in adult rats. Brain Res. 574, 131–137. Takahashi, L.K., Turner, J.G., Kalin, N.H., 1998. Prolonged stress-induced elevation in plasma corticosterone during pregnancy in the rat: implications for prenatal stress studies. Psychoneuroendocrinology 23, 571–581. Uddenberg, N., Englesson, I., 1978. Prognosis of postpartum mental disturbance: a prospective study of postpartum women and their 41/2 year old children. Acta Psychiatr. Scand. 58, 201–212. Vallee, M., Mayo, W., Dellu, F., LeMoal, M., Simon, H., Maccari, S., 1997. Prenatal stress induces high anxiety and postnatal handling induces low anxiety in adult offspring: correlation with stress-induced corticosterone secretion. J. Neurosci. 17, 2626–2636. Weinstock, M., 1997. Does prenatal stress impair coping and regulation of hypothalamic–pituitary–adrenal axis? Neurosci. Biobehav. Rev. 21, 1–10. Weinstock, M., Poltyrev, T., Schorer-Apelbaum, D., Men, D., McCarty, R., 1998. Effects of prenatal stress on plasma corticosterone and catecholamines in response to footshock in rats. Physiol. Behav. 64, 439–444. Welberg, L.A.M., Seckl, J.R., 2001. Prenatal stress, glucocorticoids and the programming of the brain. J. Neuroendocrinol. 13, 113–128. Williams, M.T., Hennessy, M.B., Davis, H.N., 1998. Stress during pregnancy alters rat offspring morphology and ultrasonic vocalizations. Physiol. Behav. 63, 337–343. Wisner, K.L., Wheeler, S.B., 1994. Prevention of recurrent postpartum major depression. Hosp. Comm. Psychiatry 45, 1191–1196. Wrate, R.M., Roony, A.C., Thomas, P.F., 1985. Postnatal depression and child development. Br. J. Psychiatry 146, 622–627.