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Brummelte, S., Lieblich, S. E., & Galea, L. A. M. (2012). Gestational and postpartum corticosterone exposure to the dam affects behavioral and endocrine outcome of the offspring in a sexually-dimorphic manner. Neuropharmacology, 62(1), 406-418.
Djavad Mowafaghian Centre for Brain Health
Brummelte, Susanne
Lieblich, Stephanie E.
Galea, Liisa A. M.
Exposure to high levels of glucocorticoids in utero and during the postpartum period has a detrimental effect on brain development. We created an animal model of postpartum stress/depression based on administering high levels of corticosterone (CORT) to the dams during the postpartum period which caused behavioral changes and reduced hippocampal cell proliferation in the offspring. As the consequences of early exposure to glucocorticoids may depend on the dose and the developmental stage of the offspring, the present study was conducted to investigate the effects of low (10mg/kg) or high levels of CORT (40mg/kg) given to dams either during gestation, postpartum or across both gestation and postpartum on the outcome of the offspring. Male and female offspring were weighed throughout the experiment, tested in a series of behavioral tests (forced swim test, open field, elevated plus maze) and basal and restraint stress CORT levels were examined in adolescence or young adulthood. Results show that maternal CORT exposure, regardless of when administered, significantly attenuated body weight gain until adulthood in the offspring. Offspring exposed to low maternal CORT, but not high maternal CORT, during the postpartum had higher basal levels of CORT as young adults. Further, male and female offspring of dams exposed to high maternal CORT in utero showed more depressive-like behavior in the forced swim test, while males of dams exposed to high maternal CORT postpartum exhibited more anxiety-like behavior in the elevated plus maze. Taken together, maternal glucocorticoid exposure have long lasting effects on male and female offspring‟s behavioral and neuroendocrine measures in adolescence and adulthood depending on the time of exposure to glucocorticoids.
1 Gestational and postpartum corticosterone exposure to the dam affects behavioral and endocrine outcome of the offspring in a sexually-dimorphic manner Susanne Brummelte1+, Stephanie Lieblich1 and Liisa A.M. Galea1,2*1 Department of Psychology and 2Brain Research Center, University of British Columbia,Vancouver, BC, Canada, *Corresponding author: Liisa A.M. Galea, Ph.D., Professor, The University of British Columbia,2136 West Mall, Vancouver, B.C. V6T 1Z4, Phone: (604) 822-6536, Fax: (604) 822-6923, Email: lgalea@psych.ubc.ca. +Present Address: Developmental Neurosciences & Child Health, Department of Pediatrics, University of British Columbia, Vancouver, BC, Canada 2 Abstract Exposure to high levels of glucocorticoids in utero and during the postpartum period has a detrimental effect on brain development. We created an animal model of postpartum stress/depression based on administering high levels of corticosterone (CORT) to the dams during the postpartum period which caused behavioral changes and reduced hippocampal cell proliferation in the offspring. As the consequences of early exposure to glucocorticoids may depend on the dose and the developmental stage of the offspring, the present study was conducted to investigate the effects of low (10mg/kg) or high levels of CORT (40mg/kg) given to dams either during gestation, postpartum or across both gestation and postpartum on the outcome of the offspring. Male and female offspring were weighed throughout the experiment, tested in a series of behavioral tests (forced swim test, open field, elevated plus maze) and basal and restraint stress CORT levels were examined in adolescence or young adulthood. Results show that maternal CORT exposure, regardless of when administered, significantly attenuated body weight gain until adulthood in the offspring. Offspring exposed to low maternal CORT, but not high maternal CORT, during the postpartum had higher basal levels of CORT as young adults. Further, male and female offspring of dams exposed to high maternal CORT in utero showed more depressive-like behavior in the forced swim test, while males of dams exposed to high maternal CORT postpartum exhibited more anxiety-like behavior in the elevated plus maze. Taken together, maternal glucocorticoid exposure have long lasting effects on male and female offspring‟s behavioral and neuroendocrine measures in adolescence and adulthood depending on the time of exposure to glucocorticoids. Keywords: gestation, prenatal, in utero, glucocorticoids, depression, stress, sex differences, early programming, development, adolescence3 1. Introduction Stress during pre- or postnatal development can have a profound impact on the long term outcome of the individual. The maternal hypothalamus pituitary adrenal (HPA) axis is downregulated during pregnancy, partly to protect the offspring in utero from harmful effects of elevated glucocorticoids (Brunton et al., 2008). However, despite the alteration of the HPA axis function of the mother, prenatal stress profoundly affects the neurodevelopment of the offspring (Talge et al., 2007). For instance, if a rat dam is exposed to psychological stress during her pregnancy, her male offspring display depressive-like behaviors in the forced swim test and altered HPA axis function compared to offspring from a non-stressed dam (Abe et al., 2007). Similarly, male and female children born to women who experienced stress during pregnancy had a higher risk for developing depressive symptoms and severe depression as young adults compared to age-matched controls from non-stressed mothers (Watson et al., 1999). Further, exposing the pregnant dam to restraint stress causes changes in anxiety, spatial learning, neuroendocrine function and receptor levels in the brain of the offspring, which are highly sex- dependent (Darnaudery and Maccari, 2008; Zuena et al., 2008). It is also well known that early postpartum stress, such as maternal separation, can have profound and sex-dependent effects on the adult phenotype of the F1 (e.g. Gross et al., 2010; Renard et al., 2007; Slotten et al., 2006), and F2 offspring generations (Franklin et al., 2010). In addition early stress can affect maternal behavior in the lactating female offspring (Bosch et al., 2007) as prenatal and postnatal exposure to stress also changes maternal behavior in the dam. For instance, chronic stress during pregnancy reduces maternal licking (Smith et al., 2004), while high maternal corticosterone (CORT) treatment postpartum reduces time spent on the nest and nursing (Brummelte and Galea, 4 2010a). Thus, early stress or exposure to glucocorticoids can impact the developmental outcome of the offspring directly and indirectly by reducing maternal care (Fleming et al., 1997; Liu et al., 1997; Nomura et al., 2002). Direct administration of glucocorticoids to the dam, rather than stress exposure, will allow us to better understand the contribution of glucocorticoids on the effects of pre- or postnatal stress. Antenatal steroids are used in threatened preterm labor to support the lung surfactant maturation (Roberts and Dalziel, 2006), but it has been suggested that multiple dosages could have a negative influence on the development of the infant (Wapner et al., 2007). From animal studies, it is well known that administration of glucocorticoids directly to the offspring during early postnatal development can cause permanent morphological, physiological, and behavioral modifications (Edwards and Burnham, 2001; Machhor et al., 2004; Theogaraj et al., 2005). These modifications include a decrease in brain weight (DeKosky et al., 1982; Ferguson and Holson, 1999) , long-term and selective down-regulation of glucocorticoid receptors (Felszeghy et al., 1996), impaired adrenocortical response to stress (Erskine et al., 1979), disruptions in learning (DeKosky et al., 1982; Vicedomini et al., 1986), and delayed development (Golub, 1982; Pavlovska-Teglia et al., 1995). However, less is known on how maternal glucocorticoid levels affect the offspring, as the offspring may be partly buffered from maternal glucocorticoids during the first two weeks of life, a period known as the „stress hypo-responsive period‟ (Sapolsky and Meaney, 1986). We have shown previously that administration of high CORT to dams postpartum leads to reduced hippocampal cell proliferation in male pre-adolescent offspring and behavioral changes in adult male and female offspring (Angelucci et al., 1983; Brummelte et al., 2006). We further showed that CORT treatment to the dam during pregnancy resulted in increased serum CORT in the offspring shortly after birth (Brummelte et al., 2010). Interestingly, maternal 5 postpartum treatment with CORT increased whole brain levels of CORT on day 7 but no significant differences were seen in CORT levels in the prefrontal cortex, hypothalamus or hippocampus in 18 day old pups from CORT-treated dams compared to controls (Brummelte et al., 2010). Further we have found that treatment with high CORT postpartum, but not during gestation, results in a depressive phenotype in the dam (Brummelte and Galea, 2010a), suggesting that the effects of elevated maternal glucocorticoids on behavior and glucocorticoid levels in the offspring may also depend on the time of exposure during gestation or postpartum. The present study was conducted to investigate the effects of two doses of CORT (10mg/kg; 40mg/kg) administered to dams during gestation, postpartum or both periods on behavioral and neuroendocrine parameters in male and female offspring. For this, dams received daily injections of either oil or CORT (low or high dose) from gestational day 10-20 and/or from postnatal day 2-24. Offspring were tested on the forced swim test (depressive-like behavior), the open field test (locomotor and anxiety-like behavior), the elevated plus maze (anxiety-like behavior), and the resistance to capture test (impulsivity/aggressiveness) during late adolescence or given restraint stress during adulthood. We hypothesized that CORT administered during the postpartum period would have a different effect on the offspring than CORT administered to dams during pregnancy. Further, we expected that high maternal CORT would have a stronger effect than low maternal CORT on the offspring and that the prolonged exposure (pregnancy and postpartum) of CORT would have a more substantial effect on the offspring than during pregnancy or the postpartum alone. We also hypothesized that males and females will show different vulnerabilities for high maternal glucocorticoid levels depending on the time of exposure. 6 2. Materials and methods2.1. Animals Forty-six female and twenty male Sprague-Dawley rats, approximately 3 month old, were obtained from the University of British Columbia Animal Care Facility (Vancouver, Canada) for breeding. Rats were housed initially in same sex pairs in opaque polyurethane bins (48 × 27 × 20 cm) with absorbent bedding and were given Purina rat chow and tap water ad libitum. Animals were maintained on a 12 h: 12 h light/dark cycle (lights on at 7:30 a.m.). The number of animals was based on previous work in our laboratory (Brummelte et al., 2006) to give us sufficient power for the analysis. All protocols were in accordance with ethical guidelines set by the Canada Council for Animal Care and were approved by the University of British Columbia Animal Care Committee. All efforts were made to minimize animal suffering and to reduce the number of animals used. For an overview of the experimental procedures see Figure 1. 2.2. Hormone Preparation An emulsion of corticosterone (CORT; Sigma-Aldrich, St. Louis, MO; USA) was prepared every few days at a concentration of 10mg/ml (low dose) or 40 mg/ml (high dose) by mixing CORT with 10% ethanol in sesame oil. The high dose was chosen because it elevates blood levels of CORT for a prolonged period of time (Johnson et al., 2006; Sapolsky, 1985), increases depressive-like behavior (Brummelte et al., 2006; Brummelte and Galea, 2010a; Gregus et al., 2005) and results in serum levels of CORT consistent with ether stress in postpartum females (Yeh, 1984). The low dose was chosen because it does not alter depressive-like behavior or baseline CORT levels in male or female rats after s.c. injections, but it has been shown to diminish their CORT-response to stress suggesting this dose mimics CORT levels of a 7 mild chronic stressor (Brummelte and Galea, 2010b; Johnson et al., 2006). However, it is important to note, that oral application of CORT hemisuccinate through drinking water equivalent to a dose of 5-7mg/kg for 20 days results in a depressive phenotype in rats and mice 2-4 weeks after the cessation of treatment (Gourley et al., 2008; Gourley and Taylor, 2009). 2.3. Procedures Detailed breeding procedures and dam treatment has been described previously (Brummelte and Galea, 2010a). Briefly, pregnant dams were either given no injection (Undisturbed) or daily subcutaneous (s.c.) injections (1ml/kg) of either oil, low CORT (10mg/kg) or high CORT (40mg/kg). Subcutaneous injections were performed daily around 10 a.m. from day 10-20 of pregnancy and day 2-24 of postpartum. Rats were randomly assigned to one of the following groups: Undisturbed (n=5); Oil (n=10): received sesame oil injections during pregnancy and postpartum; Preg10 (n=5): received low CORT during pregnancy and oil injections during postpartum; Post10 (n=5): received sesame oil during pregnancy and low CORT during the postpartum period, P+P 10 (n=5): received low CORT during pregnancy and the postpartum, Preg40 (n=5) received high CORT during pregnancy and oil during postpartum, Post40 (n=5): received oil during pregnancy and high CORT during the postpartum period and P+P 40 (n=5) received high CORT during pregnancy and the postpartum. For an overview of the groups see Supplementary Table S1. The day the litter was born was considered postnatal day (PND) 0. On PND1 all litters were weighed and male and female pups were counted before culling the litter to 5 males and 5 females per dam. If there were not enough males or females in one litter, pups were either cross-fostered from a dam within the same group who gave birth the same day (3 cases) or if there were no pups available when the litter was culled, the dam kept an uneven number of male and female pups (2 cases). The litter was weighed every 4 days starting 8 PND9 until PND20. Afterwards (including weaning) males and females were weighed separately. On postpartum days 2 to 8 maternal behavior was assessed as previously described in detail (Brummelte and Galea, 2010a). Time spent licking, nursing, or off pups was scored twice a day for 10 min. Dams were moved to maternal bins (48 x 40 x 27) with their litters on postnatal day 9. Pups were weaned on day 23 and group housed with same sex siblings for one week before they were pair-housed with same-sex siblings in standard clear polyurethane bins (Fig.1). 2.4. Behavioral testing of the offspring Starting at a mean age of 42.35 ± 0.25 days 8 male and 8 female offspring from each group for a total of 128 rats were tested in a battery of behavioral tests including the forced swim test, open field test, elevated plus maze and resistance to capture test during late adolescence (~40-60d). No more than 2 males and 2 females were taken from each litter for the behavioral tests. Behavioral tests were conducted in the same order for all animals (see Fig. 1) and were completed after ~20days with at least 2 days break between each test for each animal. In early adulthood (age ~70d) one male/female (n=5/group) pup from each litter (which had not been used for behavioral testing) underwent 1 hour of restraint stress and baseline and stress response levels of CORT were measured. 2.4.1. Forced swim test The forced swim test (FST) was used to assess depressive-like behavior in the offspring and was used as previously described (Galea et al., 2001; Hansen et al., 1997). Briefly, the apparatus consisted of a vertical cylindrical glass container (45 × 28 cm) filled to a depth of 30 cm with tap water at 25 ± 0.5°C. Session 1 of the FST lasted 15 min and was followed 24 hours 9 later by a 2nd session lasting 10 min. All test sessions were videotaped and the 2nd session was later scored by an observer blind to conditions. The behaviors scored in the forced swim test were: (1) struggling—quick movements of the forelimbs such that the front paws break the surface of the water; (2) swimming—movement of forelimbs or hind limbs in a paddling fashion; and (3) immobility— floating in the water with only movements necessary to keep the head above water. 2.4.2. Open Field Test On a separate day following the 2nd FST session and in order to assess general locomotor activity the open field test (OFT) was used as previously described (Galea et al., 2001). Briefly, the apparatus, a 120×120 cm2 arena divided into 16 squares of equal dimension with 40 cm high walls, was placed in a dimly lit room, with a video camera installed in the center above the apparatus. Rats were placed in the field facing a corner and locomotor activity was video recorded for a 10 min period. An observer blind to conditions scored the number of rears and the number of fecal boli from each animal. The videos were analyzed with Anymaze (Stoelting Co., Wood Dale, USA), software for automatic behavioral scoring to assess the total distance travelled by the animal and time spent in each zone. 2.4.3. Elevated Plus Maze On a day following OFT but still during adolescence (~50d), pups were tested in the Elevated Plus Maze (EPM) as described previously (Brummelte et al., 2006; Pellow et al., 1985). Briefly, rats were placed in the center of a plus-shaped apparatus with two open and two enclosed arms for 5 minutes. The number of entries into open or closed arms was counted as well as the 10 time spent in a closed arm, open arm or the center. The amount of time spent in a closed arm vs. time spent in open arm or center was used as an index of anxiety. 2.4.4. Resistance to capture Resistance to capture scale from Kalynchuk et al. (1997) was adjusted and performed as previously described (Brummelte et al., 2006). Briefly, a few days after the OFT animals were placed into an unfamiliar small open field (50cm x 50 cm) and were allowed to explore it freely for 2 min. Then an experimenter approached them with an unfamiliar large leather glove and tried to pick them out of the field. Behaviors were scored according to a 7-point scale (0= easy to pick up/ 7= manage to escape from grip, Supplementary Table S2) at three different time points: approach of the glove, touch and lifting. In addition, the time to capture was recorded.2.4.5. Restraint stress To assess the young adult offspring‟s CORT response to stress, separate rats were examined that had not undergone behavioral tests. Five males and 5 females from each group were subjected to 1 hour of restraint stress at age 66-73 days. A baseline blood sample was collected prior to placing the rat into the tube (t0). All samples were collected within 3 min after touching their home cage to avoid a rise in CORT levels due to the stress of tail bleeding. Further samples were taken at 30min (t30) into the stress, at the end of the stressor (t60), and 1 hour after (t120) their return to their home cage. All blood samples were collected from the tail vein and in under 3 minutes. 2.5. Corticosterone assay 11 Blood samples were stored overnight at 4°C to allow blood to clot completely, centrifuged at 10,000 × g for 10 min, serum collected and stored at -20°C until further processing. Total CORT (bound and free) was measured using the ImmuChem Double Antibody 125I radioimmunoassay Kit (MP Biomedicals, Orangeburg, NY, USA). The antiserum cross-reacts 100 % with CORT, 0.34 % with deoxycorticosterone, 0.10 % with testosterone, 0.10 % with cortisol, but does not cross-react with progesterone and estrogens (< 0.01%). The intra-assay and inter assay coefficients of variation were less than 7.1% and 6.5% respectively. All reagents were halved and samples run in duplicates. 2.6. Data analyses The average weight per pup was calculated by dividing the litter weight after culling by the number of pups per litter (n=10). Changes in body weight during the postpartum was analyzed with a repeated-measures ANOVA with group (Control, Oil, Preg10, Post 10, P+P10, Preg40, Post40, P+P40) as the between-subjects factor and age (PND1, PND9, PND13, PND17, PND20) as the within-subject factors. Analysis of FST, OFT, EPM and resistance to capture was performed using repeated-measure ANOVAs with FST behavior (struggling, swimming, immobile), OFT number of crossings and distance travelled (peripheral, center), EPM arm entries (open, closed) and time spent in each zone (open, closed, center) as the within-subject factors and group and sex (male, female) as the between-subject factors. Further, number of rears (EPM and OFT), time spent in center (OFT), central/total distance ratio (OFT), time in closed/time in open arm or center (EPM) and number of fecal boli (OFT and resistance to capture), capture behavior (cumulative score and time to capture in resistance to capture) were each analyzed separately using a one-way ANOVA. Serum CORT levels during restraint stress were analyzed using a repeated-measures ANOVA with time (t0, t30, t60, t120) as the within subject factor and group 12 as the between-subjects factor. Pearson product-moment correlations were conducted between offspring behaviors and maternal care behavior (% of time spent off the nest). Tests of homogeneity of variance (using the Brown-Forsythe‟s test), normality (using the Kolmogorov-Smirnov test) and in the cases where more than two dependent variables were analyzed in a repeated-measures ANOVA, tests of sphericity were conducted. Violations to these assumptions are noted in the text however it should be noted that the ANOVA is quite robust to violations of normality when using a fixed-effects ANOVA (Glass and Hopkins, 1995), as in the present case. When violations were noted in sphericity we used the Huynh-Feldt adjustment, while violations of homogeneity of variance used the Welch‟s test. All post-hoc tests utilized LSD. All figures show data separately for males and females regardless of significant main or interaction effects of sex. 3. Results 3.1. Body weight - Offspring from dams exposed to high CORT showed attenuation of weight gain throughout development. At adulthood, offspring from dams exposed to low CORT weighed less than controls. Litter characteristics from the current offspring have been published previously (Brummelte and Galea, 2010a) and are shown in Supplementary Table S3. Detailed results of the post-hoc analysis for body weight development during the postpartum period are shown in Table 1. There was no significant difference in body weight between the groups on postnatal day 1 (all p‟s>0.46), but starting on PND9 or PND 13 all high dose groups and Post10 showed attenuated weight gain (all p‟s<0.02). Results of the repeated-measures ANOVA were as follows: main effects for group (F(7, 38)=7.26, p<0.001) and age (F(4, 152)=2522.3, p<0.001) and a significant 13 interaction of group and age (F(28, 152)=3.56, p<0.001). There was a violation of sphericity (p<.001), however the Huynh-Feldt adjusted p value for the age by group interaction was p<0.0001). There were no other violations in the assumptions of the ANOVA. At weaning (postnatal day 23) Post10, Preg40, Post40 and P+P40 pups weighed less than undisturbed or oil controls (all p‟s <0.008; Figure 2A). The ANOVA revealed significant main effects of group (F(7, 76)=16.07, p<0.0001) and sex (F(1, 76)=6.67, p=0.01) with males being significantly heavier than females, but no significant interaction effect (p=0.80). There were no violations of the assumptions of the ANOVA. Animals were weighed again in early adulthood (day 72 ± 0.25) and body weight was analyzed with an ANCOVA correcting for age. All low CORT groups weighed significantly less compared to oil rats (all p‟s <0.01), but the high CORT groups were not different from oil controls (all p‟s >0.66) (Figure 2B). There was no significant interaction effect (F(7,75)=1.26, p=0.28), but a main effect of sex (F(1,75)=973.6, p<0.001), with males weighing more than females, and a significant group effect (F(7,75)=3.22, p= 0.005). There were no violations of the assumptions of the ANOVA. 3.2. Forced Swim Test (FST) – Offspring from dams exposed to high CORT during gestation, but not postpartum, exhibited depressive-like behavior in the FST. Due to the heterogeneity of variance the data were transformed with the arcsine transformation for the analysis. The Preg40 group showed an increase in immobility and decrease in swimming compared to undisturbed (p‟s=0.01) or oil controls (p‟s<0.002; Figure 3). The repeated measure ANOVA revealed a significant interaction effect of forced swim test behavior and group (F(14, 254)=2.16, p=0.009) and a main effect of behavior (F(2, 254)=204.1, 14 p<0.001), but no significant main effect of sex or other interaction effects (all p‟s>0.13). Because there was a violation of sphericity and the Huynh-Feldt adjusted p value for the behavior by group interaction was p<0.02. There were no other violations in the assumptions of the ANOVA. 3.3. Open Field Test (OFT): Offspring from dams exposed to CORT during gestation and the postpartum travelled greater distances in the periphery of the open field compared to controls. For peripheral distance, oil females travelled greater distances than Post10 females (p=0.004) and undisturbed controls (p=0.01). P+P10 and P+P40 females travelled significantly more in the periphery than undisturbed controls (p<0.001; p=0.005, respectively) and oil (p=0.009, P+P10 females only). For males, P+P10 travelled significantly more in the periphery compared to male oil controls (p=0.05), but Preg40 crossed significantly less than oil controls (p=0.02) or undisturbed controls (p=0.01; Figure 4). There were no significant differences between the groups for the distance travelled in the center of the open field (Table 2). Repeated-measures ANOVA for distance travelled showed significant main effects for group (F(7,121)= 2.28, p=0.03), sex (F(1,121)=28.04, p<0.001) and area (F(1, 121)=5229.2, p<0.001), significant two-way interaction effects for group and area (F(7,121)=2.17, p=0.04), sex and area (F(1,121)=12.25, p<0.001) and a significant three-way interaction for group, sex and area (F(7,121)=2.08, p=0.05). Further, ANOVAs showed that males showed significantly more fecal boli (p<0.001), less rearing (p=0.01), less time in the center (p<0.001), lower center/total distance ratio (p<0.001) and longer latency to enter the center (p=0.02) in the open field test (Table 2). There were no violations to assumptions of the ANOVA. 15 3.4. Elevated Plus Maze (EPM): Male, but not female, offspring from dams exposed to high CORT during gestation and the postpartum or just the postpartum, exhibited anxiety-like behavior in the EPM. Preg10 and Post10 rats made overall fewer entries into the open or closed arms than oil rats (p‟s<0.02) (Table 3). For the time spent in each arm, males from dams that received high CORT postpartum spent more time in closed arms compared to oil controls (Post40: p=0.03; P+P40=0.004). For females, only the Post40 group spent significantly more time in the closed arms (p=0.04), and P+P10 spent more time in the center compared to oil controls (p=0.02). The time in a closed arm vs. time in open arm or center was used as an index of anxiety with a higher ratio indicating more anxious behavior. Post40 and P+P40 males had significantly higher closed/open ratios compared to undisturbed or oil controls (p‟s <0.02), while no such effect was seen in females (p‟s >0.39, Figure 5). Repeated-measures ANOVA for the entries into the open or closed arm showed a significant main effect of group (F(7, 128)=2.66, p=0.01), sex (F(1, 128)=14.1, p<0.001) and arm (F(1,128)=1087.2, p<0.001), with more closed than open arm entries and females making more entries than males. For the time spent in each zone (closed arm, open arm or center) repeated measures ANOVA showed a significant interaction effect of group and zones (F(14, 248)=3.41, p<0.001) sex and zones (F(2, 248)=4.48, p=0.01) and a significant three-way interaction (F(14, 248)=1.78, p=0.04). Closed/open ratios showed an interaction effect of group and sex (F(7, 124)=2.51, p=0.02) main effect of sex (F(1, 124)=4.67, p=0.03) and group (F(7, 124)=3.40, p=0.002). There were no violations to homogeneity of variance but the open arm entries and time spent in open arms deviated from normality (p<.05). 3.5. Resistance to capture - Offspring from dams exposed to high CORT had higher capture scores compared to controls. 16 Compared to oil controls, P+P10 animals were caught faster (p=0.006), while Preg40 animals took longer to be caught (p=0.02), and all high CORT groups (i.e. Preg40, Post40, and P+P40) had higher cumulative capture scores compared to oil and undisturbed controls (all p‟s <0.03; Figure 6). There was a significant group effect for the time to capture the animal (F(7, 126)=3.87, p<0.001) and the cumulative capture score (F(7, 126)=3.52, p=0.002) but no sex or interaction effects (p‟s >0.05). There was heterogeneity of variance for total score (p<0.015) but not for time to capture. However, all of the effects noted above were still significant using the Welch‟s test. 3.6. Restraint stress - Offspring from dams exposed to CORT during gestation had lower basal CORT while offspring from dams exposed to low CORT postpartum had higher basal CORT. Offspring from dams exposed to CORT during gestation or low CORT during gestation and the postpartum had higher stress levels of CORT. Preg10 and Preg40 animals had lower baseline (t0) levels compared to undisturbed controls (both p‟s=0.02), but not compared to oil controls (p‟s>0.17) while Post10 and P+P10 rats had higher baseline levels (t0) compared to oil controls (p‟s<0.001). Preg10, Preg40 and P+P10 groups had higher peak levels at the end of restraint stress (t60) compared to oil controls (all p‟s<0.02). P+P10 had also significantly higher recovery (t120, one hour after end of restraint stress) levels compared to oil controls (p‟s <0.004), while all high CORT groups had lower recovery levels (t120) compared to undisturbed controls (p‟s<0.05). There was no difference between undisturbed and oil controls for any time point (all p‟s>0.12). For a full display of all significant differences see Figure 7. Repeated measures ANOVA with time (t0, t30,t60 and t120) as within-subject factors revealed significant main effects of group (F(7,71)=4.93, p<0.001), sex (F(1,71)=163.25, p<0.001) and time (F(3,213)=423.58, p<0.001) and significant interaction 17 effects between group and sex (F(7,71)=2.17, p=0.05), sex and time (F(3,213)=16.11, p<0.001), and time and group (F(7,213)=1.61, p=0.05), but not between all three factors (p=0.65). Mauchley‟s test for sphericity was significant, however the Huynh-Feldt adjustment indicated significant effects for all the within-subjects factors (adjusted p‟s all <0.014). There were no violations of homogeneity of variance, but normality was violated for t120 in males only. Table 4 gives an overview of the directions of effects of selected tests and behaviors. 3.7 Correlation of maternal care with behavioral outcome Detailed results of maternal behavior have been reported previously (Brummelte and Galea 2010a). Briefly, the P+P10, Preg40, Post40 and P+P40 groups spent more time off the nest (all p‟s< .01) and less time nursing (all p‟s< .05) than oil controls, while there was no significant difference in amount of time spent licking for any of the groups (all p‟s>.71). The peripheral distance crossed in the OFT was not associated with maternal care overall (p=0.86), but looking at the correlations by group, the peripheral distance was negatively associated with the dams‟ time spent off the nest for the Preg10 group (r= -0.58, p=0.017) and positively associated for the Post10 group (r=0.58, p=0.019). Total capture score in the resistance to capture test was slightly but significantly correlated with maternal time spent off the nest (r= 0.19, p=0.02), however there were no significant correlations for maternal behavior and resistance to capture behavior of the offspring by group (all p‟s>0.18). There was a significant correlation for the recovery CORT levels (t120: r=-0.20, p=0.05) after restraint stress and maternal time off the nest. Analysing this correlation for each group separately showed that for the oil group, offspring from mother spending more time off the nest had lower CORT levels at t120 (r= -0.61, p=0.006).There were no other significant correlations. 18 4. Discussion The results from the present study demonstrate that exposure to maternal exogenous CORT during gestation or the postpartum leads to dose-dependent and time of administration-dependent changes in the male and female offspring. To our knowledge this is the first report on consequences of elevated maternal CORT levels throughout pregnancy and the postpartum for the male and female offspring. High levels of maternal CORT during pregnancy, but not the postpartum, lead to more depressive-like behavior in the offspring, while high maternal CORT during the postpartum lead to more anxiety-like behavior in the male, but not female, offspring compared to controls. Further, all high CORT groups showed more impulsive-like behavior as measured by the resistance to capture test. Both male and female rats exposed to CORT during pregnancy had lower adult baseline levels and higher stress corticosterone levels, while rats exposed to low CORT during the postpartum had higher baseline CORT levels. Weight gain during the postpartum period was significantly attenuated in offspring exposed to maternal CORT pre- or post-natally. Interestingly, in adulthood only the low CORT groups had reduced weight compared to controls. Overall, our results demonstrate that the impact of maternal CORT on the offspring depend on the dose and time of exposure, with varying effects of antenatal, postnatal or prolonged exposure during gestation and the postpartum period. 4.1. Offspring from dams exposed to high CORT showed attenuation of weight gain throughout development; however as adults all the offspring from dams exposed to low CORT weighed less than controls. In line with other studies, our results showed that early CORT exposure can affect the weight gain of the offspring (Brummelte et al., 2006; Franko et al., 2010). Offspring from dams exposed 19 to high CORT either during gestation or postpartum showed attenuation of weight gain throughout development. However, as adults all the offspring from dams exposed to low CORT, either during gestation or postpartum, weighed less than controls. These findings suggest that different levels of CORT can differentially affect or re-program the metabolic system (Franko et al., 2010), which is in line with previous studies showing varying results of chronic CORT on weight depending on the dose, timing, route of administration and type of glucocorticoid used (Brummelte and Galea, 2010a; Catalani et al., 1993; Karatsoreos et al., 2010). Our findings are further consistent with studies examining the effect of maternal stress on offspring body weight (Baker et al., 2009; Mueller and Bale, 2006). For example, maternal stress during late pregnancy temporarily changed the offspring‟s body weight, while stress experienced earlier in pregnancy resulted in long-term increase in body weight in male offspring of stress-sensitive dams (Mueller and Bale, 2006). 4.2. High levels of maternal CORT during pregnancy leads to more depressive-like behavior in the offspring Our finding of more depressive-like behavior in the Preg40 group is in concert with many studies showing more depressive-like behavior after prenatal stress in rodents (Abe et al., 2007; Alonso et al., 2000; Secoli and Teixeira, 1998). Further, one prospective longitudinal study in humans found that adolescents who had been exposed to antenatal depression had a 4.7 times higher risk for being diagnosed with a depressive disorder compared to non-exposed adolescents (Pawlby et al., 2009). However, direct glucocorticoid administration to pregnant dams does not always produce behavioral despair in the offspring (e.g. Hauser et al., 2009) and we did not observe increased-depressive-like behavior in our Preg10 group which were also exposed to elevated CORT in utero. Intriguingly, Wilcoxon and Redei (2007) demonstrated that both 20 adrenalectomy (ADX) of the dam and ADX plus high CORT replacement lead to increased immobility in the forced swim test. Therefore, the mechanism underlying the effect of maternal glucocorticoids on depressive-like behavior in the offspring is complex and it is conceivable that there is a curvilinear relationship between maternal CORT levels and behavioral outcome. Along with increased levels of immobility in the FST, the Preg40 group also had decreased time spent swimming in the FST. Swimming behavior in the FST is mediated through serotonin levels (Detke et al., 1995), indicating that serotonin levels may be dysregulated in offspring exposed to high maternal CORT. Chronically elevated CORT levels are believed to impair serotonergic neurotransmission (Meijer and de Kloet, 1998), thus it is tempting to suggest that the decrease in swimming in the Preg40 group may be due to CORT-induced alterations in serotonergic transmissions. The fact that there was no effect of postnatal CORT exposure on immobility in the forced swim test in male or female offspring is in line with our previous study (Brummelte et al., 2006). It should be noted that while the FST is well established as a behavioral indicator of antidepressant efficacy (Cryan et al., 2005; Porsolt et al., 1978) where decreased immobility is associated with greater efficacy for antidepressant properties, many researchers have used the corollary to suggest that increased time spent immobile in the FST is indicative of depressive-like behaviors (Brummelte et al., 2006; Galea et al., 2001; Kalynchuk et al., 2004; LaPlant et al., 2009; Stoffel and Craft, 2004). However the use of FST as an index of depressive-like behavior is not without controversy as it is not clear exactly what immobility in the FST is modeling (for review see: Cryan et al., 2005). 4.3. High levels of postpartum CORT increase anxiety-like behavior in males Post40 and P+P40 males spent more time in the closed arm of the Elevated Plus Maze and had higher closed/open arm ratio both of which are indicative of more anxiety-like behavior. 21 This finding is not due to these animals showing less locomotor activity as they did not differ from controls regarding the distance travelled in the Open Field Test. This effect is in concert with the literature showing higher anxiety after early stress exposure (Lukkes et al., 2009; Wei et al., 2010) but our study is the first to demonstrate that high CORT postpartum (or during gestation and postpartum) leads to more anxiety-like behavior in the male adolescent offspring. The higher resistance to capture scores in the high CORT offspring compared to oil controls could also indicate more anxious and/or impulsive behavior in these animals (Brummelte et al., 2006; Kalynchuk et al., 1997). Stone and colleagues (1988) demonstrated increased escape behavior after chronic administration of CORT to adult rats and reduced escape behavior after adrenalectomy, suggesting that CORT may play a role in maintaining flight responses during chronic stress. Taken together our results show that early CORT exposure has a complex impact on anxiety-related behaviors, which may be due to different developmental maturation patterns of associated brain structures, such as the amygdala or the prefrontal cortex. These brain areas may be more vulnerable to excessive glucocorticoids at different time points during development. 4.4. Adult offspring exposed to low and high CORT in utero had lower basal but higher stress levels of CORT Adult offspring given one hour of restraint stress revealed changes in basal and stress levels of CORT that depended on time of maternal exposure to CORT (gestation or postpartum) and the dose of CORT. Exposure of the dam to low CORT postpartum upregulated basal serum CORT levels in the offspring, while exposure of the dam to CORT antepartum decreased basal serum CORT in the offspring. Exposure of the dam to high CORT downregulated the CORT 22 response during recovery in the offspring, resulting in CORT levels below those of the undisturbed or oil controls, which was more particularly evident in the females. However exposure of the dam to low or high CORT antepartum resulted in an upregulated CORT response during the one hour of stress. Consistent with previous literature (Matthews et al., 2004; Meaney et al., 2007) this suggests that the function of the HPA axis is programmed differently during in utero versus ex utero exposure to elevated maternal glucocorticoids. Prenatal exposure to high levels of glucocorticoids might prepare the offspring for a challenging environment by reprogramming the HPA axis to being hyposensitive to mild stressors. Exposure to high levels of maternal glucocorticoids during the postpartum on the other hand may cause different “programming” as the HPA axis is further matured at this stage and may be less plastic. In line with this, chronic low CORT exposure during adolescence attenuates the physiological stress response in adulthood (Xu et al., 2010). In addition maternal contact can inhibit stress responsiveness and that maternal stimuli play a special role in the modulation of pituitary-adrenal activity during development (Stanton and Levine, 1990), thus it is possible that the reduced maternal care/contact we observed in the dams (Brummelte and Galea, 2010a) also contributed to programming the HPA axis of the offspring during the postpartum period. 4.5. Sex differences in the effects of elevated maternal CORT on the offspring Our results show that the effects of CORT treatment in dams were not always consistent between the sexes. This is consistent with several studies that have reported sex differences after an early glucocorticoid treatment (Golub, 1982; Scheff et al., 1988; Vicedomini et al., 1986). Furthermore, Kalynchuk and her colleagues (2004) found that adult male rats treated with high CORT for three weeks exhibited stronger physiological effects than treated females. Males 23 generally appear to be more vulnerable to the effects of acute and chronic stress, depending on the nature of the stressor and the age of the animal (Falconer and Galea, 2003; Galea et al., 1997). Possible reasons for this are several sex differences in the endocrine system and HPA axis activity. It is well known that females show higher basal and stress ACTH, CORT and corticosteroid binding globulin levels than males (Duncko et al., 2001; Gala and Westphal, 1965). Further, females appear to be more resistant to the damaging effects of stress at certain periods in their life (Bowman, 2005) and show a dampened response of their HPA axis during reproduction, which may be an evolutionary mechanism to protect the offspring (Brunton et al., 2008; Young and Cook, 2004). A sex-dependent effect of stress would be in line with previous human studies showing a more pronounced impact of postnatal or antenatal depression on behavioral and cognitive development in boys compared to girls (Gerardin et al., 2011; Murray and Cooper, 1997). 4.6. Early life maternal conditions change adult phenotype: lessons from the CORT-induced model of stress and/or depression We have previously shown that chronic exposure to CORT postpartum leads to increased depressive-like behavior, reduced maternal care, reduced hippocampal neurogenesis and reduced weight gain in dams and may thus serve as an animal model of postpartum depression with high face validity (Brummelte et al., 2006; Brummelte and Galea, 2010a). The offspring from this model show sex-dependent significant changes in certain behaviors and alterations in their stress response which indicates adaptation of the pups to the early adverse conditions. In line with our results, Petropoulos and colleagues (2010) have recently demonstrated that synthetic glucocorticoid administration during pregnancy can alter levels of blood-brain barrier proteins in a complex dose- time- and sex-specific manner, indicating that all three factors may play a 24 crucial role in determining the impact of glucocorticoids on brain maturation. Glucocorticoids are indispensable for early development and essential for many peripheral and neuronal maturation processes (McEwen, 1987). Importantly, even if a single course of antenatal steroid is considered safe and does not alter HPA axis function (Dalziel et al., 2005; Gover et al., 2011), repeated doses of synthetic antenatal steroids have been associated with restricted growth and altered cardiovascular, HPA and metabolic function (for review see: Sloboda et al., 2005). An increase in endogenous glucocorticoids, as seen after prenatal stress, can lead to earlier earflap and eye opening and faster righting in rat pups (Secoli and Teixeira, 1998), which suggests that increased glucocorticoids levels accelerate maturation of the fetus beyond lung maturation. An accelerated development could represent an evolutionary mechanism to adapt to a challenging environment. Interestingly, in our study the prolonged exposure to CORT during pregnancy and the postpartum did not result in accumulated effects in the offspring. This may be due to the possibility that the exposure during pregnancy already prepared them for a more adverse postnatal environment. In line with this, King and colleagues (2004) showed that maternal malnutrition during pregnancy prevented the effects of acute postnatal stress on neurogenesis in the dentate gyrus. Overall, the offspring from our study seemed to be less affected compared to other studies using synthetic glucocorticoids or direct exposure of the offspring (e.g. DeKosky et al., 1982; Flagel et al., 2002; Hauser et al., 2009), which might be due the lower potency and better protection from natural glucocorticoid compared to synthetic ones which can for instance easily cross the placental barrier (Seckl et al., 2000). Further, we cannot exclude the possibility that CORT-induced changes in maternal care may have contributed to or caused the observed behavioral and neuroendocrine changes in the offspring. We found differences in maternal behavior between the groups (Brummelte and Galea, 2010a) and correlations between the time dams spent off the nest and resistance to capture behavior and HPA axis function of the 25 offspring. Unfortunately the present design prevents us from teasing apart the relative contributions of maternal CORT to directly or indirectly influence offspring behavior, but future studies could investigate this. Considering that high maternal CORT results in high CORT in the offspring (Brummelte et al., 2010) it is likely that the observed effects in the offspring are dependent on the multifaceted combination of glucocorticoid exposure, maternal behavior and environment. 5. Conclusions Taken together, findings from our study show that maternal CORT impacts the behavioral and endocrine outcome of the offspring which is dependent on the dose, time of exposure to maternal CORT and on the sex of the offspring. Male, but not female, offspring of dams who were exposed to high CORT postpartum exhibited greater anxiety-like behavior, while offspring of dams given high CORT during gestation exhibited greater depressive-like behavior. This indicates that there are different critical windows for early exposure to glucocorticoids to influence anxiety or depressive-like behavior in the adult offspring. The timing of exposure to maternal CORT was also important in the manifestation of basal and stress CORT levels in adulthood as offspring from dams exposed to CORT during gestation had lower basal, but higher stress levels of CORT, while offspring from dams exposed to low, but not high, CORT postpartum had higher levels of basal CORT. Intriguingly the Preg40 group which showed more depressive-like behavior also had altered HPA axis activation in response to restraint stress, while the more „anxious‟ Post40 and P+P40 males showed no such changes. It is important to better understand how early adverse experiences reprogram the offspring to prepare it for its subsequent environment as this will help to determine risk factors for neuropsychiatric diseases such as depression and anxiety disorders and health outcomes in future generations. 26 Acknowledgements We thank Lucille Hoover, Kathleen Wonch and Julia Rummel for their technical assistance. SB was funded by a postdoctoral fellowship of the Department of Foreign Affairs and International Trade Canada (DFAIT). This research was funded by an Operating Grant from CIHR to LAMG. LAMG is a Michael Smith Senior Scholar. Funding sources had no role in study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication. 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J Affect Disord (Epub ahead of print). Yeh KY (1984) Corticosterone concentrations in the serum and milk of lactating rats: parallel changes after induced stress. Endocrinology 115:1364-1370. Young BJ, Cook CJ (2004) Stress-induced modification of anxiety in rats is dependent on reproductive status. Physiol Behav 80:569-575. Zuena AR, Mairesse J, Casolini P, Cinque C, Alema GS, Morley-Fletcher S, Chiodi V, Spagnoli LG, Gradini R, Catalani A, Nicoletti F, Maccari S (2008) Prenatal restraint stress generates two distinct behavioral and neurochemical profiles in male and female rats. PLoS One 3:e2170. 34 Figure Legends Figure 1 Experiment Overview and Timeline. Figure 2 Body weight after weaning (A) and in adulthood (B). A: Body weight of male and female offspring at postnatal day 23 (weaning). Males weighed significantly more than females. Post10, Preg40, Post40 and P+P40 pups weighed less than undisturbed or oil controls. B: Body weight of male and female offspring in adulthood. Males were significantly heavier than females. All low CORT groups weighed significantly less than oil controls. However, oil controls weighed more than undisturbed controls. a: significantly different from undisturbed controls; b: significantly different from oil controls; p<0.05. Figure 3 Percentage of time spent with each behavior (struggling, swimming and immobile) during the Forced Swim Test. Offspring from the Preg40 group showed increased immobility and decreased swimming compared to undisturbed or oil offspring. a: significantly different from undisturbed controls; b: significantly different from oil controls; p<0.05. Figure 4 Peripheral distance travelled in the Open Field Test. Females were significantly more active in the Open Filed Test than males. Female oil covered more distance than female undisturbed controls (p=0.01) and Post10 females travelled 35 significantly less (p=0.004), while P+P10 females travelled significantly more than oil controls (p=0.009). Similarly, for males, P+P10 travelled significantly more peripheral distance compared to male oil controls (p=0.05), but Preg40 travelled significantly less than oil controls (p=0.02) or undisturbed controls (p=0.01).a: significantly different from undisturbed controls; b: significantly different from oil controls Figure 5 Behavior in the Elevated Plus Maze. Males exposed to high CORT during the postpartum (i.e. Post40 and P+P40) showed more anxiety-like behavior in the Elevated Plus Maze by spending more time in the closed arm and revealing a higher closed/open & center ratio. a: significantly different from undisturbed controls; b: significantly different from oil controls; p<0.05. Figure 6 Total time it took to capture the animals and the cumulative capture score based on a 7-point scale for resistance to capture during approach, touch and lift up. The P+P10 group was captured faster compared to oil controls, while the Preg40 group took longer to be captured. All high CORT groups had higher cumulative scores compared to oil or undisturbed controls. a: significantly different from undisturbed controls; b: significantly different from oil controls; p<0.05. Figure 7 Corticosterone (CORT) serum levels during restraint stress, measured before the stressor (t0, baseline), during the restraint stress (t30), at the end of the stressor (t60) and an hour after return 36 to the home cage (t120, recovery). Animals that were exposed to CORT in utero alone (Preg10 and Preg40) had lower baseline levels compared to undisturbed controls, while animals exposed to low CORT during the postpartum (Post10 and P+P10) had higher baseline levels compared to oil controls, which was particularly obvious in females. One hour after the stress, all high CORT groups had significantly lower CORT levels compared to undisturbed controls, again most evident in females, while the P+P10 groups had also higher recovery CORT levels compared to oil and undisturbed controls. a: significantly different from undisturbed controls; b: significantly different from oil controls; p<0.05. Table 1 Mean (± SEM) weight per pup during the postpartum period [grams] PND1 PND9 PND13 PND17 PND20 Undisturbed 6.9 ± 0.1 22.2 ± 1.1 33.1 ± 1.3 40.9 ± 1.9 47.1 ± 3.0 Oil 6.7 ± 0.2 21.6 ± 0.4 31.0 ± 0.8 39.6 ± 0.8 49.3 ± 0.9 Preg10 6.7 ± 0.1 22.3 ± 0.8 32.0 ± 0.9 38.1 ± 2.3 a 50.4 ± 2.3 a Post10 7.0 ± 0.3 20.3 ± 0.4 29.3 ± 0.5 a 35.2 ± 0.9 a,b 44.2 ± 1.3 a,b P+P10 6.7 ± 0.2 22.0 ± 0.8 31.0 ± 1.2 39.0 ± 1.2 48.2 ± 2.2 Preg40 6.5 ± 0.4 20.0 ± 0.9 28.3 ± 1.2 a,b 36.7 ± 1.6 a,b 45.0 ± 2.0 b Post40 6.7 ± 0.2 19.9 ± 0.2 27.6 ± 0.2 a,b 35.1 ± 0.6 a,b 45.2 ± 0.7 b P+P40 6.0 ± 0.3 17.8 ± 0.9 a,b 24.2 ± 1.1 a,b 30.4 ± 1.5 a,b 38.0 ± 1.9 a,b a significantly different (p<0.05) from Undisturbed pups, b significantly different (p<0.05) from Oil pups Table 1Table 2 Open Field Test Measures (Mean ±S.E.M.) Group # of rears Fecal boli Center entering latency [s] Time in center [s] Center distance [m] Center /total distance Males* Undisturbed 45.5 (± 4.8) 1.9 (± 0.7) 96.5 (± 67.3) 9.5 (± 3.2) 1.6 (± 0.6) .025 (±.009) Oil 40.8 (± 4.2) 1.1 (± 0.4) 112.9 (± 37.5) 17.6 (± 2.3) 3.6 (± 0.4) .058 (±.011) Preg10 48.8 (± 7.0) 3.4 (± 0.8) 138.0 (± 68.8) 14.6 (± 4.5) 3.0 (± 1.0) .042 (±.012) Post10 45.3 (± 3.0) 3.0 (± 0.7) 101.6 (± 16.0) 18.1 (± 2.3) 3.6 (± 0.5) .057 (±.008) P+P10 41.9 (± 5.6) 2.5 (± 0.8) 118.6 (± 47.3) 13.0 (± 2.9) 2.7 (± 0.5) .040 (±.007) Preg40 36.8 (± 3.4) 2.1 (± 1.0) 48.1 (± 10.1) 11.4 (± 2.2) 2.0 (± 0.4) .043 (±.007) Post40 48.0 (± 8.4) 3.3 (± 1.2) 45.1 (± 9.6) 17.4 (± 4.7) 3.6 (± 0.8) .057 (±.011) P+P40 44.1 (± 5.6) 1.6 (± 0.7) 78.3 (± 32.2) 15.0 (± 3.6) 3.3 (± 0.8) .055 (±.014) Females Undisturbed 45.8 (± 3.5) 1.3 (± 0.9) 61.4 (± 40.1) 21.9 (± 5.5) 4.2 (± 0.8) .065 (±.013) Oil 56.8 (± 4.3) 0.6 (± 0.4) 48.4 (± 11.7) 22.8 (± 2.4) 5.1 (± 0.5) .070 (±.006) Preg10 48.7 (± 6.5) 0.6 (± 0.4) 70.0 (± 23.5) 17.5 (± 1.6) 3.8 (± 0.5) .052 (±.009) Post10 43.6 (± 2.3) 0.4 (± 0.4) 24.5 (± 5.7) 25.5 (± 4.2) 5.5 (± 0.8) .080 (±.012) P+P10 51.4 (± 4.3) 0.4 (± 0.3) 66.3 (± 15.9) 30.7 (± 4.0) 5.9 (± 0.9) .072 (±.010) Preg40 51.9 (± 3.2) 0.3 (± 0.3) 68.1 (± 18.6) 20.8 (± 4.5) 5.1 (± 1.1) .068 (±.013) Post40 52.0 (± 1.7) 1.1 (± 0.7) 37.6 (± 16.6) 18.8 (± 2.8) 4.4 (± 0.5) .063 (±.006) P+P40 54.0 (± 5.2) 0.0 (± 0.0) 47.5 (± 22.2) 16.0 (± 3.6) 3.6 (± 0.8) .057 (±.013) Table 2* Males showed significantly fewer rears (p=0.01), more fecal boli (p<0.001), higher latency to enter the center (p=0.02) and less time and distance in the center (p<0.001) than females. Table 3 Entries into and time spent in closed or open arms or center during the Elevated Plus Maze (EPM) Entries Time Group Closed arm Open arm Open arm Center Undisturbed 10.5 ± 0.8 1.6 ± 0.3 17.3 ± 2.6 82.0 ± 8.0 Oil 11.2 ± 0.5 2.1 ± 0.4 19.5 ± 3.8 81.1 ± 4.2 Preg10* 9.6 ± 0.8 0.9 ± 0.2 6.8 ± 1.8 89.8 ± 8.8 Post10* 9.1 ± 0.6 1.6 ± 0.9 13.4 ± 3.3 67.7 ± 6.4 P+P10 10.3 ± 0.6 1.4 ± 0.8 11.4 ± 2.1 106.1 ± 6.5 Preg40 12.5 ± 0.8 2.2 ± 1.3 13.6 ± 3.3 80.6 ± 5.2 Post40 10.6 ± 0.9 1.3 ± 0.3 7.0 ± 2.5 65.7 ± 7.4 P+P40 11.6 ± 1.1 2.0 ± 0.8 12.1 ± 3.8 70.5 ± 7.0 *Preg10 and Post10 animals made significantly fewer overall entries compared to oil rats (p’s < 0.02). Table 3Table 4 Summary of selected effects per group compared to oil controls Preg10 Post10 P+P10 Preg40 Post40 P+P40 Weight At weaning ↓ ↓ ↓ ↓ As adults>a ↓ ↓ ↓ Forced Swim Test Immobility ↑ Open Field Test Peripheral distance F:>a F: ↓ ↑ M:↓ Elevated Plus Maze Time in closed arm ↑ M:↑ Closed / Open ratio M:↑ M:↑ Resistance to capture Time to capture ↓ ↑ Capture Score ↑ ↑ ↑ Restraint stress# Baseline ↓# ↑ ↑ ↓# End of stress ↑ ↑ ↑ Recovery ↑ ↓# ↓# ↓# >a: indicates that oil controls were significantly higher compared undisturbed controls #: for restraint stress differences are also displayed if the group was different compared to undisturbed controls; F: only in Females; M: only in Males Table 4Figure 1Click here to download Figure(s): Figure 1.pdfFigure 2Click here to download Figure(s): Figure 2.pdfFigure 3Click here to download Figure(s): Figure 3.pdfFigure 4Click here to download Figure(s): Figure 4.pdfFigure 5Click here to download Figure(s): Figure 5.pdfFigure 6Click here to download Figure(s): Figure 6.pdfFigure 7Click here to download Figure(s): Figure 7.pdfSupplementary Table 1Click here to download Supplementary Material: Table S1.docSupplementary Table 2Click here to download Supplementary Material: Table S2.docSupplementary Table 3Click here to download Supplementary Material: Table S3.doc
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Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Faculty
Postdoctoral
Gestation
Prenatal
In utero
Glucocorticoids
Depression
Stress
Sex differences
Early programming
Development
Adolescence
Gestational and postpartum corticosterone exposure to the dam affects behavioral and endocrine outcome of the offspring in a sexually-dimorphic manner
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