Stress and the pregnant female: Impact on hippocampal cell proliferation, but not affective-like behaviors

Hormones and Behavior 59 (2011) 572–580

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Stress and the pregnant female: Impact on hippocampal cell proliferation, but not affective-like behaviors Jodi L. Pawluski ⁎, Daniël L.A. van den Hove, Ine Rayen, Jos Prickaerts, Harry W.M. Steinbusch Department of Neuroscience, School of Mental Health and Neuroscience, Maastricht University, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands

a r t i c l e

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Article history: Received 4 December 2010 Revised 9 February 2011 Accepted 26 February 2011 Available online 3 March 2011 Keywords: Hippocampus Neurogenesis Dentate gyrus Pregnancy Depression Anxiety Prenatal stress Motherhood Gestation Cell proliferation

a b s t r a c t Fifteen percent of women worldwide develop postpartum depression; however, many women also suffer from mood disorders during pregnancy. Our knowledge of how these stress-related disorders affect the neurobiology of the mother is very limited. In animal models, depressive-like behavior is often associated with repeated stress and alterations in adult neurogenesis in the hippocampus. However, research has yet to investigate the effect of stress on affective-like behavior and hippocampal neurogenesis in the pregnant female. The aim of the present study was to determine whether stress during gestation alters affective-like behaviors, corticosterone levels, and hippocampal cell proliferation and new cell survival in the pregnant female, and whether these effects differ from virgin females. Age-matched pregnant and virgin Sprague– Dawley rats were divided into two conditions: 1) stress and 2) control. Females in the stress condition were repeatedly restrained during gestation, and at matched time points in virgin females. Affective-like behaviors were assessed at the end of gestation, and at matched time points in virgin females. Results demonstrate that regardless of repeated restraint stress, pregnant females have increased anxiety-like behavior, decreased depressive-like behavior, and lower corticosterone levels, compared to non-stressed, and at times stressed, virgin females. In addition, stressed virgin females have lower levels of depressive-like behavior compared to control virgin females. Interestingly, hippocampal cell proliferation was increased in both virgin and pregnant females after stress. Understanding how stress affects the female during different reproductive states will aid in improving the health and well being of the mother and child. © 2011 Elsevier Inc. All rights reserved.

Introduction The transition to motherhood is a time of distinct neural and behavioral plasticity in the mother (Kinsley and Lambert, 2006; Leuner et al., 2010; Pawluski and Galea, 2008). Unfortunately, this is also a time when a woman is vulnerable to the effects of stress and stress-related disorders. Recent estimates document that during pregnancy and the postpartum period, 10–20% of women experience mood disorders such as depression and anxiety (Bennett et al., 2004b; Gavin et al., 2005; Oberlander et al., 2006) and many women experience their first depressive-episode during pregnancy (Bennett et al., 2004a). Depression is often associated with increased stress and decreased coping ability and can have detrimental effects on the mother, child and family. Stress during gestation significantly impacts offspring development on a number of domains in humans and animal models (Glover et al., 2010; Maccari and Morley-Fletcher, 2007; Weinstock, 2008). However, very little research has investigated how gestational stress affects the neurobehavioral outcomes in the mother during pregnancy

⁎ Corresponding author. Fax: +31 43 367 1096. E-mail address: [email protected] (J.L. Pawluski). 0018-506X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2011.02.012

or the postpartum period. Recent research has shown that daily restraint stress, for the last 10 days of pregnancy, increases depressive-like behavior and corticosterone levels during the early postpartum period in the mother (Smith et al., 2004). In addition, others have shown that repeated restraint stress during the last week of pregnancy, or chronic administration of high levels of corticosterone pre- and post-partum, result in increased depressive-like behavior in the mother at the time pups are weaned (Brummelte et al., 2006; O'Mahony et al., 2006). Stress during gestation also results in increased anxiety-like behavior and decreased depressive-like behavior in the dam two weeks after weaning (Darnaudery et al., 2004). To date, only one study has aimed to investigate the effect of stress during pregnancy on anxiety-like behavior in the pregnant female (Baker et al., 2008); Baker et al. (2008) demonstrated that stressed pregnant female rats exhibited marginal increases in anxiety-like behaviors during late pregnancy compared to non-stressed controls. These data point to a significant impact of stress during pregnancy on affective behaviors in the mother, at least during the postpartum period. Stress impacts hippocampal neurogenesis, and alterations in hippocampal neurogenesis play a role in both anxiety- and depressivelike behaviors (DeCarolis and Eisch, 2010; Eisch et al., 2008; Pawluski et al., 2009a; Revest et al., 2009). In adult female rats, the effects of stress

J.L. Pawluski et al. / Hormones and Behavior 59 (2011) 572–580

on hippocampal neurogenesis appear to be dependent on the duration of the stress and the age of the new surviving cells (Pawluski et al., 2009a). For example, virgin females subject to repeated stress have increased survival of new cells in the dentate gyrus of the hippocampus a few days after the stress (Westenbroek et al., 2004), but acute stress has no effect on cell proliferation in the hippocampus of the female (Falconer and Galea, 2003; Shors et al., 2007). However, more work is needed to determine how stress alters hippocampal neurogenesis in the adult female. The impact of stress during pregnancy on hippocampal neurogenesis in the pregnant female has not been investigated. During pregnancy, there are few differences in hippocampal cell proliferation or new cell survival in pregnant females compared to diestrus virgins (Furuta and Bridges, 2005; Pawluski et al., 2010; Shingo et al., 2003). However, during the postpartum period there are marked decreases in hippocampal neurogenesis in the mother (Leuner et al., 2007; Pawluski and Galea, 2007; Darnaudery et al., 2007). Recent work has also shown that administration of high levels of corticosterone during late pregnancy and throughout the postpartum period leads to decreased hippocampal cell proliferation in the mother after weaning (Brummelte and Galea, 2010). The effects of stress during pregnancy on hippocampal neurogenesis in the pregnant female remain to be determined. The proposed study aimed to determine how stress affects anxiety- and depressive-like behaviors, corticosterone levels, and aspects of hippocampal neurogenesis in pregnant and virgin adult female rats. For this experiment, age-matched pregnant and virgin female Sprague–Dawley rats were subject to repeated restraint stress and behavioral tests were carried out near the end of pregnancy, and at matched time points in virgin females. The repeated restraint stress paradigm was chosen as previous work has shown that this paradigm leads to increased depressive-like behavior in the mother during the postpartum period (O'Mahony et al., 2006). In addition, it is a commonly used paradigm to assess the effects of stress during gestation on offspring outcomes (Weinstock, 2008). Understanding how stress affects the female during different reproductive states will aid in improving the health and well being of the mother and child. Methods Animals Forty-three adult female Sprague–Dawley rats (approximately 275–325 g, 4–5 months of age) purchased from the Charles River Laboratories (France) were used in the present study. Rats were initially housed in pairs in clear polyurethane bins with corn cob bedding and ad libitum access to rat chow (Sniff) and tap water. Rats were maintained in a 12 h:12 h light/dark cycle (lights on at 7:00 a.m.). All protocols were approved by the Animal Ethics Board of Maastricht University in accordance with Dutch governmental regulations (DEC 2009-055). All efforts were made to minimize animal suffering. All animals were age-matched and sexually-naïve prior to random assignment to the following groups: virgin (nulligravid; n = 23) and pregnant (primigravid; n = 20). Groups were further divided into 1) stress and 2) control. Females in the stress conditions were individually restrained three times a day for 45 min in transparent plastic cylinders under bright light (between 8–10 a.m., 12–2 p.m., and 4–6 p.m.) from gestational days (GD)11–17 in pregnant females and at matched time points in virgin females, as previously described (van den Hove et al., 2005; Ward and Weisz, 1984). This time period during pregnancy is when stress results in postpartum depressive-like behavior (O'Mahony et al., 2006; Smith et al., 2004) and a period of stress exposure that affects offspring outcomes (Weinstock, 2008; Darnaudery and Maccari, 2008; van den Hove et al., 2010). In addition, this period was chosen to allow for behavioral testing during late

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gestation. Females in the non-stressed groups were left undisturbed until behavioral testing. Body weight of the females was measured at Days 1, 7, 14 and 21. Litter characteristics were assessed at sacrifice. For breeding of pregnant females, one male and one female were housed in a wire mesh cage. After a vaginal plug was released, impregnated females were individually housed in clear polyurethane bins until perfusion. Gestation day 0 (GD0) was the day a vaginal plug was released. Virgin females were individually housed in clear polyurethane bins for the same duration of time as pregnant females. Three females in the stress condition did not maintain a pregnancy and were removed from the study. Behavioral testing Behavioral testing took place on GD 18, 19 and 20 and at matched time points in virgin females (see Fig. 1 for timeline). Testing occurred as follows.

Elevated Zero Maze (EZM) To assess the degree of anxiety-like behavior in virgin and pregnant females the EZM was used as previously described (Shepherd et al., 1994). The apparatus consists of a circular alley (diameter of 98 cm), with a path width of 10 cm. The maze is divided in four parts, i.e., two opposite open parts and two opposite closed parts. The sidewalls have a height of 50 cm. The open parts have borders with a height of 5 mm. Testing took place between 9 a.m. and noon, in a dimly light room (35 lux), on GD 18 and at matched time points in virgin females. For the test, a rat was placed in one of the open arms facing a closed arm of the apparatus. Once a rat entered the closed arm, the test began for 5 min and the rat was allowed to move freely. An observer blind to the groups scored the number of open and closed arm entries, and time spent in open and closed arms for each animal during testing. An entry occurred when all four paws of the animal entered an arm. An index of anxiety-like behavior can be measured by the number of entries and time spent in the open arms, where a short duration of time spent in the open arms or a few number of entries into the open arms is indicative of anxiety (Pellow, 1985). The apparatus was cleaned with 70% ethanol and dried between trials. Forced Swim Test (FST) To assess depressive-like behavior in the females, the forced swim test was used as previously described (Darnaudery et al., 2004; Galea et al., 2001; Pawluski et al., 2009c). Briefly, the apparatus consisted of a vertical cylindrical glass container (height 50 cm × diameter 20 cm) filled to a depth of 33 cm with tap water at 26 ± 1 °C. This depth was sufficient to ensure that animals could not touch the bottom of the container with their hind paws (Lucki, 1997). The forced swim test was conducted over two days, between 11 a.m. and 2 p.m., and took place at the same time point for all females; GD18 and 19, and at matched time points in virgin females. On the first day of the FST, females were introduced to cylindrical glass tank filled with water for 15 min, towel dried and returned to their home cage. Twenty-four hours later, animals were exposed to the same experimental conditions for 5 min, dried and returned to their home cage. Sessions were videotaped and scored using the Best Collection System (Educational Consulting Inc) by an observer blind to conditions. The behaviors scored in the forced swim test were: (1) immobility— floating with the absence of any movement and (2) struggling—quick movements of the forelimbs such that the front paws break the surface of the water. The number of escape attempts was also recorded. Escape was defined as swimming to the bottom of the tank and pushing off, and may be indicative of heightened motivation to escape from the task (Boccia et al., 2007; Pawluski et al., 2009c).

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on

ati

n eg

r mp

Stress

I

0

1

BrdU

2 1 ay Day sion D T M T rfu EZ FS FS Pe

17 18 19 20

11

21

Pregnancy

Fig. 1. Timeline of experiment. Females were injected with BrdU the morning after a vaginal plug was released, behaviorally tested on days GD18-20, and perfused on GD21. Agematched virgin female rats were treated at matched time points to pregnant females. Animals in the stress condition were restrained on GD11-17 and at matched time points in virgin females.

Estrous cycle

Histology

High levels of estradiol may affect anxiety- and depressive-like behaviors (Frye et al., 2000; Walf and Frye, 2007). Therefore vaginal smears were done on virgin females to determine when rats were in proestrus and to control for the effects of proestrus on behavioral measures. For smears, a cotton swab dipped in saline solution was placed in the vagina and the contents were smeared on a plain microscope slide. Slides were examined under 10× objective. Proestrus was determined when a majority of cells evident in the vaginal mucus (70%) were nucleated epithelial cells. High levels of estradiol, as seen during proestrus, increase cell proliferation in the dentate gyrus (Tanapat et al., 1999). Therefore, estradiol levels were measured from serum taken at perfusion to account for possible effects of estradiol on hippocampal cell proliferation. Blood samples were collected from intracardiac puncture, stored at 4 °C overnight and centrifuged at 10 ×g for 15 min. All samples were run in duplicate using a commercially available RIA kit for estradiol (DPC 125I radioimmunoassay kit, Siemens Healthcare Diagnostics Inc) based on (Strom et al., 2008). The average intra- and inter-assay coefficients of variation were below 10%. The assay had a sensitivity of 1.4 pg/ml.

All histological procedures were based on previous work (Pawluski and Galea, 2007). Rats were deeply anesthetized with sodium pentobarbital and then perfused with 4% paraformaldehyde. Following extraction, brains were stored at 4 °C in 4% paraformaldehyde for 24 h, and transferred to 30% sucrose for a minimum of 48 h. Brains were sliced in 40 μm sections through the entire extent of the hippocampus using a cryostat (Leica). Brain sections were stored at − 15 °C in an antifreeze solution until immunohistochemistical processing.

Stress responsivity and corticosterone assays To investigate responsivity to stress, blood samples were collected from a tail nick as previously described (Pawluski et al., 2009b) in the morning between 8 and 9:30, 2 h prior to the second day of testing in the FST, and 10 min after FST testing. Corticosterone levels in the rat are elevated after 3 min of handling (Vahl et al., 2005), therefore basal samples were taken within 3 min of disturbing the animal (to maximum 100 μl). Blood samples were stored at 4 °C overnight and centrifuged at 10 ×g for 15 min. All samples were run in duplicate using a commercially available RIA kit for rat corticosterone from MP Biomedicals (Corticosterone I25 for rats and mice, MP Biomedicals). The average intra- and inter-assay coefficients of variation for all assays were below 10%. The assay had a sensitivity of 7.7 ng/ml. Hippocampal cell proliferation and new cell survival To test whether stress and reproductive status influenced new cell survival in the granule cell layer of the hippocampus, rats were given a single i.p. injection of the cell synthesis marker 5-bromo-2-deoxyuridine (BrdU; Sigma: 200 mg/kg in 0.9% saline), on GD1, the morning after copulation (GD0 day a vaginal plug was released), and at matched time points in virgin females. All animals were perfused 20 days later, on GD21 (see Fig. 1 for timeline). New cell survival was assessed by investigating the number of BrdU-immunoreactive (−ir) cells and cell proliferation was assessed by investigating the number of Ki67-ir cells.

Ki67 staining Levels of cell proliferation were assessed in the dentate gyrus of the hippocampus using an endogenous marker, Ki67. Tissue was stained for expression of Ki67 as previously described (Epp et al., 2009). Free-floating sections were rinsed between steps with PBS. Sections were incubated in 0.3% H2O2 for 30 min at room temperature to block endogenous peroxidase activity. Tissue was then incubated overnight in rabbit anti-Ki67 immunoglobulin G primary antibody (1:500; Vector Laboratories) in 0.4% Triton-X in PBS at 4 °C. The next day, sections were incubated overnight in donkey anti-rabbit biotinylated antibody (1:500; Jackson ImmunoResearch, Suffolk, UK) at 4 °C. Brain sections were further processed for immunohistochemistry by using the avidine-biotin complex (ABC Elite kit; 1:1000; Vector laboratories) for 4 h. 3,3-diaminobenzidine (DAB; Sigma, The Netherlands) was used as a substrate to obtain a color reaction. Sections were mounted on gelatin-coated slides, dried overnight, counterstained with Cresyl Violet acetate, dehydrated and coverslipped with Permount (Fisher Scientific). To estimate cell numbers, total Ki67-ir were counted under 100× objective with oil on every 10th section in the GCL and hilus (approx. 10–12 sections per rat). The number of cells was multiplied by 10 to obtain an estimate of the total number of Ki67-ir cells in the hippocampus following procedures used previously (Epp et al., 2009). Cells were considered Ki67-ir if they were intensely stained and exhibited medium round or oval cell bodies. The areas of the GCL and hilus were measured from sections counterstained with Cresyl Violet acetate using Image J software (National Institutes of Health) and estimates of the GCL and hilus volumes were made using Cavalieri's principle (Gundersen et al., 1988). BrdU immunohistochemistry For BrdU immunohistochemistry, free-floating sections were rinsed repeatedly between steps in TBS (0.1 M). Sections were incubated in 0.3% H2O2 for 30 min at room temperature and DNA was denatured by applying 2N HCl for 30 min at 37 °C. Sections were blocked with 3.0% normal donkey serum (NDS) for 30 min and incubated overnight in mouse anti-BrdU primary antibody (1:200 + 3% NDS + 10% Triton-X; Roche) at 4 °C. The following day, sections

J.L. Pawluski et al. / Hormones and Behavior 59 (2011) 572–580

were incubated in donkey anti-mouse secondary antibody for 4 h at room temperature (1:500 + 3% NDS; Vector Laboratories). Brain sections were further processed for immunohistochemistry by using the avidine-biotin complex (ABC Elite kit; 1:1000; Vector laboratories) for 2 h. To complete the staining, 3,3-diaminobenzidine (DAB; Sigma, The Netherlands) was used as a substrate to obtain a color reaction. Sections were mounted on gelatin-coated slides, dried overnight, counterstained with Cresyl Violet acetate, dehydrated and coverslipped with Permount (Fisher Scientific). To estimate cell numbers, total BrdU-ir cells were counted under 40× objective on every 10th section (approx. 10–12 sections per rat). The number of cells was multiplied by 10 to obtain an estimate of the total number of BrdU-ir cells in the hippocampus following procedures used previously (Pawluski and Galea, 2007). BrdU-ir cells are counted in the hilus and compared to counts in the granule cell region to determine whether effects are due to generalized effects on blood brain permeability. Cells were considered BrdU-ir if they were intensely stained and exhibited medium round or oval cell bodies (Ormerod and Galea, 2001; Pawluski and Galea, 2007) (Fig. 2).

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as the between-subjects factors. ANOVAs were also done on the volume of the GCL and hilus, to ensure there were no differences between groups. Pearson moment correlations were carried out between estradiol and corticosterone levels, behavioral measures, Ki67-ir cells, BrdU-ir cells, and litter details. Post-hoc comparisons utilized the Fisher LSD test. Results Weight As expected, pregnant females gained significantly more weight than virgin females across the study (main effect of group: F(1, 36) = 330.9, p ≤ 0.00001; % Weight change, Mean ± SEM, Virgin Control = 2.0± 1.1%, Virgin Stress = 2.0± 1.6%, Pregnant Control = 40.3 ± 2.0%, Pregnant Stress = 39.3 ± 4.0%). There were no other significant main effects or interactions with regards to weight change (.2 ≤ p ≤ .8; Table 1). Litter characteristics

Data analyses Analysis of variance tests (ANOVAs) were done on weight change, behavioral measures, estradiol levels, and corticosterone response to stress as within-subjects factors and group (pregnant/virgin and control/stress) as between-subjects factors. For virgin females, additional analysis was done on scores from the EZM and FST as within-subjects factors and group (control/stress) as betweensubjects factors using proestrus stage as a covariate. In addition, analysis on behavioral measures on the FST also used weight as a covariate. ANOVAs were also calculated separately for number of Ki67-ir or BrdU-ir cells with dentate gyrus area (GCL and hilus) as the within-subjects factor and group (pregnant/virgin and control/stress)

There were no significant differences in litter size or number of female or male fetuses in pregnant females at sacrifice (.6 ≤ p ≤ .8; Table 2). Elevated Zero Maze (EZM) Virgin females spent significantly more time in the open areas of the EZM (main effect of group: F(1, 36) = 17.6, p ≤ .0002) and made significantly more open arm entries compared to pregnant females (main effect of group: F(1, 36) = 14.4, p ≤ .0006; Fig. 3). There were no other significant main effects or interactions on measures of the EZM (.2 ≤ p ≤ .4). There was also no significant effect of poestrous on time in open arms and number of open arm entries in virgin females (p's ≥ .1:5 virgin females were in proestrus at testing). There were no significant correlations between litter characteristics and EZM performance in pregnant females (.1 ≤ p ≤ .7). Forced Swim Test (FST) Percent of time spent immobile in the FST was significantly altered with stress and reproductive state (pregnant versus virgin). On the second day of FST exposure, control virgin females spent significantly more time immobile than stressed virgin and stressed pregnant females (p's ≤ .02: interaction effect: F(1, 35) = 5.5, p ≤ .03, controlling for weight, Fig. 4), but not compared to control pregnant females (p ≤ .09). On day 1 of testing, pregnant females, regardless of stress, made significantly more escape attempts compared to virgin females (F(1, 36) = 4.5, p ≤ .04; Table 3) and stressed pregnant females tended to spend less time immobile compared to virgin females (p ≤ .06; Table 3), but not control pregnant females (p ≤ .09). There was no significant effect of pro-estrus on percent of time spent immobile, struggling, or number of escape attempts in virgin females. There was

Table 1 Mean (± SEM) body weight (g) of pregnant and virgin females. Pregnant females gained significantly more weight than virgin females across the study (main effect of group: F(1, 36) = 330.9, p ≤ 0.00001). There were no other significant main effects or interactions with regards to weight change (.2 ≤ p ≤ .8).

Fig. 2. Photomicrographs of representative (A) Ki67-ir cells (black arrows) in the subgranular zone of the dentate gyrus, and (B) BrdU-ir cells (black arrows) in the granular cell layer of the dentate gyrus 21 days after BrdU injection. Scale bar = 20 μm.

Virgin control Virgin stress Pregnant control Pregnant stress

Weight (g)

Weight (g)

Weight (g)

Weight (g)

day 0

day 7

day 14

day 21

291.3 ± 5.7 290.6 ± 4.4 295.2 ± 7.7 283.9 ± 8.6

296.0 ± 5.6 296.0 ± 5.6 319.9 ± 5.2 310.1 ± 9.5

299.3 ± 8.1 292.3 ± 4.6 344.9 ± 7.9 329.7 ± 9.3

297.5 ± 8.5 296.4 ± 6.9 414.5 ± 13.5 395.0 ± 15.4

J.L. Pawluski et al. / Hormones and Behavior 59 (2011) 572–580

Table 2 Mean (± SEM) number of male and female fetuses in utero. There were no significant differences in litter size or number of female or male fetuses in pregnant females at sacrifice (.6 ≤ p ≤ .8).

Pregnant control Pregnant stressed

No. of male fetuses

No. of female fetuses

5.5 ± 1.2 6.0 ± 0.9

4.0 ± 0.7 4.4 ± 1.1

a significant main effect of group on immobility in pregnant and virgin females (main effect of group: F(1, 35) = 9.7, p ≤ .004, controlling for differences in weight). There were no other significant main effects or interactions on measures of the FST (.2 ≤ p ≤ .9; Table 3). There was a significant negative correlation between the number of male fetuses in a litter and number of escape attempts made by pregnant females on day 1 of testing on the FST (r = −.66, p ≤ .004; Fig. 4B). There were no other significant correlations between litter sex distribution or litter size and measures on the FST (.1 ≤ p ≤ .9).

A Mean (±SEM) percent of time immobile on the FST (day 2)

576

B

35 30

Control Stress

*

*

25 20 15 10 5 0

Virgin

Pregnant

12 Control Stress

Corticosterone response to stress Pregnant females had significantly lower circulating corticosterone levels compared to virgin females (main effect of group: (F(1, 32) = 4.0, p≤.03; Fig. 5). There was also a significant main effect of time (F(1, 32)= 83.8, p ≤ .0001), with corticosterone levels being significantly elevated after swimming. There were no other significant differences

Number of male fetuses

10 8 6 4 2 0

Mean (±SEM) time spent on the open arm of the EZM (sec)

A 140

0

*

Control Stress

120

80 60 40

Virgin

Pregnant

B Mean (±SEM) number of open arm entries on the EZM

6

8

10

12

14

between groups in corticosterone levels (.3 ≤ p ≤ .7) and no significant differences between groups in the overall change in corticosterone levels prior to and after swim stress (.5 ≤ p ≤ .7). There were significant correlations between basal corticosterone levels and percent of time spent immobile (r = .33, p ≤ .04) and struggling (r = − .63, p ≤ .009) on the second day of FST. There were no other

20

14

*

Control Stress

12 10 8 6

Table 3 Mean (± SEM) percent time spent in behaviors score during the FST. On day 1 of FST testing pregnant females, regardless of stress, made significantly more escape attempts compared to virgin females (p ≤ .04) and stressed pregnant females tended to spend less time immobile than virgin females (p's ≤ .06). There were no other significant main effects or interactions on percent of time spent struggling or number of escape attempts on day 2 of the FST (.23 ≤ p ≤ .94). * denotes significantly different from virgin females. n = 7–12/group. Immobility

4 2 0

4

Fig. 4. A) Mean (± SEM) percent of time spent immobile on the FST on Day 2. Control virgin females spent significantly more time immobile than stressed virgin and pregnant females (p's ≤ .02), but not compared to control pregnant females (p ≤ .09). n = 7–12/group. B) Correlation between number of male fetuses and number of escape attempts during pregnancy. There was a significant negative correlation between number of male fetuses in a litter and number of escape attempts made by pregnant females on day 1 of testing on the FST (r = − .66, p ≤ .004).

100

0

2

Number of escape attempts on the FST

Virgin

Pregnant

Fig. 3. Mean (±SEM) number of open arm entries and time in an open arm as measured on the EZM. Pregnant females A) spent significantly less time in the open areas (p ≤ .0002) and B) made significantly fewer open arm entries compared to virgin females (p ≤ .001). * denotes pregnant females significantly different from virgin females. n = 7–12/group.

Virgin control Virgin stress Pregnant control Pregnant stress

Struggling

Struggling

No. of escape No. of escape attempts attempts

(% on day 1) (% on day 1) (% on day 2) (day 1)

(day 2)

23.2 ± 3.3

18.6 ± 2.9

26.3 ± 3.0

2.2 ± 0.7

0.1 ± 0.1

23.9 ± 2.5

13.8 ± 1.9

21.5 ± 2.5

1.3 ± 0.4

0.2 ± 0.1

24.2 ± 3.4

17.9 ± 2.7

30.0 ± 5.5

3.8 ± 1.1*

0.2 ± 0.1

14.1 ± 2.5

16.5 ± 1.8

30.3 ± 4.1

2.9 ± 0.7*

0.1 ± 0.1

Mean (±SEM) total corticosterone levels (ng/mL)

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1400

Virgin Control Virgin Stress Preg Control Preg Stress

1200 1000 800 600

BrdU-ir cells There were no significant effects of reproductive state or stress on the total number of BrdU-ir cells in the GCL or hilus (p's ≥ .5; Table 4). As expected, there was a significant main effect of region with all animals having significantly more BrdU-ir cells in the GCL than in the hilus (F(1, 16) = 138.17, p ≤ .0001). There were no significant correlations between oestradiol levels at the time of perfusion and number of BrdU-ir cells in the GCL or hilus (.2 ≤ p ≤ .4).

400 200 0

*

Discussion basal

swim stress

Fig. 5. Mean (± SEM) total serum corticosterone (ng/ml). Overall, pregnant females, regardless of stress exposure, had significantly lower corticosterone levels compared to virgin females (p ≤ .03) and corticosterone levels were significantly elevated after swim stress (p ≤ .0001). *denotes pregnant females significantly different from virgin females. n = 6–11/group.

significant correlations between corticosterone levels, behavioral measures and litter characteristics (.2 ≤ p ≤ .8).

Ki67-ir cells Volume of the GCL and hilus did not differ with reproductive state or stress (.4 ≤ p ≤ .8), therefore estimates of the total number of cells were used for analysis. Stressed females, regardless of reproductive state and estradiol levels, had significantly more Ki67-ir cells in the GCL of the hippocampus compared to non-stressed females (p ≤ .01; interaction effect: F(1, 18) = 4.4, p ≤ .05; Fig. 6). As expected, there was a significant main effect of region with all animals having significantly more Ki67-ir cells in the GCL than in the hilus (F(1, 18) = 186.8, p ≤ .0001). There were no other significant differences between groups and no significant correlations between oestradiol levels at the time of perfusion or behavioral measures on the FST and number of Ki67-ir cells in the GCL or hilus (.5 ≤ p ≤ .9). There were also no significant differences between groups in serum levels of estradiol at sacrifice (.2 ≤ p ≤ .5; Preg Control, 7.9 ± 1.5 pg/ml; Preg Stress, 4.5 ± .7 pg/ml; Virgin Control, 9.1 ± 2.8 pg/ml; Virgin Stress, 8.7 ± 2.0 pg/ml).

4000

Mean (±SEM) number of Ki67-ir cells in the GCL

577

* *

Control Stress

3000

An increasing number of women experience stress and suffer from stress-related disorders during pregnancy, yet our knowledge of how stress impacts the brain and behavior of the mother during pregnancy is very limited. Therefore, the aim of the present study was to determine how repeated stress exposure during gestation, using a paradigm commonly employed to investigate effects of prenatal stress on offspring outcomes (Weinstock, 2008; Darnaudery and Maccari, 2008), may alter affective-like behaviors and hippocampal neurogenesis in pregnant and virgin females. Main findings of the present study show that pregnant females, regardless of stress exposure, have decreased depressive-like behavior, lower corticosterone levels, and increased anxiety-like behavior during late pregnancy, compared to virgin females. Furthermore, stress, regardless of reproductive state (pregnant versus virgin), significantly increased cell proliferation in the hippocampus of the adult female.

Effects of stress on immobility in pregnant and virgin female rats In the present study late pregnant females had reduced immobility in the FST compared to control virgin females, and this difference was greater in stressed pregnant females. These findings expand previous research showing that pregnant females have decreased immobility compared to post-partum females (Frye and Walf, 2004), and further document the role of stress during gestation on immobility. Interestingly, similar repeated stress paradigms during gestation, or administration of elevated levels of corticosterone pre- and postpartum, increase depressive-like behavior, as measured by the FST, during the postpartum period and at weaning (Brummelte et al., 2006; O'Mahony et al., 2006; Smith et al., 2004). It is not clear why differences exist in the effects of stress on depressive-like behavior pre- and post-partum but there are a number of physiological changes during this time that may play a role in these differences. For example, it may be that during pregnancy a female rat is less affected by stress, as this is a time of hyporesponsiveness to stress (Slattery and Neumann, 2008), and the need to ensure the birth and survival of young outweighs the response to stress. It is also possible that with a greater interval between stress exposure and testing the effect may become stronger. This ‘incubation’ effect has particularly been observed with drug exposure (Kelamangalath and Wagner, 2009), but may be relevant for other exposures as well.

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Table 4 Mean (± SEM) total number of BrdU-ir cells in the GCL and hilus. There were no significant effects of reproductive state or stress on the total number of BrdU-ir cells in the GCL or hilus, 21 days after BrdU injection (p ≥ .48). n = 5/group.

1000

0

virgin

pregnant

Fig. 6. Mean (± SEM) total number of Ki67-ir cells in the GCL of the hippocampus. Stressed females, regardless of reproductive state, had significantly more Ki67-ir cells in the GCL of the hippocampus compared to non-stressed females (p ≤ .008). * denotes stressed females significantly different from control females. n = 5–6/group.

Virgin control Virgin stress Pregnant control Pregnant stress

GCL

Hilus

804.0 ± 165.7 788.0 ± 104.4 800.0 ± 164.0 826.0 ± 92.7

250.0 ± 70.7 234.0 ± 57.7 268.0 ± 79.3 382.0 ± 72.5

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We also found that repeated restraint of virgin females decreased immobility in the FST compared to non-stressed virgin females. Although, effects of this sub-chronic restraint stress on depressivelike behavior in the virgin female have not been reported, others have found that after chronic mild stress, females show decreased immobility on the FST (Dalla et al., 2005). These changes in depressive-like behavior may be indicative of better coping strategies in stressed females or poorer memory for previous FST exposure. In fact, memory may play a role in both pregnant and virgin female's performance on the FST. However, many studies demonstrate that chronic and sub-chronic stress enhance memory in adult virgin female rats (Bowman et al., 2009), but to date little work has looked at the effect of stress during gestation on memory in the pregnant female. It is also possible that different stress paradigms, different periods of testing, and other tests of depressive-like behavior, such of the sucrose preference test, may provide more insight in to the effects of stress on the virgin and pregnant female. Fetal sex affects escape behaviors in the FST In the present study we found the number of male fetuses, but not litter size or weight, was associated with escape attempts by pregnant females. This suggests that physiological differences, via differences in testosterone levels, may mediate behaviors of pregnant females in the FST. Circulating testosterone levels in pregnant rodents have been positively associated with number of male fetuses in utero and these changes in testosterone are associated with changes in behavior of the dam (Clark et al., 1993) and neuroplasticity (Pawluski and Galea, 2006). In turn, testosterone plays a role in performance on the FST in virgin male rats (Buddenberg et al., 2009), and aged virgin male and female mice (Frye and Walf, 2009). However, the role of testosterone in maternal depression has not been assessed. Anxiety-like behavior is increased in pregnancy With regard to anxiety-like behavior, we found that late pregnant females had increased anxiety-like behavior compared to virgin females and exposure to restraint stress had little effect on anxietylike behavior in pregnant or virgin females. These findings expand previous work in pregnant females (Neumann et al., 1998; Baker et al., 2008) and replicate findings in virgin females (Bowman et al., 2009). In addition, others have found that gestational stress, via repeated restraint, as in the present study, has marginal effects on anxiety-like behavior during gestation (Baker et al., 2008) but increases anxietylike behavior after weaning in the dam (Darnaudery et al., 2004). As mentioned above, there appears to be few immediate effects of gestational stress on affective-like behaviors in the pregnant female, but marked long term effects. It may be that the physiological changes of parturition and lactation contribute to the long-term effects of gestational stress on the mother. There may also be long-term effects of stress in virgin females, however, to our knowledge this has yet to be investigated. Corticosterone levels and repeated restraint stress In line with previous work (Pawluski et al., 2009b), we found lower serum corticosterone levels in late pregnant females compared to virgin females. We did not find a marked effect of repeated stress on circulating corticosterone levels after termination of the restraint in pregnant or virgin females. Previous work has shown repeated stress throughout gestation can increase basal corticosterone levels during early pregnancy (Takahashi et al., 1998) and during the postpartum period (Smith et al., 2004). In addition, chronic stress, 3 times longer than the duration of stress in the present study, increases corticosterone levels in virgin female rats (Dalla et al., 2005); however shorter durations of stress have little effect on corticosterone levels in virgin

female rats (Anderson et al., 1996). Possible discrepancies between our findings and those of others may be due to the duration and type of the stress exposure, and when, in relation to the stressor the corticosterone levels were assessed. For example, we employed a repeated restraint stress for 1 week, based on the work of Ward and Weisz (1984) and it is possible that a chronic stress of 3 weeks or more would elicit different results. However, it should be noted that this stress paradigm does increase corticosterone during restraint in pregnant females (Ward and Weisz, 1984), but previous work has not investigated how this stress paradigm may affect basal corticosterone levels in pregnant females. It is also possible that the stress exposure in the present study altered the amount of free basal corticosterone levels, but not total serum levels of corticosterone, by decreasing serum levels of corticosteroid binding globulin (CBG). Previous work has shown that stress during gestation can decrease CBG levels, and thus, increase free corticosterone levels (Takahashi et al., 1998). Alterations in CBG levels have also been implicated in stress responsiveness (Richard et al., 2010). Further work is needed to determine the role of CBG in response to stress during pregnancy. Weight change and repeated restraint stress In the present study, we found that stressed pregnant females, on average, weighed 20 grams less than non-stressed counterparts, however this difference did not reach significance. Previous work has shown that the same stress paradigm, can result in a significant decrease the weight gain (van den Hove et al., 2010, 2005; Darnaudery et al., 2004), but this does not always occur (van den Hove et al., 2008). One major difference between the pregnant females of the present study and those of previous work is that the initial weight of the females in the present study was heavier. For example, work that has consistently shown an effect of stress during gestation on weight gain has used females that weighed less at the beginning of the experiment (250–260 g) compared to the females used in the present study (275–325 g). Therefore, the use of heavier, and likely older, females may alter the effect of restraint stress on weight gain during pregnancy. Stress increases hippocampal cell proliferation in virgin and late pregnant females Very little research has investigated how stress affects hippocampal neurogenesis in the adult female (for review see (Pawluski et al., 2009a)). Our findings demonstrate that repeated stress enhances hippocampal cell proliferation in the adult virgin and pregnant female, even when controlling for differences in estradiol levels. Previous work has shown that repeated stress increases new cell survival in female rats when a marker of cell synthesis (BrdU) is administered during the early phase of the repeated stress (days 3–7 of the stress) (Westenbroek et al., 2004) but this is the first work to demonstrated that stress increases hippocampal cell proliferation in virgin and pregnant rats. We also found that stress effects on hippocampal cell proliferation and new cell survival did not differ between pregnant and virgin females. This is perhaps surprising as during the postpartum period there are marked differences in hippocampal neurogenesis between dams and virgin females (Darnaudery et al., 2007; Leuner et al., 2007; Pawluski and Galea, 2007). In addition, pregnancy is often associated with as a hyporesponsive period to stress and there are very different hormone profiles in pregnant females compared to non-pregnant females (Neumann et al., 1998; Slattery and Neumann, 2008). However, the present data demonstrates that although the behavioral effects of stress differ between pregnant and virgin females, the neurobiological effects of stress, at least with regards to the rate of hippocampal cell proliferation and new cell survival, are the same. It

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may be that the function and survival of the proliferating hippocampal cells is quite different in pregnant and virgin females. It is also interesting to note that stress increased hippocampal cell proliferation and decreased depressive-like behavior in virgin females, and to a lesser extent in pregnant females. This relationship between increased hippocampal cell proliferation and decreased depressive-like behavior is congruent with previous literature which demonstrates that increased hippocampal neurogenesis often leads to alleviating depressive-like behavior (DeCarolis and Eisch, 2010). However, more research is needed to determine the functional role of hippocampal neurogenesis in depression. Conclusions Findings of the present study show that repeated restraint stress during pregnancy, which has many effects on offspring brain and behavior (Weinstock, 2008), has little effect on antepartum affectivelike behaviors in the female rat, but increases hippocampal cell proliferation. This data contributes to a much needed area of research investigating the neurobehavioral effects of stress during gestation on the pregnant female. Increasing our knowledge of the impact of stress and stress-related disorders on the mother during the perinatal period will promote the health of the mother and child. Acknowledgments We gratefully acknowledge the technical help from Marisela Martinez-Claros, Hellen Steinbusch, and Denise Hermes. JLP was funded by a Natural Sciences and Engineering Research Council of Canada PDF and is presently a Charge de recherché Fonds de la Recherche Scientifique (FNRS-FRS) fellow. References Anderson, S.M., et al., 1996. Effects of chronic stress on food acquisition, plasma hormones, and the estrous cycle of female rats. Physiol. Behav. 60, 325–329. Baker, S., Chebli, M., Rees, S., Lemarec, N., Godbout, R., Bielajew, C., 2008. Effects of gestational stress: 1. Evaluation of maternal and juvenile offspring behavior. Brain Res. 1213, 98–110. Bennett, H.A., Einarson, A., Taddio, A., Koren, G., Einarson, T.R., 2004a. Depression during pregnancy: overview of clinical factors. Clin. Drug Investig. 24, 157–179. Bennett, H.A., Einarson, A., Taddio, A., Koren, G., Einarson, T.R., 2004b. Prevalence of depression during pregnancy: systematic review. Obstet. Gynecol. 103, 698–709. Boccia, M.L., Razzoli, M., Vadlamudi, S.P., Trumbull, W., Caleffie, C., Pedersen, C.A., 2007. Repeated long separations from pups produce depression-like behavior in rat mothers. Psychoneuroendocrinology 32, 65–71. Bowman, R.E., Micik, R., Gautreaux, C., Fernandez, L., Luine, V.N., 2009. Sex-dependent changes in anxiety, memory, and monoamines following one week of stress. Physiol. Behav. 97, 21–29. Brummelte, S., Galea, L.A., 2010. Chronic corticosterone during pregnancy and postpartum affects maternal care, cell proliferation and depressive-like behavior in the dam. Horm. Behav. 58 (5), 769–779. Brummelte, S., Pawluski, J.L., Galea, L.A., 2006. High post-partum levels of corticosterone given to dams influence postnatal hippocampal cell proliferation and behavior of offspring: a model of post-partum stress and possible depression. Horm. Behav. 50, 370–382. Buddenberg, T.E., Komorowski, M., Ruocco, L.A., Silva, M.A., Topic, B., 2009. Attenuating effects of testosterone on depressive-like behavior in the forced swim test in healthy male rats. Brain Res. Bull. 79, 182–186. Clark, M.M., Crews, D., Galef Jr., B.G., 1993. Androgen mediated effects of male fetuses on the behavior of dams late in pregnancy. Dev. Psychobiol. 26, 25–35. Dalla, C., Antoniou, K., Drossopoulou, G., Xagoraris, M., Kokras, N., Sfikakis, A., Papadopoulou-Daifoti, Z., 2005. Chronic mild stress impact: are females more vulnerable? Neuroscience 135, 703–714. Darnaudery, M., Maccari, S., 2008. Epigenetic programming of the stress response in male and female rats by prenatal restraint stress. Brain Res. Rev. 57, 571–585. Darnaudery, M., Dutriez, I., Viltart, O., Morley-Fletcher, S., Maccari, S., 2004. Stress during gestation induces lasting effects on emotional reactivity of the dam rat. Behav. Brain Res. 153, 211–216. Darnaudery, M., Perez-Martin, M., Del Favero, F., Gomez-Roldan, C., Garcia-Segura, L.M., Maccari, S., 2007. Early motherhood in rats is associated with a modification of hippocampal function. Psychoneuroendocrinology 32, 803–812. DeCarolis, N.A., Eisch, A.J., 2010. Hippocampal neurogenesis as a target for the treatment of mental illness: a critical evaluation. Neuropharmacology 58, 884–893.

579

Eisch, A.J., Cameron, H.A., Encinas, J.M., Meltzer, L.A., Ming, G.L., Overstreet-Wadiche, L. S., 2008. Adult neurogenesis, mental health, and mental illness: hope or hype? J. Neurosci. 28, 11785–11791. Epp, J.R., Barker, J.M., Galea, L.A., 2009. Running wild: neurogenesis in the hippocampus across the lifespan in wild and laboratory-bred Norway rats. Hippocampus 19, 1040–1049. Falconer, E.M., Galea, L.A., 2003. Sex differences in cell proliferation, cell death and defensive behavior following acute predator odor stress in adult rats. Brain Res. 975, 22–36. Frye, C.A., Walf, A.A., 2004. Hippocampal 3alpha,5alpha-THP may alter depressive behavior of pregnant and lactating rats. Pharmacol. Biochem. Behav. 78, 531–540. Frye, C.A., Walf, A.A., 2009. Depression-like behavior of aged male and female mice is ameliorated with administration of testosterone or its metabolites. Physiol. Behav. 97, 266–269. Frye, C.A., Petralia, S.M., Rhodes, M.E., 2000. Estrous cycle and sex differences in performance on anxiety tasks coincide with increases in hippocampal progesterone and 3alpha,5alpha-THP. Pharmacol. Biochem. Behav. 67, 587–596. Furuta, M., Bridges, R.S., 2005. Gestation-induced cell proliferation in the rat brain. Brain Res. Dev. Brain Res. 156, 61–66. Galea, L., Wide, J., Barr, A., 2001. Estradiol alleviates depressive-like symptoms in a novel animal model of post-partum depression. Behav. Brain Res. 1–9. Gavin, N.I., Gaynes, B.N., Lohr, K.N., Meltzer-Brody, S., Gartlehner, G., Swinson, T., 2005. Perinatal depression: a systematic review of prevalence and incidence. Obstet. Gynecol. 106, 1071–1083. Glover, V., O'Connor, T.G., O'Donnell, K., 2010. Prenatal stress and the programming of the HPA axis. Neurosci. Biobehav. Rev. 35, 17–22. Gundersen, H.J., Bendtsen, T.F., Korbo, L., Marcussen, N., Moller, A., Nielsen, K., Nyengaard, J.R., Pakkenberg, B., Sorensen, F.B., Vesterby, A., et al., 1988. Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. APMIS 96, 379–394. Kelamangalath, L., Wagner, J.J., 2009. Effects of abstinence or extinction on cocaine seeking as a function of withdrawal duration. Behav. Pharmacol. 20, 195–203. Kinsley, C.H., Lambert, K.G., 2006. The maternal brain. Sci. Am. 294, 72–79. Leuner, B., Mirescu, C., Noiman, L., Gould, E., 2007. Maternal experience inhibits the production of immature neurons in the hippocampus during the postpartum period through elevations in adrenal steroids. Hippocampus 17, 434–442. Leuner, B., Glasper, E.R., Gould, E., 2010. Parenting and plasticity. Trends Neurosci. 33, 465–473. Lucki, I., 1997. The forced swimming test as a model for core and component behavioral effects of antidepressant drugs. Behav. Pharmacol. 8, 523–532. Maccari, S., Morley-Fletcher, S., 2007. Effects of prenatal restraint stress on the hypothalamus-pituitary-adrenal axis and related behavioural and neurobiological alterations. Psychoneuroendocrinology 32 (Suppl 1), S10–S15. Neumann, I.D., Johnstone, H.A., Hatzinger, M., Liebsch, G., Shipston, M., Russell, J.A., Landgraf, R., Douglas, A.J., 1998. Attenuated neuroendocrine responses to emotional and physical stressors in pregnant rats involve adenohypophysial changes. J. Physiol. 508 (Pt 1), 289–300. Oberlander, T.F., Warburton, W., Misri, S., Aghajanian, J., Hertzman, C., 2006. Neonatal outcomes after prenatal exposure to selective serotonin reuptake inhibitor antidepressants and maternal depression using population-based linked health data. Arch. Gen. Psychiatry 63, 898–906. O'Mahony, S.M., Myint, A.M., van den Hove, D., Desbonnet, L., Steinbusch, H., Leonard, B.E., 2006. Gestational stress leads to depressive-like behavioural and immunological changes in the rat. Neuroimmunomodulation 13, 82–88. Ormerod, B.K., Galea, L.A., 2001. Reproductive status influences cell proliferation and cell survival in the dentate gyrus of adult female meadow voles: a possible regulatory role for estradiol. Neuroscience 102, 369–379. Pawluski, J.L., Galea, L.A., 2006. Hippocampal morphology is differentially affected by reproductive experience in the mother. J. Neurobiol. 66, 71–81. Pawluski, J.L., Galea, L.A., 2007. Reproductive experience alters hippocampal neurogenesis during the postpartum period in the dam. Neuroscience 149, 53–67. Pawluski, J.L., Galea, L.A., 2008. The role of reproductive experience on hippocampal function and plasticity. In: Bridges, R. (Ed.), Neurobiology of the Parental Brain. Academic Press, Boston, pp. 493–508. Pawluski, J.L., Brummelte, S., Barha, C.K., Crozier, T.M., Galea, L.A., 2009a. Effects of steroid hormones on neurogenesis in the hippocampus of the adult female rodent during the estrous cycle, pregnancy, lactation and aging. Front. Neuroendocrinol. 30, 343–357. Pawluski, J.L., Charlier, T.D., Lieblich, S.E., Hammond, G.L., Galea, L.A., 2009b. Reproductive experience alters corticosterone and CBG levels in the rat dam. Physiol. Behav. 96, 108–114. Pawluski, J.L., Lieblich, S.E., Galea, L.A., 2009c. Offspring-exposure reduces depressivelike behaviour in the parturient female rat. Behav. Brain Res. 197, 55–61. Pawluski, J.L., Barakauskas, V.E., Galea, L.A., 2010. Pregnancy decreases oestrogen receptor alpha expression and pyknosis, but not cell proliferation or survival, in the hippocampus. J. Neuroendocrinol. 22, 248–257. Pellow, S., 1985. Can drug effects on anxiety and convulsions be separated? Neurosci. Biobehav. Rev. 9, 55–73. Revest, J.M., Dupret, D., Koehl, M., Funk-Reiter, C., Grosjean, N., Piazza, P.V., Abrous, D.N., 2009. Adult hippocampal neurogenesis is involved in anxiety-related behaviors. Mol. Psychiatry 14, 959–967. Richard, E.M., et al., 2010. Plasma transcortin influences endocrine and behavioral stress responses in mice. Endocrinology 151, 649–659. Shepherd, J.K., Grewal, S.S., Fletcher, A., Bill, D.J., Dourish, C.T., 1994. Behavioural and pharmacological characterisation of the elevated “zero-maze” as an animal model of anxiety. Psychopharmacology (Berl.) 116, 56–64.

580

J.L. Pawluski et al. / Hormones and Behavior 59 (2011) 572–580

Shingo, T., Gregg, C., Enwere, E., Fujikawa, H., Hassam, R., Geary, C., Cross, J.C., Weiss, S., 2003. Pregnancy-stimulated neurogenesis in the adult female forebrain mediated by prolactin. Science 299, 117–120. Shors, T.J., Mathew, J., Sisti, H.M., Edgecomb, C., Beckoff, S., Dalla, C., 2007. Neurogenesis and helplessness are mediated by controllability in males but not in females. Biol. Psychiatry 62, 487–495. Slattery, D.A., Neumann, I.D., 2008. No stress please! Mechanisms of stress hyporesponsiveness of the maternal brain. J. Physiol. 586, 377–385. Smith, J.W., Seckl, J.R., Evans, A.T., Costall, B., Smythe, J.W., 2004. Gestational stress induces post-partum depression-like behaviour and alters maternal care in rats. Psychoneuroendocrinology 29, 227–244. Strom, J.O., Theodorsson, A., Theodorsson, E., 2008. Substantial discrepancies in 17betaoestradiol concentrations obtained with three different commercial direct radioimmunoassay kits in rat sera. Scand. J. Clin. Lab. Invest. 68, 806–813. 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. Tanapat, P., Hastings, N.B., Reeves, A.J., Gould, E., 1999. Estrogen stimulates a transient increase in the number of new neurons in the dentate gyrus of the adult female rat. J. Neurosci. 19, 5792–5801.

Vahl, T.P., Ulrich-Lai, Y.M., Ostrander, M.M., Dolgas, C.M., Elfers, E.E., Seeley, R.J., D'Alessio, D.A., Herman, J.P., 2005. Comparative analysis of ACTH and corticosterone sampling methods in rats. Am. J. Physiol. Endocrinol. Metab. 289, E823–E828. van den Hove, D.L., Blanco, C.E., Aendekerk, B., Desbonnet, L., Bruschettini, M., Steinbusch, H.P., Prickaerts, J., Steinbusch, H.W., 2005. Prenatal restraint stress and long-term affective consequences. Dev. Neurosci. 27, 313–320. van den Hove, D.L., et al., 2008. Prenatal maternal paroxetine treatment and neonatal mortality in the rat: a preliminary study. Neonatology 93, 52–55. van den Hove, D.L., et al., 2010. Maternal stress-induced reduction in birth weight as a marker for adult affective state. Front. Biosci. (Elite Ed.) 2, 43–46. Walf, A.A., Frye, C.A., 2007. Estradiol decreases anxiety behavior and enhances inhibitory avoidance and gestational stress produces opposite effects. Stress 10, 251–260. Ward, I.L., Weisz, J., 1984. Differential effects of maternal stress on circulating levels of corticosterone, progesterone, and testosterone in male and female rat fetuses and their mothers. Endocrinology 114, 1635–1644. Weinstock, M., 2008. The long-term behavioural consequences of prenatal stress. Neurosci. Biobehav. Rev. 32, 1073–1086. Westenbroek, C., Den Boer, J.A., Veenhuis, M., Ter Horst, G.J., 2004. Chronic stress and social housing differentially affect neurogenesis in male and female rats. Brain Res. Bull. 64, 303–308.