The effects of prenatal stress on learning in adult offspring is dependent on the timing of the stressor

Behavioural Brain Research 197 (2009) 144–149

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The effects of prenatal stress on learning in adult offspring is dependent on the timing of the stressor Amita Kapoor a , Alice Kostaki a , Christopher Janus d , Stephen G. Matthews a,b,c,∗ a

Department of Physiology, Faculty of Medicine, University of Toronto, Medical Sciences Building, 1 King’s College Circle, Toronto, Ontario, M5S 1A8 Canada Department of Obstetrics, Faculty of Medicine, University of Toronto, Medical Sciences Building, 1 King’s College Circle, Toronto, Ontario, M5S 1A8 Canada c Department of Gynaecology and Medicine, Faculty of Medicine, University of Toronto, Medical Sciences Building, 1 King’s College Circle, Toronto, Ontario, M5S 1A8 Canada d Mayo Clinic Jacksonville, Department of Neuroscience, 4500 San Pablo Road, Birdsall Building R215, Jacksonville, FL 32224, USA b

a r t i c l e

i n f o

Article history: Received 10 March 2008 Received in revised form 12 August 2008 Accepted 16 August 2008 Available online 22 August 2008 Keywords: Prenatal stress Hypothalamic-pituitary-adrenal axis Guinea pigs Programming Morris water maze

a b s t r a c t Impaired fetal development has been linked with deficits in behavioural and emotional development during postnatal life. In order to investigate the mechanisms underlying this relationship, we studied the effect of acute stress at two different critical phases of pregnancy on cognitive function in adult guinea pig offspring. Pregnant guinea pigs were exposed to a psychological stressor (2 h/day) on gestational days (gd) 50, 51, and 52 (PS50) or 60, 61, and 62 (PS60). Male offspring were grown to adulthood and tested in the Morris water maze (MWM) to assess spatial learning and memory. Latency, path length, swim speed and the strategy used to find the platform in each session of the MWM were measured. A reverse learning trial was performed where the platform was moved to a different area of the pool and the ability of the guinea pigs to learn a new platform position was assessed. There was no effect of stress at gd50 on latency to find the platform during any of the sessions in the MWM. PS60 male offspring exhibited enhanced development of a spatial strategy during sessions 3 and 4 of the MWM, but this was not associated with decreased latency. In the reversal task PS50 male offspring demonstrated use of non-spatial strategies to find the platform during the reversal task. This would suggest decreased retention of spatial memory in these animals. In contrast, control and PS60 male offspring demonstrated no bias to a particular strategy type. In conclusion, there are subtle effects of prenatal stress on spatial learning. PS60 offspring appear to exhibit enhanced spatial learning, while PS50 male offspring exhibit impaired spatial learning. These findings are consistent with those in humans, which indicate a strong effect of maternal anxiety during pregnancy on cognition in children, and that the timing of the maternal stress is critical to determining outcome. This model will allow us to determine the mechanisms that underlie the association between prenatal stress and altered learning strategy and ability. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Prenatal stress is associated with a number of adult diseases, including cardiovascular and affective disorders [1,2]. The mechanism underlying this relationship is thought to be exposure of the fetus to components of the stress response: glucocorticoids, the end product of hypothalamic-pituitary-adrenal (HPA) axis activation or catecholamines, the product of sympathetic nervous system activation [3,4]. Indeed, a number of studies have demonstrated that prenatal stress results in alterations in HPA axis function and stressrelated behaviour in animal models, including rats, guinea pigs and

∗ Corresponding author at: Department of Physiology, Faculty of Medicine, University of Toronto, Medical Sciences Building, 1 King’s College Circle, Toronto, Ontario, M5S 1A8 Canada. Tel.: +1 416 978 1974; fax: +1 416 978 4940. E-mail address: [email protected] (S.G. Matthews). 0166-4328/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2008.08.018

non-human primates [5–7]. More recently, human studies have demonstrated deficits in cognitive and behavioural development in children whose mothers were exposed to excess glucocorticoids during pregnancy [8,9]. Importantly, it is also emerging from these studies that the timing of the stress is critical as there are differential outcomes depending on the trimester of exposure [10,11]. One specific aspect of cognition is hippocampal-dependent spatial learning and memory [12,13]. Studies in non-human primates and rodents have demonstrated alterations in structure and function of the hippocampus as a consequence of prenatal stress [14–17]. The Morris water maze (MWM) is a validated tool used to measure hippocampal-dependent spatial memory and non-spatial discrimination learning in rodent models including guinea pigs [18,19]. Results from studies examining prenatal stress and spatial learning and memory using the MWM in rats and mice offspring have been variable, and have suggested significant interaction

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between the stress associated with swimming and cognitive ability. Recent studies using the MWM have developed approaches for analyzing swim patterns in order to classify the strategies that are being used to find the platform [20,21]. In this way, it can be determined whether spatial learning is occurring or whether other types of search strategies are being utilized. In rats and mice, the phase of rapid brain growth (a period of established vulnerability) [22] occurs postnatally, unlike the human, the non-human primate and the guinea pig in which this crucial period of brain development is initiated prenatally [22]. Fetal brain growth in the guinea pig is well characterized; the period of rapid brain growth occurs around gestational day (gd) 50, whereas gd60 represents a period of rapid myelination [23]. We have previously shown that maternal plasma cortisol levels in pregnant guinea pigs rose by 20% and 40% in response to strobe light exposure on gd54 and gd60, respectively [6]. We have also shown that prenatal stress during the period of rapid brain growth (gd50) results in male offspring that exhibit increased basal HPA axis activity, increased anxiety behaviour and decreased plasma testosterone levels. In contrast, maternal stress late in gestation (gd60) results in male offspring that exhibit an increased plasma cortisol response to a stressor and decreased body weight from the time of weaning to adulthood [6]. Given that studies in humans are demonstrating alterations in cognitive function in children exposed to stress in utero and that there are differential effects depending on the timing of the prenatal stress, our aim was to determine the effect of prenatal stress on performance in the MWM and to assess the use of search strategies used in the MWM in a species that gives birth to neuroanatomically mature young. We hypothesized that prenatal stress would affect spatial learning and memory in male guinea pigs in the MWM and furthermore these changes will be dependent on the specific timing of the stressor.

2.2.2. Acquisition phase and reversal task All animals were tested over five consecutive days. On each day, animals received two sessions of testing, at 09:00 h and 13:00 h. Each session consisted of four trials with an inter-trial period of 10 min. For each trial, the animal was released from 1 of the 4 cardinal compass points (N, S, E, W). The animal was allowed a maximum of 60 s to locate and mount the escape platform with a post-trial timeout of 15 s on the platform. Animals that failed to locate the platform within the 60 s were placed on the platform. Acquisition was tested during sessions 1–6 and the platform was submerged in quadrant 1. On session 7 the platform was moved to quadrant 3 for the reversal task. The platform remained in quadrant 3 for sessions 8–10 for another round of acquisition testing. Trial 1, session 7 was considered as a probe trial. In probe trials, the time spent swimming in the quadrant where the platform had been previously, was recorded. This is considered to be the most specific test for spatial memory [25]. The swim speed and path of the guinea pig during each trial was recorded by a video camera suspended above the centre of the pool and connected to a video tracking system (HVS Image Advanced Tracker VP200, HVS Image, Buckingham, UK).

2. Methods

All data were expressed as mean ± standard error of the mean (S.E.M.). All statistical comparisons were analyzed using Graphpad Prism (Graphpad Software Inc., San Diego, CA, USA). MWM performance data was analyzed by two-way analysis of variance (ANOVA) with repeated measures and one-way ANOVA followed by Newman–Keuls method of post hoc comparison. The difference in latency between sessions 6 and 7 was analyzed by Wilcoxon matched paired-test. Strategy data was analyzed by Chi square tests. Significance was set at p < 0.05.

2.1. Animals Female guinea pigs (400–500 g) (Hartley strain, Charles River Canada, St. Constant, PQ, Canada) were mated in our animal facility as previously described [24]. This method produces accurately time-dated pregnant guinea pigs. Food (Guinea Pig Chow 5025, Ralston Purina International, Leis Pet Distributing Inc., Wellesley, ON, Canada) and water were available ad libitum. The animals were kept in a 12:12 h light–dark cycle, with lights off at 19:00 h. Room temperature was 23 ◦ C. All the studies were performed according to protocols approved by the Animal Care Committee at the University of Toronto, in accordance with the Canadian Council for Animal Care. Pregnant guinea pigs were exposed to a high frequency strobe light for 2 h, from 09:00 h to 11:00 h, on gd50, 51, and 52 (PS50) or gd60, 61, and 62 (PS60). A control group of pregnant guinea pigs was left undisturbed throughout gestation except for routine maintenance. All animals were allowed to deliver normally. Animals were weaned on postnatal day (pnd) 25, weighed and placed into individual clear polycarbonate cages. There was no significant effect of prenatal stress on body weight at pnd25 [6]. Animals were within visual, auditory and olfactory contact with at least two other animals at all times. Male offspring (control; n = 11, PS50; n = 9, PS60; n = 9) remained undisturbed except for biweekly cage maintenance and MWM testing around pnd70 (range pnd 70–76). 2.2. Morris water maze testing 2.2.1. Testing room and apparatus The water maze apparatus consisted of a circular pool (1.54 m diameter) made of white plastic. The pool was filled with water (23 ◦ C) that was made opaque by the addition of non-toxic white tempura paint. During MWM training, an escape platform (20 cm diameter) made of clear plastic with a grooved surface was submerged 5 cm under the water level. All tests were carried out in the same experimental room and the MWM testing area was isolated from the operator and computer by a white curtain. Dark boards of different shapes provided landmarks in the testing room. Three days prior to the commencement of the acquisition phase, guinea pigs underwent training to acclimatize them to the testing room and pool. The platform was placed in the centre of the pool and guinea pigs were placed on the platform three times, for 15 s.

2.2.3. Analysis of strategy The swim path for each trial during MWM testing was automatically plotted in the HVS image system. A single investigator blinded to prenatal treatment status assigned a predominant search strategy to the first trial of each session using a categorization scheme based on those previously developed [20,21]. Briefly, the strategy that best described the majority of the swim path was assigned. In a reanalysis of the categorization of search strategy by the same investigator 2 weeks later, the intra-observer agreement on strategy classification was 98%. For each session strategies were classified into three broad categories: spatial, systematic but nonspatial and repetitive looping path strategies. Spatial strategies included ‘spatial direct’ (swimming directly to the platform), ‘spatial indirect’ (swimming to the platform with at most one loop), and ‘focal: correct target quadrant’ (swimming directly to and searching intently in the quadrant containing the platform). Systematic but non-spatial strategies included ‘scanning’ (searching the interior portion of the tank without spatial bias), ‘random’ (searching the entire tank without bias towards any portion), and ‘focal: incorrect target quadrant’ (searching intently a quadrant of the tank that does not contain the platform). Strategies involving repetitive looping paths included ‘chaining’ (circular swimming at an approximately fixed distance greater than 15 cm from the wall), ‘peripheral looping’ (persistent swimming around the outer 15 cm of the pool), and ‘circling’ (swimming in tight circles). 2.3. Statistical analysis

3. Results 3.1. Acquisition and reverse learning Analysis of latency by two-way repeated measures ANOVA revealed a significant effect of session, indicating the animals were learning the platform position (p < 0.0001; Fig. 1A), however, there was no effect of prenatal stress nor was there a significant interaction between time and latency to find the platform. Wilcoxon matched pair-test for comparison of the latencies between sessions 6 and 7 revealed that both control (p < 0.01) and PS50 (p < 0.02) demonstrated a significant increase in latency from session 6 to 7 as the platform position was changed from quadrant 1 to 3. However, this was not observed in PS50 male offspring. Two-way repeated measures ANOVA of the path length to find the platform revealed a significant effect of session, as the animals exhibited a shorter path length over time (p < 0.0001; Fig. 1B). There was no significant interaction between session and prenatal stress, nor was there a significant effect of prenatal stress alone on path length. Wilcoxon matched pair-test for comparison of the path length between sessions 6 and 7 revealed that both control (p < 0.01) and PS50 (p < 0.01) demonstrated a significant increase in latency from session 6 to 7 as the platform position was changed from quadrant 1 to 3, however, this was not observed in PS50 male

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Fig. 1. (A) Latency (s; mean ± S.E.M.) and (B) path length (m; mean ± S.E.M.) to find the platform in sessions 1–10 of the Morris water maze in male offspring born to mothers left undisturbed throughout pregnancy (control, squares, n = 11) or exposed to prenatal stress on gestational day 50, 51, and 52 (PS50, triangles, n = 9) or gestational day 60, 61, and 62 (PS60, circles, n = 9). Arrow indicates move in platform position from quadrant 1 to 3 (session 7).

offspring. There was no difference in swim speed between groups or over the course of MWM testing (Table 1). 3.2. Session 7 probe trial analysis Two-way ANOVA of the percentage of time spent in each quadrant in the probe trial in session 7 revealed a significant interaction between prenatal stress and quadrant (p < 0.0001; Fig. 2A). Oneway ANOVA analysis of the percentage of time spent in quadrant 1 demonstrated a significant effect of prenatal stress (p < 0.03). Post hoc revealed this difference was between control and PS50 male offspring (p < 0.05). One-way ANOVA of the percentage of time spent in quadrant 2 revealed a significant effect of prenatal stress (p < 0.05), but further post hoc analysis did not reveal any differences in the prenatal stress groups. For quadrant 3, one-way ANOVA demonstrated a significant effect of prenatal stress (p < 0.01) and post hoc analysis revealed the difference was between control and

Fig. 2. (A) Percent time spent (mean ± S.E.M.) and (B) percent distance traveled (mean ± S.E.M.) through quadrants 1–4 in session 7 of the Morris water maze in male offspring born to mothers left undisturbed throughout pregnancy (white bars; n = 11) or exposed to prenatal stress on gestational day 50, 51, and 52 (PS50; black bars; n = 9) or gestational day 60, 61, and 62 (PS60; gray bars; n = 9). Dashed line indicates 25% probability that the animal would enter the quadrant by chance. * p < 0.05 compared to control in the same quadrant.

PS50 male offspring (p < 0.05). There was no effect of prenatal stress on the percentage of time spent in quadrant 4. Two-way ANOVA of the percentage of distance traveled in each quadrant revealed a significant interaction of prenatal stress × quadrant (p < 0.0003). One-way ANOVA revealed a significant effect of prenatal stress on the percentage distance traveled in quadrant 1 (p < 0.05). There was a significant effect of prenatal stress on percent distance traveled in quadrant 2 (p < 0.05), however, further post hoc analysis did not reveal any difference in the prenatal stress groups. For quadrant 3, one-way ANOVA revealed a significant effect of prenatal stress on the percent distance traveled in quadrant 3 (p < 0.01). Post hoc analysis demonstrated that this difference was between control and PS50 offspring (p < 0.05). There was no effect of prenatal stress on the percent distance traveled in quadrant 4.

Table 1 Swim speed (m/s; mean ± S.E.M.) during sessions 1–10 of the Morris water maze (MWM) in male offspring whose mothers were undisturbed throughout pregnancy (control) or exposed to a strobe light (2 h/day) on gd50, 51, and 52 (PS50) or gd60, 61, and 62 (PS60) Prenatal treatment Session

Control (11) PS50 (9) PS60 (9)

1

2

3

4

5

6

7

8

9

10

0.305 ± 0.02 0.278 ± 0.02 0.324 ± 0.01

0.299 ± 0.01 0.311 ± 0.02 0.295 ± 0.02

0.323 ± 0.01 0.311 ± 0.01 0.270 ± 0.02

0.276 ± 0.01 0.279 ± 0.01 0.268 ± 0.02

0.282 ± 0.01 0.290 ± 0.01 0.269 ± 0.01

0.284 ± 0.01 0.289 ± 0.01 0.256 ± 0.01

0.303 ± 0.01 0.289 ± 0.01 0.284 ± 0.01

0.296 ± 0.01 0.303 ± 0.01 0.293 ± 0.01

0.283 ± 0.01 0.287 ± 0.02 0.302 ± 0.01

0.296 ± 0.01 0.291 ± 0.02 0.293 ± 0.02

Numbers in brackets indicate number of animals in each group.

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3.3. Strategy analysis The breakdown of strategies used in all sessions between prenatal treatment groups is presented in Fig. 3. Chi square analysis demonstrated that by session 6, a significant proportion of control male offspring was using a spatial strategy to find the platform (p < 0.05). In session 7 (reverse learning), there was no bias to a particular strategy type. Indeed, 18% of animals attempted to use a spatial strategy, 55% of control animals reverted to a repetitive looping path strategy and 27% were using a systematic, but nonspatial strategy. In sessions 8–10 there was a comparable use of all 3 types of strategies. No PS50 animals (Fig. 3B) used a spatial strategy in session 1. Similar to controls, by session 6, a significant proportion of PS50 male offspring were using a spatial strategy to find the platform (p < 0.05). Chi square analysis also demonstrated in session 7, that the majority of the PS50 offspring used a systematic, but non-spatial strategy, consistent with the results of the percentage time spent and distance traveled through each quadrant in session 7 (p < 0.05; Fig. 2A and B). By session 10, the majority of PS50 males were again using a spatial strategy (p < 0.05). All PS60 males (Fig. 3C) used a repetitive looping path strategy in session 1. In sessions 3 (p < 0.05) and 4 (p < 0.05), Chi square analysis demonstrated that the majority of PS60 males were using a spatial strategy to find the platform. However, the spatial strategy was not retained overnight (i.e. in session 5). By session 6 again, the majority of PS60 male offspring were using a spatial strategy (p < 0.05). For session 8, the majority of PS60 males employed a systematic, but non-spatial strategy to find the platform (p < 0.05). 4. Discussion The present study is the first to assess and compare the effect of prenatal stress during different critical windows of brain development on MWM performance in adult offspring. Specifically, we determined differences in latencies and strategies used to find the hidden platform. Maternal stress in late gestation (gd60, 61, and 62) resulted in adult male offspring that were faster to develop a spatial strategy, however, this was not associated with a decrease in latency. The reversal task, as tested in session 7, is designed to assess animal’s ability to learn a new platform location. Use of the focal search strategy, searching for the platform in the previous platform location, is indicative of development of spatial memory. In session 7, 36% of control male offspring used a focal strategy, but no PS50 male offspring attempted to search for the platform in quadrant 1. In contrast however, the majority of PS60 male offspring employed use of a focal search strategy of the previous platform location quadrant. This strategy analysis is further confirmed by the distance traveled and passes through each quadrant in the first 30 s of session 7 (Fig. 2). As we have previously demonstrated with endocrine data [6], the timing of the stress is crucial when considering outcome. We have shown that male offspring whose mothers were exposed to stress during the period of rapid brain growth (gd50, 51, and 52) did not employ use of a spatial strategy in the reversal task, which resulted in no significant increase in latency when the position of the platform was changed. Relatively few studies have examined the effects of prenatal stress on learning and memory in the MWM. In rats, exposure to daily stress during the final week of gestation resulted in offspring that exhibited increased latency in the reversal task [15,26]. Strategy use in a prenatal stress model has been considered in two studies in rats. However, neither study undertook systematic analysis of strategy such as that defined by Janus [21]. Young adult rats born to mothers exposed to prenatal stress exhibited decreased flexibility in their strategy in solving the reversal task, as demonstrated by behavioural perseverance in persistently searching for

Fig. 3. (A) Search strategy (% animals exhibiting specific strategy within treatment group) in sessions 1–10 of the Morris water maze (MWM). Black bars indicate spatial strategies (focal direct, focal indirect, focal: correct target quadrant). Open bars indicate systematic, but non-spatial strategies (scanning, random, focal: incorrect quadrant). Gray bars indicate repetitive looping path strategies (peripheral looping, chaining, circling). (A) Male offspring born to mothers left undisturbed throughout pregnancy (control, n = 11). (B) Male offspring born to mothers exposed to a strobe light (2 h/day) on gestational day 50, 51, and 52 (PS50, n = 9). (C) Male offspring born to mothers exposed to a strobe light (2 h/day) on gestational day 60, 61, and62 (PS60, diagonal bars, n = 9). * Significant deviation from the random use of any one type of strategy.

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the platform in its previous location [27]. In a more recent study, prenatally stressed male and female rat offspring demonstrated increased latencies to find the platform in the MWM which was associated with the use of a random search strategy as opposed to use of a straight strategy [28]. However, this study failed to classify the strategies according to the sessions and did not perform a reversal task. Therefore, development of the use of a spatial strategy is unclear. If we presume that use of a straight strategy is indicative of use of a spatial strategy, then it is consistent with the results obtained for PS50 male offspring in the current study, where the majority of offspring used a non-spatial strategy in the reversal task. The molecular mechanism underlying learning and memory in the MWM is thought to be long-term potentiation (LTP), particularly in the hippocampus although there is increasing evidence for the involvement of other brain regions including the amygdala and cortical structures [29,30]. The studies that have examined the effects of prenatal stress on LTP have found changes in LTP to be associated with deficits in spatial learning and memory. In rats, prenatal stress during late gestation increased latency in the MWM and this was associated with decreased LTP in hippocampal CA1 region slices [31]. Adult mice offspring whose mothers were exposed to prenatal stress exhibit deficits in the 8-arm radial maze, another test of spatial learning and memory, as well as decreased NMDAdependent LTP in the CA1 region of the hippocampus. However, as demonstrated in the present study, changes in latency may also reflect differences in the use of non-spatial strategies. While the previous studies have been useful in characterizing learning and memory, none have performed detailed analysis of search strategies. In the present study, we have shown that the use of spatial strategies is highly efficient for the acquisition phase of the MWM when compared with the use of repetitive looping paths. However, the use of systematic but non-spatial strategies is also efficient during acquisition and a better strategy for the reversal task. This is crucial when considering the results of other MWM studies, as alterations in search strategies may not necessarily be reflected by latencies. Male rat offspring whose mothers were exposed to a series of mixed stressors during the last week of pregnancy demonstrated an increased latency to find the platform. Consistent with previous studies, these animals also exhibited impaired hippocampal LTP in the CA1 region and this was associated with reduced hippocampal NR2B subunit expression and increased expression of the NMDA scaffolding protein PSD95 [32]. Together, these data indicate that prenatal stress affects the expression and function of NMDA receptors which are critical for LTP, however, whether these molecular changes are reflecting a difference in spatial learning is unknown. Further studies are required to determine if the NMDA receptor signal transduction is altered in our model of prenatal stress. A number of studies have classified the use of strategies in the MWM in normal animals [20,21,33–37], and these have provided some insight into the mechanisms that may be involved in the use of strategies. Adenosine receptor knockout mice exhibit increased peripheral looping, compared to other types of repetitive looping paths, but no difference in the frequency of use of a spatial strategy compared to wild-type mice. Further, administration of antagonists to block cholinergic transmission demonstrated that the central cholinergic systems were important in the use of spatial mapping, but not for other types of strategies such as the systematic but non-spatial strategies and the repetitive looping paths [36]. These studies indicate that the adenosine receptor and cholinergic systems are not likely to be involved in differences in strategy associated with maternal stress. Transgenic mice over-expressing human amyloid protein which is associated with an increased risk of Alzheimer’s disease, exhibited increased use of repetitive looping and systematic but non-spatial compared to spatial strategies [20].

Similarly, another transgenic Alzheimer’s mouse model, expressing amyloid beta plaques, displayed no difference in latency in the MWM, but analysis of search strategy revealed that transgenic mice were using systematic but non-spatial strategies [21]. In apolipoprotein E knockout mice, another model of Alzheimer’s disease, stress prior to MWM testing increased the use of a spatial strategy compared to wild-type mice, indicating an interaction between HPA axis activity and the use of a strategy [34]. There is currently no data on a potential relationship between Alzheimer’s disease and prenatal stress as the prospective human cohorts are school-age. However, this is an outcome that should be considered in the human studies. There is a sparse literature on the brain areas contributing to the differences in search strategies. It has been demonstrated that hippocampal lesions in rats lead to impairment in all measures of the MWM, again highlighting the importance of this brain region in learning and memory [38]. However, perirhinal cortex and amygdala lesioned rats displayed decreased perseverance in the probe trial with no difference in the acquisition phase, suggesting that these regions may be involved in strategy selection [38]. We have not determined the effect of prenatal stress on hippocampal structure, but other studies have demonstrated differences in hippocampal neurogenesis as a result of prenatal stress in primates [14,39]. Indeed, there is evidence for both components of the stress response, the HPA axis and the sympathetic nervous system, affecting hippocampal neurogenesis [40,41]. Glucocorticoids and testosterone have been shown to affect MWM performance [42,43]. Glucocorticoids affect the hippocampus in a U-shaped manner with acute high levels facilitating hippocampal neurogenesis, synaptogenesis and dendritic remodeling, but chronically elevated levels impairing these processes [44,45]. In addition, chronic elevation of corticosterone levels in rats impairs performance on the Barnes maze, another test of spatial mapping [43]. We have previously shown that basal plasma cortisol levels are elevated in PS50 males and that HPA responsiveness to stress is increased in PS60 males [6]. The possibility exists that the chronically high basal plasma cortisol levels in the PS50 males contributed to the impairment of spatial learning, but the acute increases in plasma cortisol in PS60 males in response to swim stress enhanced development of spatial learning. There is also recent evidence for a role of the mineralocorticoid receptor (MR) to enhance learning and memory [46,47]. For the current study, any differences in MR mRNA or protein in the hippocampi in PS50 or PS60 male offspring remains to be determined. We have previously demonstrated significantly decreased plasma testosterone levels in PS50 males [6]. Administration of testosterone to castrated male rats significantly decreased latency to find the platform in the MWM [42]. However, it is unknown whether the testosterone affected the search strategy used by these animals or whether MWM performance was improved by another mechanism. There is also evidence for effects of testosterone on memory retention as castrated male rats performed significantly worse in the MWM as the inter-trial length was increased [48]. Interestingly, the current study has demonstrated that 67% of PS50 males were using a spatial strategy by session 6, however, in session 7, which was approximately 4 h after session 6, no animals used spatial memory of the platform location in session 7. Given these findings, it is highly likely that the reduced plasma testosterone levels in the PS50 animals are affecting performance, specifically spatial learning in the MWM, however, the specific mechanisms involved will require further investigation. In conclusion, this is the first study to investigate the effects of prenatal stress on learning and memory and search strategy use in guinea pigs, which represent a species in which the profile of fetal brain development is comparable to the human. We

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