Physiology & Behavior 129 (2014) 104–109
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Prenatal stress decreases spatial learning and memory retrieval of the adult male offspring of rats Fatemeh Modir, Mahmoud Elahdadi Salmani ⁎, Iran Goudarzi, Taghi Lashkarboluki, Kataneh Abrari School of Biology, Damghan University, Damghan, Iran Institute of Biological Sciences, Damghan University, Damghan, Iran
H I G H L I G H T S • • • • •
Heterogeneous sequential stress (HSS) used as a model for prenatal stress (PS) PS applied before pregnancy (BPS), during first (PS1) and second (PS2) half of it HSS increased serum corticosterone of BPS and PS1 adult offspring and PS2 mothers. Latency and distance of BPS and PS1 increased in acquisition and retrieval tests. HSS decreased learning and memory of adult offspring in BPS and PS1, prominently.
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Article history: Received 9 September 2013 Received in revised form 25 January 2014 Accepted 18 February 2014 Available online 28 February 2014 Keywords: Prenatal stress Corticosterone Learning and memory Adult offspring Morris water maze
a b s t r a c t Introduction: Early life or prenatal stress induces many lifelong, mostly cognitive, homeostatic alterations in the behavior of the offspring. Purpose: We investigated the effect of heterogeneous sequential stress (HSS) at three separate periods, before and during the first and second half of pregnancies on spatial learning and memory retrieval of adult male offspring. Method: HSS is composed of several stressors, each in a day, during nine consecutive days including; restraint, swimming, isolation, and water and food deprivation on Wistar rats. The offspring were studied in a Morris water maze (MWM) apparatus to explore the latency, distance, proximity and target to opposite area as measures of learning and memory. Serum corticosterone was measured as a criterion of stress application. Results: HSS increased blood corticosterone in dams of PS2 (Pregnancy Stress second half), and also in adult male offspring from BPS (Before Pregnancy Stress) and PS1 (Pregnancy Stress first half) groups. The weight of the offspring decreased in the PS1 and PS2 groups. While distance traveled and latency to locate the hidden platform were increased in BPS and PS1 acquisition trials, swimming speed was unchanged during the acquisition and retrieval tests. Moreover, time to platform location was increased in BPS and PS1 during retention tests. While control rats spent more time in the target quadrant, stressed animals spent a longer duration in the opposite quadrant. Furthermore, proximity measure was increased in all stress treated rats. Conclusion: It is concluded that prenatal stress, around the beginning of the pregnancy, increases corticosterone in adult male offspring, which might be the basis for spatial learning and memory retrieval deficits in this study. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Prenatal stress (PS), or adverse life events, during gestation have many deleterious effects on the development and behavior of the offspring [1]. PS may result in adverse effects on social and sexual Abbreviations: HPA, hypothalamic–pituitary–adrenal; MWM, Morris water maze; HSS, heterogeneous sequential stress; BPS, Before Pregnancy Stress; PS1, Pregnancy Stress first half; PS2, Pregnancy Stress second half. ⁎ Corresponding author at: School of Biology, Damghan University, Cheshme Ali Road, Damghan, Iran. Tel./fax: +98 2325247146. E-mail addresses: [email protected], [email protected] (M. Elahdadi Salmani).
http://dx.doi.org/10.1016/j.physbeh.2014.02.040 0031-9384/© 2014 Elsevier Inc. All rights reserved.
behaviors and may increase the risk for anxiogenic and depressivelike behaviors, as well as some schizophrenic features. Earthquakes, as natural source of stressors during pregnancy displayed a more depressive state in newborns [2]. Furthermore, PS along with other uncontrolled and prolonged stress, raises the HPA (hypothalamic– pituitary–adrenal axis) activities and leads to higher cortisol or corticosterone in mother's blood [3–7]. In this regard, maternal corticosterone can cross the placental barrier and influence the fetal brain [8], as HPA develops toward the end of pregnancy. However, fetal corticosterone alterations following a mother's response to stress depend on the time that stress is applied during pregnancy [9].
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The timing of stress experience is an important factor determining the severity of structural and functional outcomes [10]. Most studies show PS induced learning and memory deficits during the second half of pregnancy [11–14]. For instance, prenatal stress during the second half, decreased spatial learning which may be the consequence of a deficit in neurogenesis correlated with dysfunction of the HPA axis [15]. On the other hand, there are a number of human studies highlighting the effect of stress on the early days of pregnancy specifically during the early gestational period, which leads to lower birth weight, preterm birth labors and more severe motor impairments [1,16]. Moreover, stress before or around pregnancy severely affects the future developmental fate of a fetus [16]. For example, Schachar-Dadon et al. showed that unpredictable variable stress before conception alters affective and social behavior in the adult offspring [17]. PS imposes some behavioral and neurobiological effects related to hippocampal function [18]. As such, deficits in learning and memory of adult offspring are important consequences of stress experience [19]. For instance, Sahu et al. showed that PS from gestation day 11 until delivery increased escape latency in water maze test [20]. Similarly, daily varied gestational stress on gestational days (GD) 17–21 [14] and also GD 15–19 [7] impaired spatial learning in the MWM [7,14]; however there are few reports of enhancing learning capability due to PS [21,22], which indicates the controversy of the stress effect during gestation. Since PS has short and long term effects on learning and memory, and stress during or before pregnancy has developmental effects on HPA axis and other neurodevelopmental cognitive outcomes, it seems likely that prenatal or preconception stress will affect learning and memory in the adult offspring. Thus, in the current study, we are investigating the effect of PS on spatial learning and memory retrieval of offspring, and how corticosterone changes concomitantly after chronic PS application in three time periods; before pregnancy (BPS), during the first half (PS1) and second half of pregnancy (PS2). This will help determine the role of gestational corticosterone on learning and memory changes in adult offspring. 2. Methods 2.1. Animals Virgin female Wistar (Iran Pasteur Institute) rats (16 dams) weighing 200–220 g at the beginning of experiments were used and paired randomly with male Wistar rats. To ensure pregnancy, vaginal smears were checked on a slide every day at 7:00 am. The day of sperm detection was considered GD 0, males were taken out of the breeding cage and delivery was expected 21 days later. Each dam plus its litter was maintained in the home cage on wood flaked bedding in standard lab conditions, until weaning at 30 days postnatal. Then, male pups were selected (two male pups per mother to avoid litter effect), and grown 5 per cage normally until 2 months old, when they were used for water maze procedure. All experiments were done in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 23-80, revised 1996) and were approved by the research ethical standards for the care and use of animals in Damghan University. Rats were kept 5 per cage, except pregnant dams which were kept alone in the cage; all were under normal 12–12 light–dark cycle, with lights on at 07:00 am, and food and water ad libitum. 2.2. Experimental groups Female rats were randomly assorted into four experimental groups (8 animals in each group): (1) Control; (2) Before Pregnancy Stress (BPS); (3) First half Pregnancy Stress (PS1); and (4) Second half Pregnancy Stress (PS2). Adult male offspring of these control and stressed mothers were used to investigate spatial learning and memory retrieval in water maze task. The timeline of experiments is illustrated in Fig. 1.
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2.3. Stress protocol We used the heterogeneous sequential stress (HSS) model to investigate learning and memory changes in stressed mother's offspring. HSS was applied in 9 days [23] and included several daily stress paradigms as follows: 1. Forced swimming (22 ± 2 °C) for 10 min. 2. 3. 4. 5. 6. 7. 8. 9.
Restraint stress for 3 h, Water deprivation for 24 h, Restraint at 4 °C for 1.5 h, Isolation from others for 24 h, Food deprivation for 24 h, Water deprivation for 24 h, Restraint at 4 °C for 2 h, Food deprivation for 24 h [23]. The timing of HSS is outlined below:
Treatment
Start of treatment
BPS PS1 PS2
10 days before mating GD 0 GD 11
The rate of successful mating was 7 out of 10 and stressed female rats with unsuccessful mating for 48 h were excluded from the study. Neither the mothers nor the pups received stress in the control nonstressed group and they were left undisturbed in the colony cage until MWM test. 2.4. Corticosterone assay Blood samples were taken from mothers one day after the termination of HSS application and from adult offspring after finalizing MWM task, all through the tail lateral vein in conscious restrained animal by an expert lab technician. To avoid stress to neonates, sampling was performed from its male sibling from home cage on the second day of delivery (2 days old) by heart puncture. Sampling from the control dams was similar to the PS2 group. All of the blood samples were transferred to an anticoagulant coated tube using sodium citrate. After mixing, the specimen was centrifuged in 10,000 rpm for 15 min and the resulted plasma was stored in −80 °C freezers until assay. Concentration of corticosterone in plasma was measured using a corticosterone EIA competitive assay kit (Cayman Chemical; cat, 500655). On the testing day, supernatants (10 μl from 100 μl) were incubated with reaction mix solution for 30 min at 37 °C. Absorption was determined at 450 nm using a spectrophotometer and amounts of corticosterone were determined based on the standard curve. The sensitivity of the kit was 150 pg/ml. Intra-assay and inter-assay coefficients of variation were 6.6% and 5.7%, respectively. 2.5. Water maze test A circular tank (140 cm in diameter, 60 cm high) filled with water at a temperature of 24 ± 2 °C to depth of 30 cm, was used for the procedure. An escape invisible platform (15 cm in diameter) was placed 2 cm below the water surface, midway between the center and rim of the pool in the same quadrant throughout the task with the spatial cues available around the pool room. One day before the behavioral testing, each animal was habituated for 1 min on the platform to explore the pool. During the learning task, animals had to navigate the hidden platform, given four trials a day for 5 days, while the starting positions changed randomly over trials. Each trial began with the animal in the pool facing the sidewalls and ended either after 60 s of swimming or reaching the platform. The rat was then allowed to remain on the platform for 10 s after each trial. A probe task was performed 24 h
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Fig. 1. Timeline of experiments (see “Experimental groups” for details). Asterisks are the time of blood sampling. BPS, Before Pregnancy Stress; PS1, Pregnancy Stress first half; PS2, Pregnancy Stress second half; MWM, Morris water maze.
after the last day of learning trials with the platform removed from the pool. All of the learning and memory tasks were recorded with a CCD camera connected to a computer with Ethovision-XT tracking software (Noldus Company) designed for data acquisition and analysis. The parameters measured in learning and memory tasks, were the distance and the latency to escape onto the platform and velocity of swimming. In the probe task, total distance traveled during 60 s and the time swam to locate the platform area (platform location latency), where the platform had been located during the learning task, were measured. The proximity, average of the swimming distance from the center of platform location, and target to opposite target area were also considered. Proximity is a sensitive measure for better detection of group differences and target to opposite area examines how the precision of spatial searches changes over the course of the probe trial [24]. The results were analyzed as the individual mean of the four daily trials.
Mother generation revealed different corticosterone levels in all groups (F3,48 = 4.137, P b 0.01). Mothers that underwent HSS during the late pregnancy, PS2, displayed significantly higher levels of corticosterone compared with respective control (P b 0.01). Further, PS2 mothers showed an enhanced level of corticosterone compared with other HSS treated groups; BPS (P b 0.05) and PS1 (P b 0.01) (Fig. 2B). Analysis of offspring groups showed that treatment with BPS, PS1 or PS2 had no significant effect on corticosteroid levels in the infant rats. Adult animals however, displayed a significant increase in corticosterone levels in the BPS group (P b 0.005) and PS1 group (P b 0.05) compared with related controls. In contrast, PS2 treatment displayed no significant difference in adult offspring (Fig. 2B).
2.6. Statistical analysis Results of the experiments in MWM (escape latencies, distance traveled and swimming velocity) were analyzed using two-way ANOVA during acquisition days. Plasma corticosterone levels as dependent variable and prenatal stress before, during the first and second half of gestation as independent variables in both control and stress-exposed groups, were analyzed using two-way ANOVA for groups and generations (mother, neonate and adult offspring). Probe trial analysis using the following factors; platform location latency, total path length, swimming velocity, and proximity or target time in different groups was performed by one-way ANOVA. Post-hoc comparison of group means was performed with the Tukey or LSD test when needed. All of the statistical tests were performed by statistica v.16 software. Data presented as mean ± S.E.M. Minimum significant level was set at P b 0.05. 3. Results 3.1. Prenatal stress decreased birth weight of offspring Analysis of birth weight in neonates, on the second postnatal day using one-way ANOVA showed a prominent difference between groups (F3,52 = 11.203, P = 0.000, n = 56). Tukey post hoc operation revealed that birth weight was lower in PS1 (P b 0.01, n = 17) and PS2 (P b 0.001, n = 11) compared with the control (Fig. 2A). There was no significant difference in birth weights between stress treatment groups during pregnancy. 3.2. Prenatal stress increased corticosterone in adult offspring Statistical analysis employing two-way ANOVA revealed a significant difference between groups (F3,48 = 5.428, P = 0.003, n = 60) and also between generations (F2,48 = 41.286, P = 0.000, n = 60), as well as a significant group ∗ generation interaction (F6,48 = 7.833, P = 0.000, n = 60).
Fig. 2. Effect of stress on birth weight and corticosterone. A. HSS decreased birth weight. HSS decreased birth weight in PS1 (P b 0.01) and PS2 (P b 0.001), but not in BPS. B. HSS increased plasma corticosterone level in late pregnancy stressed mothers and adults. Maternal corticosterone was increased only in PS2 compared to control (P b 0.01), PS1 (P b 0.01) and BPS (P b 0.05). Adult male corticosterone levels were prominently increased in BPS (P b 0.001) and PS1 (P b 0.05). Asterisks denote comparison to control of the same group and crosses show comparison with maternal PS2 group. BPS, Before Pregnancy Stress; PS1, Pregnancy Stress first half; PS2, Pregnancy Stress second half; *P b 0.05, **P b 0.01, *** P b 0.001 compared with control.
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Statistical analysis showed a difference in plasma corticosterone of the BPS group between generations of mother and neonate (P b 0.05), with a parallel difference between generations of mother and adult offspring (P b 0.001). 3.3. HSS decreased water maze learning acquisition in prenatally stressed adult offspring Investigating the effect of HSS on acquisition of spatial learning in BPS, PS1 and PS2 using two-way ANOVA, showed a significant statistical difference in escape latency over the days (days 1–5) of learning (F4,135 = 25.474, P = 0.000, n = 155), and between groups (F3,135 = 11.658, P = 0.000, n = 60), without significant group ∗ generation interaction (F12,135 = 0.83, P = 0.61, n = 60). Tukey post hoc operation showed group differences during the first (P b 0.001) and last (P b 0.01) days of the learning paradigm (Fig. 3). Further, two-way ANOVA revealed a meaningful difference of distance traveled over 5 days of learning (F4,135 = 19.110, P = 0.000, n = 155), with a significant difference between groups (F3,135 = 14.868, P = 0.000), but not significant group ∗ generation interaction (F12,135 = 0.28, P = 0.99, n = 60). There was a difference in the distance traveled by different groups on the first (F3,135 = 3.476, P = 0.018), second (F3,135 = 5.556, P = 0.001), third (F3,135 = 2.949, P = 0.035) and fourth (F3,135 = 2.930, P = 0.036) days, using Tukey followed by the fifth day (F3,135 = 2.010, P b 0.05) which employed the LSD test. Meanwhile, swimming
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velocity did not change significantly over days (F4,147 = 0.494, P = 0.7, n = 155) or between groups (F4,147 = 1.945, P = 0.125) in the learning paradigm. 3.4. HSS decreased water maze memory retrieval in prenatally stressed adult offspring One day after learning acquisition, rats were allotted to retrieval (probe) test in MWM, which showed significant difference in platform location latency (F3,27 = 4.458, P = 0.01, n = 30) by using one way ANOVA test. LSD post hoc test indicated an increase of latency in BPS (P b 0.05, n = 7) and PS1 (P b 0.01, n = 9) compared with the control (Fig. 4). There was no statistical difference in total path length during 60 s of swimming (F 3,27 = 1.719, P = 0.18, n = 30) along with swimming velocity (F3,27 = 1.769, P = 0.17, n = 30) between different groups. Proximity (the average distance swam from the platform) in the probe trial was analyzed using one way ANOVA, revealing a prominent difference between groups (F3,28 = 3.671, P = 0.024, n = 31). LSD test showed an increase of proximity measure in BPS (P b 0.01), PS1 (P b 0.05) and PS2 (P b 0.05) compared with the control. There was a strong positive correlation between platform location latency and proximity, which was statistically significant (r = 0.685, n = 31, P b 0.0005). The time spent in the target area (platform zone) to the opposite area differed between groups (F3,27 = 4.14, P b 0.01, n = 31). The LSD post hoc test showed a decrease in the time spent in the target area as opposed to that of the opposite area in BPS (P b 0.05), PS1 (P b 0.05) and PS2 (P b 0.001) when they are compared with the control. There was a negative correlation between platform location latency and target to opposite area navigation, which was statistically significant (r = 0.485, n = 31, P b 0.004), and also between target to opposite area and proximity with a significant correlation (r = 0.592, n = 31, P b 0.0005). 4. Discussion
Fig. 3. Effect of HSS on spatial learning acquisition. A. HSS increased latency to locate the platform. Latency to find the platform was increased in BPS during first (P b 0.001) and last (P b 0.05) days and in PS1 during first (P b 0.01) and fifth (P b 0.01) days compared with the same day control. Latency did not change in PS2 group. B. HSS increased distance traveled in water maze swimming. Rats traveled a longer path in BPS during first (P b 0.05), second (P b 0.01), third (P b 0.05), fourth (P b 0.05) and fifth (P b 0.05) days and in PS1 during first (P b 0.05), second (P b 0.01), third (P b 0.05) and fifth (P b 0.05) days. Bar charts show the differences between each group and control. All of the P values are compared with control. BPS, Before Pregnancy Stress; PS1, Pregnancy Stress first half; PS2, Pregnancy Stress second half; HSS, heterogeneous sequential stress; D, Day; *P b 0.05, **P b 0.01, ***P b 0.001.
The present study investigated the effect of prenatal stress on spatial learning and memory retrieval in adult male offspring. The results of the study showed that prenatal stress increased plasma corticosterone, while learning capability and memory retrieval were decreased in adult male offspring. Spatial learning acquisition and memory retrieval were decreased following BPS and PS1 treatments, whereas only limited features of retrieval were reduced in PS2. HSS (relating to prenatal stress) is a chronic stress in nature, which induces prolonged activation of the HPA axis [6,25]. During normal pregnancy, cortisol levels gradually increase [26,27] and reach maximum levels with pregnancy progress of 2–3 times the level in non-pregnant females [27]. Our data showed stress induced increase of corticosterone in the late pregnancy compared to concurrent control. Further, stress induced late pregnancy increase of corticosterone was higher compared to the early or before pregnancy stressed groups. However, the early stressed animals did not develop higher corticosterone compared to control (data not shown). Based on these findings, it is theorized that placental corticotropin releasing hormone (CRH) may be behind this corticosterone increase. It stimulates cortisol release directly from the adrenal cortex, which in turn stimulates CRH release from the placenta, in a positive feedback [28]. This feedback probably enhances progressive corticosterone increase during gestation, which was demonstrated in mothers of PS2 treatment. Conversely, after crossing from the placenta, corticosterone is converted to cortisone and with the notion of the necessity of fetal HPA development in responding to stress from mid-gestation [6], it is reasonable that corticosterone increases more progressively in the second half of pregnancy, not in early pregnancy. Our data also showed that stress negatively impacted birth weight during pregnancy progression (Fig. 2A). This was
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Fig. 4. Effect of HSS on spatial memory. A. Platform location latency increased in BPS (P b 0.05) and PS1 (P b 0.01) but not PS2 groups. B. Total path length did not change in different groups. C. Animals of BPS group (P b 0.01), PS1 (P b 0.05) and PS2 (P b 0.05) swam a greater distance to platform area (proximity) than the control animals. D. The time spent in target platform quadrant to opposite quadrant was shorter in BPS (P b 0.05), PS1 (P b 0.05) and PS2 (P b 0.001) compared to control. BPS, Before Pregnancy Stress; PS1, Pregnancy Stress first half; PS2, Pregnancy Stress second half; HSS, heterogeneous sequential stress; *P b 0.05, **P b 0.01, ***P b 0.001.
especially observed during the second half of pregnancy, demonstrating a correlation between corticosterone secretion and low birth weight in stressed pregnant animals. In this regard, Wadhwa et al. showed a similar correlation between gestational stress and birth weight, which they related to high circulating CRH in the second trimester in humans [29–31]. In contrast to corticosterone elevation toward the late pregnancy, our data showed an increase in corticosterone of adult offspring after early gestational and pre-conceptional stressing adverse conditions. This negative correlation might be because of changes in epigenetic programming and later mis-development of fetal HPA [15,32,33]. Our experiments demonstrated HSS induced increase in escape latency and path length, which indicates the reduction of spatial learning during the water maze test. This data is accompanied with the increase of corticosterone in male adults. There are reports supporting the detrimental effects of chronic stress on learning and memory of rats [34,35]. For instance, Diamond et al. illustrated deficits of working memory due to an unfamiliar environment, indicative of hippocampal memory dysfunction [36]. In addition, high levels of corticosterone in prenatally stressed adult male rats might result in learning deficit along with hippocampal damage [37–39]. We also observed a decline in retrieval memory due to prenatal stress which is also confirmed by others in the chronic [34,35] and prenatally stressed models [13,40–44]. Thus, our data confirms the outcomes of stress application during the second half of gestation and the fetal HPA axis development [6,9,45]. While our data confirm previous studies regarding learning and memory impairments in adult male offspring following late pregnancy stress, a more pronounced decline has been observed in mothers stressed before or during the first half of pregnancy (Figs. 3 and 4). Corticosterone changes cannot justify this effect, since HPA axis and stress related hypothalamic nuclei are developed during the second gestational half [1], as our data of preconception and early pregnancy corticosterone confirmed, and even maternal corticosterone is metabolized to ineffective compounds [6] during the first half. The candidate mechanism which mirrors stress influence in fetal brain development might be stress induced alteration of epigenetic mechanisms. For
instance, anxiogenic behavior of PS males and females and concomitant learning deficit in males were completely abolished by adrenalectomy [46]. This and our BPS regime might modulate HPA axis development which leads to changes of hippocampal neurogenesis [15] and even alteration in brain CRF1 receptors [47] or other targeted epigenetic influences. The latter might imply trans-generational transmission of stress vulnerability [48,49] such as an increase of ova and testis CRF1 receptor expression [47]. Therefore, the HPA axis in this group may be affected by changes in epigenetic programming [50,51] of different stress related structures due to stress to mothers prior to pregnancy. This conclusion highlights the difference between preconception and prenatal stresses, especially the second half. The latter impacts the fetus and its environment directly, whereas pregestational stress does not [17]. The exact programming mechanism requires further investigation in future experiments.
5. Conclusions It is concluded that late pregnancy stress increases corticosterone secretion in pregnant mothers, but not prior to, or in early pregnancy. In contrast, preconception and early pregnancy stress experiences impair offspring maze performance. Furthermore, the effective level of detrimental stress depends on the time of stress application, more prominently effected before and during the first half of pregnancy. This study emphasizes the importance of pre-gestational and pre-mating stresses and may open a new hope of better and enriched gestational developmental outcomes even in humans. However, one of the constraints of this trans-generational type of study is changing seasons from the selection of male and female rats to adolescence of their offspring in different groups, and further studies are required to better understand the implication of these results. Meanwhile, it is needed to establish other experiments in the future to better understand the mechanisms by which early stress and epigenetic risk factors promote learning disabilities in animals as well as in humans.
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Acknowledgment This study was supported by Damghan University, Damghan, Iran. Authors declare no conflict of interest with any other research, studies or publications.
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Prenatal stress decreases spatial learning and memory retrieval of the adult male offspring of rats Fatemeh Modir, Mahmoud Elahdadi Salmani ⁎, Iran Goudarzi, Taghi Lashkarboluki, Kataneh Abrari School of Biology, Damghan University, Damghan, Iran Institute of Biological Sciences, Damghan University, Damghan, Iran
H I G H L I G H T S • • • • •
Heterogeneous sequential stress (HSS) used as a model for prenatal stress (PS) PS applied before pregnancy (BPS), during first (PS1) and second (PS2) half of it HSS increased serum corticosterone of BPS and PS1 adult offspring and PS2 mothers. Latency and distance of BPS and PS1 increased in acquisition and retrieval tests. HSS decreased learning and memory of adult offspring in BPS and PS1, prominently.
a r t i c l e
i n f o
Article history: Received 9 September 2013 Received in revised form 25 January 2014 Accepted 18 February 2014 Available online 28 February 2014 Keywords: Prenatal stress Corticosterone Learning and memory Adult offspring Morris water maze
a b s t r a c t Introduction: Early life or prenatal stress induces many lifelong, mostly cognitive, homeostatic alterations in the behavior of the offspring. Purpose: We investigated the effect of heterogeneous sequential stress (HSS) at three separate periods, before and during the first and second half of pregnancies on spatial learning and memory retrieval of adult male offspring. Method: HSS is composed of several stressors, each in a day, during nine consecutive days including; restraint, swimming, isolation, and water and food deprivation on Wistar rats. The offspring were studied in a Morris water maze (MWM) apparatus to explore the latency, distance, proximity and target to opposite area as measures of learning and memory. Serum corticosterone was measured as a criterion of stress application. Results: HSS increased blood corticosterone in dams of PS2 (Pregnancy Stress second half), and also in adult male offspring from BPS (Before Pregnancy Stress) and PS1 (Pregnancy Stress first half) groups. The weight of the offspring decreased in the PS1 and PS2 groups. While distance traveled and latency to locate the hidden platform were increased in BPS and PS1 acquisition trials, swimming speed was unchanged during the acquisition and retrieval tests. Moreover, time to platform location was increased in BPS and PS1 during retention tests. While control rats spent more time in the target quadrant, stressed animals spent a longer duration in the opposite quadrant. Furthermore, proximity measure was increased in all stress treated rats. Conclusion: It is concluded that prenatal stress, around the beginning of the pregnancy, increases corticosterone in adult male offspring, which might be the basis for spatial learning and memory retrieval deficits in this study. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Prenatal stress (PS), or adverse life events, during gestation have many deleterious effects on the development and behavior of the offspring [1]. PS may result in adverse effects on social and sexual Abbreviations: HPA, hypothalamic–pituitary–adrenal; MWM, Morris water maze; HSS, heterogeneous sequential stress; BPS, Before Pregnancy Stress; PS1, Pregnancy Stress first half; PS2, Pregnancy Stress second half. ⁎ Corresponding author at: School of Biology, Damghan University, Cheshme Ali Road, Damghan, Iran. Tel./fax: +98 2325247146. E-mail addresses: [email protected], [email protected] (M. Elahdadi Salmani).
http://dx.doi.org/10.1016/j.physbeh.2014.02.040 0031-9384/© 2014 Elsevier Inc. All rights reserved.
behaviors and may increase the risk for anxiogenic and depressivelike behaviors, as well as some schizophrenic features. Earthquakes, as natural source of stressors during pregnancy displayed a more depressive state in newborns [2]. Furthermore, PS along with other uncontrolled and prolonged stress, raises the HPA (hypothalamic– pituitary–adrenal axis) activities and leads to higher cortisol or corticosterone in mother's blood [3–7]. In this regard, maternal corticosterone can cross the placental barrier and influence the fetal brain [8], as HPA develops toward the end of pregnancy. However, fetal corticosterone alterations following a mother's response to stress depend on the time that stress is applied during pregnancy [9].
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The timing of stress experience is an important factor determining the severity of structural and functional outcomes [10]. Most studies show PS induced learning and memory deficits during the second half of pregnancy [11–14]. For instance, prenatal stress during the second half, decreased spatial learning which may be the consequence of a deficit in neurogenesis correlated with dysfunction of the HPA axis [15]. On the other hand, there are a number of human studies highlighting the effect of stress on the early days of pregnancy specifically during the early gestational period, which leads to lower birth weight, preterm birth labors and more severe motor impairments [1,16]. Moreover, stress before or around pregnancy severely affects the future developmental fate of a fetus [16]. For example, Schachar-Dadon et al. showed that unpredictable variable stress before conception alters affective and social behavior in the adult offspring [17]. PS imposes some behavioral and neurobiological effects related to hippocampal function [18]. As such, deficits in learning and memory of adult offspring are important consequences of stress experience [19]. For instance, Sahu et al. showed that PS from gestation day 11 until delivery increased escape latency in water maze test [20]. Similarly, daily varied gestational stress on gestational days (GD) 17–21 [14] and also GD 15–19 [7] impaired spatial learning in the MWM [7,14]; however there are few reports of enhancing learning capability due to PS [21,22], which indicates the controversy of the stress effect during gestation. Since PS has short and long term effects on learning and memory, and stress during or before pregnancy has developmental effects on HPA axis and other neurodevelopmental cognitive outcomes, it seems likely that prenatal or preconception stress will affect learning and memory in the adult offspring. Thus, in the current study, we are investigating the effect of PS on spatial learning and memory retrieval of offspring, and how corticosterone changes concomitantly after chronic PS application in three time periods; before pregnancy (BPS), during the first half (PS1) and second half of pregnancy (PS2). This will help determine the role of gestational corticosterone on learning and memory changes in adult offspring. 2. Methods 2.1. Animals Virgin female Wistar (Iran Pasteur Institute) rats (16 dams) weighing 200–220 g at the beginning of experiments were used and paired randomly with male Wistar rats. To ensure pregnancy, vaginal smears were checked on a slide every day at 7:00 am. The day of sperm detection was considered GD 0, males were taken out of the breeding cage and delivery was expected 21 days later. Each dam plus its litter was maintained in the home cage on wood flaked bedding in standard lab conditions, until weaning at 30 days postnatal. Then, male pups were selected (two male pups per mother to avoid litter effect), and grown 5 per cage normally until 2 months old, when they were used for water maze procedure. All experiments were done in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 23-80, revised 1996) and were approved by the research ethical standards for the care and use of animals in Damghan University. Rats were kept 5 per cage, except pregnant dams which were kept alone in the cage; all were under normal 12–12 light–dark cycle, with lights on at 07:00 am, and food and water ad libitum. 2.2. Experimental groups Female rats were randomly assorted into four experimental groups (8 animals in each group): (1) Control; (2) Before Pregnancy Stress (BPS); (3) First half Pregnancy Stress (PS1); and (4) Second half Pregnancy Stress (PS2). Adult male offspring of these control and stressed mothers were used to investigate spatial learning and memory retrieval in water maze task. The timeline of experiments is illustrated in Fig. 1.
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2.3. Stress protocol We used the heterogeneous sequential stress (HSS) model to investigate learning and memory changes in stressed mother's offspring. HSS was applied in 9 days [23] and included several daily stress paradigms as follows: 1. Forced swimming (22 ± 2 °C) for 10 min. 2. 3. 4. 5. 6. 7. 8. 9.
Restraint stress for 3 h, Water deprivation for 24 h, Restraint at 4 °C for 1.5 h, Isolation from others for 24 h, Food deprivation for 24 h, Water deprivation for 24 h, Restraint at 4 °C for 2 h, Food deprivation for 24 h [23]. The timing of HSS is outlined below:
Treatment
Start of treatment
BPS PS1 PS2
10 days before mating GD 0 GD 11
The rate of successful mating was 7 out of 10 and stressed female rats with unsuccessful mating for 48 h were excluded from the study. Neither the mothers nor the pups received stress in the control nonstressed group and they were left undisturbed in the colony cage until MWM test. 2.4. Corticosterone assay Blood samples were taken from mothers one day after the termination of HSS application and from adult offspring after finalizing MWM task, all through the tail lateral vein in conscious restrained animal by an expert lab technician. To avoid stress to neonates, sampling was performed from its male sibling from home cage on the second day of delivery (2 days old) by heart puncture. Sampling from the control dams was similar to the PS2 group. All of the blood samples were transferred to an anticoagulant coated tube using sodium citrate. After mixing, the specimen was centrifuged in 10,000 rpm for 15 min and the resulted plasma was stored in −80 °C freezers until assay. Concentration of corticosterone in plasma was measured using a corticosterone EIA competitive assay kit (Cayman Chemical; cat, 500655). On the testing day, supernatants (10 μl from 100 μl) were incubated with reaction mix solution for 30 min at 37 °C. Absorption was determined at 450 nm using a spectrophotometer and amounts of corticosterone were determined based on the standard curve. The sensitivity of the kit was 150 pg/ml. Intra-assay and inter-assay coefficients of variation were 6.6% and 5.7%, respectively. 2.5. Water maze test A circular tank (140 cm in diameter, 60 cm high) filled with water at a temperature of 24 ± 2 °C to depth of 30 cm, was used for the procedure. An escape invisible platform (15 cm in diameter) was placed 2 cm below the water surface, midway between the center and rim of the pool in the same quadrant throughout the task with the spatial cues available around the pool room. One day before the behavioral testing, each animal was habituated for 1 min on the platform to explore the pool. During the learning task, animals had to navigate the hidden platform, given four trials a day for 5 days, while the starting positions changed randomly over trials. Each trial began with the animal in the pool facing the sidewalls and ended either after 60 s of swimming or reaching the platform. The rat was then allowed to remain on the platform for 10 s after each trial. A probe task was performed 24 h
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Fig. 1. Timeline of experiments (see “Experimental groups” for details). Asterisks are the time of blood sampling. BPS, Before Pregnancy Stress; PS1, Pregnancy Stress first half; PS2, Pregnancy Stress second half; MWM, Morris water maze.
after the last day of learning trials with the platform removed from the pool. All of the learning and memory tasks were recorded with a CCD camera connected to a computer with Ethovision-XT tracking software (Noldus Company) designed for data acquisition and analysis. The parameters measured in learning and memory tasks, were the distance and the latency to escape onto the platform and velocity of swimming. In the probe task, total distance traveled during 60 s and the time swam to locate the platform area (platform location latency), where the platform had been located during the learning task, were measured. The proximity, average of the swimming distance from the center of platform location, and target to opposite target area were also considered. Proximity is a sensitive measure for better detection of group differences and target to opposite area examines how the precision of spatial searches changes over the course of the probe trial [24]. The results were analyzed as the individual mean of the four daily trials.
Mother generation revealed different corticosterone levels in all groups (F3,48 = 4.137, P b 0.01). Mothers that underwent HSS during the late pregnancy, PS2, displayed significantly higher levels of corticosterone compared with respective control (P b 0.01). Further, PS2 mothers showed an enhanced level of corticosterone compared with other HSS treated groups; BPS (P b 0.05) and PS1 (P b 0.01) (Fig. 2B). Analysis of offspring groups showed that treatment with BPS, PS1 or PS2 had no significant effect on corticosteroid levels in the infant rats. Adult animals however, displayed a significant increase in corticosterone levels in the BPS group (P b 0.005) and PS1 group (P b 0.05) compared with related controls. In contrast, PS2 treatment displayed no significant difference in adult offspring (Fig. 2B).
2.6. Statistical analysis Results of the experiments in MWM (escape latencies, distance traveled and swimming velocity) were analyzed using two-way ANOVA during acquisition days. Plasma corticosterone levels as dependent variable and prenatal stress before, during the first and second half of gestation as independent variables in both control and stress-exposed groups, were analyzed using two-way ANOVA for groups and generations (mother, neonate and adult offspring). Probe trial analysis using the following factors; platform location latency, total path length, swimming velocity, and proximity or target time in different groups was performed by one-way ANOVA. Post-hoc comparison of group means was performed with the Tukey or LSD test when needed. All of the statistical tests were performed by statistica v.16 software. Data presented as mean ± S.E.M. Minimum significant level was set at P b 0.05. 3. Results 3.1. Prenatal stress decreased birth weight of offspring Analysis of birth weight in neonates, on the second postnatal day using one-way ANOVA showed a prominent difference between groups (F3,52 = 11.203, P = 0.000, n = 56). Tukey post hoc operation revealed that birth weight was lower in PS1 (P b 0.01, n = 17) and PS2 (P b 0.001, n = 11) compared with the control (Fig. 2A). There was no significant difference in birth weights between stress treatment groups during pregnancy. 3.2. Prenatal stress increased corticosterone in adult offspring Statistical analysis employing two-way ANOVA revealed a significant difference between groups (F3,48 = 5.428, P = 0.003, n = 60) and also between generations (F2,48 = 41.286, P = 0.000, n = 60), as well as a significant group ∗ generation interaction (F6,48 = 7.833, P = 0.000, n = 60).
Fig. 2. Effect of stress on birth weight and corticosterone. A. HSS decreased birth weight. HSS decreased birth weight in PS1 (P b 0.01) and PS2 (P b 0.001), but not in BPS. B. HSS increased plasma corticosterone level in late pregnancy stressed mothers and adults. Maternal corticosterone was increased only in PS2 compared to control (P b 0.01), PS1 (P b 0.01) and BPS (P b 0.05). Adult male corticosterone levels were prominently increased in BPS (P b 0.001) and PS1 (P b 0.05). Asterisks denote comparison to control of the same group and crosses show comparison with maternal PS2 group. BPS, Before Pregnancy Stress; PS1, Pregnancy Stress first half; PS2, Pregnancy Stress second half; *P b 0.05, **P b 0.01, *** P b 0.001 compared with control.
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Statistical analysis showed a difference in plasma corticosterone of the BPS group between generations of mother and neonate (P b 0.05), with a parallel difference between generations of mother and adult offspring (P b 0.001). 3.3. HSS decreased water maze learning acquisition in prenatally stressed adult offspring Investigating the effect of HSS on acquisition of spatial learning in BPS, PS1 and PS2 using two-way ANOVA, showed a significant statistical difference in escape latency over the days (days 1–5) of learning (F4,135 = 25.474, P = 0.000, n = 155), and between groups (F3,135 = 11.658, P = 0.000, n = 60), without significant group ∗ generation interaction (F12,135 = 0.83, P = 0.61, n = 60). Tukey post hoc operation showed group differences during the first (P b 0.001) and last (P b 0.01) days of the learning paradigm (Fig. 3). Further, two-way ANOVA revealed a meaningful difference of distance traveled over 5 days of learning (F4,135 = 19.110, P = 0.000, n = 155), with a significant difference between groups (F3,135 = 14.868, P = 0.000), but not significant group ∗ generation interaction (F12,135 = 0.28, P = 0.99, n = 60). There was a difference in the distance traveled by different groups on the first (F3,135 = 3.476, P = 0.018), second (F3,135 = 5.556, P = 0.001), third (F3,135 = 2.949, P = 0.035) and fourth (F3,135 = 2.930, P = 0.036) days, using Tukey followed by the fifth day (F3,135 = 2.010, P b 0.05) which employed the LSD test. Meanwhile, swimming
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velocity did not change significantly over days (F4,147 = 0.494, P = 0.7, n = 155) or between groups (F4,147 = 1.945, P = 0.125) in the learning paradigm. 3.4. HSS decreased water maze memory retrieval in prenatally stressed adult offspring One day after learning acquisition, rats were allotted to retrieval (probe) test in MWM, which showed significant difference in platform location latency (F3,27 = 4.458, P = 0.01, n = 30) by using one way ANOVA test. LSD post hoc test indicated an increase of latency in BPS (P b 0.05, n = 7) and PS1 (P b 0.01, n = 9) compared with the control (Fig. 4). There was no statistical difference in total path length during 60 s of swimming (F 3,27 = 1.719, P = 0.18, n = 30) along with swimming velocity (F3,27 = 1.769, P = 0.17, n = 30) between different groups. Proximity (the average distance swam from the platform) in the probe trial was analyzed using one way ANOVA, revealing a prominent difference between groups (F3,28 = 3.671, P = 0.024, n = 31). LSD test showed an increase of proximity measure in BPS (P b 0.01), PS1 (P b 0.05) and PS2 (P b 0.05) compared with the control. There was a strong positive correlation between platform location latency and proximity, which was statistically significant (r = 0.685, n = 31, P b 0.0005). The time spent in the target area (platform zone) to the opposite area differed between groups (F3,27 = 4.14, P b 0.01, n = 31). The LSD post hoc test showed a decrease in the time spent in the target area as opposed to that of the opposite area in BPS (P b 0.05), PS1 (P b 0.05) and PS2 (P b 0.001) when they are compared with the control. There was a negative correlation between platform location latency and target to opposite area navigation, which was statistically significant (r = 0.485, n = 31, P b 0.004), and also between target to opposite area and proximity with a significant correlation (r = 0.592, n = 31, P b 0.0005). 4. Discussion
Fig. 3. Effect of HSS on spatial learning acquisition. A. HSS increased latency to locate the platform. Latency to find the platform was increased in BPS during first (P b 0.001) and last (P b 0.05) days and in PS1 during first (P b 0.01) and fifth (P b 0.01) days compared with the same day control. Latency did not change in PS2 group. B. HSS increased distance traveled in water maze swimming. Rats traveled a longer path in BPS during first (P b 0.05), second (P b 0.01), third (P b 0.05), fourth (P b 0.05) and fifth (P b 0.05) days and in PS1 during first (P b 0.05), second (P b 0.01), third (P b 0.05) and fifth (P b 0.05) days. Bar charts show the differences between each group and control. All of the P values are compared with control. BPS, Before Pregnancy Stress; PS1, Pregnancy Stress first half; PS2, Pregnancy Stress second half; HSS, heterogeneous sequential stress; D, Day; *P b 0.05, **P b 0.01, ***P b 0.001.
The present study investigated the effect of prenatal stress on spatial learning and memory retrieval in adult male offspring. The results of the study showed that prenatal stress increased plasma corticosterone, while learning capability and memory retrieval were decreased in adult male offspring. Spatial learning acquisition and memory retrieval were decreased following BPS and PS1 treatments, whereas only limited features of retrieval were reduced in PS2. HSS (relating to prenatal stress) is a chronic stress in nature, which induces prolonged activation of the HPA axis [6,25]. During normal pregnancy, cortisol levels gradually increase [26,27] and reach maximum levels with pregnancy progress of 2–3 times the level in non-pregnant females [27]. Our data showed stress induced increase of corticosterone in the late pregnancy compared to concurrent control. Further, stress induced late pregnancy increase of corticosterone was higher compared to the early or before pregnancy stressed groups. However, the early stressed animals did not develop higher corticosterone compared to control (data not shown). Based on these findings, it is theorized that placental corticotropin releasing hormone (CRH) may be behind this corticosterone increase. It stimulates cortisol release directly from the adrenal cortex, which in turn stimulates CRH release from the placenta, in a positive feedback [28]. This feedback probably enhances progressive corticosterone increase during gestation, which was demonstrated in mothers of PS2 treatment. Conversely, after crossing from the placenta, corticosterone is converted to cortisone and with the notion of the necessity of fetal HPA development in responding to stress from mid-gestation [6], it is reasonable that corticosterone increases more progressively in the second half of pregnancy, not in early pregnancy. Our data also showed that stress negatively impacted birth weight during pregnancy progression (Fig. 2A). This was
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Fig. 4. Effect of HSS on spatial memory. A. Platform location latency increased in BPS (P b 0.05) and PS1 (P b 0.01) but not PS2 groups. B. Total path length did not change in different groups. C. Animals of BPS group (P b 0.01), PS1 (P b 0.05) and PS2 (P b 0.05) swam a greater distance to platform area (proximity) than the control animals. D. The time spent in target platform quadrant to opposite quadrant was shorter in BPS (P b 0.05), PS1 (P b 0.05) and PS2 (P b 0.001) compared to control. BPS, Before Pregnancy Stress; PS1, Pregnancy Stress first half; PS2, Pregnancy Stress second half; HSS, heterogeneous sequential stress; *P b 0.05, **P b 0.01, ***P b 0.001.
especially observed during the second half of pregnancy, demonstrating a correlation between corticosterone secretion and low birth weight in stressed pregnant animals. In this regard, Wadhwa et al. showed a similar correlation between gestational stress and birth weight, which they related to high circulating CRH in the second trimester in humans [29–31]. In contrast to corticosterone elevation toward the late pregnancy, our data showed an increase in corticosterone of adult offspring after early gestational and pre-conceptional stressing adverse conditions. This negative correlation might be because of changes in epigenetic programming and later mis-development of fetal HPA [15,32,33]. Our experiments demonstrated HSS induced increase in escape latency and path length, which indicates the reduction of spatial learning during the water maze test. This data is accompanied with the increase of corticosterone in male adults. There are reports supporting the detrimental effects of chronic stress on learning and memory of rats [34,35]. For instance, Diamond et al. illustrated deficits of working memory due to an unfamiliar environment, indicative of hippocampal memory dysfunction [36]. In addition, high levels of corticosterone in prenatally stressed adult male rats might result in learning deficit along with hippocampal damage [37–39]. We also observed a decline in retrieval memory due to prenatal stress which is also confirmed by others in the chronic [34,35] and prenatally stressed models [13,40–44]. Thus, our data confirms the outcomes of stress application during the second half of gestation and the fetal HPA axis development [6,9,45]. While our data confirm previous studies regarding learning and memory impairments in adult male offspring following late pregnancy stress, a more pronounced decline has been observed in mothers stressed before or during the first half of pregnancy (Figs. 3 and 4). Corticosterone changes cannot justify this effect, since HPA axis and stress related hypothalamic nuclei are developed during the second gestational half [1], as our data of preconception and early pregnancy corticosterone confirmed, and even maternal corticosterone is metabolized to ineffective compounds [6] during the first half. The candidate mechanism which mirrors stress influence in fetal brain development might be stress induced alteration of epigenetic mechanisms. For
instance, anxiogenic behavior of PS males and females and concomitant learning deficit in males were completely abolished by adrenalectomy [46]. This and our BPS regime might modulate HPA axis development which leads to changes of hippocampal neurogenesis [15] and even alteration in brain CRF1 receptors [47] or other targeted epigenetic influences. The latter might imply trans-generational transmission of stress vulnerability [48,49] such as an increase of ova and testis CRF1 receptor expression [47]. Therefore, the HPA axis in this group may be affected by changes in epigenetic programming [50,51] of different stress related structures due to stress to mothers prior to pregnancy. This conclusion highlights the difference between preconception and prenatal stresses, especially the second half. The latter impacts the fetus and its environment directly, whereas pregestational stress does not [17]. The exact programming mechanism requires further investigation in future experiments.
5. Conclusions It is concluded that late pregnancy stress increases corticosterone secretion in pregnant mothers, but not prior to, or in early pregnancy. In contrast, preconception and early pregnancy stress experiences impair offspring maze performance. Furthermore, the effective level of detrimental stress depends on the time of stress application, more prominently effected before and during the first half of pregnancy. This study emphasizes the importance of pre-gestational and pre-mating stresses and may open a new hope of better and enriched gestational developmental outcomes even in humans. However, one of the constraints of this trans-generational type of study is changing seasons from the selection of male and female rats to adolescence of their offspring in different groups, and further studies are required to better understand the implication of these results. Meanwhile, it is needed to establish other experiments in the future to better understand the mechanisms by which early stress and epigenetic risk factors promote learning disabilities in animals as well as in humans.
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Acknowledgment This study was supported by Damghan University, Damghan, Iran. Authors declare no conflict of interest with any other research, studies or publications.
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