Physiology & Behavior 139 (2015) 459–467

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Effects of different timing of stress on corticosterone, BDNF and memory in male rats Maryam Radahmadi, Hojjatallah Alaei ⁎, Mohammad Reza Sharifi, Nasrin Hosseini Department of Physiology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

H I G H L I G H T S • • • • •

Recovery period restored chronic stress-induced memory deficit. The effects of chronic stress on memory were time-dependent. Only very long duration of stress (over 21 days) had adaptive effects on memory. The decrease in BDNF level directly continued with duration of stress. The changes in BDNF level were slower and more permanent than the CORT changes.

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Article history: Received 20 July 2014 Received in revised form 25 November 2014 Accepted 2 December 2014 Available online 4 December 2014 Keywords: Stress Memory Passive avoidance Corticosterone BDNF Body weight Rat

a b s t r a c t Learning and memory seem to be affected by chronic stress. Previous reports have considered chronic stress as a precipitating factor of different neuropsychological disorders, while others reported neurobiological adaptations following stress. The present study investigated the effects of chronic stress before, after, and during learning on the changes of learning and memory, on serum and hippocampal levels of corticosterone (CORT), brain-derived neurotrophic factor (BDNF) and body weight in rats. Male Wistar rats were randomly divided into four groups (n = 10) including Control (Co), Stress-Learning-Rest (St-L-Re), Rest-Learning-Stress (Re-L-St), and Stress-Learning-Stress (St-L-St) groups. The chronic restraint stress was applied 6 h/day for 21 days. Moreover, the passive avoidance test was used to assess memory deficit, 1, 7, and 21 days after training. At the end of experiments, CORT and BDNF levels were measured. The findings did not support adaptation in chronic stress conditions. The acquisition time as well as the short and mid-term memories was significantly impaired in the St-L-Re group. Short, mid, and long-term memories were significantly impaired in the Re-L-St and St-L-St groups compared with the Co group, as a result of the enhancement of CORT and reduction of BDNF levels. In the St-L-St group, changes in memory functions were less pronounced than in the Re-L-St group. Also, body weight declined following the chronic stress, while recovery period enhanced the body weight gain in stressed rats. It can be concluded that a potential time-dependent involvement of stress and recovery period on the level of BDNF. Longer duration time of chronic stress might promote adaptive effects on memory and CORT level. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Stressful experiences alter brain function and regulate memory processing by activating glucocorticoids (corticosterone in rats) [1]. The negative impact of chronic stress on neuronal plasticity, learning, and memory functions is suggested to be modulated by various mechanisms, possibly, by involving glucocorticoids [2], brain derived neurotrophic factor (BDNF), and other contributors [3,4]. BDNF, the most ubiquitous neurotrophin in the rodents' brain, is affected by glucocorticoids [5], and is considered as an important factor regulating synaptogenesis and ⁎ Corresponding author. E-mail address: [email protected] (H. Alaei).

http://dx.doi.org/10.1016/j.physbeh.2014.12.004 0031-9384/© 2014 Elsevier Inc. All rights reserved.

synaptic plasticity mechanisms underlying learning and memory functions [6,7]. On the other hand, the hippocampus is one of the most important regions crucially involved in the processing of memory [8], mood, cognition, and neuroendocrine stress reactivity. According to literature, however, chronic stress is thought to affect such functions [9–11]. For instance, Luine et al. [12] showed that chronic restraint stress induces a reversible memory impairment in the eight-arm radial maze performance and Y maze task [13]. As such, severe and/or long-term stresses, not only affect the emotional responses and cognition [14], but also alter the normal brain structure and function [15]. Hence, it seems that the hippocampal corticosterone (CORT) and BDNF levels may play a crucial role in behavioral changes following stress. While some studies have

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considered the chronic stress as a precipitating factor of various neuropsychological disorders [16], other reports have suggested that stress may cause neurobiological adaptations [17]. Given this, one may explain different effects of stress ranging from adaptation to illness. It seems that the duration of stress period may be the most important factor that can influence the cognitive function as well as some related hormonal and molecular mechanisms. As far as some studies investigated timelines of stress on spatial memory impairment [18,19], there are few reports of chronic stress studies evaluating memory function by passive avoidance test; that is hippocampus-dependent learning and memory tasks [20]. The aim of the present investigation was to assess the effects of chronic stress (before, after and during the learning process), recovery period after the stress and different timing of induced stress on learning and memory functions. Further, it aimed at studying the concentration of CORT and BDNF in the serum and the hippocampus, evaluating conversion of short-term memory to long-term memory, and investigating body weight in rats. This study, also, tried to show the correlation between these variables in experimental rats which received chronic stress.

2. Materials and methods 2.1. Experimental animals Experiments were performed on 40 male Wistar rats with an initial weight of 250–300 g and initial age of 55–70 days which were obtained from the Jondishapour Institute, Ahvaz, Iran. All experimental protocols were approved by the Ethical Committee of Isfahan University of Medical Science (Isfahan, Iran) in compliance with the “Principles of Laboratory Animal Care” and the European Community Council Directive of 24 November 1986 (86/609/EEC). Rats were maintained under light-controlled condition (12-h light/dark; lights on 07:00–19:00) in a room with a temperature of 22 ± 2 °C. Food and water were available ad libitum, except during the stress conditions. All behavioral experiments were carried out between 14:00–15:00. The experiments lasted 42 days. And, passive avoidance test was performed day 21 in all groups (Fig. 1). Rats were randomly assigned to four groups (n = 10 in each) as follows: (1) Control group (Co) in which rats were transported to the laboratory room and handled similar to the experimental animal throughout the study period receiving no special treatment, (2) Stress-Learning-Rest group (St-L-Re; Stress before learning) in which restraint stress was applied 6 h/day for 21 days, and then rats remained undisturbed for 21 days, (3) Rest-Learning-Stress group (Re-L-St; Stress after learning) in which rats had no special treatment for 21 days, and then chronic restraint stress was applied, 6 h/day for 21 days, and (4) Stress-Learning-

Stress group (St-L-St; Stress during the learning or continual stress) in which rats were under restraint stress 6 h/day for 42 days (Fig. 1). 2.2. Experimental procedures 2.2.1. Stress paradigms Rats were placed and tightly fitted in Plexiglas cylindrical restrainers for 6 h/day (8:00–14:00) for 21 or 42 days in the chronic stress model. The fact that moving or turning around was not possible for rats, while restrained, made this procedure a potent emotional stressor [21,22]. 2.2.2. Behavioral apparatus and method Evaluation of learning and memory was assessed by a step-through a passive avoidance test [23]. The passive avoidance learning (PAL) test involves cognitive memory [24]; it is a hippocampus-dependent learning and memory task [20]. Also, hippocampus-dependent learning and memory tasks are sensitive to corticosterone disruption [25]. Therefore, this paradigm was used in the current study. The passive avoidance apparatus was divided into two compartments (light and dark) which were separated by a sliding guillotine door. On day 20 of the experiment, each rat was placed in the apparatus without the electric shock for 5 min to habituate to the apparatus. On a later day (after 24 h, on day 21), a single acquisition trial was performed. During this trial rats were individually placed in the light compartment for 1 min after which the guillotine door was raised. When the animal entered the dark compartment, the door was closed and an inescapable scrambled single foot electric shock (50 Hz, 0.2 mA, 3 s) was delivered through the grid floor by an isolated stimulator. The initial latency of entrance into the dark compartment (IL, acquisition latency) was recorded. Rats with initial latency greater than 60 s were excluded from the study. Each rat underwent three trial sessions (retention), 1, 7, and 21 days after receiving the foot shock (on days 22, 28, and 42) in passive avoidance test [26]. During the probe trials, rats were placed in the light compartment again, with access to the dark compartment without any shock. The delay before entering the dark compartment from the light compartment was recorded as latency (up to a maximum of 300 s). When an animal refrained from entering the dark compartment within 300 s, the trial was terminated. Comparison of acquisition and retention after 1 day showed learning, and three trial sessions indicated retrieval of memory [27]. The passive avoidance task determined the ability of the animal to remember the delivered foot shock. Lack of entry into the dark compartment or a longer duration of stay in the light compartment indicated a positive response. 2.2.3. Assessment of serum corticosterone levels At the end of the experiments, animals were anesthetized with urethane (1.5 g/kg, i.p.) and sacrificed at 14:00–15:00 by decapitation

Fig. 1. Experimental protocol for different groups and the days on which memory acquisition and retention were tested. Co: Control group; St-L-Re: Stress-Learning-Rest; Re-L-St: Rest-LearningStress group; St-L-St: Stress -Learning-Stress group; PAL: Passive avoidance learning test.

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on day 43. Their blood samples were obtained from the trunk blood; serum was separated by centrifugation (6000 rpm, 20 min) and stored at −80 °C until analysis. The commercial enzyme-linked immunosorbent assay (ELISA) kits (DRG Co., Marburg, Germany) were used to assess the serum corticosterone levels. 2.2.4. Assessment of hippocampal corticosterone and BDNF levels Following decapitation of animals, their brains were immediately removed from skull and the hippocampi were instantly dissected and kept on dry ice. Each hippocampus was immersed in Problock™-50, EDTA free (Gold Bio Co.; USA), and phosphate buffer solution (PBS buffer, 0.01 M, pH 7.4). Indeed, this solution contained complete protease inhibitor cocktail. The hippocampi were homogenized and centrifuged in a cooled centrifuge (4 °C, 10,000 g for 20 min). The supernatant was collected and stored at −80 °C until assessment. The BDNF levels in the hippocampal homogenate were measured by BDNF ELISA kit (Promega Co.; Sweden). Similarly, the commercial ELISA kit (DRG Co., Marburg, Germany) was used to assess the hippocampal corticosterone levels. The amounts of BDNF and CORT were, respectively, determined in pg/ml and nmol/l of supernatant solution [28–30]. 2.2.5. Measurement of body weight Animals' body weights were measured on days 1, 21, and 42 of the experiment. The body weight differences [(BWDInitial = BWDay21 − BWDay1) and (BWDFinal = BWDay42 − BWday21)] were, also, evaluated. 3. Data analysis The latency of entrance to the dark compartment of the passive avoidance test (between group comparison) was analyzed using the Kruskal–Wallis nonparametric one-way analysis of variance (ANOVA), followed by a two-tailed Mann–Whitney U test. The comparisons of acquisition and retention times in 1, 7, and 21 days after electrical foot shock (within groups) were analyzed using the Friedman test, followed by a Wilcoxon signed rank test. Body weight differences, hippocampal BDNF levels, serum and hippocampal corticosterone levels were analyzed by ANOVA followed by Tukey's post-hoc test for multiple groups. In addition, Pearson's correlation analysis was performed to find the correlation between the variables. In this research, values are reported as mean ± SEM, where P b 0.05 is considered statistically significant. 4. Results 4.1. The latency of entrance to the dark compartment Statistical analysis on the initial latency (IL) data in Stress-LearningRest (St-L-Re; stress before learning) and Stress-Learning-Stress groups (St-L-St; stress during learning) revealed a significant decrease (Kruskal–Wallis; Mann–Whitney: P b 0.01) compared with the control group (Co). It indicated that stressor exposure before and during learning had interfered with learning processes (Fig. 2). Meanwhile, the IL in the Rest-Learning-Stress group (Re-L-St; stress after learning) was not significantly different from the IL in the Co group. Moreover, there was no significant difference (P N 0.05) regarding IL between the St-LRe and St-L-St groups (Fig. 2.A). In the St-L-Re group, latencies in retention trials turned to be significantly (P b 0.05, in both trials 1 and 7 days) lower than latencies in the Co group except on the trial of 21 days after receiving an electrical foot shock (Fig. 2.A). Taken together, the short and mid-term memories were shown to be significantly impaired in this group. As shown in Fig. 2.A, the latency values of all trials in the Re-L-St and St-L-St groups were significantly lower than that of the Co group (P b 0.05, P b 0.001, P b 0.01, in day 1, day 7 and day 21 trials, respectively). In the St-L-St group, the latencies of day 7 and day 21 were significantly lower (P b 0.01 and P b 0.001, respectively) than that of the St-L-Re

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group, after receiving the electrical foot shock. The latency of day 1 in this group was not significantly different (P N 0.05) from that of the St-L-Re group (Fig. 2.A). Latencies of the initial and each three trials were analyzed by the related samples to evaluate within group latency differences. In the current study, the IL vs. day 1; day 1 vs. day 7; day 7 vs. day 21; and day 1 vs. day 21 were compared (Fig. 2.B). The comparison of IL and retention trial after 1 day show learning and other trial sessions indicated memory changes. The data revealed significant differences (Friedman test; Wilcoxon: P b 0.01) between IL and latency after 1 day in all groups, indicating that learning happened in all groups and the chronic stress induced learning deficits in stressed groups, especially in the Re-L-St group (Fig. 2.B). There were no significant differences in latencies of day 1 vs. day 7, day 7 vs. day 21, and day 1 vs. day 21 after receiving the electrical foot shock in the Co group. However, in the St-L-Re, Re-L-St and St-L-St groups, a significant difference (P b 0.01) was noted upon similar comparisons (Fig. 2.B). The retention latencies of days 1, 7, and 21 showed a descent trend in all groups. This was, mainly, due to time passage, because there were no repeated shocks. This descent trend was more serious in the Re-L-St group (stress after learning) (Fig. 2.B).

4.2. Assessment of corticosterone level in serum and hippocampus Based on the ANOVA and post-hoc Tukey's results, there was no significant difference (P N 0.05) in CORT levels of both the serum and hippocampus between Co and St-L-Re groups. This suggested that the recovery period after the chronic stress reduced the CORT levels of the serum and hippocampus (Fig. 3). As shown in Fig. 3, in both the Re-L-St and St-L-St groups, there was a significant increase in CORT levels of the serum (P b 0.01, for both groups) and hippocampus, when compared with Co (P b 0.001 and P b 0.01, respectively). As shown in Fig. 3, significant differences were noted in CORT levels of the serum and hippocampus in the Re-L-St and St-L-St groups compared with the St-L-Re group (P b 0.01 and P b 0.05, respectively). Following an increase in the duration of the stress period, CORT levels of the serum and hippocampus did not significantly decrease in the St-L-St group compared with that of the Re-L-St group. It was, probably, due to increased habituation to the stress, because the St-L-St animals had undergone a longer exposure (Fig. 3). Furthermore, these data indicated that the reduction of CORT levels was more pronounced in the hippocampus than serum in all groups (Fig. 3). It indicated that although serum CORT crossed from the blood brain barrier (BBB), it limited the crossing of CORT to the brain.

4.3. Assessment of hippocampal BDNF levels The obtained results demonstrated no significant difference in the hippocampal BDNF level between the St-L-Re and Co groups (ANOVA; Tukey's test: P N 0.05). Therefore, the recovery period after the chronic stress is suggested to improve BDNF levels (Fig. 4). As shown in Fig. 4, there were significant decreases in BDNF levels in the Re-L-St and St-L-St groups as compared with the Co group (P b 0.01 and P b 0.001, respectively). Furthermore, the BDNF levels of the Re-L-St and St-L-St groups were significantly different from that of the St-L-Re group (P b 0.05 and P b 0.01, respectively). Based on the abovementioned, although hippocampal BDNF levels in the St-L-St (continual stress) and Re-L-St (stress after learning) groups were, nearly, identical, the changes of BDNF levels followed stress duration. In other words, a slightly more pronounced decline was observed in the BDNF levels with the enhancement of stress duration (as compared with the transitional stress period) (Fig. 4).

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Fig. 2. (A) Latencies to entrance to the dark compartment in the passive avoidance apparatus during memory acquisition (Initial latency; IL), and retention test, 1, 7, and 21 days after receiving electrical foot shock in different groups (between groups, n = 10). Results are expressed as mean ± SEM (Kruskal–Wallis test, Mann–Whitney U test; ⁎P b 0.05, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 when compared with the Co group; θθP b 0.01 and θθθP b 0.001 when compared with the St-L-Re group in each trial). (B) Trend line of latency before and after receiving electrical foot shock (within groups). Results are expressed as mean ± SEM (Friedman test, Wilcoxon signed ranks test; ¤¤P b 0.01, ++P b 0.01, $$P b 0.01 and ££P b 0.01 for the latency values in day 1 vs. IL, day 1 vs. day 7, day 7 vs. day 21, and day 1 vs. day 21, respectively).

4.4. Correlation between serum and hippocampal CORT levels The analysis revealed no significantly positive correlation between the serum and hippocampal CORT levels in Co and St-L-Re groups. However, significant positive correlations were found between the serum and hippocampal CORT levels in the Re-L-St (Pearson's correlation; r = 0.822, P b 0.01) and St-L-St (r = 0.703, P b 0.05) groups (Fig. 5). These findings supported the idea of using serum CORT levels as a surrogate to estimate the hippocampal CORT levels in stressed groups.

4.5. Correlation between the hippocampal CORT and BDNF levels The findings demonstrated no significant correlation between the hippocampal CORT and BDNF levels in the Co and St-L-Re groups. However, negative significant correlations were observed between the hippocampal CORT and BDNF levels in the Re-L-St (Pearson's correlation; r = − 0.847, P b 0.01) and St-L-St groups (r = − 0.681, P b 0.05)

(Fig. 6). These findings revealed the direct effect of serum CORT levels on the changes of hippocampal BDNF levels in stressed groups. 4.6. Correlations between the behavioral and physiological parameters Results of the present study demonstrated no significant correlation between the latency of day 21 and hippocampal CORT and/or the BDNF levels in all groups (not presented as a graph here). The findings suggested that multiple factors (e.g., CORT and BDNF levels, and other variables) were involved in long-term memory deficit in stressed groups. Consequently, it seems that many different mechanisms are involve in memory process. The levels of CORT and BDNF, and other factors may synergistically affect the chronic stress-induced memory deficits. 4.7. Body weight difference The initial body weight differences (BWDInitial = BWDay21 −BWDay1) in the St-L-Re and St-L-St groups, were significantly (ANOVA, Tukey's

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Fig. 3. Effects of chronic restraint stress on serum and hippocampal corticosterone (CORT) levels (nmol/l) in different groups (n = 10). Results are expressed as mean ± SEM (ANOVA test, Tukey's post-hoc test; ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 when compared with the Co group; θP b 0.05 and θθP b 0.01 when compared with the St-L-Re group).

test: P b 0.001) lower than the Co group. Meanwhile, no significant difference was observed between the Re-L-St and Co groups in terms of the BWDInitial (Fig. 7). The results indicated no significant difference between the St-LRe and Co groups with regard to the final body weight differences (BWDFinal = BWDay42 − BWDay21) (Fig. 7). As shown in Fig. 7, the BWDFinal was significantly lower in the Re-LSt- and St-L-St groups (P b 0.01 and P b 0.001, respectively) compared with the Co group. The BWDFinal in the Re-L-St and St-L-St groups, was significantly (P b 0.01) lower than that in the St-L-Re group (Fig. 7). In addition, the assessments of the BWDInitial and the BWDFinal showed that, there was a significant enhancement in BWDFinal compared with BWDInitial in the St-L-Re group (P b 0.01), indicating a severe compulsive effect of a recovery period after the chronic psychical stress on body weight gain (Fig. 7). The BWDFinal was significantly lower than the BWDInitial in the Re-L-St group (P b 0.001) (Fig. 7). The BWDFinal did

Fig. 4. Effects of chronic restraint stress on the hippocampal BDNF levels (nmol/l) in different groups (n = 10). Results are expressed as mean ± SEM (ANOVA test, Tukey's post-hoc test; ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 when compared with the Co group; θP b 0.05 and θθ P b 0.01 when compared with the St-L-Re group).

not significantly decrease compared with BWDInitial in the St-L-St group (Fig. 7). 5. Discussion Present findings demonstrated that the chronic stress caused memory deficits in all stressed groups (Fig. 2). In line with the results of this study, other studies have shown that chronic restraint stress makes deleterious impacts on learning and memory process [2,31]. According to the results of the present study, chronic restraint stress before learning (St-L-Re group) caused a severe memory deficit on days 1 and 7, but not on day 21 after the electrical foot shock (Fig. 2.A). Therefore, harmful effects of stress remained on short and mid-term memory consolidation. Hence, there was no adaptation under the chronic restraint stress until 7 days after the passive avoidance test. These results were in agreement with the previous studies [31–33]. In other words, the obtained data suggested that stressor exposure had interfered with learning and memory processes, as was expressed before where the decreased retention scores at day 7 in the stressed group that had stress before and during learning (St-L-Re and St-L-St groups) were discussed (Fig. 2). In the present investigation, long-term recovery period (21 days) after the chronic stress had beneficial effects on the improvement of memory deficit in stressed rats. Indeed, rest after stress prevented further cognitive impairment because the latency to enter the dark compartment on day 21 was, still, significantly lower than on day 1. Moreover, the levels of CORT and BDNF in the serum and hippocampus were returned to basal levels by such recovery period after the chronic stress (Figs. 3 and 4). Previous reports have demonstrated similar findings in CA3 of the hippocampus, but not basolateral amygdala on BDNF level [34]. The above observation substantiated that the recovery period after chronic stress could abrogate the effects of the chronic stress on memory impairment. In support of this view, Sapolsky et al. [35] has reported similar results in young rats. In contrast, other reports indicated that recovery for 21 days was not enough for changing behavior and other parameters after stress in female rats [36]. This difference might be related to different factors such as kind of stress, intensity and duration of exposure to stressors, behavioral test, and especially gender differences [37–40]. On the other hand, the obtained results revealed that the chronic restraint stress after learning (Re-L-St) and stress during learning

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Fig. 5. Correlation analysis of the serum and hippocampal corticosterone (CORT) levels in different groups (n = 10). Results are expressed as mean ± SEM (Pearson's correlation test; ⁎P b 0.05, ⁎⁎P b 0.01 in St-L-Re and St-L-St groups).

(St-L-St) impaired all types of short, mid and long-term memory functions, especially the mid-term memory (Fig. 2.A). In line with present results, other forms of stress have been shown to impair long-term memory [41]. In this regard, Shoji and Mizoguchi [42] reported that chronic stress enhanced the emotional and stress-related responses in animals. Other studies have demonstrated that stress is a significant factor potentially altering the brain cell properties and disturbing some critical cognitive processes including learning and memory [9], especially spatial memory function in rats [31]. In contrast with our results, Dallman [43] reported that animals were able to habituate to the chronic stress condition. In the current study, the induced chronic stress after learning had a significant effect on the enhancement of the serum and hippocampal CORT levels and on the reduction of BDNF concentration in the hippocampus (Figs. 3 and 4). Some researchers reported that the reduction of BDNF mRNA and enhancement of glucocorticoid levels might have implications for brain plasticity and behavioral changes following the stress in rat's hippocampus [44–46]. Other reports revealed that CORT levels in stressed rats were adapted after three weeks of restraint stress [14], and no changes were observed in BDNF levels in the hippocampus after four weeks of stress [47]. These differences, possibly, depend on a variety of parameters including kind of stress, the duration and intensity of stress condition [38], sex, age, strain, and the employed behavioral tests [37,38]. Accordingly, although CORT and BDNF levels are proposed to involve in memory deficits in stressed rats, the results of this study demonstrated no significant negative correlation between the latency of day 21 (the long-term memory) and hippocampal CORT and/or the BDNF

levels in the stressed group (Fig. 6). However, there was more negative correlation in the Re-L-St and St-L-St groups compared with the St-L-Re group. It seems that many different mechanisms and multiple factors, apart from CORT and BDNF levels, are involved in long-term memory deficit in stress conditions; among these factors are changes in neurotransmitters' release, neurotrophic factors, leptin, and oxidative stress [48–50]. Another possibility is that the restraint stress, as a potent of emotional stressor [21], activated the limbic system and released numerous neurotransmitters in some regions of the brain involved in the regulation of stress responses [51]. Consequently, it seems that multiple systems extensively interact with cognitive memory function during the chronic stress conditions. The present findings revealed that memory deficits were slightly modulated in the St-L-St as compared with the Re-L-St group (Fig. 2.B). Therefore, it was found that two-fold (42 days) of stress showed adaptive mechanisms in rats compared with 21 days chronic stress. Whereas, Jeong et al. [52] reported that chronic stress accelerates the onset and severity of cognitive dysfunctions. It, probably, confirms that although chronic stress may increase the brain damage in individuals and promote other neurocognitive disorders, the very long duration of the stress period has direct modulating effect on memory deficit. Hence, it is substantial for such detrimental stress effects. Since the serum and hippocampal CORT and BDNF levels were showed to be significantly enhanced and decreased, respectively, in both the Re-L-St and St-L-St groups as compared with the control group, the memory deficit in these groups may, at least partly, be attributed to the increased CORT and reduced BDNF levels (Figs. 3 and 4). Since, negative correlations were observed between the hippocampal

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Fig. 6. Correlation analysis of the hippocampal corticosterone (CORT) and BDNF levels in different groups (n = 10). Results are expressed as mean ± SEM (Pearson's correlation test; ⁎P b 0.05, ⁎⁎P b 0.01 in St-L-Re and St-L-St groups).

Fig. 7. Comparison of body weight differences (BWDInitial = BWDay21 − BWDay1 and BWD Final = BWDay42 − BWDay21) in all groups (n = 10). Results are expressed as mean ± SEM (ANOVA test, Tukey's post-hoc test; ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 when compared with the Co group; θθP b 0.01 when compared with the St-L-Re group in BWDInitial and BWDFinal, separately; ¢¢ P b 0.01, ¢¢¢P b 0.001 when BWDInitial was compared with the BWDFinal in similar groups).

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CORT and BDNF levels, it seems that CORT levels have direct effect on the changes of hippocampal BDNF levels. In line with the present results, stress is shown to increase the glucocorticoid level, and reduce the hippocampal BDNF level [44,46]. Its mechanism may be via downregulating the expression of the BDNF [45] and, as a result, leading to structural changes in the hippocampus [53]. Therefore, stress results in a range of biochemical, physiological, and behavioral changes [54] that might play a critical role in the development and maintenance of memories. According to the results of this study, hippocampal BDNF levels and serum CORT levels were nearly identical in the St-L-St (continual stress) and Re-L-St (stress after learning) groups. Moreover, a few reduction in BDNF level followed stress duration; and it had a decreased trend with enhancement of stress duration (after 42 days stress compared with 21 days stress) (Fig. 4). Whereas, there were no changes in CORT level as following stress duration (reverse response) which led to habituation phase. There was a reduction instead of increase on CORT level with enhancement of duration of stress in the St-L-St group, especially in the hippocampus (Fig. 4). It indicated the protective and adaptive effects of the brain on hippocampal CORT level in very long duration of stress. Therefore, the continual stress (more than 21 days) was shown to promote the adaptive/restorative effects on memory deficits and the CORT, but not BDNF levels. According to some investigations, when animals were, repeatedly, exposed to the same stressor, some behavioral and physiological consequences of stress exposure reduced [55], suggesting the adaptation of animals to the stimulus. Other findings showed that the hippocampal CORT concentration was less than the serum CORT level, while they had direct and significant correlation with each other in chronic stressed groups without recovery period (Fig. 5). Consistent with the present findings, Qian et al. [56] reported the synchronous rhythms of CORT levels between plasma and tissue by using microdialysis technique. The findings of this study indicated that homogenized technique (in vitro) showed similar results with microdialysis technique (in vivo), although the hippocampal CORT level was less than the serum CORT level (Fig. 3) through homogenized technique. It indicated the role of the blood barrier brain (BBB). It seems that although the serum CORT level crossed from the BBB, this barrier limited the access of CORT to the brain [57] and/or the regulation of glucocorticoid receptors in the hippocampus subject to the consequent changes [58]. On the other hand, it indicated that a homogenized technique is an easier method for the investigation of changes in CORT levels in the hippocampus. Since the peripheral CORT concentration could change the central CORT levels, the evaluation of CORT levels in serum may be a surrogate to estimate the concentration of hippocampal CORT levels. In addition, high circulating levels of corticosterone were observed in all groups that can be due to anesthesia. The anesthesia is well known for its effect on activating the hypothalamic–pituitary– adrenal axis [59–63]. According to the findings of this piece of research, although both stressed and unstressed rats had similar weights at the beginning of the study, body weight declined following the emotional stress conditions (Fig. 7). Contrary to present findings, no weight loss has been reported as a result of the restraint stress [64]. The mechanism of weight loss in the restraint stress conditions may be decreased food intake and fatty mass. This can be well-explained by the stress-induced release of catabolic hormones such as glucocorticoids [54] and serum leptin [65] based on the intensity of the stressor [54]. As stated earlier, other factors such as leptin are involved in body weight changes and memory deficit [66]. Unfortunately, the serum leptin levels were not estimated in this study. In the current study, when BWDFinal was compared with BWDInitial, it was noted that the recovery period significantly enhanced the body weight gain in stressed rats; whereas body weight loss partially continued in the St-L-St group. The increased food intake in the stressed group during the recovery period allowed the animals to regain body weight. Consistent with this finding, Martí et al. [54] reported that factors such as the severity and duration of the chronic exposure to

stressors determine the magnitude of the response of various physiological variables to the chronic stress. In conclusion, current findings confirmed that the chronic stress, especially the stress after learning, not only plays an important role in memory function, but also changes biochemical, physiological, and behavioral factors. It seems that the time of induced chronic stress and its duration are important factors in neurobiology responses. Very long duration of restraint stress (over 21 days) probably promotes adaptive effects on memory. It, possibly, modulates memory deficit, and CORT, but not BDNF levels in rats. The decrease in BDNF level directly continues with duration of stress; hence, there is no adaptation trend. Therefore, it can be concluded that the changes of decreased BDNF are slower and more permanent than the CORT changes. Furthermore, since the serum CORT crosses the BBB, the peripheral CORT concentrations may directly affect the central CORT levels. Moreover, chronic stress' condition led to a notable enhancement in the CORT levels as well as a reduction in BDNF levels. It seems that although changes in CORT and BDNF levels may be helpful in investigating the impact of chronic stress on memory, they are not sufficient variables to be considered. Therefore, additional works are required to further explain the possible mechanism(s) involved in stress consequences. Evaluating other factors which are possibly involved in memory processes is highly suggested. Acknowledgments The authors would like to thank Dr. Mehdi Hedayati and Dr. Shaghayegh Haghjoo Javanmard for their valuable assistance. Conduction of the present research was made possible through the supports received from Isfahan University of Medical Sciences (390022), Isfahan, Iran. References [1] J.L. McGaugh, B. Roozendaal, Role of adrenal stress hormones in forming lasting memories in the brain, Curr. Opin. Neurobiol. 12 (2002) 205–210. [2] C. Sandi, M.T. Pinelo-Nava, Stress and memory: behavioral effects and neurobiological mechanisms, Neural Plast. 2007 (2007) 1–20. [3] T. Falkenberg, A.K. Mohammed, B. Henriksson, H. Persson, B. Winblad, N. Lindefors, Increased expression of brain-derived neurotrophic factor mRNA in rat hippocampus is associated with improved spatial memory and enriched environment, Neurosci. Lett. 138 (1992) 153–156. [4] K. Yamada, T. Nabeshima, Brain-derived neurotrophic factor/TrkB signaling in memory processes, J. Pharmacol. Sci. 91 (2003) 267–270. [5] M.J. Schaaf, E.R. De Kloet, E. Vreugdenhil, Corticosterone effects on BDNF expression in the hippocampus. Implications for memory formation, Stress 3 (2000) 201–208. [6] C. Cunha, R. Brambilla, K.L. Thomas, A simple role for BDNF in learning and memory? Front. Mol. Neurosci. 3 (2010) 1–14. [7] A. Gomez-Palacio-Schjetnan, M.L. Escobar, Neurotrophins and synaptic plasticity, Curr. Top. Behav. Neurosci. 15 (2013) 117–136. [8] J.J. Kim, R.A. Rison, M.S. Fanselow, Effects of amygdala, hippocampus, and periaqueductal gray lesions on short- and long-term contextual fear, Behav. Neurosci. 107 (1993) 1093–1098. [9] J.J. Kim, D.M. Diamond, The stressed hippocampus, synaptic plasticity and lost memories, Nat. Rev. Neurosci. 3 (2002) 453–462. [10] B.S. McEwen, D. Albeck, H. Cameron, H.M. Chao, E. Gould, N. Hastings, et al., Stress and the brain: a paradoxical role for adrenal steroids, Vitam. Horm. 51 (1995) 371–402. [11] H. Eichenbaum, How does the brain organize memories? Science 277 (1997) 330–332. [12] V. Luine, M. Villegas, C. Martinez, B.S. McEwen, Repeated stress causes reversible impairments of spatial memory performance, Brain Res. 639 (1994) 167–170. [13] C.D. Conrad, L.A. Galea, Y. Kuroda, B.S. McEwen, Chronic stress impairs rat spatial memory on the Y maze, and this effect is blocked by tianeptine pretreatment, Behav. Neurosci. 110 (1996) 1321–1334. [14] G.E. Wood, L.T. Young, L.P. Reagan, B.S. McEwen, Acute and chronic restraint stress alter the incidence of social conflict in male rats, Horm. Behav. 43 (2003) 205–213. [15] B.S. McEwen, Physiology and neurobiology of stress and adaptation: central role of the brain, Physiol. Rev. 87 (2007) 873–904. [16] S. Dronjak, L. Gavrilović, Activity of pituitary–adrenal axis in rats chronically exposed to different stressors, Acta Vet. 55 (2005) 121–129. [17] D.L. Pitman, J.E. Ottenweller, B.H. Natelson, Plasma corticosterone levels during repeated presentation of two intensities of restraint stress: chronic stress and habituation, Physiol. Behav. 43 (1988) 47–55. [18] K.J. McLaughlin, J.L. Gomez, S.E. Baran, C.D. Conrad, The effects of chronic stress on hippocampal morphology and function: an evaluation of chronic restraint paradigms, Brain Res. 1161 (2007) 56–64.

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