Journal of Neuroscience Research 87:831–843 (2009)

Enriched Environment Restores Hippocampal Cell Proliferation and Ameliorates Cognitive Deficits in Chronically Stressed Rats J. Veena, B. N. Srikumar, K. Mahati, V. Bhagya, T. R. Raju, and B. S. Shankaranarayana Rao* Department of Neurophysiology, National Institute of Mental Health and Neuro Sciences, Bangalore, India

Adult neurogenesis, particularly in the subgranular zone, is thought to be linked with learning and memory. Chronic stress inhibits adult hippocampal neurogenesis and also impairs learning and memory. On the other hand, exposure to enriched environment (EE) is reported to enhance the survival of new neurons and improve cognition. Accordingly, in the present study, we examined whether short-term EE after stress could ameliorate the stress-induced decrease in hippocampal cell proliferation and impairment in radial arm maze learning. After restraint stress (6 hr/day, 21 days) adult rats were exposed to EE (6 hr/day, 10 days). We observed that chronic restraint stress severely affected formation of new cells and learning. Stressed rats showed a significant decrease (70%) in the number of BrdU (5-bromo20 -deoxyuridine)-immunoreactive cells and impairment in the performance of the partially baited radial arm maze task. Interestingly, EE after stress completely restored the hippocampal cell proliferation. On par with the restoration of hippocampal cytogenesis, short-term EE after stress resulted in a significant increase in percentage correct choices and a decrease in the number of reference memory errors compared with the stressed animals. Also, EE per se significantly increased the cell proliferation compared with controls. Furthermore, stress significantly reduced the hippocampal volume that was reversed after EE. Our observations demonstrate that short-term EE completely ameliorates the stress-induced decrease in cell proliferation and learning deficit, thus demonstrating the efficiency of rehabilitation in reversal of stress-induced deficits and suggesting a probable role of newly formed cells in the effects of EE. VC 2008 Wiley-Liss, Inc. Key words: neurogenesis; enriched environment; partially baited radial arm maze task; hippocampal volume; cell proliferation

The generation of new neurons in the adult hippocampus is shown in a wide range of species, including mammals (Altman, 1969; Kaplan and Hinds, 1977; ' 2008 Wiley-Liss, Inc.

Cameron et al., 1993; Doetsch and varez-Buylla, 1996; Eriksson et al., 1998). The new cells produced in the subgranular zone (SGZ) attain functional significance on integrating into the existing neural circuit of the dentate gyrus (DG) (van Praag et al., 2002). The role of hippocampus in learning and memory is well known, and neurogenesis could possibly participate in hippocampal functions, especially those related to learning and memory (Leuner et al., 2006). Studies show that learning and neurogenesis in the hippocampus (Gould et al., 1999; Madsen et al., 2003) and olfactory bulb (Gheusi et al., 2000; Rochefort et al., 2002) have a positive effect on each other. Stress is known to alter the normal physiological homeostasis. Exposure to chronic stress leads to long-term deleterious effects including dendritic atrophy and cell death associated with memory impairments and behavioral abnormalities (Luine et al., 1994; Sunanda et al., 1995, 1997, 2000a; Sapolsky, 1996; McEwen, 2000; Vyas et al., 2002; Pawlak et al., 2005; Govindarajan et al., 2006; Ramkumar et al., 2008). Recent studies from our laboratory showed impaired performance of The first two authors contributed equally to this article. B.N. Srikumar’s current address is Laboratoire Physiologie Cellulaire de la Synapse, CNRS Universite´ Bordeaux 2, 33077 Bordeaux, Cedex, France. Contract grant sponsor: Research Fellowship from Council of Scientific and Industrial Research (CSIR), Government of India (to J.V.). Contract grant sponsor: Research grant from Department of Science and Technology (DST), Government of India; (to B.S.S.); Contract grant sponsor: Research Fellowship from University Grants Commission (UGC), Government of India (to K.M). *Correspondence to: B. S. Shankaranarayana Rao, Department of Neurophysiology, National Institute of Mental Health and Neuro Sciences, PB #2900, Hosur Road, Bangalore, 560 029, India. E-mail: [email protected] Received 29 May 2008; Revised 31 July 2008; Accepted 27 August 2008 Published online 11 November 2008 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jnr.21907

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stressed rats in a partially baited radial arm maze (RAM) task (Srikumar et al., 2006, 2007). The stressful events detrimental to hippocampal integrity are found to affect neurogenesis and learning (Gould et al., 1997; Malberg et al., 2000; Tanapat et al., 2001; Czeh et al., 2002; Pham et al., 2003). An enriched environment (EE) provides increased opportunities for sensory, social, cognitive, and physical stimulations (Rosenzweig et al., 1962; Rosenzweig, 1966; van Praag et al., 2002). The favorable changes in the brain after exposure to EE include increased hippocampal thickness with both increased dendritic arborization and number of glial cells (Walsh et al., 1969). Further, EE is shown to enhance spatial memory in the Morris water maze and increase synaptic strength in the perforant pathway (Kempermann et al., 1997). EE not only modifies brain chemistry and physiology, but also enhances adult neurogenesis by increasing the survival of newborn neuronal cells in both mice and rats (Kempermann et al., 1997, 1998, 2000; Nilsson et al., 1999; Hattori et al., 2007). These new neurons in the DG are shown to be involved in the enhanced longterm memory after EE (Bruel-Jungerman et al., 2005). In addition to enhancing the brain’s normal structure and function, EE is ameliorative of motor dysfunctions after focal ischemia in mice (Nygren and Wieloch, 2005). Furthermore, EE reversed the aging-induced changes including neurogenesis in the DG and glutamate and GABA (gamma-aminobutyric acid) levels in the CA3 area of the hippocampus (Segovia et al., 2006), reduced Ab levels and amyloid deposition in transgenic mice (Lazarov et al., 2005), and improved motor behavior in 6-OHDA rat model of Parkinson’s disease (Steiner et al., 2006). Thus, although stress is detrimental to learning, EE favors learning and memory. Two earlier studies demonstrate that either prenatal or early life stress-induced impairment of hippocampal long-term potentiation and learning in the Morris water maze are reversed by EE (Cui et al., 2006; Yang et al., 2007). And although stress on a larger scale is deleterious to neurogenesis, EE is found to augment the proliferation and survival of new cells. However, whether EE can reverse the stressinduced impaired hippocampal cell proliferation is unknown. Further, the effects of EE on chronic restraint stress-induced learning impairment in the RAM task have not been examined thus far. Consequently, in the present study, we have evaluated the effects of EE (6 hr/ day, 10 days) after 21 days of restraint stress on hippocampal cell proliferation, DG and hippocampal volumes, and performance in the partially baited RAM task. MATERIALS AND METHODS Subjects and Experimental Groups Adult male Wistar rats (200–220 g, 2–2.5 months old) obtained from Central Animal Research Facility; National Institute of Mental Health and Neuro Sciences, Bangalore, were used for all experiments. Rats were housed three per cage in polypropylene cages (35.5 cm long 3 22.5 cm wide 3 15

cm high) in a temperature- (25 6 28C), humidity- (50–55%), and light- (12 hr light–dark cycle) controlled environment with access to food and water ad libitum except during the periods of stress. The experiments were carried out in accordance with the National Institute of Health Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research (2003), and our institutional animal ethics committee approved the experimental protocols. All efforts were made to minimize both the suffering and the number of animals used. The animals were randomly divided into five groups: normal control (n 5 4 for cell proliferation and n 5 12 for behavior), stress (n 5 6 for cell proliferation and n 5 9 for behavior studies), stress (no gap) (n 5 4 for cell proliferation studies and n 5 9 for behavior studies), environment enriched (EE) (n 5 4 for cell proliferation studies and n 5 13 for behavior studies), and stress 1 environment enrichment (stress 1 EE) (n 5 4 for cell proliferation studies and n 5 9 for behavior studies). Normal control rats did not undergo any stress or EE. The stress group was subjected to 21 days of restraint stress followed by 10 days of standard housing. In the stress (no gap) group, immediately after the cessation of stress, cell proliferation or RAM learning was assessed. The EE group of animals was subjected to enrichment per se for 10 days. In the stress 1 EE group, rats were exposed to 21 days of restraint stress followed by exposure to 10 days of EE housing. For the cell proliferation analysis, a single dose of BrdU (5-bromo-20 -deoxyuridine) (300 mg/kg) was administered, and rats were killed 2 hr later. The stress and stress 1 EE groups underwent BrdU administration and were killed on the 32nd day from the start of stress, and the EE group underwent the same on the 11th day from the initiation EE. For the behavioral study, a separate set of animals were subjected to RAM task after the stress and enrichment protocols. Restraint Stress In the stress group, rats were encaged in rodent restrainers for 6 hr/day for 21 days as described earlier (Shankaranarayana Rao et al., 2001; Srikumar et al., 2006, 2007; Ramkumar et al., 2008). The restraint tubes were 12.5 cm long 3 5.5 cm wide 3 6.0 cm high and made of wire mesh on a wooden support. This form of restraint stress produces gastric ulcers and increases the adrenal weights (Sunanda et al., 1997, 2000b) and plasma corticosterone levels (Luine et al., 1996). Environmental Enrichment The animals exposed to an EE (6 hr/day for 10 days) had access to objects such as toys, wooden pieces of different shapes, rearrangeable tunnels, and pipes that stimulated their exploratory behavior and permitted social interaction (6 to 10 rats per cage as opposed to 3 in standard conditions). The cage used for the enrichment protocol is similar to the one used in a previous study from our laboratory (Bindu et al, 2005). It was a specially designed cage 102 cm long 3 64 cm wide by 3 61 cm high, with the walls made of metal wire mesh and the bottom a wooden platform. The objects were rearranged every day, and different objects were placed on alternate days for novelty. Food and water were available ad libitum during the Journal of Neuroscience Research

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period of enrichment. A sliding door was provided for replacing the bedding material and placing the rats. Evaluation of Cell Proliferation in the Hippocampus BrdU injections. BrdU (Sigma, St. Louis, MO) was prepared in saline containing 0.007 M NaOH to a concentration of 37.5 mg/ml. To examine the proliferation of precursor cells, rats were given a single injection of BrdU (300 mg/kg, i.p.) and killed 2 hr after the injection. This is a sufficient amount of time for cells in S phase to incorporate BrdU but not to complete mitosis (Packard et al., 1973; Cameron and McKay, 2001; Coskun and Luskin, 2002). Histological procedure. Two hours after the BrdU administration, the animals were transcardially perfused with cold saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer under a high dose of anesthesia. The brains were postfixed in paraformaldehyde for 48 hr. For stereological analysis, serial coronal sections of the brains (
40 lm) were taken through the entire hippocampus (approximately 21.8 mm to 25.8 mm bregma) (Paxinos and Watson, 1986) on a vibratome (Leica, Wetzlar, Germany), and every sixth section was processed for immunohistochemistry. Immunohistochemistry. Free-floating sections were incubated for 2 hr in 50% formamide/23 saline sodium citrate (SSC) at 658C, followed by washing with 23 SSC buffer. Sections were then incubated in 1:1 phosphate-buffered saline (PBS)/methanol containing 3% H2O2 for 30 min to eliminate endogenous peroxidases. After washing in PBS, sections were incubated in 2 N HCl for 30 min and washed thoroughly with borate buffer (pH 8.5). After blocking with 3% normal horse serum in 0.1 M PBS (pH 7.4), sections were incubated with mouse anti-BrdU (1:200; Sigma Aldrich, St. Louis, MO) overnight at 48C. Sections were then incubated for 3 hr with biotinylated universal secondary antibody followed by amplification with an avidin–biotin complex (Vector Laboratories Inc., Burlingame, CA), and cells were visualized with 3,30 -diamino benzidine tetrahydrochloride (Sigma Aldrich) (Malberg et al., 2000). Cell counting. Quantification of bromodeoxyuridine-immunoreactive (BrdU-ir) cells within the DG was performed by using a modified unbiased stereology protocol (Peterson, 1999, 2004; Malberg et al., 2000). Sections were coded and quantified by an experimenter blinded to the code. Sections spanned the entire rostrocaudal extent of the hippocampus, and every sixth hippocampal section was processed for quantification. BrdU-ir cells were counted in the subgranular zone/granule cell layer (SGZ/GCL) and in the hilar region of the hippocampus. The SGZ includes the layer lying at the interface between the GCL and hilus and the newly formed cells lying in the two-nucleus-wide band below the apparent border between the GCL proper, and the hilus was considered to be the cells in SGZ (Malberg et al., 2000; Kempermann et al., 2003). Counting of cells was done at 403 with a light microscope (Olympus BX51, Tokyo, Japan), omitting cells in the outermost focal plane. The total number of BrdU-ir cells in the SGZ/GCL and hilus was estimated by multiplying the total number of BrdU-ir cells per SGZ/GCL and hilus counted from every sixth section by the section peJournal of Neuroscience Research

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riodicity (6) and reported as the total number of BrdU-ir cells per region (Malberg et al., 2000). Evaluation of Hippocampal and DG Volume Volume of the DG and the entire hippocampus was calculated by unbiased stereology by Stereo Investigator (MBF Bioscience, Microbrightfield Inc.) software. Coronal sections (40 lm) through the entire extent of the hippocampus were taken in a vibratome (Leica, Wetzlar, Germany), and every sixth section was stained with cresyl violet. Images of the hippocampus were captured by a video camera (Optronics, Microfire) installed in an Olympus BX51 microscope interfaced with software (Stereo Investigator, MBF Bioscience, Microbrightfield, Inc.). Images of the hippocampus were displayed onto a monitor, and its boundaries were outlined for area measurement. The boundaries of the DG and hippocampus were defined in accordance with the atlas of Paxinos and Watson (1986). Cavalieri’s principle was used to calculate the DG and hippocampal volumes. A 50-lm grid was drawn across the entire section, and the grid points lying within the contour were selected. If the grid points fell onto the outline, the points were selected if their right upper corner was located inside the outline. The program was used to estimate the area of the contours on the basis of the number of selected grid points. This process was repeated on every sixth section of the entire rostrocaudal extent of the hippocampus, with the software totaling the volume to yield the final volume. The total volume of the DG or the hippocampus was obtained by multiplying the final volume by 6. Evaluation of Behavior in the RAM Learning and memory in the RAM were assessed as described earlier (Devi et al., 2003; Srikumar et al., 2006, 2007). The eight-arm radial maze (RAM) consisted of a computer monitored maze (Columbus Instruments, Ohio), with equally spaced arms (42 cm long 3 11.4 cm wide 3 11.4 cm high) radiating from an octagonal central platform, and the maze was kept elevated 80 cm off the ground. Two days before the beginning of the training in the RAM task, the rats were maintained on a restricted diet, and body weight was maintained at 85% of their free feeding weight, with water available ad libitum. Acclimatization. To facilitate familiarization of the maze to the rats, they were subjected to two sessions of acclimatization on consecutive days during which all the arms were baited and rats were allowed to explore the maze for 10 min. Acquisition. Ethanol (70%) was used to thoroughly clean the entire maze before each trial to eliminate any olfactory cues, and four of the arms (2, 3, 6, and 8) were baited with a chocolate reinforcement (Kellogg’s Planets and Stars, Kellogg India Ltd., Mumbai, India). The rat was placed in the center of the octagon and was allowed a free choice of the arms. An arm choice was recorded when a rat ate bait or reached the end of an arm. Only the first entry into the baited arm was recorded as a correct choice, and the maze arms were not rebaited within a particular trial. The trial continued until the rat entered all the four baited arms or 5 min had

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Fig. 1. Representative photomicrographs of coronal sections through the dentate gyrus of the hippocampus of rats showing BrdU-ir cells in normal control (A), stress (B), EE (C), and stress 1 EE (D) groups. Stress, rats subjected to 21 days of restraint stress followed by 10 days of standard housing; EE, rats exposed to 10 days of enriched environment; stress 1 EE, rats subjected to 21 days of restraint stress

followed by exposure to 10 days of enriched environment. Note the presence of BrdU-ir cells in the subgranular zone, the granule cell layer and within the hilus. Scale bars 5 10 lm in B; 20 lm in the inset. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

elapsed. At the end of the trial, the rat was returned to the home cage and was given the second trial after an intertrial interval of 1 hr. Training was continued until the rats attained the criteria of 80% correct choice (at least four correct entries out of five). Ten days after acquisition, rats were evaluated for retention of the task. Rats were given two trials, and the average was taken for analysis. Evaluation criteria. Data from four trials were averaged and expressed as blocks. The data were analyzed for percentage correct choice, reference, and working memory errors. An entry into an unbaited arm was considered a reference memory error, and any reentry was considered as a working memory error. A reentry into a baited arm or an unbaited arm was considered as working memory error correct or working memory error incorrect, respectively.

havioral experiments. Either two-way analysis of variance (ANOVA) or one-way ANOVA followed by Tukey’s or least significant difference post hoc test was used to compare the means. P < 0.05 was considered statistically significant.

Statistics Data are expressed as mean 6 standard error of the mean (SEM). The number of rats in each group was 4–6 for cell proliferation and volume experiments and 9–12 for be-

RESULTS Exposure to EE Reverses Chronic Restraint Stress-induced Decrease in Hippocampal Cell Proliferation The effect of chronic restraint stress on the proliferation of adult hippocampal progenitors was examined by the mitotic marker BrdU. After 21 days of restraint stress, the number of newly formed cells was significantly reduced in the SGZ/GCL (t8 5 11.47; P < 0.001) and hilus (t8 5 7.86; P < 0.001) compared with normal control (Fig. 1). Chronic exposure to stress decreased total number of BrdU-ir cells in the DG of adult animals by approximately 70% when compared with the normal control (normal: 1963 6 119; stress: 564 6 66.54). To assess whether there was any spontaneous recovery in Journal of Neuroscience Research

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TABLE I. Effect of Stress and EE on Dentate Gyrus and Total Hippocampal Volume in Adult Rats* Groups Normal control Stress EE Stress 1 EE

Dentate gyrus volume (mm3) 21.94 20.24 21.80 25.29

6 6 6 6

0.97 0.99 0.89 0.43#,***

Hippocampal volume (mm3) 73.64 63.79 73.01 83.99

6 6 6 6

1.8 3.65# 4.39 1.96#,***

*Stress: rats subjected to 21 days of restraint stress followed by 10 days of standard housing (n 5 6). EE, rats exposed to 10 days of enriched environment (n 5 6); stress 1 EE, rats subjected to 21 days of restraint stress followed by exposure to 10 days of enriched environment (n 5 5). Note a reduction in the hippocampal volume with no effect on the dentate gyrus volume in stressed rats and the reversal in the stress 1 EE group. Data are expressed as mean 6 SEM. # P < 0.05 vs. normal control (n 5 6). *** P < 0.001 vs. stress. One-way ANOVA followed by LSD post hoc test.

levels (Fig. 2). Also, after EE, the total number of BrdU-ir cells in the DG was restored to normal levels (stress 1 EE: 1849.5 6 115.44).

Fig. 2. Effect of chronic restraint stress and exposure to enriched environment on the cell proliferation in SGZ/GCL (A) and hilus (B). Stress (no gap), rats subjected to 21 days of restraint stress and immediately killed on the 22nd day (n 5 4); stress, rats subjected to 21 days of restraint stress followed by 10 days standard housing (n 5 6); EE, rats exposed to 10 days of enriched environment (n 5 4); stress 1 EE, rats subjected to 21 days of restraint stress followed by exposure to 10 days of enriched environment (n 5 4). Data are expressed as mean 6 SEM. #P < 0.05, ###P < 0.001 vs. normal control (n 5 4), ***P < 0.001 vs. stress; one-way ANOVA followed by Tukey’s post hoc test.

the 10-day period, cell proliferation was evaluated immediately after the cessation of stress in a separate group of animals. Interestingly, there was no difference in cell proliferation between the two groups in the SGZ/GCL (stress followed by 10 days of standard housing: 402 6 45.16; stress without gap: 399 6 61.99; t8 5 0.04, P > 0.05; Fig. 2A). Similarly, in the hilus, there was no significant difference between the two groups (stress followed by 10 days of standard housing: 100.5 6 16.96; stress without gap: 75 6 15.78; t8 5 0.95, P > 0.05; Fig. 2B). After 21 days of restraint stress, the animals were subjected to enrichment (6 hr/day for 10 days) and killed 2 hr after BrdU injection. Analysis of BrdU-ir cells in the DG of animals subjected to enrichment after stress showed a complete restoration of stress-induced decrease in cell proliferation the SGZ/GCL (F4,17 5 65.12; P < 0.001) and hilus (F4,17 5 37.18 P < 0.001) to normal Journal of Neuroscience Research

Exposure to EE Per Se Increases the Number of Newly Formed Cells in the DG In the animals that were exposed to EE (6 hr/day, 10 days) per se, there was a significant increase (32%) in the number of proliferating cells in the SGZ/GCL (F4,17 5 65.12; P < 0.001) but not in the hilus when compared with the normal control reared under standard conditions (Fig. 2). Effect of Stress and EE on the DG and Total Hippocampal Volumes Twenty-one days of restraint stress did not alter the volume of the DG. EE (6 hr/day, 10 days) after stress notably increased the volume of the DG (F3,18 5 5.42, P < 0.001). However, EE per se did not significantly alter the volume of the DG (Table I). Restraint stress significantly decreased the hippocampal volume compared with the normal control with a reduction of 13.37% in the stressed rats. The animals subjected to 10 days of EE after stress showed a significant increase (F3,19 5 6.12, P < 0.001) in the volume of the hippocampus. Not only was the volume restored when compared with the stressed animals, but it was significantly (P < 0.05) more than their normal counterparts. However, EE per se did not alter the volume of the hippocampus and was comparable to normal levels (Table I). Stress-induced Impairment in RAM Learning Is Reversed by Exposure to EE Rats subjected to restraint stress for 21 days showed impaired acquisition of the partially baited RAM task. The percentage of correct choice to obtain the food pellet from the end of arms was 67.73 6 3.48 in block

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Fig. 3. Effect of stress and exposure to enriched environment on percentage correct choice (mean 6 SEM) in a partially baited RAM task. A: Acquisition of the RAM task across trials (expressed as block of 4 trials; see methods for details). B: Performance in blocks 7 and 8. Groups are as described in Figure 1. #P < 0.05, ###P < 0.001 vs. normal control, ***P < 0.001 vs. stress; two-way ANOVA with repeated measures on one factor followed by Tukey’s post hoc test. Normal control (n 5 12), stress (no gap, n 5 9), stress (n 5 9), EE (n 5 13), and stress 1 EE (n 5 9).

8 with a 24.22% decrease compared with the normal control (Fig. 3A,B). EE after stress restored RAM learning (Fig. 3A,B). Two-way ANOVA with repeated measures on one factor revealed a significant interaction between the groups and learning (F28,315 5 2.87; P < 0.001) and a significant effect of the groups (F4,315 5 5.84; P < 0.001). Similar to the cell proliferation experiments, to rule out any spontaneous recovery during the 10 days of EE in the stressed animals, another group of rats was subjected to the RAM task immediately after cessation of stress. There was no difference in performance between the stress and the stress (no gap) groups (Fig. 3A,B). Although the performance of the stress 1 EE group was better compared with the stress from the fourth block onward, it was statistically significant (P < 0.001) in the seventh and eighth blocks and was comparable to that of the normal control (Fig. 3B). The performance of the group of rats exposed to EE per se was comparable to the normal control animals (Fig. 3A,B). Reversal of Reference Memory Impairment by EE An entry into an unbaited arm was considered a reference memory error. Rats subjected to chronic restraint stress showed an increase in the number of ref-

Fig. 4. Effect of stress and exposure to enriched environment on the number of reference memory errors (mean 6 SEM) in a partially baited RAM task. A: Acquisition of the RAM task across trials (expressed as block of four trials; see Methods for details). B: Performance in blocks 7 and 8. Groups are as described in Figure 3. #P < 0.05, ##P < 0.01, ###P < 0.001 vs. normal control, **P < 0.01 vs. stress; two-way ANOVA with repeated measures on one factor followed by Tukey’s post hoc test.

erence memory errors compared with the normal control (Fig. 4A,B). On an average from the block 5 onward, there was a 114% increase in the number of reference memory errors in the stress group in comparison to the normal control. There was a significant effect of the group on the number of reference memory errors (F4,315 5 4.87; P < 0.01). Similar to the percentage correct choice, there was no significant difference between the stress and stress (no gap) groups (P > 0.05). Exposure to 10 days of EE after stress significantly restored the reference memory in stressed rats. In the block 7 and 8, the number of reference memory errors in the stress 1 EE group was significantly (P < 0.01) less compared with the stress group and was comparable to the normal control (Fig. 4B), while EE per se did not affect the reference memory. In contrast to reference memory, stress and EE did not affect working memory. Although the number of correct working memory errors was less in the EE group Journal of Neuroscience Research

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during the initial days of training, it was comparable to the other groups in the eighth block. The number of correct working memory errors in the normal control, stress, stress (no gap), EE and stress 1 EE groups was 0.16 6 0.06, 0.23 6 0.08, 0.18 6 0.06, 0.15 6 0.05 and 0.06 6 0.04, respectively in the eighth block. Similarly, the number of incorrect working memory errors was not statistically significant between groups and was zero for all the groups in the block 8. Effect of EE on the Retention of the RAM Task Because EE reversed the stress-induced deficit in learning, we wanted to examine whether the effects would be limited to acquisition or extend to long-term memory formation. In the retention test, performance of the stress 1 EE rats was comparable to the normal controls and there was a statistically significant difference in the percent correct choice (F4,44 5 8.63; P < 0.001; Fig. 5A) and the number of reference memory errors (F4,44 5 12.98; P < 0.001; Fig. 5B) between the stress and stress 1 EE groups. However, the number of correct and incorrect working memory errors was zero in all the groups in the retention test. DISCUSSION In the present study, we report that 21 days of restraint stress decreases hippocampal cell proliferation in adult male Wistar rats and impairs learning in the partially baited RAM, a hippocampal-dependent spatial memory task. We also show a decrease in volume of the hippocampus with no significant change in the DG volume. EE (6 hr/day, 10 days) restored the cell proliferation and hippocampal volume, and ameliorated stressinduced deficit in the RAM performance. Interestingly, animals subjected to enrichment after stress showed increased DG volume compared with normal control and stressed rats. A large body of evidence has reported that stress decreases neurogenesis in the DG of the hippocampus. Stress-induced inhibition of cell proliferation in the DG appears to occur in a wide range of manipulations from physical stressors like resident-intruder stress (Gould et al., 1998), foot shock (Malberg and Duman, 2003; Vollmayr et al., 2003), restraint stress (Pham et al., 2003; Bain et al., 2004), cold immobilization, cold swim (Heine et al., 2004), and psychological stressors including subordination stress (Gould et al., 1997), isolation (Dong et al., 2004) and predator odor (Tanapat et al., 2001; Falconer and Galea, 2003; Mirescu et al., 2004). Further, stress is shown to differentially affect the survival and differentiation of the newly formed cells. Although a few studies support suppression in new neuron production (Czeh et al., 2002; Pham et al., 2003; Westenbroek et al., 2004), others report enhanced survival and unaffected neuronal maturation (Tanapat et al., 2001; Malberg and Duman, 2003) after stress. Our results, in accordance with previous studies, show a significant Journal of Neuroscience Research

Fig. 5. Effect of stress and exposure to enriched environment on percentage correct choice (A) and number of reference memory errors (B) in the retention test of the RAM task. Ten days after the last day of the acquisition of the RAM task, rats were subjected to two trials, and the results were averaged. Groups are as described in Figure 3. Data are presented as mean 6 SEM. #P < 0.05, ##P < 0.01, ###P < 0.001 vs. normal control, **P < 0.01, ***P < 0.001 vs. stress; one-way ANOVA followed by Tukey’s post hoc test.

reduction in the hippocampal cell proliferation after chronic restraint stress. EE increases the thickness of the hippocampus (Walsh et al., 1969), dendritic arborization (Fiala et al., 1978; Ip et al., 2002) and the number of glial cells (Walsh et al., 1969). EE experience resulted in larger synapses (Rosenzweig and Bennet, 1996) and changes in spine density (Kolb and Whishaw, 1998). It is shown to improve spatial memory in Morris water maze and also increase the synaptic strength in the perforant pathway (Kempermann et al., 1997). In addition to all these changes, studies in the last few years have shown that enrichment can increase the survival of newborn cells in the hippocampus (Kempermann et al., 1997; Kempermann and Gage, 1999; Nilsson et al., 1999). These studies report that EE did not significantly affect the proliferation but had a survival-promoting effect selective to neurons (Will et al., 2004). However, in our study, short-term EE exposure of 6 hr/day for 10 days showed a significantly increased proliferation in the SGZ/GCL region when compared with animals reared in the standard laboratory conditions. On similar lines, increase in cell proliferation and neurogenesis is seen in adult DG of

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mice subsequent to enrichment (Kempermann and Gage, 1999). Young et al. (1999) report a 45% reduction in spontaneous apoptotic cell death in the rat hippocampus after enrichment. Also, it induces expression of glialderived neurotrophic factor and brain-derived neurotrophic factor (BDNF), and increases phosphorylation of the transcription factor cyclic-AMP response element binding protein (CREB), indicating transcription factor activation and induction of growth factor expression to bring about the resistance to insults and spontaneous apoptosis (Young et al., 1999). Failure to up-regulate BDNF levels accompanied the lack of a neurogenic response in enriched BDNF heterozygous mice emphasizing the requirement of BDNF for the environmental induction of neurogenesis (Rossi et al., 2006). In the present study, the objects in the enrichment cage were rearranged every day. The novelty and complexity of the environment, as well as changes to it, could trigger the neurogenic response in the animals exposed to EE, and similar mechanisms recruiting the trophic support could be speculated to bring about the increase in cell proliferation seen in our study and need to be addressed in the future studies. A few studies have explored the effect of enrichment on reversal of stress-induced deficits. For the first time, we report here that the restraint stress-induced decrease in hippocampal cell proliferation is reversed by short-term EE and was associated with behavioral recovery. Cui et al. (2006) studied the effect of EE on early life stress and report that EE on postnatal days 22–52 significantly facilitated long-term potentiation in control rats, and completely overcame the effects of early life stress on young adult rats. However, there was no effect on spatial learning/memory and depressive-like behavior in control rats exposed to enrichment, which may be due to relative short period of EE experience or that hippocampal long-term potentiation may be more sensitive to EE (Cui et al., 2006). Similarly, in our study, EE per se did not enhance RAM learning but reversed the stress-induced learning impairment (Figs. 2 and 3). In another study by Yang et al. (2007), prenatal stress in Wistar rats was induced by foot shocks at gestational day 13–19. The electrophysiological and Morris water maze test performed at 8 weeks of age showed that prenatal stress impaired long-term potentiation but facilitated long-term depression in the hippocampal cornu ammonis 1 region in the slices and impaired spatial learning and memory. These deficits induced by prenatal stress were recovered by EE (Yang et al., 2007). In the current study, we observed that EE after restraint stress to adult rats restored the cytogenesis and learning. This assumes importance in the clinical situation and emphasizes the role of rehabilitation after exposure to stressful situations. It may be argued that there was spontaneous recovery in the stressed rats during the period of enrichment. To test this possibility, hippocampal cytogenesis was assessed in a separate group of rats immediately after the cessation of stress. Interestingly, there was no differ-

ence between the stress (no gap) and stress groups (Fig. 2A). Earlier studies have demonstrated recovery after a stress-free period (Heine et al., 2004, 2005). The stressfree period resulted in complete recovery in acute stress, whereas in chronically stressed animals, 3 weeks of stress-free period resulted in partial recovery of hippocampal cytogenesis. The lack of recovery seen in our results could be due to the shorter duration of the stressfree period (10 days vs. 3 weeks). It can be speculated that the stress paradigm (chronic restraint stress vs. chronic unpredictable stress) could also contribute to the difference. Importantly, clinically, it may not be always possible to provide a stress-free period or allow spontaneous recovery to occur without any therapeutic intervention. In this context, our result that short-term exposure to EE reverses the stress-induced inhibition of cytogenesis is noteworthy. A myriad of studies have shown that stress impairs learning in various tasks (Luine et al., 1994; Conrad et al., 1996; McLay et al., 1998; Diamond et al., 1999; Sunanda et al., 2000a; Yang et al., 2007). A recent study from our lab demonstrated that chronic stress impairs reference memory component of spatial learning in the RAM task (Srikumar et al., 2006). Similar to our earlier report, stress and EE appeared to affect only the reference memory component without affecting the working memory. This is in contrast to some of the earlier findings (Luine et al., 1994; Diamond et al., 1996; Galani et al., 2002; He et al., 2002; Pothuizen et al., 2004) on stress and learning. The reasons for such differences are unclear. However, because the species, strain, stress paradigm, or behavioral test was different, these factors could contribute to the contrasting effect obtained (Srikumar et al., 2006). Another important factor that could be playing a role is the corticosterone levels. Stress, by and large, is known to increase the levels of corticosterone and affects hippocampal neurogenesis. Adrenalectomy (Gould et al., 1991; Cameron and Gould, 1994; Cameron and McKay, 1999) or inhibiting hypothalamo-pituitary-axis activity by blocking CRF-1 and V1b receptors (Alonso et al., 2004) increases cell proliferation and adult neurogenesis. By contrast, increased exogenous corticosterone suppresses neurogenesis, both during the early postnatal period and in adulthood (Gould et al., 1991; Cameron and Gould, 1994). Dihydroepiandrosterone modulates the action of glucocorticoids, stimulates neurogenesis, and enhances the survival of newly formed DG neurons (Karishma and Herbert, 2002). On the other hand, living in an EE (Benaroya-Milshtein et al., 2004; Moncek et al., 2004) and training on learning paradigms (Leuner et al., 2004) appear to increase circulating glucocorticoid levels. Yet both EE living and learning have been associated with enhanced neurogenesis (Kempermann et al., 1997; Nilsson et al., 1999; Leuner et al., 2006). However, both EE and learning impart a plethora of changes in the brain, above and beyond the effects of corticosteroids (Mirescu et al., 2004). It would definitely be interesting to examine the role of adrenal steroids in the Journal of Neuroscience Research

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effects of stress and EE on hippocampal cell proliferation and performance in the RAM in the future. A large body of evidence correlates new neurons in the DG with learning (Leuner et al., 2006). Gould et al. (1999) have reported the potential involvement of hippocampal neurogenesis in associative memory formation. Mice with reduced hippocampal neurogenesis performed worse than controls in the hippocampus-dependent learning tasks, including place recognition (Madsen et al., 2003), trace conditioning (Shors et al., 2001, 2002), basic nonmatching-to-sample, and spatial learning in the Barnes maze (Raber et al., 2004), whereas they were not impaired in hippocampus-independent learning like object-recognition task or elevated plus maze. Similarly, administration of antimitotic agents was associated with deficits on a fear memory task (Shors et al., 2002), and irradiation impaired learning in a hippocampal-dependent place recognition task, but not in an object recognition task, (Madsen et al., 2003; Rola et al., 2004). On the other hand, EE increased the survival of newborn cells and improved the performance in a spatial learning task (Kempermann et al., 1997; Nilsson et al., 1999). Thus, the evidence favoring the association between learning and neurogenesis suggests the possible involvement of a restored cell proliferation after EE in the behavioral recovery of stressed rats in the present study. However, it has to be noted that several investigators have argued against a causal relationship between neurogenesis and learning or the effects of EE. Exposure to the methyl azoxy methanol treatment, which significantly reduced the population of new cells, did not result in a spatial navigation deficit in the Morris water maze, nor was there any effect on the expression of contextual fear conditioning (Shors et al., 2001, 2002). Similarly, others have found no effect of irradiation-induced depletion on spatial learning in the water maze task (Madsen et al., 2003; Raber et al., 2004; Snyder et al., 2005). Further, in the same model, it is also shown that enrichment alters performance in Morris water maze regardless of their hippocampal neurogenic capability, suggesting that the newborn cells do not mediate the learning effects of enrichment (Meshi et al., 2006). Although these studies suggest that newborn cells are not required for these tasks, it could also be that these learning tasks may not be sufficiently sensitive to the loss of newly generated hippocampal neurons. Nevertheless, given the equivocal nature of the evidence for learning and neurogenesis, the data in the present study should be interpreted after giving due consideration to the reports arguing against the role for cytogenesis or neurogenesis in learning. Yet the current study does indicate an important possibility for the recruitment of newborn cells in the reversal of stress-induced cognitive deficits by EE. Further detailed studies are warranted to unequivocally establish the causal relationship between the effects of stress and EE on neurogenesis and learning. Although the restored hippocampal cell proliferation could contribute the reversal of the stress-induced Journal of Neuroscience Research

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learning deficits, it may also be argued that the effects on cell proliferation are because of a global effect on the DG and not because of a specific effect on the progenitor population of cells. Further, it is also possible that the changes in the cell proliferation alter the volume of the DG. To address these questions, several earlier studies have estimated the volume of the DG or GCL in addition to assessment of cell proliferation. van Praag et al. (2002) showed that the hippocampal neurogenesis can be influenced by EE or running with no change in the DG volume. Further, in several models of psychosocial stress, it has been shown that the cell proliferation is affected specifically with the hippocampal volume being unaltered (Czeh et al., 2001; van der Hart et al., 2002). Three weeks of restraint stress decreased the hippocampal cell proliferation but not the GCL volume, while 6 weeks of stress decreased the cell proliferation and GCL volume (Pham et al., 2003). Further, treatment with clomipramine or tianeptine reversed the stress-induced decrease in hippocampal cell proliferation but did not affect the DG volume (Czeh et al., 2002; van der Hart et al., 2002). Thus, stress and reversal paradigms may exhibit differential effects on the hippocampal cell proliferation and DG volumes. Accordingly, in the current study, we estimated the DG volume for a better understanding of the specific effects of stress and the reversal modalities on proliferation rate in relation to the alterations in volume. Although stress decreased the cell proliferation and EE increased their numbers significantly, results from the current study showed that there were no major alterations in the volume of the DG, emphasizing the direct effect of the stress and EE on hippocampal cell proliferation. These results are in accordance with earlier studies showing the specific effect of stress and EE on hippocampal neurogenesis (van Praag et al., 2002; Pham et al., 2003). The hippocampus is one of the brain structures that has been extensively studied with regard to the actions of stress, depression, and antidepressant actions (McEwen, 1999, 2000; Czeh et al., 2001; van Praag et al., 2002). Recent imaging studies in humans revealed that the hippocampus undergoes selective volume reduction in stress-related neuropsychiatric disorders (Lindauer et al., 2006; Gianaros et al., 2007; Lupien et al., 2007) such as recurrent depressive illness (Sheline, 1996; Bremner et al., 2000; Sapolsky, 2000). To determine whether chronic restraint stress influences the hippocampal volume, volumetry of the entire hippocampus was performed using Cavalieri’s principle, and we observed a reduction in the hippocampal volume after restraint stress. In animals, chronic psychosocial stress leads to cellular changes in subregions of the hippocampus that decrease its overall volume. In several species, chronic stress decreases the length and arborization of apical dendrites in the CA3 subfield (Sunanda et al., 1995, 1997; Magarinos et al., 1996; Shankaranarayana Rao et al., 2001; Vyas et al., 2002). These changes at the cellular level could decrease the hippocampal volume after restraint stress. Exposure of stressed rats to EE not

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only restored the hippocampal volume, but also enhanced it beyond the normal control level. To our knowledge, this is the first report demonstrating a reversal of the stress-induced decrease in hippocampal volume and impaired learning by EE. It is not clear why poststress EE enhanced the hippocampal volume while EE per se did not affect it. It may be speculated that because the brain is already subjected to changes in stressed animals, exposure to EE, which has a favorable effect on the levels of trophic factors (Pham et al., 1999), brought about the changes more robustly in stressed rats compared with the normal animals. Augmentation of neurotrophin synthesis after the termination of stress has also been reported (Smith et al., 1995; Sousa et al., 2000). An earlier study demonstrated that postlesion EE restores the hippocampal dendritic arborization while the same duration of EE in the absence of the lesion did not have any effect (Bindu et al., 2005). In another study, about 4 months of EE enhanced the hippocampal cornu ammonis 1 volume with no significant changes in the DG volume (Faherty et al., 2003). Similar to these results, we found that poststress enrichment enhanced total hippocampal volume without affecting the DG volume. However, in contrast to their findings, here, EE per se did not have any effect on the hippocampal or DG volumes. It may be speculated that the short-term EE (in our study) vs. 4 months of EE could be the reason for such differences. Further, our findings on the effects of EE per se on hippocampal and DG volume is in accordance with an earlier report on the effect of EE on the morphological consequences of neonatal hypoxia–ischemia (Pereira et al., 2007). More detailed experiments would be required in the future to understand the subtle changes produced by stress and EE. In addition to stress, the beneficial effects of EE have been documented after other insults like focal ischemia (Nygren and Wieloch, 2005), aging (Segovia et al., 2006), Alzheimer’s disease (Lazarov et al., 2005), and Parkinson’s disease (Steiner et al., 2006). Thus, EE is emerging as a major therapeutic tool in treating several neurodegenerative diseases, and in this context, our current study emphasizes the efficiency of EE in the amelioration of stress-induced cognitive dysfunction, thus providing initial evidence for the possible involvement of hippocampal cytogenesis in restoration of stress-induced deficits. However, further studies showing the effects on survival and differentiation of the newborn cells and specific design of experiments would be required to show the involvement of neurogenesis in the stress-induced learning deficits and its reversal by exposure to EE. ACKNOWLEDGMENTS The project was partially funded by the Department of Science and Technology, New Delhi, India. This work was supported from research fund from National Institute of Mental Health and Neuro Sciences (NIMHANS), Bangalore, India. We thank Prof. Gerd Kempermann, Max Delbru¨ck Center for Molecular

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