Physiology & Behavior 86 (2005) 21 31

The effects of chronic unpredictable stress on male rats in the water maze
Angela M. Gouirand, Leslie Matuszewich *
Department of Psychology, Northern Illinois University, DeKalb, IL 60115, United States Received 25 March 2005; received in revised form 7 June 2005; accepted 20 June 2005

Abstract Exposure to chronic stress can affect cognitive processes in a complex manner depending upon the intensity and duration of the stressors. The current study investigated the effects of chronic unpredictable stress (CUS), a procedure thought to use moderate stressors, on acquisition of and performance in the Morris Water Maze (MWM). Separate behavioral tests were also used to determine whether the stress-induced changes in MWM were due to general changes in locomotor activity or preference for a rewarding stimulus. Adult male rats were exposed to 10 days of different stressors applied at various times. Following the last stressor, stressed and non-stressed rats began training in the MWM, were tested in an open field box, or were tested for sucrose preference. In the MWM, rats exposed to stress had shorter latencies to reach the hidden platform during training. The path lengths on day 2 of training, trials 2 and 4, were shorter in CUS rats compared to controls, with the stressed rats traveling less in the outer portion of the maze. During the probe trial, CUS rats also traveled less overall and less in the outer portion of the maze, although all other measures were the same. The facilitation in learning the platform location was not due to a change in other behavioral components that could contribute to the measures, such as general activity, sensorimotor processing or the preference for a 2% sucrose solution. Thus, chronic unpredictable stress selectively appears to affect the search strategies in the water maze. D 2005 Elsevier Inc. All rights reserved.
Keywords: Spatial memory; Learning; Chronic stress; Locomotion; Sucrose consumption

1. Introduction The effects of chronic stress on cognition are complex. In humans and animals, chronic stress exposure is associated generally with impaired performance in learning and memory tasks [32]. Previous studies found that adults exposed to chronic life stress, either related to social economic class or mental illness, show decreases in cognitive functions, such as performance on memory tasks [29,48]. Likewise, rodents that experience repeated stress demonstrate deficits on tasks assessing learning or memory. Rats restrained daily for 21 days had impaired performance on the 8-arm radial maze, Y-maze and object recognition task [4,8,28]. Exposure to other repeated stress paradigms for 4 12 weeks resulted in deficits on the 8-

* Corresponding author. Tel.: +1 815 753 7072; fax: +1 815 753 8088. E-mail address: lmatusze@niu.edu (L. Matuszewich). 0031-9384/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2005.06.027

arm radial maze and the T-maze delayed alteration task [34,38,41]. In a similar manner, daily exposure to a variety of physical stressors for 28 days [51] or 6 months of social stress [5] resulted in impaired learning and performance on a spatial navigation task, the Morris Water Maze (MWM). On the other hand, other studies have found that chronic stress exposure enhances the acquisition or performance components of memory tasks. Thirteen days of daily restraint stress improved the performance of rats in the radial arm maze [27]. Likewise, chronic social stress in tree shrews enhanced spatial memory performance in the hole board task [3] and reducing food availability for chickadees improved their performance on a spatial memory task to search for food [44]. Juvenile rodents exposed to variable social or physical stressors for 28 days had decreased latencies to locate the hidden platform in the MWM when training began 1 day after the last stressor [25]. Thus, although some types of

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chronic stress exposures decrease performance on memory tasks, other stress exposures facilitate acquisition or performance components of memory tasks. These discrepant findings have been characterized by an inverted Ushaped curve with milder or transient stress exposure augments learning, while more severe chronic stress exposure disrupts learning [47,49]. However, it is difficult to determine which of the above stress exposures should be considered mild, moderate or severe because comparable measures (e.g., plasma hormone levels, hippocampal morphology) have not been assessed following each exposure. One stress procedure that appears to cause a moderate stress response is chronic unpredictable stress. The chronic unpredictable stress (CUS) protocol exposes rodents to 10 days of unpredictable stressors, both physical and psychological in nature [18,23,30,40]. Rats exposed to CUS show reduced body weight gain and increased adrenal gland weight, but not changes in basal or restraint stressstimulated plasma corticosterone levels [23,31]. A previous report found that rats exposed to CUS show some dendritic atrophy of CA3 neurons (13% loss), but not to the same degree as repeated immobilization stress (29% loss [54]). To our knowledge, there have been no behavioral tests of the effects of 10 days of CUS on the acquisition or performance of a cognitive task. Therefore, the purpose of this paper was to investigate the effects of CUS as a moderate stress protocol on a spatial memory task, the MWM. One inherent difficulty in studying cognition is the number of factors that contribute to successful acquisition and performance of a task. An appropriate response on most rodent cognitive tasks requires general motor abilities, intact sensorimotor processing and an assessment of a rewarding stimulus, in addition to the memory capacity being tested. Exposure to stress could alter the contribution of these non-memory based factors and affect the measures of the cognitive task without altering memory processes. Many of the chronic stress procedures shown to alter cognition did not assess other factors related to the behavioral test [25,27,28,34,44,53]. To assess whether altered performance on the MWM could be attributed to other factors, separate groups of stressed and non-stressed rats were tested in an open field arena to assess general motor ability and for consumption of a 2% sucrose solution to assess the valence of a rewarding stimulus. Exposure to other chronic stress paradigms for 2 to 3 weeks decreases consumption of the sucrose water [58,59], suggesting a dysfunction of normal reward systems. Sucrose consumption has not been assessed following the 10 day CUS and will therefore be measured to evaluate whether CUS alters the processes associated with rewarding stimuli, which could influence acquisition of platform location in the MWM. Due to the moderate level of stress duration and variety of physical and psychological stressors applied in CUS, we hypothesized that CUS

would facilitate acquisition of the platform location in the MWM.

2. Materials and methods 2.1. Animals and housing Male Sprague Dawley rats (Charles River derived) from the Northern Illinois University psychologys animal colony (45 60 days old) were housed in groups of two in polycarbonate cages and maintained on a 12-h light/dark cycle (lights on at 06:00 h). The room temperature was maintained at 22 T 2 -C with low humidity and food and water freely available. All procedures were in adherence to the National Institutes of Healths Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, revised 1996) and approved by the local institutional animal care committee. All rats were randomly assigned to one of two groups: chronic stress or no stress. Stressed and non-stressed rats were weighed daily (09:00 h) to monitor their overall health. Rats assigned to the chronic stress group were exposed to the following chronic unpredictable stress protocol (CUS): Day 1 11:00 a.m. 50 min cold room (4 -C), and 12:00 p.m. 60 min cage rotation (the rats are placed in a polycarbonate cage and then onto a homemade platform that vibrates the cage); Day 2 1:00 p.m. 4 h wet bedding (400 ml tap water in home cage), and 6:00 p.m. lights on overnight; Day 3 12:00 p.m. 3 h lights off, and 3:00 p.m. 60 min restraint stress (6 21.6 cm Plexiglas commercial restrainer; Harvard Apparatus, Inc., Holliston, MA); Day 4 6:00 p.m. 50 min cage rotation, and food and water deprivation overnight (15 h); Day 5 3:00 p.m. 15 min cold room isolation, and 4:00 p.m. isolation housing overnight (17 h); Day 6 11:00 a.m. 4 h wet bedding, and 3:00 p.m. 2 h lights off; Day 7 1:00 p.m. 30 min cage rotation, and 6:00 p.m. 1 h lights on; Day 8 10:00 a.m. 20 min cage rotation, and 3:00 p.m. 60 min restraint stress; Day 9 10:00 a.m. 4 h wet bedding, and 6:00 p.m. food and water deprivation; Day 10 6:00 p.m. isolation housing and lights on overnight. Immediately after the last stressor, the animals were videotaped in the open field (CUS n = 13; control n = 14), tested for sucrose consumption (CUS n = 12; control n = 12) or began training in the Morris Water Maze (CUS n = 16; control n = 20). The order of the stressors was slightly modified for rats tested for sucrose consumption to have the 2nd food and water deprivation on day 10. CUS rats used in the sucrose test experienced wet bedding and lights on overnight on day 9. Then, both rats exposed to CUS and non-stressed controls were isolated and food and water deprived on day 10 and tested for sucrose consumption the following evening (18:00 h). Each rat was tested in only one behavioral test. The open field and MWM were conducted during the light cycle between 12:00 3:00, while sucrose consumption was tested at the start of the dark cycle.

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2.2. Procedures 2.2.1. Morris Water Maze The water maze consisted of a metal pool (170 cm in diameter 58 cm tall) filled with tap water (25 -C, 40 cm deep) divided into 4 quadrants. In the center of 1 quadrant (44 cm from wall) was a removable escape platform below the water level and covered with a white athletic sock to disguise its presence, as well as to create traction for the rat to climb onto the top of the platform. One 16 oz jar of white, water-based Tempura paint was added to the tap water, causing the platform to be undistinguishable from the water. The placement of the pool in the room and the placement of the platform in the pool were the same for all of the acquisition trials, maintaining constant distal spatial cues throughout all swim sessions. Immediately after the last day of CUS (day 11), acquisition training began in the MWM and consisted of 4 trials per day. The rat was placed in the water facing the perimeter of the pool at a pre-determined, randomly selected quadrant (north, south, east, or west). For subsequent trials, the rat was released in each of the other three quadrants. For each trial, the rat swam in the pool until climbing onto the platform or for 60 s. If the rat did not find the platform within the 60 s, he was led by the experimenter with a metal strainer to the platform. The rat remained on the platform for 30 s in all trials and was then removed from the pool, dried in a clean cage without bedding, and returned to its home cage. The pool was strained of feces and stirred gently between each swim session to disrupt any scent left behind from the rats path taken to find the platform. Eight rats were trained per day and all 8 received their 1st training trial before the 2nd training trial occurred for that day (ITI = 15 min). The training continued for 4 days. On day 5, there was one probe trial in which the platform was removed from the pool and the animal swam for 120 s. All swim sessions, acquisition training and probe trials, were videotaped for later behavioral analysis. For the acquisition trials, the following measures were recorded: the animals latency to climb onto the hidden platform, the time spent and number of crossings made into the quadrant with the platform and the total distance traveled in meters. Statistical analysis of training measures was performed on the average of each training day computed for the 4 trials. For the probe, the following measures were recorded: the time spent and number of crossings into the target quadrant and total distance traveled. A subset of control (n = 12) and stressed (n = 12) rats were randomly selected for further analysis of distance traveled in the outside 50% and inside 50% of the maze during the probe trial, 1 trial of training day 2 near the beginning of the training day (trial 2) and 1 trial of training day 2 at the end of the training day (trial 4). The distance traveled was calculated by digitizing the swim paths using the True-Lite 100 and then Bioscan Optimas 3.01 software (3rd ed., 1990). All data was standardized for the dimensions of the tank. Finally, the swim speed was

calculated for acquisition and probe trials (distance traveled/ time in water). 2.2.2. Open-field test A large plywood box (75 75 29 cm) painted grey with a black grid (15 15 cm squares) on the floor was used for exploration testing. The rat was placed into a corner of the box and allowed to explore freely for 10 min. The box was thoroughly cleaned between subjects with a disinfectant solution. All test sessions were videotaped and the following measures were later recorded: number of rears (animal on hind limbs), number of grid boxes entered (front 2 paws over a line), time in center 9 squares, and latency to leave the corner box initially. 2.2.3. Sucrose consumption All rats tested for sucrose consumption were isolated housed and given 2% sucrose water for 48 h in place of their regular drinking water in their home cages, prior to being divided into groups for stress. Rats were then randomly assigned to stress or control groups. The stress group went through 10 days of the CUS with the last stressor being food and water deprivation for 23 h. Rats in the control group were weighed daily but not stressed, and then on day 10, were food and water deprived for 23 h (identical to the CUS group). Both groups were offered 2% sucrose water and regular drinking water at the start of the dark period (18:00 h) on the 11th day. The bottles were weighed prior to being given to the rats and at the conclusion of the test (19:00 h). 2.2.4. Measurement of adrenal gland weight One day after completion of the MWM, all rats were euthanized with CO2 (09:00 h) and their adrenal glands removed and weighed immediately. The adrenal weight was reported as ratio of 100 mg of the animals total body weight [21]. Four of the rats were used for other research following completion of the MWM and therefore, those adrenal glands weight were not included in the analysis. 2.2.5. Statistics The following measures of stressed and non-stressed control rats were compared with an independent t-test in the MWM: for the probe trial, total time in the target quadrant, frequency of crossings into the target quadrant, total path length and calculated swim rate; for total path lengths on acquisition day 2, trials 2 and 4, and for latency to climb onto the hidden platform on each training day. Independent t-tests were also used to compare stressed and non-stressed rats on all measures in the open field test, on preference of sucrose solution (sucrose consumed / total fluid consumed), adrenal gland weights and body weight difference during the MWM (day 15 day 11). In the sucrose test, a 2 Way ANOVA was used to analyze differences between the amount of fluid consumed by group (stress or non-stressed) and type of fluid (2% sucrose or water).

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Fig. 1. Exposure to CUS decreased the latency to locate the hidden platform during training in the MWM. Time to reach the platform was significantly shorter in CUS rats than in control rats overall ( F(1, 102) = 4.28, p < 0.05). When groups were compared by day, CUS rats had shorter latencies compared to the control rats on days 2 and 3 of training (* = p < 0.05). Both CUS and non-stressed controls showed a decreased latency to the hidden platform over the four days of training ( F(3, 102) = 32.8, p < 0.001). Each day of training represents the mean of 4 trials conducted on that training day. Error bars represent standard error of the mean.

Repeated Measures mixed ANOVA (group time) was used to analyze data collected during the acquisition of the platform location in the MWM. These measures included the latency to climb onto the hidden platform, number of

crossings into the quadrant with the platform and proportion of time spent in the quadrant with the platform. Body weight data was also compared with a Repeated Measures ANOVA (group day). The distance swam in the inner 50% of the

Fig. 2. Training increased the time spent in the target quadrant in the MWM. Both CUS and non-stressed controls spent more time in the quadrant with the platform (target quadrant) following the first day of training ( F(3, 102) = 13.8, + = p < 0.05 from training day 1). CUS rats tended to spend more time in the target quadrant, but this measure was not significant between groups ( F(1, 102) = 3.1, p < 0.09). Each day of training represents the mean of 4 trials conducted on that training day. Error bars represent standard error of the mean.

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Fig. 3. Exposure to CUS decreased the total frequency of crossings into target quadrant during training in the MWM. CUS rats had fewer crossings into the target quadrant than control rats ( F(1, 102) = 4.6, p < 0.05). Both CUS and non-stressed controls showed a decrease in the total number of crossings into the target quadrant by training day ( F(3, 102) = 9.9, + = p < 0.05 from training day 1). Each day of training represents the mean of 4 trials conducted on that training day. Error bars represent standard error of the mean.

Fig. 4. (A) Exposure to CUS decreased swimming in outer portion of maze on the 2nd trial of training day 2. CUS rats swam significantly less than controls in the outer portion of the maze ( F(1, 44) = 6.4, * = p < 0.05 compared to control rats). Both CUS and non-stressed controls swam a greater distance in the outer portion of the maze ( F(1, 44) = 7.4, p < 0.01). Error bars represent standard error of the mean. (B) Exposure to CUS decreased swimming in outer portion of maze on the 4th trial of training day 2. CUS rats swam significantly less than controls in the outer portion of the maze overall ( F(1, 44) = 4.0, p < 0.05), but there was no significant interaction ( F(1, 44) = 2.44, n.s.). Both CUS and non-stressed controls swam a greater distance in the outer portion of the maze ( F(1, 44) = 7.2, p < 0.05). Error bars represent standard error of the mean.

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MWM compared to the outer 50% were compared with a 2 Way ANOVA (location group). All significant main effects and interactions were further investigated with Tukey post hoc comparisons.

3. Results 3.1. Morris Water Maze 3.1.1. Acquisition phase There was a significant effect of group and time on the latency to find the hidden platform during training in the MWM. The latency for both stressed and non-stressed rats to find the hidden platform (escape latency) decreased with increasing trials ( F(3, 102) = 32.8, p < 0.001, Fig. 1). The rats exposed to CUS had shorter latencies to reach the platform in comparison to non-stressed rats ( F(1, 102) =4.28, p < 0.05). The interaction was not significant ( F(3, 102) = 1.14, n.s.), however t-tests indicated a difference between groups on days 2 and 3 of training (day 2: t(34) = 2.5, p < 0.05; day 3: t(34) = 2.1, p < 0.05), but not day 1 (t(34) = 1.1, n.s.) or day 4 (t(34) = 0.8, n.s.). There were also significant effects of time spent in and crossings into the correct quadrant. Both groups spent a greater proportion of the time in the quadrant with the platform after the 1st training day ( F(3, 102) = 13.8, p < 0.05, Fig. 2). The stress group tended to spend more time swimming in the quadrant with the platform compared to control group, however this measure did not reach statistical significance for group ( F(1, 102) = 3.1, p < 0.09). There was also a significant decrease in the number of crossings into the quadrant with the platform during training in the MWM for both stressed and control rats ( F(3, 102) = 9.9, p < 0.001, Fig. 3). Rats exposed to CUS had a lower average number of crossings overall ( F(1, 102 = 4.6, p < 0.05), perhaps suggesting a more efficient strategy in locating the platform once entering the target quadrant. To characterize more carefully the swim paths of the subjects, a subset of CUS (n = 12) and control rats (n = 12) were analyzed for day 2 of training, a trial at the beginning of the training day (trial 2) and a trial at the end of the training day (trial 4). On training trial 2, rats exposed to CUS showed shorter overall path lengths to the hidden platform (t(22) = 2.2, p < 0.05). When the distance of the path lengths were separated into the outer half of the maze (from walls of maze toward center by 50% distance) compared to the inner half of the maze, stressed and nonstressed rats traveled a greater distance in the outer portion ( F(1, 44) = 7.4, p < 0.01, Fig. 4A). Rats exposed to CUS traveled significantly less than control rats in the outer portion of the maze as indicated by a significant interaction ( F(1, 44) = 6.4, < 0.05) followed by a Tukey post hoc test. On training trial 4 of day 2, there was a similar increase in distance traveled in the outer portion compared to the inner

portion of the maze for stress and control rats ( F(1, 44) = 7.2, p < 0.05, Fig. 4B). Control rats swam a greater distance compared with rats exposed to CUS ( F(1, 44) = 4.0, p < 0.05), but the interaction did not reach significance ( F(1, 44) = 2.44, n.s.). The increase in path distance did not reflect a change in calculated swim rate between the groups ( F(1, 66) = 0.002, n.s.). 3.1.2. Probe trial During the probe trial, rats exposed to CUS traveled significantly shorter distances (t(22) = 4.4, p < 0.01, Table 1). Stressed rats traveled significantly less than controls in the outer portion of maze, but not in the inner portion of the maze as indicated by a significant interaction ( F(1, 44) = 26.9, p < 0.001, Fig. 5) followed by Tukey post hoc tests. Both CUS and non-stressed rats swam longer distances within the outer half of the maze than they did within the inner half ( F(1, 44) = 135.9, p < 0.001). The control rats swam at a faster rate than CUS rats during the 120 s probe trial (t(22) = 4.4, p < 0.001, Table 1). The exposure of rats to CUS had no effect on behaviors related to the target quadrant in the probe trial. There was no difference between groups for the number of crossings into the correct quadrant (t(34) = 1.5, n.s., Table 1) or time in the correct quadrant (t(34) = 1.3, n.s.). 3.2. Open field test There was no effect of CUS on the number of boxes entered during the 10 min test overall (t(25) = 0.32, n.s.;
Table 1 The effects of chronic unpredictable stress (CUS) on measures from the probe trial of the Morris Water Maze, an open field test or a 2 bottle sucrose preference test Non-stressed Rats exposed control to CUS Probe trial measure Total distance traveled (m) 3.5 T 0.2 2.3 T 0.2* Calculated swim rate (cm/sec) 2.9 T 0.6 1.9 T 0.5* Number of crossings into target quadrant 7.7 T 0.8 9.1 T 0.3 Time spent in target quadrant (sec) 35.5 T 2.1 40.3 T 3.1 Open field measures Number of boxes entered in 10 min 185.4 T 20.1 194.4 T 20.4 Number of rears in 10 min 34.9 T 4.7 50.8 T 6.2* Latency to inside of portion of the 109.3 T 38.1 103.5 T 33.0 field (sec) Duration of time in the inside portion 12.2 T 2.1 12.8 T 3.2 of the field (sec) Sucrose consumption Water intake (g) 3.8 T 0.9 2.6 T 0.4 2% sucrose solution (g) 15.5 T 1.5 17.2 T 2.2 Percent preference for 2% sucrose solution 79.9 T 5.6 84.5 T 2.5 Stressed rats were exposed to 10 days of CUS, while control rats were weighed daily. The rats exposed to CUS traveled significantly shorter distances and swam at slower rates on the probe trial of the MWM compared to non-stressed control rats. There was a significant increase in the number of rears exhibited by CUS rats during the 10 min open field test (* p < .05).

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Fig. 5. Exposure to CUS decreased swimming in outer portion of maze during the 120 s probe trial. In the probe trial, without a platform present, CUS rats swam significantly less than controls in the outer portion of the maze ( F(1, 44) = 26.9, * = p < 0.05 compared to control rats). Both CUS and non-stressed controls swam a greater distance in the outer portion of the maze ( F(1, 44) = 135.9, p < 0.001). Error bars represent standard error of the mean.

Table 1) or when the first 3 min were compared at 30 s intervals ( F(5, 125) = 0.09, n.s., data not shown). There was a significant increase in the number of rears exhibited by stressed rats during the 10 min test (t(25) = 2.23, p < 0.05), but not when the first 3 min were compared at 30 s intervals ( F(5, 125) = 1.6, n.s., data not shown). There was also no difference in the time spent in the center of the open field

box or the latency to leave the corner when first placed into the box (Table 1). 3.3. Sucrose consumption Stressed and control rats drank a greater amount of sucrose water than regular drinking water in the 1 h test

Fig. 6. Exposure to CUS decreased body weight gain. During the stress exposure (days 1 9), CUS rats gained significantly less weight over time than controls as indicated by a significant interaction ( F(8, 656) = 44.0, * = p < 0.05 compared to control rats). Error bars represent standard error of the mean.

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( F(1, 43) = 76.7, p < 0.01). However, there was no difference between stress and control rats in the amount of regular water or sucrose water drank ( F(1, 43) = 0.04, n.s., Table 1) or in the preference for the sucrose water over the total fluid consumption (t(21) = 0.95, n.s.). There was no significant effect on sucrose consumption even when weighted by body weight (t(22) = 1.44, n.s., control: 0.48 mg/g; CUS: 0.61 mg/g). 3.4. Body and adrenal gland weights Rats exposed to CUS showed less body weight gain than controls in all experiments. There was a significant interaction between group (CUS and non-stressed rats) and time ( F(8, 656) = 44.0, p < 0.05, Fig. 6). Multiple comparison procedures showed differences between stressed and non-stressed rats after day 5 of the stress procedures. The body weights of rats trained in the MWM were monitored after stress ended and were compared at day 1, day 11 and day 15. Although stressed rats had decreased weight gain between day 1 and day 11 compared to controls (control: 65.1 T 2.3 g; CUS: 32.8 T 3.0 g), the two groups gained similar amounts of weight during MWM training (day 15 compared to day 11)(control: 18.2 T 1.5 g; CUS: 16.8 T 2.0 g) and the groups did not differ when compared at the last time point only (t(38) = 0.55, n.s.). The adrenal glands of rats exposed to CUS were significantly greater when compared to non-stressed control rats when measured as a proportion of body weight (control: 11.9 + 0.5 mg/100 g body weight; CUS: 13.6 + 0.4 mg/100 g body weight, t(27) = 2.6, p < 0.05).

4. Discussion The present study found that exposure to unpredictable stress for 10 days facilitated acquisition of the platform location in the Morris Water Maze. Rats exposed to CUS had shorter latencies to the hidden platform overall during training compared to non-stressed rats. The control rats spent an increased amount of time in the outer portion of the MWM compared to CUS rats and had predictably longer paths to the hidden platform during the 2nd day of training. The difference in search strategies was also evident in the probe test, when the platform was not present; rats exposed to CUS traveled less distance than the control rats in the outer portion of the maze during the probe trial, although the duration of the time spent in the correct quadrant did not differ. The effects of unpredictable stress on training in the MWM does not appear to be due to a change in general activity as assessed by an open field task or in processing rewarding stimuli. Thus, chronic unpredictable stress selectively appears to affect the search strategies in the water maze. The faster latencies to the hidden platform in the MWM by the CUS group were most noticeable on days 2 and 3 of

acquisition. The latencies of CUS and control rats differed significantly on days 2 and 3 when compared through independent t-tests, but not on days 1 and 4 (Fig. 1). The similar latencies on day 1 indicate no difference between groups at the start of the experiment. In fact, on the first trial of the first day, only 1 of the 36 rats found the platform within the 60 s trial and most demonstrated high levels of thigmotaxis. By training day 2, however, CUS rats were faster to locate the platform and traveled shorter path lengths in the outer portion of the maze (Fig. 4), which suggests that rats exposed to CUS are more effective at locating the platform earlier in training. By the end of training, there were again no statistical differences in latency to locate the platform. On the probe trial, stressed and non-stressed rats showed similar behaviors related to the quadrant that had contained the platform during training. They had similar frequency of crossings into and time spent in the target quadrant, which further suggests that they were trained to a comparable level by day 4 (Table 1). However, there were differences between the groups in the probe trial that suggest the use of different strategies to locate the platform. Non-stressed control rats swam greater distances in the outer portion of the maze than the CUS rats in the probe trial, as they did during day 2 of training (Fig. 5). It is possible that non-stressed rats, without the platform present in the probe trial, used less direct swim paths similar to their earlier training trials, while the CUS rats targeted their swim paths more toward the previous location of the platform. Alternatively, the non-stressed rats may have learned to swim faster, resulting in decreased latencies during acquisition training but longer path lengths in the probe trial. The non-stressed rats swam a greater distance during the probe trial and therefore, a faster calculated swim rate (distance/120 s). Previous research classified natural swim patterns in mice using several behavioral parameters collected on a video tracking program. One of the classifications, nonspatial concentric, had a faster swim speed than the other types and a pattern of swimming similar to the non-stressed control group in the present experiment [11]. Future studies are planned to determine whether there are consistent differences in the overall swim patterns of stressed and non-stressed rats similar to those observed in mice. The neural mechanisms responsible for the improvement in cognitive function following exposure to chronic moderate stress are not known. One mechanism may be related to hippocampal function. The integrity of the hippocampus is very important for many cognitive tasks, especially for spatial memory. Lesions of the hippocampus or destruction of inputs to the hippocampus disrupts acquisition of the MWM, as well as other cognitive tasks (for review see [6,7,14,57]). Exposure to chronic stress may alter the structure or function of hippocampus neurons and subsequently alter performance on cognitive tasks. For example, juvenile rodents exposed to physical or social stress for 28 days showed hypertrophy in the CA1 region

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of the hippocampus and decreased escape latencies in the MWM [25]. The behavioral effects were very similar to the current study and suggest that the improved acquisition of MWM in adult rats following CUS may be due to increased hippocampal volume as well. However, Vyas and colleagues (2002) reported that adult rats exposed to the CUS showed slight decreases in dendritic length and number of branch points on CA3 neurons [54]. In a separate study, adult rats showed improved performance on a radial arm maze following 13 days of restraint stress, but no differences in the length or number of branch points in the CA3 region of the hippocampus [27]. Thus, the hippocampus may mediate the stress-induced changes on cognitive tasks, but the relationship between morphology and function may not be linear. Another factor, such as glucocorticoids, may modulate the relationship between chronic stress and hippocampal function. Removal of the adrenal glands causes damage to the dentate gyrus of the hippocampus and impaired performance on the MWM, which suggests some level of glucocorticoids are necessary to support hippocampal function and spatial memory [9,10,39,50]. Short-term increases in corticosterone due to an acute stressor or a single administration of exogenous corticosterone can enhance some forms of learning (for review see [46,47,49]) [1,26,45,60]. Likewise, chronic exposure to moderate levels of plasma corticosterone facilitated food retrieval in chickadees [43] and enhanced prime burst stimulation and LTP in hippocampal tissue [15,42]. On the other hand, chronic exposure to high levels of glucocorticoids contributes to hippocampal atrophy [51,56,61], reduced neurogenesis [25], impaired performance on several cognitive tasks [5,8,33,51] and suppressed LTP and prime burst stimulation of the hippocampus [5,15,42]. The inverted U-shaped curve is frequently used to explain the complex relationship between stress or glucocorticoid levels and learning [47,49]. The chronic unpredictable stress protocol used in the present study could be considered to induce a moderate level of stress based on the curve. Male rats exposed to CUS show enhanced acquisition of the MWM, have relatively limited remodeling effects on hippocampal neurons [54] and do not show differences in basal plasma corticosterone [31]. In addition to the importance of the hippocampus in stress-related effects, the hippocampus is critical to many aspects of spatial navigation assessed by the MWM (for review see [7,14,16,35,57]). The hippocampus is thought to contribute to the use of efficient navigational strategies. Lesions of the hippocampus or fimbria fornix disrupts acquisition of the hidden platform location in the MWM [20,36,37,52], which the CUS rats were more efficient at locating in the present study. Moreover, lesioned rats trained to locate the platform have more difficulty changing their navigational strategies if the release position into the maze was altered [17]. The current study provided many distal cues during the training and probe trials, such as

different colored walls (black, off-white, brown cabinets and open space) and posters on one wall, and also trained the rats from different release sites in the maze. Therefore, it is not clear whether the CUS rats were more efficient at learning the relative location of the extra maze cues to the hidden platform, at adapting their search strategies to different release points, or some combination of the 2 approaches. Alternatively, CUS may alter other navigational strategies dependent upon the hippocampus, such as path integration and location estimation. These navigational strategies would allow the rat to locate the platform without the use of extra maze cues through continually estimating its location in the maze based on the release point and the distance traveled [55,57]. Acquisition of the platform location in the MWM task depends upon several behavioral components, such as motor skills, sensorimotor processing and assessment of rewarding stimuli. Exposure to CUS did not alter any of these behavioral components, and therefore the components cannot account for the observed differences in learning the location of the hidden platform in the MWM. Rats exposed to CUS did not differ in general locomotor activity in an open field test as indicated by number of entries into squares, although the frequency of rears increased. There were also no differences between groups on the first day of training as discussed above and the calculated swim rates did not differ during the acquisition trials. Thus, general swimming ability and sensorimotor processing do not appear to cause the differences between groups in the MWM. Finally, CUS and non-stressed control rats did not differ on the sucrose consumption, indicating similar assessment of a rewarding stimulus. Previous studies using other chronic unpredictable stress protocols have found decreases in sucrose consumption [12,13,58,59,62], but other investigators have not replicated those effects using the same stress protocols [19,22,24] or have questioned the meaning of the measure [2]. Although no effects of CUS were observed in preference for a sucrose solution, CUS has been shown to alter behavioral and neurochemical responses to drug rewards, such as cocaine and methamphetamine [23,31]. In particular to measures of rewarding stimuli, rats exposed to CUS increased their preference for moderate doses of cocaine, but decreased their preference for high doses of cocaine [23]. A similar dose response shift for sucrose water was not tested in the current study, but the data do not suggest a similar response. Taken together, these findings suggest that stressed and non-stressed rats had similar swimming abilities, sensorimotor skills, reward processing and even spatial memory at the start of MWM training. Understanding the relationship between different stress exposures and cognitive function is an important but complex task. The current study exposed male rats to 10 days of unpredictable, moderate stressors and found that the acquisition of the platform location in the MWM occurred more quickly, possibly due to more efficient search strategies. CUS was selected because it appears to mimic

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A.M. Gouirand, L. Matuszewich / Physiology & Behavior 86 (2005) 21 31 [14] DHooge R, De Deyn PP. Applications of the Morris Water Maze in the study of learning and memory. Brain Res Rev 2001;36:60 90. [15] Diamond DM, Bennett MC, Fleshner M, Rose GM. Inverted U relationship between the level of peripheral corticosterone and the magnitude of hippocampal primed burst potentiation. Hippocampus 1992;2:421 30. [16] DiMattis BD, Kesner RP. Spatial cognitive maps: differential role of parietal cortex and hippocampal formation. Behav Neurosci 1988; 102:471 80. [17] Eichenbaum H, Stewart CA, Morris RGM. Hippocampal representation in place learning. J Neurosci 1990;10:2531 42. [18] Fitzgerald LW, Ortiz J, Hamedani AG, Nestler EJ. Drugs of abuse and stress increase the expression of GluR1 and NDMAR1 glutamate receptor subunits in the rat ventral tegmental area: common adaptations among cross-sensitizing agents. J Neurosci 1996;16: 274 82. [19] Forbes NF, Stewart CA, Matthews K, Reid IC. Chronic mild stress and sucrose consumption: validity as a model of depression. Physiol Behav 1996;60:1481 4. [20] Galani R, Weiss I, Cassel J-C, Kelche C. Spatial memory, habituation, and reactions to spatial and nonspatial changes in rats with selective lesions of the hippocampus, the entorhinal cortex or the subiculum. Behav Brain Res 1998;96:1 12. [21] Garcia AM, Octavi, Valles Astrid, Dal-Zotto Silvina. Recovery of the hypothalamic pituitary adrenal response to stress. Neuroendocrinology 2000;72:114 25. [22] Hagan JJ, Hatcher JP. Revised CMS model. Psychopharmacology 1997;134:354 6. [23] Haile CN, GrandPre T, Kosten TA. Chronic unpredictable stress, but not chronic predictable stress, enhances the sensitivity to the behavioral effects of cocaine in rats. Psychopharmacology 2001; 154:213 20. [24] Harris RB, Zhou J, Youngblood BD, Smagin GN, Ryan DH. Failure to change exploration or saccharin preference in rats exposed to chronic mild stress. Physiol Behav 1998;63:91 100. [25] Isgor C, Kabbaj M, Akil H, Watson SJ. Delayed effects of chronic variable stress during peripubertal juvenille period on hippocampal morphology and on cognitive and stress axis functions in rats. Hippocampus 2004;14:636 48. [26] Kovacs GL, Telegdy G, Lissak K. Dose-dependent action of corticosteroids on brain serotonin content and passive avoidance behavior. Horm Behav 1977;8:155 65. [27] Luine V, Martinez C, Villegas M, Magarinos AM, McEwen BS. Restraint stress reversible enhances spatial memory performance. Physiol Behav 1996;59:27 32. [28] Luine V, Villegas M, Martinez C, McEwen BS. Repeated stress causes reversible impairments of spatial memory performance. Brain Res 1994;639:167 70. [29] Lynch JW, Kaplan GA, Shema SJ. Cumulative impact of sustained economic hardship on physical, cognitive, psychological and social functioning. N Engl J Med 1997;337:1889 95. [30] Matuszewich L, Yamamoto BK. Long-lasting effects of chronic stress on DOI-induced hyperthermia in male rats. Psychopharmacology 2003;169:169 75. [31] Matuszewich L, Yamamoto BK. Chronic stress augments the longterm and acute effects of methamphetamine. Neuroscience 2004;124: 637 46. [32] McEwen BS, Sapolsky RM. Stress and cognitive function. Curr Opin Neurobiol 1995;5:205 16. [33] McLay RN, Freeman SM, Zadina JE. Chronic corticosterone impairs memory performance in the Barnes Maze. Physiol Behav 1998;63: 933 7. [34] Mizoguchi K, Yuzurihara M, Ishige A, Sasaki H, Chui D-H, Tabira T. Chronic stress induces impairment of spatial working memory because of prefrontal dopaminergic dysfunction. J Neurosci 2000;20:1568 74. [35] Morris R. Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods 1984;11:47 60.

the pattern of daily life stressors experienced by humans and length of stress exposure is especially appropriate for students in college (e.g., 7 10 days of final exams). The physiological effects in the current study, decreased weight gain and increased adrenal gland weight, do indicate that rats exposed to stress differed from controls as suggested by earlier studies using this protocol [23,31]. The physiological and behavioral data, taken together, suggest that spatial memory can be improved given a moderate level of stress exposure.

Acknowledgements This research was supported by the Department of Psychology at Northern Illinois University. The authors would like to thank Douglas Wallace for his guidance in the Water Morris Maze, Steven Janasik for his assistance on the open field test, and Ross Friedman for the helpful comments on the manuscript.

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