Eighty-four adult male Sprague-Dawley rats (260±20 g) were obtained from the laboratory animal's center of Shiraz University of Medical Sciences, Shiraz, Iran. The Ethics Committee of the University approved the animal experiment (Approval No: 91-6124). The male rats were randomly assigned to experimental and control groups. Each group included 12 rats that were housed under standard conditions, room temperature (22~24℃), and a 12:12 h light-dark schedule and had free access to water and food.

The animals were divided into seven groups each containing 12 rats: (I) stress +water group daily receiving stress and distilled water, (II) stress +olive oil group daily receiving stress and olive oil, (III) stress+curcumin group daily receiving stress and curcumin (100 mg/kg/day) solved in olive oil [22, 32-34], (IV) stress+sertraline group daily receiving stress and sertraline (10 mg/kg/day) solved in distilled water [35], (V) control group, (VI) curcumin group receiving curcumin (100 mg/kg/day) solved in olive oil [22, 32-34], and (VII) sertraline group receiving sertraline (10 mg/kg/day) solved in distilled water [35]. All the animals were treated by gavages every day for 8 weeks.

Dose of curcumin was determined according to our previous study. It showed 100 mg/kg/day as the appropriate dose of curcumin with no side effects on the liver, kidney, and blood levels of aspartate aminotransferase, alanine aminotransferase, urea nitrogen, and creatinine [21, 32-34]. Dose of sertraline was selected according to the previous studies [25]. Since it is chronically used in our practice, this period and dosage has been accepted as the period of usage for displaying its chronically effects [35].

The 84 study animals were randomly divided into seven groups (n=12). The animals were submitted to a chronic variable stress regime over a 56-day period or remained in their home cages without stress manipulation [36]. The chronic variable stress is described in Table 1. The schedule of the different manipulation exerted on the animals subjected to the stress and treatments are presented in Table 2.

All the behavioral tests were performed in an experimental room, constant noise, and temperature controlled environment. It should be noted that the animals were allowed to adapt to the experimental room. It should be mentioned that the individual who was responsible for performing the assessment was blind to the experimental conditions of each animal. 84 rats in seven groups (n=12) were tested for anxiety and anhedonia and 70 male rats (n=10) were subjected to the memory and learning tests.

Working memory can be explained as a memory for an object, stimulus, or location that is used within the evaluation session, but not typically between the sessions [5]. Reference memory that would typically be attained with repeated training and would maintained from days to months [37, 38]. In the present study the Radial Arm Maze (RAM) has been designed to evaluate the learning and memory in rats. The apparatus consists of eight equidistantly spaced arms (42×12×12 cm³). The arms were radiated from a central octagonal platform. The animals were assessed for learning and memory in a partially baited RAM. Prior to the training, the animals were kept on a restricted regime; so that their body weight would reach 85% of that prior to the training [37, 38].

The animals were allowed to have two sessions of adaptation on two continuous days prior to the beginning of the learning period. During these adaptation sessions, the rats were allowed to explore the baited arms of the maze for 10 min.

During the acquisition session (learning period), the animals were given two acquisition trials per day until they attained the learning criteria. The learning criteria were defined as follows. The trial was continued for 5 min and the training was continued until the rats reached the criteria of 80% correct choice; i.e., at least four correct entries out of five. This session lasted up to fifteen days. At the beginning of each trial, the maze was cleaned with ethanol (70%) and thereafter four of the arms (2, 3, 5, and 7) were baited with nourishment. The rat was placed on the central platform and was allowed to move freely. The arm choice was recorded when the rat ate bait or reached the end of an arm. Only the first entrance to the baited arm was recorded as a correct choice and the maze arms were not nourished again. Entrances into the unbaited arms and reentrances into the baited arms were recorded as Reference Memory Errors (RME) and Working Memory Errors (WME), respectively. Each rat was given two trials daily and the data obtained from the two trials were averaged and entered into the final data analysis. The rats' actions were scored by the percentage of the correct choices, RME, and WME [5, 37, 38].

Ten days after acquisition, the rats were examined for retention of the task. The rats were given two trials, and the mean scores of the percentage of the correct choices, WME, and RME was used for analysis [5, 37, 38].

The elevated plus maze is a rodent model of anxiety. The test setting consists of a plus-shaped apparatus with two open and two enclosed arms, each with an open roof. The model is based on rodents' disgust of open spaces. Anxiety reduction in the plus-maze is defined by an increase in the time spent in the open arms and an increase in the entries into the open arms. Total number of arm entries and number of closed-arm entries are usually employed as measures of general activity [39]. In the present study for the elevated plus maze test, the rats were placed at the intersection of the four arms of the maze. The maze consisted of two open arms (50×10 cm²) without walls and two opposite enclosed arms of the same size with 40 cm high walls. The maze was placed 50 cm above the floor in a quiet and dimly lit room. The rats got acclimatized to the testing room for at least 1 h before starting the test. The maze was cleaned after each testing. The animals were placed in the center of the maze facing an enclosed arm. Then, the number of entries into the enclosed or open arms and time spent in both open and enclosed arms were recorded for 5 minutes. The total number of entries into both enclosed and open arms was considered as the general motor activity [11, 39, 40].

To evaluate anhedonia in the rats, during the sucrose consumption test, the rats were given. The rats were allowed to adapt to the two bottles of water for 7 days and further experimental manipulations were started after 7 days. The rats had a free choice between two bottles filled with tap water and 1%, 2%, 4%, 8%, 16%, 32%, and 64% sucrose in 7 consecutive days. The acceptable criterion for sucrose preference was consumption of 60% of total liquid intake. Non-preferring animals (10%) were excluded from the study. The consumption of water and sucrose solution was estimated simultaneously in the control and experimental groups by weighing the bottles. The sucrose intake was calculated as the amount of consumed sucrose in mg/gram body weight. In addition, the preference for sucrose was calculated as the percentage of consumed sucrose solution out of the total amount of the liquid which was drunk [41].

The data were analyzed using analysis of variance (ANOVA) followed by Tukey's test. All the values are expressed as mean±SD. Besides, p≤0.05 was considered as statistically significant.

On days 1~3, all the animals in each group showed similar learning performance in the radial maze (RAM), while they seemed to gradually improve their ability to learn the location of the baited arm during the learning phase from the 4^th to the 8^th day (Fig. 1).

A two-way repeated measures ANOVA was performed with training day as within-subjects factor and experimental group as between-subjects factor. A significant different was found for Day [(p<0.001); F (7, 483)=29.79)], Group [(p<0.001); F (6, 63)=17.55)], and the Day by Group interaction [(p<0.001); F (42, 483)=8.22)].

This suggests that the performance of the animals changed during the acquisition and that this change in performance differed between the groups (p<0.001) (Fig. 1). Performance on 8^th day was further analyzed to assess the group differences in learning and a significant effect was found for groups at this time point [(p<0.001); F (6, 63)=7.66] (Fig. 2). The score of the correct choices were increased in the rats of the stress+curcumin, stress+sertraline groups in comparison to the stress+water and stress+olive oil groups (Fig. 1).

In comparison to the non-stressed rats, stress+water and stress +olive oil groups showed a significantly lower percent of correct choices from the 1^st to 8^th day (Fig. 1). However, no significant differences were found among the stress+curcumin, stress+sertraline and both compounds improved the rats' performance after stress (Fig. 1).

A significant effect was observed for Day [(p<0.001); F (7, 483)=8.02)], Group [(p<0.001); F (6, 63)=11.23)], and the Day by Group interaction [(p<0.001); F (42, 483)=5.67)].

This suggests that the RME during the acquisition phase changed with the days of training and these changes were different between the groups (Fig. 3).

Reference memory errors on 8th day were further analyzed to assess the group differences in learning and a significant effect was found for groups at this time point [(p<0.001); F (6, 63)=45.29] (Fig. 2). Moreover, stress+water and stress+olive oil groups revealed significantly more RME from the 1^st to 8^th day compared to non-stressed, stress+curcumin and stress+sertraline rats (p<0.001). Fewer errors were observed in evaluation of the reference memory in the rats of stress+curcumin and stress+sertraline groups in comparison to the stress+water and stress+olive oil groups (Fig. 3). Nevertheless, no significant differences were found among the stress+curcumin, stress+sertraline and both compounds improved the rats' performance after stress (Fig. 3).

A two-way repeated measures ANOVA was performed with training day as within-subjects factor and experimental group as between-subjects factor. A significant effect was observed for Day [(p<0.001); F (7, 483)=7.86)], group [(p<0.001); F (6, 63)=13.53)] as well as the day by group interaction [(p<0.001); F (42, 483)=4.50)] (Fig. 4). Working memory errors on day 8 were further analyzed to assess the group differences in learning and a significant main effect was found for groups at this time point [(p<0.001); F (6, 63)=24.87)] (Fig. 2). Furthermore, stress+water and stress+olive oil groups showed a significantly more WME from the 1^st to 8^th day compared to non-stressed rats (p<0.001). Fewer errors were observed in evaluation of the working memory in the rats of stress+curcumin and stress+sertraline groups in comparison to the stress+water and stress+olive oil groups (Fig. 4). Nonetheless, no significant differences were found among the stress+curcumin, stress+sertraline and both compounds improved the rats' performance after stress (Fig. 4).

A one-way ANOVA was used to analyze the percentage of the correct choices during the retention session. A significant difference was found among the study groups regarding the percentage of correct choices [(p<0.001); F (6, 63)=16.17] (Fig. 5). Furthermore, the stress+water and stress+olive oil groups revealed a significant reduction in the percentage of the correct choices compared to the non-stressed rats (p<0.001). Nevertheless, no significant differences were found among the stress+curcumin, stress+sertraline and both compounds improved the rats' performance after stress (Fig. 5). This demonstrated that concomitant treatment of curcumin or sertraline during stress prevented the reduction of scores of the correct choices in the retention session.

A significant difference was observed among the groups regarding the number of reference memory errors [(p<0.001); F (6, 63)=19.75], and the number of working memory errors (p<0.001). Besides, in comparison to the other groups, stress+water and stress+olive oil showed more RME and WME during the retention testing in comparison to the non-stressed rats (p<0.001) (Fig. 5). The concomitant treatment of curcumin or sertraline during stress causes fewer errors during evaluation of the reference and working memories in the retention session and they have similar effects (Fig. 5).

ANOVA was performed on Open Arms Time (OAT) and Open Arms Entries (OAE), as measures of anxiety, and revealed a significantly lower percent of the OAT and OAE by the stress+water and stress+olive oil groups compared to the non-stressed rats [(p<0.001); F(6, 77)=11.48]. Nevertheless, no significant differences were found among the stress+curcumin, stress+sertraline, and both compounds improved the rats' performance in OAT and OAE after stress (Fig. 6). ANOVA was also performed on closed arms entries and open arms entries, as a parameter reflecting the changes in the general motor activity, and showed no significant differences among the study groups (Fig. 6).

The rats consumed almost less water in these tests and sucrose solution intake varied according to its concentration. Analysis of the data revealed that the rats drank more sucrose solution than water at the 4, 8, 16, 32, and 64% concentrations, while the difference was not significant at 1 and 2% concentrations. In contrast, the stress+water and stress+olive oil groups consumed more water than sucrose solution at 1~4% concentrations compared to 8, 16, 32, and 64% concentrations. Overall, as the concentration increased from 1% to 64%, the rats' total intake increased, as well. The results revealed no difference among stress+curcumin and stress+sertraline groups regarding sucrose preference. However, a significant decrease of sucrose preferences was observed in stress+water and stress+olive oil groups compared to the non-stressed groups (Fig. 7). Moreover, the results revealed a significant difference between stress+water and stress+olive oil groups compared to stress+curcumin and stress+sertraline groups; [(p<0.001) F(6, 77)=95.00].