We have previously shown that 21-day chronic restraint stress impacts instrumental learning, but overall few studies have examined sex differences on the impact of stress on learning. We further examined sex differences in response to extended 42-day chronic stress on instrumental learning, as well as recovery from chronic stress. Rats were tested in aversive training tasks with or without prior appetitive experience, and daily body weight data was collected as an index of stress. Relative to control animals, reduced body weight was maintained from day 22 through day 42 across the stress period for males, but not for females. Stressed males had increased response speed and lower learning efficiency during appetitive acquisition and aversive learning. Males overall showed slower escape shaping times and more shock exposure. In contrast, stressed females showed slower appetitive response speeds and higher appetitive and aversive efficiency but overall reduced avoidance rates during acquisition and maintenance for transfer animals and during maintenance for aversive-only animals. These tasks reveal important nuances on the effect of stress on goal-directed behavior and further highlight sexually divergent effects on appetitive versus aversive motivation. Furthermore, these data underscore that systems are temporally impacted by chronic stress in a sexually divergent pattern.
Chronic stress can lead to learning deficits in humans [
We have previously used the instrumental appetitive-to-aversive transfer task to investigate a variety of manipulations [
In a subsequent study we specifically investigated the impact of 21-day chronic restraint stress and sex differences on the appetitive-to-aversive transfer task and found task by sex by stress specific deficits [
The current study is a follow-up to our previous publication and examines several questions. Are there impairments with 42 days of chronic restraint stress? Can male and female rats recover from the impact of 42 days of chronic restraint stress? We hypothesized that the extended duration of six weeks of chronic restraint stress would produce learning deficits relative to control animals and that the avoidance learning deficits would be exacerbated relative to those previously seen with three-week chronic restraint stress for males and females. Finally, we hypothesized that both males and females that were restrained for 42 days would recover with three weeks of nonrestraint prior to training.
Adult Sprague-Dawleyrats weighing between 175 and 275 g (females) and 275 and 375 g (males) were randomly assigned to one of twelve groups. There were three experimental conditions (control, restraint, or recovery), two learning tasks (appetitive-to-aversive transfer or aversive-only), and two sexes (male or female). Animals were housed socially until experiment onset. Due to food restriction for the appetitive training, all animals were housed individually five days prior to experiment onset and were maintained at 85% of their free-feeding weight throughout all phases of instrumental training. Weighing occurred immediately prior to training and subsequently the animal was returned to its home when food availability began. Food restriction began five days before the first day of shaping onset for all animals. The rats had access to water
Animals were transported to a separate room and restrained for six hours per day between the hours of 10 a.m. to 4 p.m., seven days per week, for 42 consecutive days as previously described in detail [
To examine the impact of recovery, a group of animals underwent the same 42-day chronic restraint protocol, but testing did not occur immediately following the last day of restraint. Instead rats were given three weeks of additional “recovery” time to sit in their home cages prior to beginning appetitive or escape shaping. Depending upon whether the animal was in the transfer group or the aversive-only group, appetitive or aversive shaping began the day immediately after the 21-day recovery period.
Separate male and female litter-matched animals underwent the same experimental restraint procedures to assess body weight changes (the first 21 days of these data were published in [
Animals were appetitively shaped using a method of successive approximations to barpress for food reward (sugar pellet), which began one day immediately following the last day of restraint or recovery. Beginning on a fixed ratio (FR-1) schedule, when the animal barpressed 100 times within 30 minutes it was advanced to a fixed ratio-4 (FR-4) schedule of reinforcement. On the FR-4 schedule animals had to barpress 400 times to receive 100 food rewards within 40 min for two consecutive days in order to advance. After meeting the FR-4 schedule animals advanced to appetitive tone training in which a 3 sec tone (2000 Hz, 90 dB SPL) signaled the availability of food reward if the bar was pressed within the 3 sec tone period. One tone-signaled training session consisted of 100 trials (for details see [
On the day immediately following appetitive training transfer animals began the aversive learning task. Additional groups were placed directly into the escape shaping task with no prior appetitive experience. In order to directly compare the impact of chronic restraint between transfer and AV-only animals, the average number of days transfer animals spent in the appetitive paradigm was calculated. AV-only animals waited the average number of days after restraint prior to beginning escape shaping. The aversive shaping program consisted of a shock that could be escaped with a lever press. The shock intensity was maintained between 0.8 and 1.0 mA and the shock pulses (250 ms at 1.33 Hz) were presented continuously until the bar was pressed. If the animal did not press the bar within 30 pulses, the current was turned off manually for a rest period of 10 seconds; however, when the animal barpressed, a rest period of 30 seconds was initiated. The escape shaping criterion was a barpress within 5 shock pulses or fewer for 15 consecutive escape trials. After meeting the escape criterion, animals were then advanced to tone-signaled (same as above) trials on the subsequent training day. On tone-signaled trials, four foot shock pulses of 250 ms separated by 500 ms could be avoided with a barpress during the first 3 seconds of tone presentation or escaped by a barpress in the latter 3 seconds after the shock pulses began. To prevent the animals from adopting a strategy of constantly holding the bar down to avoid shock, shock pulses (off-bar shocks, OBSs) were delivered after 5 seconds of holding and continued until the animal released the bar (for details see [
Data are presented as mean ± standard error of the mean (SEM). A condition (3) × sex (2) analysis of variance (ANOVA) model was used to test all appetitive shaping data, all appetitive tone-signaled data, aversive efficiency ratios (ERs), aversive off-bar shocks (OBSs), and aversive difference scores. A condition (3) × sex (2) × task (2) ANOVA model was used to test escape shaping (number of days to criterion) data. Pearson’s chi-square analysis was used to analyze group differences in the ability to meet the escape shaping criteria. A condition (2) × time (21) repeated measure analysis of variance (RM-ANOVA) test was used to evaluate body weight data for males and females and a condition (3) × sex (2) × days (10) RM-ANOVA was used to test avoidance learning. When assumptions of sphericity were violated, the degrees of freedom were adjusted using the Huynh-Feldt correction. To determine what was driving any overall significant effects, post hoc tests (more than two groups) or pairwise comparisons that were significant were reported. Additionally, repeated contrasts looked at learning improvement via day-by-day comparisons for all repeated measures. Multiple comparisons were adjusted using the Bonferroni correction and statistical significance was set at
Figure
It shows body weight for males (a) and females (b) from training day 22 through day 42. Stressed males (black) gained weight at an attenuated rate (−1.2%) relative to controls (gray), whereas females gained weight at comparable rates.
Figure
It shows appetitive shaping data for the fixed ratio-1 task (a) and the fixed ratio-4 task (b) for control (gray), stressed (black), and recovered (checkered) animals. There was significant sex by condition interaction on the FR-1 task with females and recovered animals taking longer to meet the criterions. There was a main effect of sex on the FR-4 task with females taking longer to meet the appetitive shaping criterion.
Figure
It shows appetitive tone-signaled data for the number of days to criterion (a), efficiency ratios during acquisition (b), and the latency to barpress during acquisition (c) for control (gray), stressed (black), and recovered (checkered) animals. There was a significant sex by condition interaction on all three measures. Whereas chronic stress reduced learning and increased response speed in males, it increased learning and reduced response speed in females. Recovery from stress showed variable results across measures.
Efficiency ratios (number of correct responses/total number of barpresses) were also measured during acquisition of the first two days of appetitive tone-signaled learning (see Figure
We also examined latency to barpress during the first two days of acquisition on the appetitive tone-signaled task (Figure
All animals except for one restrained male advanced to aversive learning to begin escape shaping. One index of escape shaping is whether or not they meet criterion. For control animals, 100% of males for both the transfer and AV-only tasks reached criterion, but for females 94% of the transfer animals met criterion and 90% of the AV-only animals met criterion. For restrained animals, 78% of males met criterion for the transfer task whereas 75% met criterion for the AV-only task. In contrast, 100% of females met criterion for both tasks. For the recovery animals, 100% of transfer males met criterion, while 75% of AV-only males met criterion. Finally, 100% of females met both criterions for recovery animals. A chi-square analysis revealed a weak trend for sex differences
Figure
It shows escape shaping for aversive transfer (a) and aversive-only (b) animals for control (gray), stressed (black), and recovered (checkered) animals. There were no significant effects on escape shaping for any transfer animal. There was a main effect of sex for the AV-only animals. Males took longer to reach the escape criterion than females.
Animals that successfully reached the escape shaping criterion were advanced to tone-signaled avoidance training. Figure
It shows ten-day avoidance rates for transfer males (a) and females (b) and AV-only males (c) and females (d) for control (gray), stressed (black), and recovered (gold) animals. 42-day restraint stress resulted in significant reductions in avoidance rate acquisition and maintenance for transfer females and for maintenance only for AV-only females. However, all recovered animals had avoidance % at rates equal to controls. Day-by-day comparisons for significant differences between control and stressed animals are denoted with
In addition to analyzing the avoidance percentage, the average avoidance efficiency ratio (number of correct responses/total number of barpresses, ER; see Figure
It shows tone-signaled avoidance efficiency ratios (ERs) for transfer (a) and AV-only (b) animals for control (gray), stressed (black), and recovered (checkered) animals. There was a main effect of sex for transfer animals with females avoiding more efficiently. There was also an effect of condition for females with stressed females avoiding significantly less efficiently than control females. A similar pattern was seen for AV-only; however the effect was less robust and showed only a moderate sex effect.
It shows the average number of off-bar shocks (OBSs) across all ten days of avoidance training for transfer (a) and AV-only animals (b). There was an overall main effect of sex for both learning tasks. Males averaged significantly more OBSs.
Figures
Efficiency ratios (ERs) and off-bar shocks (OBSs) were also calculated for AV-only animals. The ten-day average ER for male animals was
We evaluated the impact of the extended duration of restraint on avoidance learning relative to 21-day restraint for transfer and AV-only animals. Daily difference scores were averaged across animals for each restraint condition: (1) 21-day restraint minus control, (2) 42-day restraint minus 21-day restraint, and (3) 42-day restraint minus control. For transfer animals there was a significant main effect of condition
Analyses for AV-only animals showed only an overall trend for condition
The current paper is a follow-up to our manuscript published last year [
In our previous publication, we indexed the physiological impact of the commonly used 21-day chronic restraint stress model with daily body weight measurement, which has been previously utilized as an index of stress [
The two primary findings from the appetitive shaping task found a significant interaction of sex by condition on the FR-1 task and a main effect of sex on the FR-4 task. In both tasks, females took longer to reach the shaping criterions suggesting that the males were either more motivated to barpress for food reward or simply faster at it which matches previous reports in the literature [
The data show multiple effects on appetitive tone-signaled learning. Although the impact of restraint on females still enhanced their performance similar to that previously seen in 21-day restrained females relative to control females, the effect was not significant for 42-day restrained females again suggesting the onset of anhedonia and an impact on striatal circuitry. However, we did find significant sex by condition interactions for the number of days to meet criterion, for efficiency ratios during acquisition, and for the latency to barpress during acquisition. Females were overall more efficient than males during acquisition which matches our previous report [
In contrast to 21 days of restraint stress [
Similar to our previous findings [
Following advancement to tone-signaled learning, the 42-day restrained transfer animals showed exacerbated deficits relative to those previously seen with 21-day restraint [
Relative to the 21-day restrained animals, different patterns emerged on the AV-only task for both males and females. For males, there was increased variability in barpressing which was the highest during the AV-only task. This variability was likely driven by the overall increase in off-bar shocks (OBSs) during AV-only relative to aversive transfer. There was little impact of chronic restraint on overall avoidance % for males, but there was an effect on response efficiency. Males continued to show overall high but inefficient levels of responsiveness with the impact of restraint being an exacerbation on inefficiency. Given that restrained males were significantly faster responders it is likely that they were experiencing heightened arousal which resulted in proactive interference. Males had significantly higher numbers of OBSs alongside their nonefficient but robust response pattern which lead to an overall increase in response variability within animal on a single session as well as across sessions especially on the AV-only task. The current set of results suggests that on the AV-only task the males’ change in responsiveness is more drastic representing an overall shift in performance strategy. On days 4 through 6, restrained males reduced their average number of barpresses, but their ERs did not improve indicating that they did not have an increased understanding of the task demands but rather they suffered “response maintenance fatigue.” It is not clear if this fatigue was related to a physical demand overload or a cognitive strain or potentially some combination.
In contrast, females appeared to suffer from an overall decreased motivation which impacted their avoidance rates on the AV-only task as well. However, the magnitude of the deficit was smaller relative to the transfer task and existed only on postasymptotic training days relative to the larger transfer deficit which was also present during learning acquisition and transition. The combined results from the appetitive, transfer, and AV-only tasks suggest that 22 to 42 days of chronic restraint stress may be a sensitive time period for impacting motivation for females and that higher cognitive tasks may be the most vulnerable to these effects, but not exclusively so.
The impact of the extended restraint revealed a dose-dependent response on female transfer learning. After analyzing difference scores between groups, we saw a significant effect for 21-day restraint minus control versus 42-day restraint minus control which suggest that the impact of each of these durations is significantly different from each other with the 42-day restraint minus control having the larger impact. However, when directly comparing the effect sizes for the first three weeks versus the final three weeks (21-day restraint minus control versus 42-day restraint minus 21-day restraint) there was no significant difference. This suggests that while the impact of chronic restraint stress is progressive, the added contribution of the final three weeks of restraint did not substantially differ from the initial three weeks.
There is little research investigating recovery from the impact of 42 days of restraint stress. The concept of recovery encapsulates the idea of undoing harm such that system homeostasis is reestablished [
There are a few limitations of this study to mention. First, although 21-day chronic restraint stress has been well characterized, we did not specifically assess the neural correlates underlying the behavioral effects seen in the paradigm. Secondly, in addition to gonadal hormones moderating the effects of chronic stress on learning, menstrual phase has been shown to do this as well [
The primary impact of chronic restraint stress revealed heightened responsiveness for males leading to further response variability and inefficient responding which interfered with task demands. Although we also saw high variability in male avoidance rates with 21-day restraint, we did not see an effect on response speed until 42-day restraint indicating a progressive impact on males. For stressed females, there was a decrease in responding and in response speed, but no decrease in response efficiency indicating an overall pattern of decreased motivation which was reversible. Given that both sexes are experiencing performance deficits, albeit opposing ones, one utility of this study was to shed light on areas that can be targeted for effective learning intervention techniques which vary according to sex, stressor duration, and task demands. Finally, it is important to note that, even with an extended stressor duration, animals were able to recover from the avoidance deficits.
None of the authors have any conflict of interests to declare.
The authors of this study would like to acknowledge Indiana University Clinical Science Training Grant NIH T32 MH 017146 for support.