Background
The experience of social stressors such as bullying during adolescence places individuals at high risk for developing various psychiatric disorders both acutely and later in life, and include depression [
1,
2], anxiety [
1,
3], attention deficit hyperactivity disorder (ADHD) [
4,
5], and substance abuse [
6,
7]. While these disorders can present with widely varying symptoms, each is characterized by deficits in executive function [
8], thus providing a common variable by which to study the consequences of adolescent stress across a range of separate diagnoses. Broadly defined, executive function encompasses cognitive processes that allow for organized and adaptive behavior, including the ability to utilize and maintain task-relevant information, otherwise known as spatial working memory [
9]. Of particular relevance to adolescent social stress, both executive function ability and its major associated brain region, the prefrontal cortex (PFC), continue to mature during the adolescent period [
10]. The maturation of the adolescent PFC is delayed compared to both subcortical structures [
11] and to other cortical regions [
12,
13]. This appears to render the adolescent PFC susceptible to long-lasting neural disruption from social stressors [
14], which may contribute to the cognitive deficits seen in stress-associated disorders. The susceptibility of the developing PFC to stress is reflected by the finding of one pilot study that emotional abuse during childhood is associated with decreases in human adult spatial working memory [
15]. Furthermore, structural variations in the PFC appear to mediate the relationship between early life stressors and later spatial working memory deficits [
16], suggesting that early life stressors may alter the structure of the developing PFC to contribute to cognitive dysfunction later in life. While such studies provide important retrospective data on the potential consequences of early life stressors on cognitive ability, the direct effects of adolescent social stress on executive function and its underlying neural mechanisms have yet to be explored.
We have developed a paradigm using social defeat of adolescent rats to model the victimization and imbalance of power inherent in human social stress experiences, including teenage bully-victim relationships [
17,
18]. Following exposure to adolescent social defeat, several significant behavioral and neural alterations emerge in young adulthood [
18‐
21]. In particular, rats defeated in adolescence show decreased dopamine (DA) content in the medial PFC (mPFC) in adulthood at baseline [
18] and blunted extracellular mPFC DA release when challenged with acute amphetamine [
19,
22]. Adolescent defeat also results in increased expression of DA transporters in the adult mPFC [
21], which our preliminary data suggest enhances DA clearance and contributes to lowered mPFC DA activity [
23]. In addition, previously defeated rats show several behaviors indicative of mPFC DA hypofunction including increased locomotor responses to novel environments [
18,
19] as well as increased conditioned place preference for amphetamine [
20]. These results are noteworthy given that mesocortical DA plays a critical role in mediating executive function [
9]. Depletions of mPFC DA are known to produce deficits in spatial working memory tasks in both primates and rats [
24‐
26], and this appears to modulated by a lack of dopaminergic activity at the DA D1 receptor [
27,
28]. However, the relationship between spatial working memory performance and DA D1 stimulation appears to be based on an inverted-U function, as performance deficits are noted following both D1 agonism [
29] and blockade [
28]. Importantly, mPFC DA release decreases as the task delay is prolonged, and this decay in release correlates with increased spatial working memory errors [
30]. Pharmacological D1 stimulation can rescue memory performance during either long delays or following poor baseline performance, but is detrimental during shorter delays or when baseline performance is already high [
29,
31‐
33]. These studies suggest that an optimal level of mPFC DA activity is needed to maintain and utilize task-specific information across varying delay periods.
Previous research has demonstrated that stress in the form of water restraint [
32] or benzodiazepine antagonism [
34] in adult animals leads to acute disruptions to DA-dependent spatial working memory. While evidence suggests that adolescent social stressors may result in long term disruption to hippocampal-dependent spatial memory [
35‐
37], we are currently unaware of any research that has tested protracted changes in mPFC DA-dependent spatial working memory as a result of stress in adolescence. Given our previous findings of mPFC DA hypofunction following adolescent social defeat, we hypothesized that previously defeated rats would demonstrate disruptions in spatial working memory performance in adulthood. Therefore, we employed two separate spatial working memory tasks that differ in terms of delay period used. In the delayed win-shift task, rats acquire spatial information that then must be utilized to guide search behavior across delays that typically range from 5 minutes to 6 hours [
34]. The second task, the delayed alternating T-maze, also requires memory to guide search behavior but commonly utilizes shorter delays of 0 to 90s [
26]. Furthermore, because these tasks use food reward (sweetened condensed milk [SCM]), baseline SCM consumption and conditioned place preference for SCM were tested following adolescent social defeat to rule out potential differences in task motivation that could confound results of the spatial working memory tests [
38,
39]. As predicted, adolescent social defeat caused impairments in both spatial working memory paradigms, which were dependent on the delay period used. Moreover, these deficits appear to be independent of differences in motivation for the food reward used in the memory tasks.
Discussion
The results of the present study demonstrate that exposure to repeated social defeat stress during adolescence leads to performance deficits on two separate tasks of spatial working memory. These deficits appear to be dependent on delay duration, with defeated rats making significantly more errors than controls only following a 5 min delay on the delayed win-shift task and only following a 90 s delay on the delayed alternating T-maze task. These results add to previous findings demonstrating that social stress in adolescence also results in deficits in hippocampal-based spatial memory [
35‐
37] and suggest that adolescent stress can have an extensive impact on cognitive function.
Conditioned place preference for task reward (SCM) was similar between groups, suggesting that differences in spatial working memory performance were not due to alterations in the motivation to seek out palatable food. Control rats did demonstrate a significant increase in SCM consumption over time during the conditioning procedure that was not exhibited by previously defeated rats. Given the weak trend for defeated rats to show an increase in consumption over four conditioning sessions (see Figure
1), it is possible that additional sessions would be necessary for this effect to become significant, but further experiments would be required to determine this definitively. However, both total consumption across all conditioning sessions and home-cage consumption did not differ between groups in the current study. Combined with the conditioned place preference results, this further supports the notion that differences in motivation to consume reward did not contribute to memory task performance. Interestingly though, adult rats subjected to adolescent social defeat do show heightened conditioned place preference for amphetamine-paired cues [
20]. Therefore, the current finding that similar adolescent social defeat does not augment conditioned place preference for SCM-paired cues may suggest that this social stress during adolescence increases drug but not food reward-related processes. In agreement with the present findings, other studies have demonstrated that male rats subjected to stressors in adolescence do not show later changes in sucrose consumption [
49‐
51], although long term effects on drug reward processes have been more equivocal [
20,
52,
53]. It is known that more chronic social defeat procedures in adulthood can induce anhedonia for both food and drugs [
54,
55]. However, the adolescent social defeat procedure employed presently may have more in common with episodic as opposed to chronic social defeat paradigms in adulthood, as episodic defeat has been found to increase psychostimulant self-administration without disrupting sucrose preference [
55].
Analysis of spatial working memory performance revealed that on the delayed win-shift task, control rats demonstrated an expected increase in errors as the delay was prolonged. This pattern was absent in previously defeated rats, with performance at the 5 min delay similar to that at the 480 min delay. Thus, it appears that in the win-shift task, not only do defeated rats commit more errors compared to controls at the shortest delay used, but their performance also appears to asymptote across subsequent increased delay periods. This is in contrast to the pattern seen on the delayed alternating T-maze, in which defeated rats showed impairments after a 90 s delay, but not after shorter delays of 30 and 60 s. It should be noted that while the delayed alternating T-Maze and delayed win-shift task are similar in respects to their association with mPFC DA and assessment of memory-guided behavior, we are unaware of any studies that have compared the same delay times between these two spatial working memory paradigms. Additional testing would therefore be required to determine definitively the span of delay periods within the same test for which adolescent defeat causes impaired spatial working memory performance.
Because spatial working memory performance on these delayed tasks is known to be dependent upon optimal PFC DA activity [
27‐
33], it is possible that the mPFC DA hypofunction caused by adolescent social defeat [
18,
22] is contributing to the observed differences in the two spatial working memory tasks. Interestingly, rats experiencing an 85% reduction in mPFC DA content via 6-OHDA lesion exhibit spatial working memory deficits at a 90 s delay on the alternating T-maze, but not at shorter delays of 30 and 60 s [
26], similar to the performance of defeated rats in the current study. Additional spatial working memory impairments on the delayed alternating T-maze have been found in rats with deficits in PFC DA activity resulting from either chronic stress [
32] or aging [
33]. Thus, while other mechanisms cannot be ruled out, disruptions to the developing mPFC DA system during adolescence caused by social defeat stress may contribute to long term deficits in spatial working memory. However, further experiments in which mPFC DA activity in previously defeated rats is restored prior to working memory assessment are required to support such a hypothesis definitively.
Given that increased levels of DA activity are needed to support spatial working memory performance with more prolonged delays [
30,
31], it might be expected that defeated rats with lower mPFC DA activity would have shown spatial working memory deficits during the 30, 60, and 480 min delay on the delayed win-shift task compared to controls. The lack of differences at these delay intervals between control and previously defeated rats may be due to the role of other brain regions in spatial working memory, specifically at longer delay periods. For example, it is known that an intact connection between the hippocampus and the mPFC is necessary for performance on the delayed win-shift at a 30 min delay, but not during a non-delayed task [
34]. A separate study also found that an intact hippocampus became essential for spatial working memory performance at a 5 min delay, but not a 10 s delay [
56]. It is therefore possible that the multiple regions involved in spatial working memory performance at longer delays in the delayed win-shift task allowed for compensation in previously defeated rats despite deficits in mPFC DA activity.
An unexpected finding was that despite showing an increased number of spatial working memory errors at 5 min, rats defeated in adolescence required fewer training trials (without a delay) to reach criterion on the delayed win-shift task. It is unlikely that this difference is due to varying levels of motivation for food reward, as both defeated rats and controls showed similar conditioned place preference for and consumption of SCM. One possible explanation for faster criterion acquisition for this task by defeated rats may be an increased preference for novelty. It has been postulated that both alternation behavior on the T-maze and win-shift behavior on the radial arm maze is reminiscent of a rat’s tendency to seek out environmental information in an efficient manner by exploring new places rather than returning to previously visited ones (see reviews [
57,
58]). Wistar rats selected for naturally high locomotor responses to novelty show an increase in learning performance on a radial arm maze task two days earlier than their low responding peers [
59]. Although no differences in locomotion were noted during initial habituation to the apparatus (results not shown), we previously demonstrated increases in adult locomotor behavior in a novel open field and on an elevated plus maze following adolescent social defeat [
18,
19], as well as upon initial exposure to a CPP apparatus [
20]. However, if an increased preference for novelty drove time to reach criterion in the current study, it might be expected that defeated rats would have also required fewer training trials in the alternating T-Maze, which was not the case (although there was a non-significant trend towards faster acquisition in the defeated group). Similar performance between groups during T-Maze training might have been due to the necessary “forced alternation” trials that all rats went under prior to training, which may have served to equalize any potential differences in criterion acquisition in this particular task, negating any effects of increased novelty preference caused by adolescent social defeat that were apparent in the delayed win-shift paradigm. Another potential explanation for faster task acquisition may have to do with differences in learning strategies employed by rats exposed to adolescent social defeat. On maze tasks, rats are known to utilize either a place based/spatial strategy dependent on the hippocampus, or a response based/egocentric strategy dependent on the nucleus accumbens [
59]. For win-shift tasks in particular, spatial strategies dependent on the hippocampus are favored to reach initial task acquisition [
59,
60]. Therefore, while previously defeated rats showed impairment in delayed working memory performance that may be due to deficits in mPFC DA, it is possible that differences in hippocampal monoaminergic activity caused by adolescent defeat [
18] promoted enhanced utilization of spatial strategies to acquire the task initially during training.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
AMN carried out the spatial working memory tasks, helped with the defeat procedure, and drafted the manuscript. LCM carried out the conditioned place preference tests and helped with defeat procedure. GLF oversaw design of the conditioned place preference test and statistical analysis for the entire study, as well as helping to draft the manuscript. MJW conceived the defeat procedure and oversaw design of the entire study and writing of the manuscript. All authors read and approved the final manuscript.