Adolescence has gained increasing attention as a sensitive period of development, a period in which pubertal transitions may increase the vulnerability to stressors (McCormick et al., 2017). Adolescence is defined as a transition between childhood and adulthood, and puberty is thought to be an important hallmark of it. Adolescence is also of great clinical importance; it is the time in which many mental health problems such as mood disorders emerge. Further, the risk of adult drug abuse and addiction is greater in adults who were exposed to drugs in their adolescence, rather than those who started drug use in adulthood (Odgers et al., 2008). Animal studies has confirmed the effect of stress during adolescence to be deleterious for adult affective and cognitive functions as well as stress reactivity (Pohl et al., 2007; McCormick et al., 2008; Arnett et al., 2015). However, these effects of adolescent stress were found to be sex dependent (McCormick and Mathews, 2007; Weintraub et al., 2010). It is often thought that females are more vulnerable to the effects of adolescent stress, particularly in their depressive behavior (Mathews et al., 2008; Bourke and Neigh, 2011). In contrast, differences have been observed in anxiety-like behaviors of adult males after acute adolescent stress, with no or very limited effect in females (Lovelock and Deak, 2019).

In animal studies, stressors can be administered to rodents at postnatal day (PD) 22–32 during early adolescence (EA) or peripubertal period, later in adolescence up to PD 45, or post-pubertally (Lo Iacono and Carola, 2018). Stress experienced in the peripubertal phase is of clinical importance, as it is associated with the development of disorders such as depression, anxiety, and post-traumatic stress disorder in adulthood (Spear, 2000; Bale et al., 2010; McCrory et al., 2012; Turecki, 2012; Brown and Spencer, 2013). It is known that acute stress produces more prolonged activation of the hypothalamic-pituitary-adrenal (HPA) axis in peripubertal animals than in adults, thus, the consequence of acute stress during this developmental period could be long lasting (Lui et al., 2012). However, how genetic predisposition to increased stress-reactivity interacts with a mild developmental challenge is less explored. Specifically, animals of differing stress-reactivity that encounter a mild, acute stress during the prepubertal EA period may produce lesser or differential affective behaviors in adulthood.

Two inbred rat strains with differential stress-reactivity were employed in this study. These strains were generated by bidirectional selection from the Wistar Kyoto (WKY) parental strain using immobility behavior in the forced swim test (FST) as a functional selector (Will et al., 2003). The by now inbred Wistar Kyoto More Immobile (WMI) strain consistently show greater immobility compared to the inbred WKY Less Immobile (WLI) strain (Andrus et al., 2012; Mehta et al., 2013). Adult WMI males have decreased anxiety-like behavior in the open field test (OFT) compared to WLI males, while females of both strains have shown the same high levels of anxiety in adulthood (Mehta et al., 2013). WMI males also show greater behavioral stress reactivity than WLI males, indicated by their increased fear memory after stress (Lim et al., 2018).

Our hypothesis was that EA stress would exacerbate the behavioral deficits of WMI rats and generate a vulnerability in the WLI strain in adulthood. Additionally, we hypothesized that the genetic differences in stress-reactivity between the strains would interact with the sex differences observed in other rodent studies in adult behaviors after EA stress. To test these hypotheses, males and females of the more stress-reactive WMI, and their nearly isogenic less stress-reactive control WLIs were exposed to a stressor during early adolescence. This stressor was a mild foot-shock received during the contextual fear conditioning (CFC) test. In adulthood, we tested remote fear memory (RM) to determine the EA stress effects. After RM, we tested affective behaviors that are known to differ between the strains and sexes in adult WMI and WLIs. Previously, strain and sex differences have been seen in the OFT and in the FST, the latter being the functional selector for the original selective breeding (Will et al., 2003; Mehta et al., 2013). After adult stress, social interaction and recognition also differed within the strains by sex (Schaack et al., 2021).

Expression of the brain-derived neurotrophic factor (BDNF) has been shown to be region-specifically responsive to stress and thought to be a marker of neuronal plasticity (Bath et al., 2013). The BDNF stress-sensitivity hypothesis posits that disruption in the endogenous BDNF activity potentiates sensitivity to stress (Notaras and van den Buuse, 2020). Since the WMI strain show enhanced sensitivity to stress, Bdnf expression in relevant brain regions, specifically the hippocampus and the prefrontal cortex, has been studied. Although the nature of the BDNF-glucocorticoid connection is very complex (Choy et al., 2008; Numakawa et al., 2013), a brain region and development-dependent connection has been suggested between them (Choy et al., 2008; Daskalakis et al., 2015). Therefore, we examined the glucocorticoid receptor (Nr3c1) expression in the same brain regions.

The purpose of this study was to determine whether genetic hypersensitivity to stress would alter behaviors in adulthood after a limited mild stress during early adolescence. Additionally, sex differences in the effects of EA stress could also be determined, and thereby ascertain the significance of sex in the consequences of genetic—environmental interactions.

The goals of the study were to identify the role of genetic stress-reactivity in the long-lasting behavioral effects of stress during early adolescence. The adolescent stress was a contextual fear memory test, in which no strain and sex differences were found. The remote memory of this stress was affected in adult males, specifically in WLI males, but not in females. Similarly, adult females were not affected by EA stress in their anxiety and social behaviors, while this EA stress brought positive changes in these behaviors to adult males. However, the enhanced immobility of the less stress-reactive WLI males and females after EA stress in the FST suggest that similarly to other strains, EA stress enhances depression-like behavior or passive coping in adulthood. The opposite effect of EA stress on WMI females question whether this mild stress can generate a resilience in the stress-reactive strain, particularly in females that were not affected by EA stress in any of the other measures. The lack of effect of EA stress on Nr3c1 expression in either brain region, but the WMI-specific changes in Bdnf transcript levels propose that glucocorticoid independent processes regulate these latter changes.

Adult-like CFC emerges by PD 24 (Casey et al., 2015), therefore, the lack of strain and sex differences in fear memory at EA suggests no differences in developmental trajectory between the strains. Still, despite this lack of differences in EA responses to CFC, major strain and sex differences were found in remote memory, particularly in WLI males. The increased percent freezing of adult EA-stressed WLI males is seemingly contradictory with their increased time spent in the center of the OFT. Would this mean a clear separation between fear and anxiety as increased percent freezing suggests increased fear, while increased time spent in the anxiety-provoking center of the OFT indicates decreased anxiety?

In neurobiological research, the distinction between anxiety and fear is ambiguous, and often one is used to define the other (Perusini and Fanselow, 2015). Fear is thought to be specific to responses directed to a present threat associated with specific cues or contexts, while anxiety is in preparation for threats that are future-oriented (Davis et al., 2010). The distinct definition of fear and anxiety are supported by opposing changes found after environmental challenges in these two facets of danger-induced behaviors (Heinz et al., 2021). However, human and animal studies support the opposing hypothesis showing correlations between these behaviors (Indovina et al., 2011; Ahn et al., 2013). Since anxiety measurements are based on the rodents’ natural tendency to avoid open areas, it is thought to measure intrinsic anxiety-related characteristics of the animal. Since male WMIs already show less anxiety than WLIs (Mehta et al., 2013), the exaggerated effect of EA stress on decreasing anxiety of the adult WMI males could be due this innate difference. Indeed, the genetically high anxiety HR rats also show increased contextual fear memory compared to the low anxiety LR animals (Lehner et al., 2010), just like the EA-stressed WLI males.

The finding of decreased anxiety-like behaviors in males of both strains after EA stress is similar to some, but dissimilar to other findings in the literature. Acute lipopolysaccharide injection at PND 26 shown to be anxiolytic in male, but not in female, Wistar rats as measured by time spent in the open arm of the elevated plus maze (EPM) (Ariza Traslaviña et al., 2014). No other acute stressor applied at the same developmental period showed this response in the study. Sex specific effect is also reported for Wistar rats, when acute stress during adolescence increases anxiety behavior in male Sprague-Dawley rats, while females are not affected (Lovelock and Deak, 2019). Most studies report increased anxiety-like behaviors in adult male rodents after a single or repeated mild stress during the same developmental period (Tsoory et al., 2007; Mancini et al., 2021; Meyer et al., 2021). In contrast, a 3-day stress in the same EA period increases anxiety-like measures in the OFT and the EPM in both adult male and female rats (Jacobson-Pick and Richter-Levin, 2010).

The current findings of decreased anxiety-like behaviors in adult males after EA stress is novel and at variance with other studies. The cause of these differing results is likely related to the strains of the animals employed. The parental strain of the WLI and WMI, the WKY strain, show increased anxiety and depression-like behavior as measured by immobility in the FST. However, immobility in the FST is also considered to be more of a measure of passive coping with stress (de Kloet and Molendijk, 2016). The selective breeding was based on the immobility behavior, and anxiety behavior in the OFT did not segregate consistently between the strains. In the present study, males of both strains showed decreased adult anxiety-like behavior after the relatively mild acute stress during early adolescence, suggesting a resilience generated by the EA stress. In contrast, only adult WMI females had a positive effect of adolescent stress on immobility in the FST. Interestingly, in a recent study we found that stress during adolescence in another strain of rats with genetic predisposition to increased passive coping led to increased anxiety in males, but decreased immobility in the FST in females (Ji et al., 2021). Would females genetically predisposed to increased passive coping be more resilient to the effects of adolescent stress than males of the same strain? Would the resilience generated in females after the double hit of adolescent stress and the stress of FST in adulthood be like the resilience generated by social defeat in adolescence to the single stress in adulthood (Mancini et al., 2021)? Future studies modifying the nature of the stressor in adolescence and the strain of animals used could in part answer this question.

The increased social interaction and recognition observed in adult male rats after EA stress may be related to the decreased anxiety-like behavior observed. This hypothesis could be confirmed if other genetically anxious or stress-prone strains show similar phenomena. The Roman Low Avoidance (RLA) strain has consistently shown high anxiety compared to the Roman High Avoidance (RHA) animals (Giorgi et al., 2019). However, after neonatal handling, social interaction increases in males of both strains in parallel with their decreased anxiety (Sampedro-Viana et al., 2021). The authors of that study argue that the effects of neonatal handling on social interaction seem to be independent from the reduction of anxiety. In a similar vein, we could argue that these phenotypes are also independent in our study as anxiety-like behavior as measured by distance traveled in the center of the OFT is less improved in EA-stressed WLI males compared to WMI males. Still social interaction is improved by EA stress to a similar degree in males of both strains.

Females of either strain were resistant to the behavioral changes caused by EA stress in agreement with many studies, using similar or dissimilar stressors and prepubertal or adolescent developmental time frame (Klinger et al., 2019). Other studies have found no effect in females (different strains, stressors and ages of exposure to stress) in the EPM and/or in the OFT (Barna et al., 2003; Slotten et al., 2006; Prusator and Greenwood-Van Meerveld, 2015; Guadagno et al., 2018). Although examined at earlier time points (i.e., PD3, PD9, or PD11) and different stressors, others have also found no impact of early life stress on female rats with respect to the EPM, NOR, and object recognition location test (Loi et al., 2017). A recent study conducted in female MAM rats also found no differences in anxiety-like behavior in the EPM (Perez et al., 2019).

The passive coping measure of immobility responded to EA stress in both adult males and females. EA stress increased immobility in the adult WLI males, similarly to our findings after chronic stress in adulthood (Mehta-Raghavan et al., 2016). However, it did have the opposite effect on adult females; increased immobility in WLIs, but decreased immobility in WMI females after EA stress. While one can argue that in the case of males, control WMI males show a ceiling effect in immobility, this is clearly not the case for females, as immobility of EA-stressed WLI females is higher than that of control WMI females. The exaggerated immobility of stressed WLI females is inverse of their decreased climbing behavior, confirming this finding. As the WLI and WMI inbred strains were originally selected for immobility behavior in the FST, these changes induced by EA stress suggest that in the absence of genetic predisposition, even a minor stress during early adolescence could have adverse long-lasting effects on the individual. However, the same minor stress could generate resilience in the genetically predisposed individual. A recent very illuminating review of resilience discuss novel interpretation of resilience, and pathways that can lead to it (Malhi et al., 2019).

Brain-derived neurotrophic factor is a growth factor acting through TrkB tyrosine kinase receptors to promote neuronal survival and differentiation. The evidence for a critical role of BDNF in resilience during development is largely based on animal models of chronic stress (Taliaz et al., 2011), which significantly implicate hippocampal BDNF mediation. BDNF genes are also involved in the development of neural circuits that control coping mechanisms (Cattaneo et al., 2015). BDNF is also involved in social behaviors as demonstrated by animal models of social behaviors (Branchi et al., 2013). It is also critical for experience-dependent synaptic plasticity and memory, including fear learning (Chao, 2003; Lee et al., 2003; Andero and Ressler, 2012; Casey et al., 2015). Loss of Bdnf expression in adult genetic knockout mice leads to impaired fear learning and increased anxiety-related behaviors (Chen et al., 2006). Furthermore, Bdnf expression in the hippocampus is linearly correlated with time spent in the center of the OFT (Bahi, 2017). Thus, we expected that at least some of the behavioral phenotypes show correlations with Bdnf expression. This was not the case. It is proposed that decreased Bdnf does not cause depressive behaviors but does hamper the effect of antidepressant drugs (Martinowich et al., 2007), therefore, the lack of correlation between Bdnf expression and the behavioral phenotypes may only reflect that. There was also no correlation found between the expression of the glucocorticoid receptor and that of Bdnf in the same brain regions. However, both Nr3c1 and Bdnf expression were measured after the series of behavioral tests, which could clearly obscure any effects of EA stress on the expression of these genes.

This study is novel, as for the first time it demonstrates the complex effect of early adolescent stress on two nearly isogenic inbred strains exhibiting high, and low stress-reactivity. The sex-specific findings are also very illuminating, particularly the major sex differences in vulnerability to EA stress. One caveat of our study is the nature and the timing of the stress employed. Stronger and different, for example metabolic, stressors may lead to different outcomes. Additionally, despite our efforts to eliminate carryover effects of the different behavioral measurements, a cumulative stress affecting the adult animals could not be excluded. Future studies can investigate these questions and characterize whether the observed sex differences are related to differences in the early adolescent neurodevelopmental stages between males and females. Additionally, further exploration is needed to determine if the resilience caused by the EA stress can be generated at different time frames or by different stressors in both sexes.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

The animal study was reviewed and approved by Institutional Animal Care and Use Committee of Northwestern University.

SK, MM, and SG: experimental work, analyzing, interpreting data, and editing the manuscript. ER: conceptualization, writing editing of manuscript, analyzing, and interpreting data. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.