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Adolescent Stress Confers Resilience to Traumatic Stress Later in Life: Role of the Prefrontal Cortex

Open AccessPublished:March 07, 2022DOI:https://doi.org/10.1016/j.bpsgos.2022.02.009

      Abstract

      Background

      Adolescent brains are sensitive to stressors. However, under certain circumstances, developmental stress can promote an adaptive phenotype, allowing individuals to cope better with adverse situations in adulthood, thereby contributing to resilience.

      Methods

      Sprague Dawley rats (50 males, 48 females) were subjected to adolescent chronic variable stress (adol CVS) for 2 weeks at postnatal day 45. At postnatal day 85, a group was subjected to single prolonged stress (SPS). After a week, animals were evaluated in an auditory-cued fear conditioning paradigm, and neuronal recruitment during reinstatement was assessed by Fos expression. Patch clamp electrophysiology (17–35 cells/group) was performed in male rats to examine physiological changes associated with resilience.

      Results

      Adol CVS blocked fear potentiation evoked by SPS. We observed that SPS impaired extinction (males) and enhanced reinstatement (both sexes) of the conditioned freezing response. Prior adol CVS prevented both effects. SPS effects were associated with a reduction of infralimbic (IL) cortex neuronal recruitment after reinstatement in males and increased engagement of the central amygdala in females, both also prevented by adol CVS, suggesting different neurocircuits involved in generating resilience between sexes. We explored the mechanism behind reduced IL recruitment in males by studying the intrinsic excitability of IL pyramidal neurons. SPS reduced excitability of IL neurons, and prior adol CVS prevented this effect.

      Conclusions

      Our data indicate that adolescent stress can impart resilience to the effects of traumatic stress on neuroplasticity and behavior. Our data provide a mechanistic link behind developmental stress-induced behavioral resilience and prefrontal (IL) cortical excitability in males.

      Keywords

      Understanding factors that affect the brain during adolescence has substantial health relevance, given the onset of numerous affective conditions during this developmental period (e.g., depression, anxiety disorders) (
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      ). Previous work from our lab indicates that chronic variable stress (CVS) during adolescence can evoke specific effects later in life that may determine either risk or resilience (
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      ), processes linked to the prelimbic and infralimbic (IL) divisions of the rodent medial prefrontal cortex (mPFC) (
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      ). Learned fear has an obvious adaptive value, increasing the chance of survival in life-threatening situations (
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      Rodents are widely used to study how stress affects learned fear memories. Stress-enhanced fear models usually combine exposure to one or more stressors, with fear responses tested in a conditioning paradigm (
      • Blouin A.M.
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      The potential of epigenetics in stress-enhanced fear learning models of PTSD.
      ). One of the most widely used and reproduced models is the single prolonged stress (SPS) protocol developed by Liberzon (
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      ). Exposure to SPS impairs extinction and extinction recall of a fear-conditioned response 1 week later (
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      ), comprising a late-emerging enhancement of fear, as is characteristic of PTSD.
      Prefrontal activity and neuronal intrinsic excitability are associated with stress resilience and vulnerability (
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      ). For example, in humans, the aberrant fear response in PTSD is associated with ventromedial PFC (homolog to the rodent IL) (
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      ), hypoactivity, and loss of top-down control over the amygdala (
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      ). In rodents, SPS also reduces neuronal activation in the IL (
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      ), which may play a role in the abnormal fear extinction deficits associated with SPS. Conversely, optogenetic drive of the mPFC can promote stress resilience, and successful stress coping is linked to elevated mPFC activation after social defeat stress (
      • Covington 3rd, H.E.
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      ). However, the circuitry underlying vulnerability and resilience are largely unknown (
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      ).
      In this study, we assess the impact of adolescent CVS (adol CVS) on stress vulnerability or resilience to subsequent SPS in adulthood. Our data indicate that the experience of stress during adolescence disproportionately blocks fear potentiation following SPS in males, leading to resilience, a phenomenon that can be linked to decreases in intrinsic excitability of IL mPFC glutamatergic pyramidal neurons in male rats.

      Methods and Materials

      Detailed methods are described in the Supplement.

      Animals

      Male and female Sprague Dawley rats were bred in-house, with group assignments controlled for litter. All procedures and care performed in the animals were approved by the University of Cincinnati Institutional Animal Care and Use Committee.

      Experimental Design

      Experiment 1

      Experimental groups were control (no CVS–no SPS), adol CVS (only chronic stress in adolescence), SPS (only SPS in adulthood), and CVS-SPS or double-hit group (adol CVS + SPS in adulthood) of both sexes.

      Experiment 2

      Experimental groups were control (no adol CVS) and adol CVS for both sexes.

      Experiment 3

      Experimental groups were control, adol CVS, SPS, and CVS-SPS in only male rats.

      Adolescent Chronic Variable Stress

      Rats were subjected to 14 days of CVS exposure during late adolescence (postnatal day 45 ± 2). CVS consisted of a set of unpredictable variable stressors applied twice daily (AM and PM). Following CVS, animals were allowed to recover for 4 weeks and then subjected to the SPS protocol evaluated during adulthood. Timeline shown in Figure 1.
      Figure thumbnail gr1
      Figure 1(A) Experimental timeline. (B) Fear conditioning paradigm. (C, D) Previous adolescent stress prevented SPS extinction deficit in male rats and prevented reinstatement of tone-conditioned freezing after SPS in both male and female rats. Multiple planned comparisons: ∗p < .05 compared with same-sex Ctrl group, #p < .05 compared with same-sex adol CVS–SPS (extinction) or SPS (reinstat.) group. Φ = sex effect. (E, F) Context conditioning was tested as freezing during the 2 minutes prior to the first tone each day of the extinction procedure. Previous adolescent stress reduced conditioned response to context in SPS, animal re-exposure (day 1) in both sexes, and enhanced extinction of this response in male rats. Male rats had higher freezing than females (p < .05Φ). Individual planned comparisons: ∗p < .05 compared with same-sex Ctrl group, #p < .05 compared with same-sex adol CVS–SPS, °p < .05 compared with all other same-sex groups. Data represented as mean ± SEM. Image created with BioRender.com. adol CVS, adolescent chronic variable stress; Ctrl, control; PND, postnatal day; reinst., reinstatement; SPS, single prolonged stress.

      Single Prolonged Stress

      Four weeks after recovering from adol CVS, half of the CVS and control animals were subjected to SPS following Liberzon’s protocol (
      • Liberzon I.
      • Krstov M.
      • Young E.A.
      Stress-restress: Effects on ACTH and fast feedback.
      ) consisting of 2-hour restraint, 20-minute swim (25 ± 2 °C), and ether anesthesia until loss of consciousness. The other half served as control animals (no SPS). Animals were left to recover for 1 week before being tested.

      Cued Fear Conditioning Paradigm

      After 1 week, SPS animals were subjected to an auditory tone–cued fear conditioning protocol to evaluate their performance during the conditioning, extinction, and reinstatement sessions. The conditioned response evaluated was freezing behavior, considered as general absence of movement, which was scored using a video tracking system (EthovisionXT-Noldus).

      Context Fear Conditioning Test

      As a way of quantifying the conditioned response to the context, we evaluated the initial freezing response exerted every day of the extinction procedure before the first tone was presented and analyzed the progression of this response over 3 days.

      Immunohistochemistry

      At 90 minutes after reinstatement, rats were euthanized and perfused with 4% paraformaldehyde, and brains were processed for immunodetection of Fos, a marker of neuronal activation. We quantified the number of Fos-positive nuclei/unit area in the IL and prelimbic subdivisions of the mPFC, the basolateral amygdala complex, and the central lateral (CeL) and central medial divisions of the central amygdala, following coordinates from a brain atlas (
      • Paxinos G.
      • Watson C.
      The Rat Brain in Stereotaxic Coordinates.
      ). For logistical reasons, to reduce the variability of immunoreactivity, the tissue was processed in batches from same-sex animals; therefore, corresponding analysis was performed within sexes (see the Supplement for more details). We performed secondary analysis of sex differences using z scores.

      Electrophysiology

      Whole-cell patch clamp recordings were obtained from layer V pyramidal neurons in the IL PFC, which were easily identifiable in the slice on the basis of somal morphology and the presence of a prominent apical dendrite. A warm slicing protocol was used to prepare healthy adult rat brain slices as previously described (
      • Ting J.T.
      • Daigle T.L.
      • Chen Q.
      • Feng G.
      Acute brain slice methods for adult and aging animals: Application of targeted patch clamp analysis and optogenetics.
      ). All measurements of intrinsic membrane excitability were taken from the resting membrane potential in the current clamp mode once a stable membrane potential was observed.

      Statistical Analysis

      Fear conditioning data were analyzed by repeated measurements analysis of variance (ANOVA) (adol CVS × SPS × sex × time), with a level of significance of p < .05. Fos data were analyzed by two-way ANOVA (2 × 2 design: adol CVS × SPS) within each sex with a level of significance p < .05. Electrophysiology data were analyzed by 2 × 2 ANOVA (adol CVS × SPS). Details of data structure and number of cells used for electrophysiology data analysis are outlined in Table S1. In cases where significant differences and interactions were found, the Bonferroni test was used for post hoc analysis. In cases where there were only main effects of the factors but no significant interaction between them, we performed planned comparisons to evaluate individual differences. Data were analyzed using Statistica 7.0 (Stat Soft, Inc) and Prism 8 (GraphPad Software). Outliers were detected using the Grubbs’ test and removed from analysis.

      Results

      Experiment 1

      Cued Conditioned Response

      Figure 1C–F illustrates the conditioned freezing response throughout the different sessions of the fear conditioning paradigm in animals that were submitted to chronic variable stress during adolescence (adol CVS) and later subjected to SPS in adulthood. Animals were submitted to a tone-conditioned paradigm as shown in Figure 1B. Figure 1C, D shows the effects for each phase of paradigm evaluated.

      Conditioning

      None of the treatments had effects on the conditioning phase. There was an interaction between adol CVS and SPS (F1,79 = 5.075, p = .027) but no individual differences in the Bonferroni test. The significant effect of time (F5,395 = 469.308, p < .0001) confirmed conditioning of the response. There was main effect of sex (F1,79 = 7.724, p = .007), with Bonferroni comparisons indicating a general higher expression of freezing in male rats.

      Three-Day Extinction

      There were significant effects of SPS (F1,76 = 6.698, p = .012) and time (F2,152 = 475,661, p < .0001) and an adol CVS × SPS interaction (F1,76 = 7.414, p = .008), with no effect of sex. The CVS × SPS post hoc analysis confirmed that in general, SPS groups had higher freezing levels than control groups over the whole extinction procedure regardless of sex. When performing planned comparisons by sex, only male rats showed statistically significant effects, with the SPS group having higher freezing than the control group on all 3 days (p < .05) (the CVS only group had enhanced freezing only on day 2 [p < .05]). Prior adol CVS prevented SPS effects, because the double-hit group remained at control levels on all testing days and had significantly less freezing than the SPS group on day 2 (p < .05). Sex differences were observed only on day 3, with SPS evoking higher freezing in male rats (p < .05).

      Recall

      The levels of extinction attained were stable for both sexes as tested in the recall phase. In this case, 5 days after extinction, animals received a brief extra extinction session (three tones) to test for possible spontaneous recovery of the conditioned response and to corroborate that the levels of freezing in all the groups were equal before reinstatement. We observed a main effect of sex (F1,76 = 7.958, p = .006), with males expressing more freezing in general, and a triple interaction of sex × adol CVS × SPS (F1,76 = 4.792, p = .032), with no group differences emerging for any individual Bonferroni comparison.

      Reinstatement

      We observed a significant adol CVS × SPS interaction (F1,76 = 11.8095, p = .001). Post hoc comparison indicated that regardless of sex, the SPS group expressed increased freezing compared to the control group (p < .05), while the double-hit group prevented the effect of SPS, remaining at control freezing levels and expressing significantly less freezing time than the SPS group (p < .05). When analyzing the individual responses by sex (planned comparisons), we observed that in female rats, only the SPS group differed from the control group (p < .05). In the case of male rats, the SPS group had higher freezing than the control (p < .05) and adol CVS–SPS (p < .05) groups.

      Context-Conditioned Response

      To quantify the conditioned response to the conditioning context, we evaluated the freezing response evoked every day of the extinction procedure before the first tone was presented and analyzed the progression over 3 days (Figure 1E, F). We observed a main effect of adol CVS (F1,76 = 4.097, p = .046) and significant adol CVS × SPS interaction (F1,76 = 11.729, p = .001). The post hoc analysis indicated that animals subjected to SPS expressed higher freezing when re-exposed to the conditioning context compared with all other groups (p < .05). There was a sex effect (F1,76 = 6.971, p = .01), with male rats having more freezing time. There was also an effect of time (F2,152 = 172.946, p < .00001), indicating reduction of freezing to subsequent exposure. Finally, there was a time × sex × SPS interaction (F2,152 = 9.253, p = .0002). Planned comparisons showed that male rats subjected to SPS alone expressed higher freezing than the control group on day 2, while the group subjected to the double-hit model of stress had less context freezing compared with all other groups on day 1 (p < .05). This difference was maintained against the SPS group on the rest of the days tested (p < .05). In the case of female rats, the SPS group had higher freezing to the context than all other groups on day 1 (p < .05), and the adol CVS–SPS group also had more freezing than control animals on that day (p < .05).

      Fos Expression After Reinstatement

      Figure 2 summarizes Fos activation (Fos immunoreactivity) in the mPFC and amygdala assessed following the reinstatement trial (90 min after the onset of the session; see Methods and Materials for details on statistical analysis). In the case of male rats, we observed a significant effect of SPS in the IL cortex (F1,16 = 7.706, p = .0135). Planned comparisons confirmed that the SPS groups had significantly less Fos immunoreactivity than the control and CVS animals (p < .05), while the other groups did not differ from control animals (Figure 2A). In female rats, the effects of adol CVS and SPS were only observed in the central nucleus of the amygdala, particularly in the CeL, but not medial, subdivision of the central amygdala (SPS: F1,23 = 5.945, p = .0229; adol CVS × SPS: F1,23 = 7.241, p = .0130). Bonferroni’s test confirmed that SPS significantly increased neuronal recruitment (p < .05), and this was prevented in the double-hit group (p < .05) (Figure 2B). Indirect z score analysis on Fos to account for possible differences between sexes supports the idea of sexes processing stress effects in different brain regions (Figure S1).
      Figure thumbnail gr2
      Figure 2Fos immunoreactivity in the medial prefrontal cortex (A) and amygdala (B) of the animals 90 minutes after reinstatement (see timeline in ). There was significant effect of SPS in the IL division of male rats. Planned comparisons confirmed less Fos immunoreactivity in the SPS group compared with the Ctrl group (∗p < .05). None of the other groups differed from Ctrl animals. In females, effects of adol CVS and SPS were only observed in the central nucleus of the amygdala, particularly in CeL and not in the CeM divisions. Bonferroni’s test confirmed that SPS significantly increased neuronal recruitment (∗p < .05), and this was prevented in the double-hit group (#p < .05). Data represented as mean ± SEM. adol CVS, adolescent chronic variable stress; BLA, basolateral amygdala; CeL, central lateral; CeM, central medial; Ctrl, control; IL, infralimbic; PL, prelimbic.

      Experiment 2

      Electrophysiology

      We next investigated the potential cellular mechanisms underlying how adolescent stress can prevent SPS-induced changes in fear behavior and Fos activation in the IL in male rats. Male rats were selected based on the clear effects of both SPS and adol CVS–SPS on extinction learning, a process linked to the IL. We measured the intrinsic membrane properties and firing frequency of IL pyramidal neurons in layer V, the major source of subcortical output from the IL (
      • Baker A.
      • Kalmbach B.
      • Morishima M.
      • Kim J.
      • Juavinett A.
      • Li N.
      • Dembrow N.
      Specialized subpopulations of deep-layer pyramidal neurons in the neocortex: Bridging cellular properties to functional consequences.
      ). We found that prior experience of adol CVS prevented SPS-mediated changes in intrinsic excitability of IL pyramidal neurons. There was a significant main effect of SPS (F1,99 = 32.3, p < .0001) (Table S1) and adol CVS (F1,99 = 8.5, p = .005) on rheobase (Figure 3D). SPS significantly increased rheobase compared with the control group (p < .05), which was prevented by prior adol CVS (p < .05 compared with SPS). There was a significant main effect of SPS (F1,98 = 41.96, p < .0001) and adol CVS (F1,98 = 21.7, p = .0002) and a significant adol CVS × SPS interaction (F1,98 = 6.7, p = .01) on membrane resistance (Figure 3E). Bonferroni’s test indicated that SPS significantly decreased membrane resistance (p < .05), which was prevented by prior adol CVS (p < .05 compared with SPS). Statistical analysis for membrane resistance was performed on log-transformed data. There were significant main effects of SPS (F1,92 = 19, p < .0001) and adol CVS (F1,92 = 14.3, p = .0003) and a significant adol CVS × SPS interaction (F1,92 = 5.0, p = .02) on action potential (AP) threshold (Figure 3F). Bonferroni’s test indicated that SPS significantly increased AP threshold compared with control animals (p < .05), with prior adol CVS preventing the effect (p < .05 compared with SPS). There were main effects of SPS (F1,97 = 20, p < .001) and adol CVS (F1,97 = 25.9, p < .0001) and a significant adol CVS × SPS interaction (F1,97 = 6.5, p = .01) on AP amplitude (Figure 3G). Bonferroni’s test indicated that SPS significantly lowered AP amplitude compared with control animals (p < .05), and prior experience adol CVS prevented it (p < .05 compared with SPS). There was a significant effect of SPS (F1,97 = 8.3, p = .005) and adol CVS (F1,97 = 42.2, p < .0001) on AP50 (Figure 3H). Planned comparisons indicated a decrease in AP50 following SPS compared with control animals (p < .05), and prior adol CVS prevented that effect (p < .05 compared with SPS). Increase in AP50 was also observed after adol CVS only (p < .05 compared with control group). Resting membrane potential was unaltered among the groups (Figure 3I). Two-way ANOVA revealed no significant main effect of SPS (F1,95 = 1.3, p = .3) or adol CVS (F1,95 = 0.02, p = .9) or an SPS × adol CVS interaction on resting membrane potential (F1,95 = 0.1, p = .7).
      Figure thumbnail gr3
      Figure 3Adol CVS prevents SPS effects on membrane properties and intrinsic excitability of infralimbic pyramidal neurons. (A) Experimental timeline. (B) Schematic of coronal brain sections through the prefrontal cortex where recordings were performed; blue boxes indicate the infralimbic region of the prefrontal cortex. (C) Pyramidal neurons were identified based on somal morphology and presence of prominent apical dendrite. Arrows indicate pyramidal neurons. (D, E) SPS increased rheobase (D) and decreased membrane resistance (E), whereas prior experience of adol CVS was able to prevent these effects. (F, G) SPS increased the threshold for AP firing (F) and decreased AP amplitude (G), both of which were prevented by prior adol CVS. SPS also reduced the duration of AP (AP 50), which was also blocked by prior adol CVS. (H) It should be noted that adol CVS alone increased AP duration. (K) Finally, adol CVS was also able to attenuate the reduction in peak firing frequency observed following SPS. (I, J) No changes in resting membrane potential (I) or membrane capacitance (J) were observed. (L) Representative traces of action potentials evoked by 20-pA current injection for the respective groups. For (D), (H), and (K), × and # represent planned comparison effects compared with control and SPS, respectively. For (E), (F), and (G), ∗ and # represent post hoc Bonferroni effects compared with control and SPS, respectively. Data represented as mean ± SEM. adol CVS, adolescent chronic variable stress; AP, action potential; IL, infralimbic; PND, postnatal day; SPS,single prolonged stress.
      Membrane capacitance was unaltered among groups (Figure 3J), indicating that the treatments did not likely affect cell size. Two-way ANOVA of membrane capacitance revealed no significant main effect of SPS (F1,97 = 1.8, p = .2) or adol CVS (F1,97 = 3.6, p = .06) or an SPS × adol CVS interaction (F1,97 = 2.9, p = .09). Analysis of peak firing frequency revealed a significant main effect of SPS (F1,36 = 13.4, p = .0008). Planned comparisons indicated that SPS significantly reduced peak firing frequency compared with control animals (p < .05), whereas the prior adol CVS–SPS group did not differ from the control group (Figure 3K). Figure 3L shows representative traces of APs evoked by 20-pA current injection for the respective groups. Together, these data indicate that prior experience of adolescent stress is able to prevent a reduction in intrinsic excitability and the firing rate of IL layer V pyramidal neurons following SPS in male rats.

      Discussion

      Our results strongly suggest that prior experience with stress during adolescence can evoke a resilient phenotype in the adult, characterized by prevention of the effects of SPS in a fear conditioning paradigm on IL pyramidal cell excitability. Our data indicate that the adaptations resulting from exposure to chronic stress during adolescence buffer the behavioral impact of a model of traumatic stress in adulthood, blocking known effects of SPS on subsequent fear potentiation.
      While prior CVS is able to block enhancement of reinstatement in both sexes, it appears to do so by distinct neuronal mechanisms. Reversal of SPS-induced reinstatement was accompanied by IL hypoactivity in males and CeL recruitment in females, suggesting differential engagement of cortical regions regulating extinction (males) versus fear expression (females) across the sexes. Extinction deficits were only observed in males, consistent with the known role of the IL in extinction of conditioned fear. A role for the IL in CVS-induced resilience in males is further supported by hypoactivity of layer V pyramidal cells following SPS, which is blocked by prior adolescent CVS. In females, the Fos data suggest a distinct mechanism involving the amygdala and, although we acknowledge the limitations of our data due to only within-sex analysis of the Fos data, our data suggest strong avenues for future research of the mechanisms underlying resilience after stress (see summary in Figure 4).
      Figure thumbnail gr4
      Figure 4Summary of the effects observed. Prior exposure to adol CVS prevented the behavioral outcomes evoked by SPS in male and female rats. In the case of males, it also counteracted the effects of SPS on IL excitability, indirectly inferred by Fos staining after reinstatement and confirmed by electrophysiology on pyramidal neurons from layer V. These central effects of adol CVS on SPS outcomes highlight the relevance of the IL cortex as a hub for neurodevelopmental plasticity that could lead to resilience to stressful events in adulthood. In female rats, the absence of SPS effects on neuronal recruitment in the IL cortex, accompanied by an increase of it in the central lateral division of the amygdala, suggests a marked difference in the circuitry affected by SPS in both sexes. This points out to a possible mechanism involving excitability in that area that has yet to be confirmed. adol CVS, adolescent chronic variable stress; IL, infralimbic; PFC, prefrontal cortex; SPS, single prolonged stress.
      Stress during development is generally thought to evoke negative behavioral effects later in life (
      • Cotella E.M.
      • Morano R.L.
      • Wulsin A.C.
      • Martelle S.M.
      • Lemen P.
      • Fitzgerald M.
      • et al.
      Lasting impact of chronic adolescent stress and glucocorticoid receptor selective modulation in male and female rats.
      ,
      • Cotella E.M.
      • Scarponi Gómez A.
      • Lemen P.
      • Chen C.
      • Fernández G.
      • Hansen C.
      • et al.
      Long-term impact of chronic variable stress in adolescence versus adulthood.
      ,
      • Begni V.
      • Zampar S.
      • Longo L.
      • Riva M.A.
      Sex differences in the enduring effects of social deprivation during adolescence in rats: Implications for psychiatric disorders.
      ,
      • Bourke C.H.
      • Neigh G.N.
      Behavioral effects of chronic adolescent stress are sustained and sexually dimorphic.
      ,
      • Green M.R.
      • Barnes B.
      • McCormick C.M.
      Social instability stress in adolescence increases anxiety and reduces social interactions in adulthood in male Long-Evans rats.
      ,
      • Negrón-Oyarzo I.
      • Pérez M.Á.
      • Terreros G.
      • Muñoz P.
      • Dagnino-Subiabre A.
      Effects of chronic stress in adolescence on learned fear, anxiety, and synaptic transmission in the rat prelimbic cortex.
      ,
      • Wilkin M.M.
      • Waters P.
      • McCormick C.M.
      • Menard J.L.
      Intermittent physical stress during early- and mid-adolescence differentially alters rats’ anxiety- and depression-like behaviors in adulthood.
      ,
      • Wulsin A.C.
      • Wick-Carlson D.
      • Packard B.A.
      • Morano R.
      • Herman J.P.
      Adolescent chronic stress causes hypothalamo-pituitary-adrenocortical hypo-responsiveness and depression-like behavior in adult female rats.
      ). However, prior studies also support the ability of adolescent stress to confer stress resilience in adulthood, using a number of stress models, e.g., intermittent predator stress (
      • Kendig M.D.
      • Bowen M.T.
      • Kemp A.H.
      • McGregor I.S.
      Predatory threat induces huddling in adolescent rats and residual changes in early adulthood suggestive of increased resilience.
      ) and predictable chronic mild stress (
      • Suo L.
      • Zhao L.
      • Si J.
      • Liu J.
      • Zhu W.
      • Chai B.
      • et al.
      Predictable chronic mild stress in adolescence increases resilience in adulthood.
      ). Adolescent predictable chronic mild stress enhances extinction and prevents reinstatement and spontaneous recovery in a fear conditioning model evaluated immediately and 1 week following predictable chronic mild stress (
      • Deng J.H.
      • Yan W.
      • Han Y.
      • Chen C.
      • Meng S.Q.
      • Sun C.Y.
      • et al.
      Predictable chronic mild stress during adolescence promotes fear memory extinction in adulthood.
      ). Consistent with our results, these suggest that adolescent stress enhancement of resilience endures well beyond the time of exposure. The impact of adolescent stress differs from that of stress imposition earlier in life, where the data generally report detrimental effects of stress (
      • Johnson D.C.
      • Casey B.J.
      Easy to remember, difficult to forget: The development of fear regulation.
      ,
      • Lukkes J.L.
      • Mokin M.V.
      • Scholl J.L.
      • Forster G.L.
      Adult rats exposed to early-life social isolation exhibit increased anxiety and conditioned fear behavior, and altered hormonal stress responses.
      ,
      • McEwen B.S.
      Physiology and neurobiology of stress and adaptation: Central role of the brain.
      ,
      • Vyas A.
      • Mitra R.
      • Shankaranarayana Rao B.S.
      • Chattarji S.
      Chronic stress induces contrasting patterns of dendritic remodeling in hippocampal and amygdaloid neurons.
      ,
      • Yee N.
      • Schwarting R.K.W.
      • Fuchs E.
      • Wöhr M.
      Juvenile stress potentiates aversive 22-kHz ultrasonic vocalizations and freezing during auditory fear conditioning in adult male rats.
      ).
      Although some authors proposed that the resilient phenotype is promoted by the predictability of the stressors (
      • Deng J.H.
      • Yan W.
      • Han Y.
      • Chen C.
      • Meng S.Q.
      • Sun C.Y.
      • et al.
      Predictable chronic mild stress during adolescence promotes fear memory extinction in adulthood.
      ), the general unpredictable nature of CVS suggests that the resilience mechanism is independent of response habituation. In our study, the adol CVS paradigm uses exposure to swim and restraint, albeit in an isolated and time-attenuated fashion relative to SPS. Nonetheless, the length and consecutive application of the stressors during SPS represents a distinct and intense unpredictable experience. This contention is supported by a recent report demonstrating behavioral resilience to SPS using exposure to completely different stressors during adolescence (
      • Chaby L.E.
      • Sadik N.
      • Burson N.A.
      • Lloyd S.
      • O’Donnel K.
      • Winters J.
      • et al.
      Repeated stress exposure in mid-adolescence attenuates behavioral, noradrenergic, and epigenetic effects of trauma-like stress in early adult male rats.
      ).
      Timing combined with stressor modality seems to be an important factor as well. In this sense, prior work indicates adult resilience even after a single intense stressor protocol at postnatal day 37 (
      • Moore N.L.T.
      • Gauchan S.
      • Genovese R.F.
      Adolescent traumatic stress experience results in less robust conditioned fear and post-extinction fear cue responses in adult rats.
      ) or following 3 days of predator-related stressors at postnatal day 33 to 35 (
      • Chaby L.E.
      • Sadik N.
      • Burson N.A.
      • Lloyd S.
      • O’Donnel K.
      • Winters J.
      • et al.
      Repeated stress exposure in mid-adolescence attenuates behavioral, noradrenergic, and epigenetic effects of trauma-like stress in early adult male rats.
      ). In contrast, a 3-day prepubertal exposure to variate stressors failed to attenuate exaggeration of fear responses in adulthood (
      • Yee N.
      • Schwarting R.K.W.
      • Fuchs E.
      • Wöhr M.
      Juvenile stress potentiates aversive 22-kHz ultrasonic vocalizations and freezing during auditory fear conditioning in adult male rats.
      ,
      • Tsoory M.M.
      • Guterman A.
      • Richter-Levin G.
      Juvenile stress” alters maturation-related changes in expression of the neural cell adhesion molecule L1 in the limbic system: Relevance for stress-related psychopathologies.
      ), indicating that developmental timing is critical for establishment of resilience.
      Hypoactivity of the mPFC is observed in several mental health disorders, including PTSD (
      • Hains A.B.
      • Arnsten A.F.T.
      Molecular mechanisms of stress-induced prefrontal cortical impairment: Implications for mental illness.
      ). Results from our group and others indicate that stress during adolescence reduces neuronal recruitment (Fos expression) to adult stressors in the mPFC (
      • Cotella E.M.
      • Morano R.L.
      • Wulsin A.C.
      • Martelle S.M.
      • Lemen P.
      • Fitzgerald M.
      • et al.
      Lasting impact of chronic adolescent stress and glucocorticoid receptor selective modulation in male and female rats.
      ,
      • Cotella E.M.
      • Scarponi Gómez A.
      • Lemen P.
      • Chen C.
      • Fernández G.
      • Hansen C.
      • et al.
      Long-term impact of chronic variable stress in adolescence versus adulthood.
      ,
      • Ishikawa J.
      • Nishimura R.
      • Ishikawa A.
      Early-life stress induces anxiety-like behaviors and activity imbalances in the medial prefrontal cortex and amygdala in adult rats.
      ). In humans, PTSD has been associated with a reduction in prefrontal drive, leading to abnormal extinction of conditioned fear (
      • Milad M.R.
      • Pitman R.K.
      • Ellis C.B.
      • Gold A.L.
      • Shin L.M.
      • Lasko N.B.
      • et al.
      Neurobiological basis of failure to recall extinction memory in posttraumatic stress disorder.
      ,
      • Milad M.R.
      • Orr S.P.
      • Pitman R.K.
      • Rauch S.L.
      Context modulation of memory for fear extinction in humans.
      ,
      • Milad M.R.
      • Rauch S.L.
      • Pitman R.K.
      • Quirk G.J.
      Fear extinction in rats: Implications for human brain imaging and anxiety disorders.
      ,
      • Rauch S.L.
      • Shin L.M.
      • Phelps E.A.
      Neurocircuitry models of posttraumatic stress disorder and extinction: Human neuroimaging research–past, present, and future.
      ). Similarly, reduced IL mPFC activity following SPS in male rats may underlie abnormal extinction of fear responses (
      • Nawreen N.
      • Baccei M.L.
      • Herman J.P.
      Single prolonged stress reduces intrinsic excitability and excitatory synaptic drive onto pyramidal neurons in the infralimbic prefrontal cortex of adult male rats.
      ,
      • Piggott V.M.
      • Bosse K.E.
      • Lisieski M.J.
      • Strader J.A.
      • Stanley J.A.
      • Conti A.C.
      • et al.
      Single-prolonged stress impairs prefrontal cortex control of amygdala and striatum in rats.
      ). Consistent with these data, our results indicate reduced IL recruitment following SPS in male rats accompanied by higher freezing during extinction and reinstatement. The reduced engagement of the IL in response to conditioned cues during the reinstatement procedure in the SPS male group also suggests a possible reduction of IL activity occurring during the prior extinction procedure, which would explain the impairment of extinction learning previously observed only in male rats. Remarkably, in the case of female rats, Fos expression was only affected by SPS in the CeL division of the central amygdala (with no changes in the PFC), and this area also exhibited resilience in the double-hit group. CeL is associated with expression of conditioned fear responses (
      • Marek R.
      • Strobel C.
      • Bredy T.W.
      • Sah P.
      The amygdala and medial prefrontal cortex: Partners in the fear circuit.
      ,
      • Marek R.
      • Sah P.
      Neural circuits mediating fear learning and extinction.
      ). Our data indicate that SPS female rats exhibit enhanced freezing expression during reinstatement with minimal effect during extinction [consistent with (
      • Keller S.M.
      • Schreiber W.B.
      • Staib J.M.
      • Knox D.
      Sex differences in the single prolonged stress model.
      )], further aligning with the involvement of the CeL and lack of involvement of the IL in the resilient effect in female rats. These results suggest likely sex differences in response to stress and fear-related behaviors and highlight involvement of sex-specific brain regions, particularly the IL in males and CeL in females, in evoking a resilient phenotype (Figure 4), results suggested by within-sex analysis of Fos expression and supported by secondary analysis using z score comparisons across sex and stress. Additional research is needed to establish mechanisms underlying fear processing in females exposed to adolescent stress.
      Neurons in the mPFC are specifically activated during stressful situations and modulate their responses to subsequent exposure to the same stressor experience (
      • Jackson M.E.
      • Moghaddam B.
      Distinct patterns of plasticity in prefrontal cortex neurons that encode slow and fast responses to stress.
      ), thus playing a critical role in eliciting adaptive responses to aversive stimuli (
      • Milad M.R.
      • Quirk G.J.
      Neurons in medial prefrontal cortex signal memory for fear extinction.
      ). Modification of PFC responses to the same stimulus can be mediated through altered glutamatergic or dopaminergic drive onto the mPFC projection neurons (
      • Bagley J.
      • Moghaddam B.
      Temporal dynamics of glutamate efflux in the prefrontal cortex and in the hippocampus following repeated stress: Effects of pretreatment with saline or diazepam.
      ,
      • Jackson M.E.
      • Moghaddam B.
      Stimulus-specific plasticity of prefrontal cortex dopamine neurotransmission.
      ). Adolescent social defeat decreases adult NMDA receptor expression in the IL PFC and also reduces freezing to fear conditioning (
      • Novick A.M.
      • Mears M.
      • Forster G.L.
      • Lei Y.
      • Tejani-Butt S.M.
      • Watt M.J.
      Adolescent social defeat alters N-methyl-D-aspartic acid receptor expression and impairs fear learning in adulthood.
      ). Thus, the enhanced excitability we observed in SPS rats with prior history of adol CVS might be a long-term adaptation to the reduced excitatory drive during adol CVS.
      Intrinsic membrane properties play an important role in determining the prefrontal excitatory/inhibitory balance, because they directly shape neuronal output by influencing the probability of a neuron firing an AP in response to synaptic inputs (
      • Anderson E.M.
      • Gomez D.
      • Caccamise A.
      • McPhail D.
      • Hearing M.
      Chronic unpredictable stress promotes cell-specific plasticity in prefrontal cortex D1 and D2 pyramidal neurons.
      ). Our data indicate that the IL intrinsic excitability changes do not manifest at baseline conditions under adol CVS alone, consistent with prior work in mice resilient to social defeat (
      • Friedman A.K.
      • Walsh J.J.
      • Juarez B.
      • Ku S.M.
      • Chaudhury D.
      • Wang J.
      • et al.
      Enhancing depression mechanisms in midbrain dopamine neurons achieves homeostatic resilience.
      ,
      • Han M.H.
      • Nestler E.J.
      Neural substrates of depression and resilience.
      ). Thus, it is possible that prior adol CVS may serve to prime the pyramidal cells to react appropriately when faced with the second hit of SPS, compensating for reduced excitability associated with SPS. The exact mechanism underlying the altered excitability of IL pyramidal neurons observed in our study is yet to be determined. Possibilities include lasting alteration in ion channel function (e.g., G protein–gated inwardly rectifying K+ channels) (
      • Anderson E.M.
      • Gomez D.
      • Caccamise A.
      • McPhail D.
      • Hearing M.
      Chronic unpredictable stress promotes cell-specific plasticity in prefrontal cortex D1 and D2 pyramidal neurons.
      ,
      • Hearing M.
      • Kotecki L.
      • Marron Fernandez de Velasco E.
      • Fajardo-Serrano A.
      • Chung H.J.
      • Luján R.
      • Wickman K.
      Repeated cocaine weakens GABA(B)-Girk signaling in layer 5/6 pyramidal neurons in the prelimbic cortex.
      ) or modulation on excitability by hyperpolarization-activated cyclic nucleotide–gated channels (
      • Shah M.M.
      Cortical HCN channels: Function, trafficking and plasticity.
      ). Further work is needed to identify the specific ionic mechanisms by which adolescent stress can protect against future stressors during adulthood (
      • Matovic S.
      • Ichiyama A.
      • Igarashi H.
      • Salter E.W.
      • Sunstrum J.K.
      • Wang X.F.
      • et al.
      Neuronal hypertrophy dampens neuronal intrinsic excitability and stress responsiveness during chronic stress.
      ), as well as to determine mechanisms underlying reduced resilience in females.

      Conclusions

      Our results support the idea that certain combinations of stressful situations during adolescence can be beneficial, evoking resilience to stress in adult life. We propose that in male rats, CVS during late adolescence determines differential activation or recruitment of the IL in response to intense stress in adulthood. This rearrangement of prefrontal activity results in a phenotype that is resilient to stress-enhanced fear learning, reducing contextual response, facilitating extinction, and preventing reinstatement of the fear-conditioned response following trauma, findings that may lend insight into understanding susceptibility of resilience to PTSD. Furthermore, our data guide our next steps to understand the sex-specific effects in behavioral resilience following adolescent stress, suggestive of fundamental sex differences in stress-reactive brain regions and their involvement in resilience. It would be important as well to determine which stressor type might result in a positive emotional valence and whether that ultimately evokes a resilience response. For example, the exercise (swim) or social component (crowding) of the adol CVS regimen might help individuals cope with subsequent stress in adulthood (
      • Herring M.P.
      • O’Connor P.J.
      • Dishman R.K.
      The effect of exercise training on anxiety symptoms among patients: A systematic review.
      ,
      • Ozbay F.
      • Johnson D.C.
      • Dimoulas E.
      • Morgan C.A.
      • Charney D.
      • Southwick S.
      Social support and resilience to stress: From neurobiology to clinical practice.
      ). The next challenge is to find the most efficient developmental triggers for the generation of resilience to the effects of adult stress, possibly including positive developmental interventions, with the goal of reducing the incidence of stress-related affect conditions, including PTSD.

      Acknowledgments and Disclosures

      This project was funded by the National Institutes of Health (Grant Nos. R01MH101729 , R01 MH049698 , and R01 MH119814 [to JPH]; Grant No. T32 DK059803 [to EMC and SEM]; Grant No. F31MH123041 [to NN]), U.S. Department of Veterans Affairs (Grant No. I01BX003858 [to JPH]), and a NARSAD Young Investigator Award from the Brain and Behavior Research Foundation (to RDM).
      We thank other members of Dr. Herman’s laboratory for their assistance in data collection and general discussion of the results. Images were created with BioRender.com.
      A previous version of this article was published as a preprint on bioRxiv: https://www.biorxiv.org/content/10.1101/2021.03.16.435691v2.
      The authors report no biomedical financial interests or potential conflicts of interest.

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