The role of glucocorticoid receptor-dependent activity in the amygdala central nucleus and reversibility of early-life stress programmed behavior

Abstract

Early-life stress (ELS) leads to sustained changes in gene expression and behavior, increasing the likelihood of developing a psychiatric disorder in adulthood. The neurobiological basis for the later-in-life psychopathology is relatively unknown. The current study used a mouse model of ELS, achieved by daily maternal separations during the first 2 weeks of postnatal life, to test the role of amygdalar glucocorticoid receptor (GR) function in mediating the persistent increase in risk-taking behaviors. ELS produced a decrease in GR mRNA in the brain, with a notable reduction in the amygdala that was associated with sustained alterations in anxiety, fear and sociability-like behaviors. Lentiviral-mediated restoration of the GR mRNA deficit, specifically within the adult central nucleus of the amygdala (CeA), reversed the enduring changes in anxiety and social behavior after ELS. These results provide evidence of lasting changes in CeA GR neural circuitry following ELS and suggest a mechanistic role for GR-regulated processes in the CeA in mediating the lifelong maladaptive behaviors of ELS. We demonstrate that the long-lasting behavioral effects of ELS are reversible later in life and implicate the involvement of CeA GR-dependent activity in the sustained dysregulation of emotion following ELS.

Figure 1

(a) LVGR restores GR expression in the adult CeA after early-life stress. Representative images show accurate targeting of LVGR to the CeA (a). Quantification of GR expression indicated that LVGR was efficiently able to express GR in vivo as indicated by the restoration of GR mRNA in the CeA of adult MS mice following ELS (one-way analysis of variance (ANOVA), P=0.019, a). LVGR stereotaxic injections significantly increased GR mRNA in the bilateral CeA of adult MS mice (MS LVGR) above MS LVGFP mice (P=0.008, a) as measured by in situ hybridization. LVGR restored GR mRNA to nearly control levels (MS LVGR CeA versus DLW LVGFP, P=0.55, a). Two bilateral injected CeA sections per animal were measured with N=3 animals per group. (b) LVGR reverses the ELS dysregulation of anxiety in the EZM. Bilateral stereotaxic injections of LVGR in the adult CeA of MS mice (N=7) produced a significant decrease in the time spent (seconds) in the open arms of the EZM (P=1.49 × 10⁻⁹, one-way ANOVA, compared with MS LVGFP (P=5.65 × 10⁻¹⁰, N=9), control LVGFP (P=0.0002, N=7) and DLW LVGFP (P=0.0005, N=9) mice. (c and d) LVGR reverses the social disinhibition of ELS. Stereotaxic LVGR injections in the adult amygdala of MS mice (MS LVGR, N=30) reverses the abnormal social function observed after early-life stress by significantly decreasing the number of active social behaviors (P=1.88 × 10⁻¹⁶ one-way ANOVA; MS LVGFP, P=7.37 × 10⁻⁷, N=32 animals, c) and the duration of time spent engaged in performing active social behaviors (P=7.15 × 10⁻¹⁹ one-way ANOVA; MS LVGFP, P=1.30 × 10⁻⁸, N=25, d), and restores sociability to levels similar to that exhibited by control LVGFP and DLW LVGFP mice in the number of active social behaviors (control LVGFP, P=0.52; DLW LVGFP, P=0.003, c) and the duration of active social behaviors (control LVGFP, P=0.0003; DLW LVGFP, P=9.5 × 10⁻⁶, d). CeA, central nucleus of the amygdala; DLW, delayed weaning; ELS, early-life stress; EZM, elevated zero maze; GFP, green fluorescent protein; GR, glucocorticoid receptor; LVGFP, lentivirus expressing GFP; LVGR, lentivirus expressing GR; MS, maternal separation. **P≤0.01, ***P≤0.001, ****P≤0.0001.

Figure 2

GR mRNA is decreased in adult mouse brain following early-life stress. In situ hybridization was used to measure GR expression in adult 8–10-week-old MS, Cntl and DLW mice. A one-way analysis of variance revealed a significant difference in GR expression between MS, control and DLW mice in the CeA (P=0.005, a), BLA (P=0.002, b), CA1 (P=0.015, c) and the PVN (P=0.007, e) but not in the SSC (P=0.084, d). Next, a Student's t-test was used to determine significant differences between groups in each brain nuclei. ELS reduced GR mRNA in the CeA (P=0.037, P=0.0003, a), BLA (P=0.030, P=0.006, b), CA1 (P=0.060, P=0.0009, c), SSC (P=0.070, P=0.006, d) and the PVN (P=0.336, P=0.005, e) compared with control and DLW mice, respectively. In addition, we found that control mice express less GR mRNA in the CeA (P=0.023, a), BLA (P=0.008, b), CA1 (P=0.053, c) and the PVN (P=0.005, e) compared with DLW mice. GR mRNA was measured in two sections per brain for N=3 animals per group. BLA, basolateral nucleus of the amygdala; CA1, hippocampus; CeA, central nucleus of the amygdala; Cntl, control; DLW, delayed weaning; ELS, early-life stress; GR, glucocorticoid receptor; mRNA, messenger RNA; MS, maternal separation; PVN, paraventricular nucleus; SSC, somatosensory cortex. *P≤0.05, **P≤0.01, ***P≤0.001.

Figure 3

Early-life stress decreases anxiety-like behavior in the elevated zero maze (EZM) and open field (OF) tasks. A one-way analysis of variance (ANOVA) revealed a significant difference in the time spent in the open arms of the EZM between MS, Cntl and DLW adult (8–10 weeks) mice (P=0.005). MS mice show a significant increase in the time spent (seconds) in the open arms of the EZM compared with DLW mice (P=0.0001, a). Similarly, control mice spent significantly more time in the open arms compared with DLW mice (P=0.030, a). In the OF, a one-way ANOVA revealed a significant difference in the time spent in the center zone of the OF between groups (P=0.030). MS mice spent a significantly greater total percentage of time (seconds, P=0.006) and traveled a significantly greater distance (meters) in the open center zone (P=0.005) compared with DLW mice (b). No changes were found in locomotor parameters (total distance traveled) measured in both the EZM and OF tests between treatment groups (P=0.319 (EZM); P=0.836 (OF)), N=13 mice per group. Cntl, control; DLW, delayed weaning; MS, maternal separation. *P≤0.05, **P≤0.01, ****P≤0.0001.

Figure 4

Early-life stress alters fear conditioning behavior. ELS did not produce any significant changes in freezing behavior between groups during training (pre-cue and shock (P =0.391), cue and shock (cue and shock (P=0.505) and postshock (P=0.201), a) or contextual testing (P=0.709, b). However, there was a significant difference in the time spent freezing between groups, in the presence of the auditory cue, in auditory cued conditioning (pre-cue (P=0.702) and cue on (P=0.022), c). MS mice showed a significant deficit in fear behavior (freezing) in the auditory-cued phase of Pavlovian fear conditioning compared with control (P=0.03) and DLW (P=0.004) mice (c). N=13 (MS), N=12 (control and DLW). Cntl, control; DLW, delayed weaning; ELS, early-life stress; MS, maternal separation. *P≤0.05, **P≤0.01.

Figure 5

Early-life stress alters sociability-like behavior. A one-way analysis of variance revealed a significance between the number of active counts (P=0.0002) and active duration (P=0.0002) between groups. MS mice (N=14) performed a significantly greater number of active social behaviors, that is, behaviors initiated toward the partner mouse by the subject mouse, compared with control (P=0.003, N=13) and DLW (P=0.003, N=10) mice (a). Likewise, there is a significant increase in the amount of time spent (seconds) engaged in active social behavior in MS mice in comparison with control (P=0.01) and DLW (P=0.0005) mice (b). Social behavior was also measured in control mice paired with DLW partners to determine the effect of partner treatment group on the subject's social behavior. Neither the number of active social behaviors performed (control subject paired with MS partner versus control subject paired with DLW partner, P=0.531, a) nor the time spent engaged in performing active social behaviors (control subject paired with MS partner versus control subject paired with DLW partner, P=0.343, b) differed in control mice paired with either a MS or DLW partner. Cntl, control; DLW, delayed weaning; MS, maternal separation. **P≤0.01, ***P≤0.001.