The basolateral amygdala regulates adaptation to stress via β-adrenergic receptor-mediated reductions in phosphorylated extracellular signal-regulated kinase

Abstract

The reactivity of physiological systems and behavior to psychological stress is reduced with increasing familiarity with a repeated stressor. This reduced reactivity, termed habituation, is a crucial adaptation limiting negative health consequences of stress and can be disrupted in psychopathology. We hypothesized that the ability to habituate physiologically and behaviorally to previously experienced stressors depends on β-adrenergic receptor activation (β-AR) in the basolateral amygdala (BLA), a specific neural substrate important for the consolidation of multiple types of memories. We observed that administration of the β-AR antagonist propranolol into the BLA after each of four daily exposures to restraint stress prevented the normal development of neuroendocrine and behavioral habituation measured during the fifth restraint in adult male rats. In contrast, the β-AR agonist clenbuterol administered into the BLA after each restraint on days 1-4 enhanced neuroendocrine habituation at the lowest dose but attenuated behavioral habituation at high doses. We then explored intracellular signaling mechanisms in the BLA that might be a target of β-AR activation during stress. β-AR activation post restraint is necessary for the alteration in basal phosphorylated ERK (pERK) levels, as daily post-stress β-AR blockade on days 1-4 prevented repeated stress from leading to decreased pERK in the BLA on day 5. Finally, we examined the effect of blocking ERK phosphorylation in the BLA after each restraint on days 1-4 with the MEK (MAPK/ERK kinase) inhibitor U0126, and found that this was sufficient to both mimic neuroendocrine habituation in stress-naive animals and to enhance it in repeatedly stressed animals during restraint on day 5. Together, the results suggest that an individual's ability to habituate to repeated stress is regulated by activation of BLA β-AR, which may have these effects by transducing subsequent reductions in pERK. Individual variations in β-AR activation and intracellular signaling in the BLA may contribute significantly to adaptation to psychological stress and consequent resilience to stress-related psychopathology.

Figure 1

A) General experimental design. All experiments were conducted over 5 days in male rats with cannulae bilaterally implanted into the BLA, with the exception of the data in Figure 6 A-C which was conducted over 5 days in intact animals. All drugs were administered during the first 4 days only, after each daily 30 minute restraint (RR) or at the same time of day in unstressed control animals (CTL). On day 5, all animals received the same treatment under drug free conditions. One of four endpoints was measured on day 5: 1) HPA activity during restraint was assessed by repeated blood sampling at 0, 15, 30, and 60 minutes after onset of 30 minute restraint; 2) struggling behavior was video acquired during undisturbed 30 minute restraint; 3) animals were sacrificed at the end of 30 minute restraint to measure gene expression; or 4) sacrificed at 0 min or at the end of 30 minute restraint for protein measures. B–D) Representative cannulae placement in the BLA in the current experiments. B) plate from the rat brain atlas by Paxinos and Watson depicting the BLA, which is outlined in C and D. C) representative photomicrograph of a cannula directed at the BLA from the current experiments. D) representative photomicrograph of a misplaced cannula directed outside of the BLA from the current experiments.

Figure 2

Daily post-restraint administration of β–AR antagonist in the BLA prevents habituation of ACTH activity to restraint. A) The β–AR antagonist propranolol (PROP) given immediately after daily restraint prevented HPA habituation to repeated restraint (RR) on day 5. ACTH habituated in RR VEH on day 5 compared to CTL groups but did not habituate in repeatedly restrained animals given propranolol in the BLA (RR PROP). n’s: CTL VEH=7–8, CTL PROP=5–9, RR VEH=4, RR PROP=5–9. B) PROP delivered at a 4h delay after restraint did not affect habituation of HPA activity in the same manner as PROP delivered immediately after restraint. The experiment in 2A included 2 additional groups where intra-BLA injections of VEH or PROP were delayed until 4 hours post-restraint (RR DELAY); these groups are graphed in (1B) and (1D) with RR PROP and RR VEH from 1A and C. Both RR DELAY VEH and RR DELAY PROP demonstrated habituation similar to RR VEH. n’s: RR DELAY VEH=5–9, RR DELAY PROP=5–9. C and D) Corticosterone from the same animals depicted in 2A and 2B. In contrast to ACTH, corticosterone did not significantly habituate to 30 minute restraint by day 5 in the current experiments, preventing us from analyzing whether PROP prevented habituation of HPA activity downstream of ACTH. E) β–AR blockade in the BLA given after daily restraint prevented the habituation of struggling behavior to restraint. Animals previously exposed to repeated restraint struggle significantly less during the first 5–10 minutes of restraint compared to animals undergoing a first restraint, providing a behavioral measure of habituation to repeated restraint. Intra-BLA propranolol after daily repeated stress (RR PROP) prevented behavioral habituation as seen in repeatedly restrained animals given vehicle (RR VEH). F) Struggling behavior from the first 10 minutes of restraint depicted in 2E, depicting the minute-by-minute changes in behavior during the first third of restraint. n’s: CTL VEH=12, CTL PROP=17, RR VEH=15, RR PROP=15. * signifies p≤0.05 between RR VEH and RR PROP. # signifies p≤0.05 between RR VEH and CTL VEH. All data is mean ± SEM.

Figure 3

Intra-BLA β–AR blockade each day after stress prevented the increased AVP mRNA in the paraventricular hypothalamus (PVN) elicited by repeated restraint. A) Representative autoradiographs of AVP mRNA in the PVN, outlining the medial parvocellular region of the PVN centered at approximately bregma −1.8, which regulates HPA activity. The non-outlined area in the images is the posterior magnocellular AVP-expressing cells of the PVN, which are not involved in HPA regulation. B) RR VEH animals demonstrated a significant increase in AVP mRNA in the PVN compared to CTL VEH. However, daily post-restraint propranolol prevented stress-induced increases in AVP message in the hypothalamus. C) Representative autoradiographs of CRF mRNA in the PVN, outlining the medial parvocellular region of the PVN centered at approximately bregma −1.8. D) There were no effects of stress or drug administration on CRF expression in the PVN. * signifies p≤0.05. All data is mean ± SEM.

Figure 4

Intra-BLA β–AR blockade each day after stress prevents repeated-stress induced changes in brain-derived neurotrophic factor (BDNF) gene expression in the basal BLA. A) Representative autoradiographs of BDNF mRNA in the BLA at approximately bregma −3.1. The BLA is comprised of two nuclei: the lateral nucleus (outlined in the figure as the dorsal triangular shape) and the basal nucleus (outlined in the figure as the ventral rectangular shape). Because we observed that BDNF mRNA expression in the BLA was not uniformly distributed but appeared to be located primarily in the basal nucleus, we subdivided these regions for our analyses. B) RR VEH animals demonstrated a significant increase in BDNF mRNA in the basal BLA as compared to CTL VEH animals, but β–AR blockade after each daily restraint prevents this increase in neurotrophin message in the BLA. C) No differences in BDNF mRNA expression were seen in the lateral BLA as a result of stress or drug administration. ns: CTL VEH=6, CTL PROP=6, RR VEH=4, RR PROP=7. * signifies p≤0.05. + signifies p≤0.1. All data is mean ± SEM.

Figure 5

Intra-BLA clenbuterol, a β–AR agonist, accelerates HPA habituation to repeated restraint at the lowest dose but attenuates behavioral habituation at higher doses. We tested a dose-response curve of clenbuterol (1 ng, 3 ng, 10 ng) injected after daily restraint as different doses of clenbuterol have been found to be effective at enhancing memory consolidation for different paradigms. Because we hypothesized that clenbuterol might accelerate the development of habituation, we analyzed struggling behavior on days 1, 2, and 4 in repeatedly restrained groups only, and we took a single blood sample at the end of restraint on day 3 in repeatedly restrained groups only. A and B) There were no differences between groups in struggling behavior on day 1 (A) or 2 (B). C) Compared to RR VEH animals on day 3, RR 1ng animals exhibit significantly further reduced ACTH activity on day 3. n’s: RR VEH=12, RR 1ng=10, RR 3ng=11, RR 10ng=12.). D) On day 4, animals given 3ng clenbuterol after restraint on days 1–3 struggled significantly more during the first 5 minutes than repeatedly restrained animals given vehicle, and animals given 10ng clenbuterol tended (p=0.06) to struggle more than repeatedly restrained vehicle animals during the first 5 minutes. 1ng clenbuterol had no effect. E) Intra-BLA administration of 1ng clenbuterol enhanced HPA habituation to restraint on day 5. We observed a Main Effect of Stress indicating significant habituation of all RR groups compared to all CTL groups; comparison within RR groups given vehicle, 1 ng, 3 ng, or 10 ng clenbuterol revealed that RR 1ng animals enhanced ACTH habituation at 30 minutes as compared to RR VEH animals. F and G) On day 5, there were no differences in HPA habituation between repeatedly restrained animals given daily post-restraint vehicle and either 3ng clenbuterol (F) or 10ng clenbuterol (G). n’s: CTL VEH=12–14, CTL 1ng=7–8, CTL 3ng=8–9, CTL 10ng=8–10, RR VEH=8–12, RR 1ng=7–10, RR 3ng=7–11, RR 10ng=8–12. * signifies p≤0.05. + signifies p≤0.1. All data are shown as mean ± SEM.

Figure 6

ERK phosphorylation (pERK) in the BLA is reduced in response to β-AR activation by repeated stress, and inhibition of pERK is sufficient to reduce HPA activity to repeated stress. A) Representative western blots from two different experiments, comparing levels of phosphorylated ERKs 1 and 2 in the BLA of naïve (CTL 0) animals, animals at the end of a first restraint (CTL 30), animals at the end of their 5^th restraint (RR 30), and separately, CTL 0 and repeatedly stressed animals under basal conditions (RR 0). B) pERK 1 levels normalized to CTL 0. In the BLA, pERK 1 is significantly decreased in response to a first restraint (CTL 30) and after 4 prior restraint exposures remains significantly decreased under basal conditions on day 5 (RR 0) as well as after 5^th restraint (RR 30). C) pERK 2 levels normalized to CTL 0. ERK 2 phosphorylation is significantly decreased in response to first restraint (CTL 30) and in response to a 5^th restraint (RR 30), and tends (p=0.07) to be decreased under basal conditions on day 5 (RR 0). ns: CTL 0′ =7, CTL 30′ =6, RR 0′ =5, RR 30′ =9. D)Representative western blots, comparing levels of phosphorylated ERKs 1 and 2 in the BLA of unstressed (CTL) or repeatedly stressed animals sacrificed basally (RR) on day 5 which received intra-BLA injections on days 1–4. E and F) pERK1 (E) and pERK 2 (F) normalized to CTL VEH (100%). Phosphorylation of both ERKs in the BLA was significantly reduced in RR VEH animals compared to CTL VEH, similarly to the effect shown in 6A–C. Intra-BLA propranolol delivered after daily restraint (RR PROP) prevented repeated restraint-induced reductions in basal pERK levels seen in RR VEH. ns: CTL VEH=11, CTL PROP=11, RR VEH=10, RR PROP=11. G–H) Daily post-restraint intra-BLA infusion of the MEK inhibitor U0126 (MEKI), which prevents MEK from phosphorylating ERK, is sufficient to reduce HPA activity to restraint regardless of prior stress experience. G) Animals received MEKI or vehicle after restraint (RR) or under unstressed (CTL) conditions each day on days 1–4, and ACTH responses to restraint were measured on day 5. RR groups demonstrated significant habituation in ACTH responses to restraint at 15 and 30 minutes as compared to CTL groups, and there was a trend (p≤0.1) towards reduced ACTH in both MEKI groups. ns: CTL VEH=11–12, CTL MEKI=8–9, RR VEH=10–12, RR MEKI=12–13. H) Integrating ACTH values, to obtain a weighted average HPA response to restraint on day 5, revealed that overall ACTH activity was reduced in RR groups regardless of drug treatment, indicating significant habituation of HPA activity, and in MEKI groups regardless of prior stress, indicating that inhibition of ERK phosphorylation in the BLA on days prior to restraint on day 5 reduced HPA activity to this test stress regardless of prior stress history. ns: CTL VEH=11, CTL MEKI=8, RR VEH=10, RR MEKI=12.* signifies p≤0.05. + signifies p≤0.1. All data are shown as mean ± SEM.

Figure 7

Schematic of the role of BLA β-AR activity and pERK activity on habituation or adaptation to repeated stress. A) The current findings support an inverted-U function to describe the effects of BLA β-AR activation immediately post-stress on the habituation of HPA activity and struggling behavior. However, the data indicate that the curve for HPA activity should be placed rightward of the curve for struggling behavior, as the habituation of HPA activity can be enhanced by the administration of a low dose of β-AR agonist, but struggling behavior cannot be enhanced beyond the levels induced by endogenous β-AR activation. A remaining question regards whether stress habituation can be completely prevented by intra-BLA administration of a high dose of β-AR agonist after stress. B) The current data indicate that reductions in pERK in the BLA below that seen in control animals is a key mechanism by which β-AR activation regulates stress adaptation. Daily administration of the MEK inhibitor U0126, which prevents ERK phosphorylation, significantly reduced HPA activity, whereas blockade of β-AR by propranolol prevented habituation and prevented decreased pERK in the BLA. However, it is not known how β-AR agonist such as clenbuterol affects pERK in the BLA, or how intracellular signaling in the BLA might be altered as a result of high levels of β-AR activation.