Endocannabinoid signaling within the basolateral amygdala integrates multiple stress hormone effects on memory consolidation

Abstract

Glucocorticoid hormones are known to act synergistically with other stress-activated neuromodulatory systems, such as norepinephrine and corticotropin-releasing factor (CRF), within the basolateral complex of the amygdala (BLA) to induce optimal strengthening of the consolidation of long-term memory of emotionally arousing experiences. However, as the onset of these glucocorticoid actions appear often too rapid to be explained by genomic regulation, the neurobiological mechanism of how glucocorticoids could modify the memory-enhancing properties of norepinephrine and CRF remained elusive. Here, we show that the endocannabinoid system, a rapidly activated retrograde messenger system, is a primary route mediating the actions of glucocorticoids, via a glucocorticoid receptor on the cell surface, on BLA neural plasticity and memory consolidation. Furthermore, glucocorticoids recruit downstream endocannabinoid activity within the BLA to interact with both the norepinephrine and CRF systems in enhancing memory consolidation. These findings have important implications for understanding the fine-tuned crosstalk between multiple stress hormone systems in the coordination of (mal)adaptive stress and emotional arousal effects on neural plasticity and memory consolidation.

Figure 1

Glucocorticoids interact with the endocannabinoid system of the BLA in enhancing memory consolidation of inhibitory avoidance training. (a) Representative photomicrograph illustrating placement of a needle tip within the BLA, basolateral amygdala. Arrow points to the needle tip. MeA, medial amygdala; CeA, central amygdala. (b) Retention latencies of rats given posttraining intra-BLA infusions of the GR agonist RU 28362 (1, 3, or 10 ng in 0.2 μl) alone or together with the CB1 receptor antagonist AM251 (0.14 ng). **P<0.01 vs vehicle; ♦♦P<0.01 vs RU 28362 only (n=9–12/group). (c) Retention latencies of rats given posttraining intra-BLA infusions of the membrane-impermeable glucocorticoid Cort:BSA (1, 3, or 10 ng in 0.2 μl) alone or together with AM251 (0.14 ng). *P<0.05; **P<0.01 vs vehicle; ♦P<0.05 vs Cort:BSA alone (n=11–14/group). (d) Retention latencies of rats given posttraining intra-BLA infusions of the cannabinoid agonist WIN55,212-2 (10, 30, or 100 ng in 0.2 μl) alone or together with the GR antagonist RU 38486 (1 ng). **P<0.01 vs vehicle; (n=10–14/group). Data depict 48-h retention latencies (mean+SEM) in seconds(s).

Figure 2

Glucocorticoids require endocannabinoid signaling to induce a training-associated increase in CREB phosphorylation within the BLA. (a) The number of pCREB-positive neurons in the BLA as assessed 1 h after inhibitory avoidance training followed by a systemic injection of corticosterone (CORT, 3 mg/kg) or/and the CB1 receptor antagonist SR141716 (1 mg/kg). **P<0.01 vs vehicle; ♦♦P<0.01 vs CORT alone (n=5–6/group). Data presented as percentages relative to vehicle. (b1) Photomicrographs of pCREB immunostaining within the BLA 1 h after inhibitory avoidance training and systemic injection of CORT (3 mg/kg), SR141716 (1 mg/kg), or drug combination. BLA, basolateral amygdala, CeA, central amygdala, MeA, medial amygdala. (b2) Confocal images illustrating cells positive for pCREB (left), CAMKII (middle), and double labeling (right) in the BLA. (c) Forty-eight hours retention latencies (mean+SEM) in seconds(s) of rats given immediate posttraining systemic injection of CORT (3 mg/kg) alone or together with SR141716 (1 mg/kg). **P<0.01 vs vehicle; ♦♦P<0.01 vs corticosterone alone (n=8–10/group).

Figure 3

The endocannabinoid system enables glucocorticoid effects on the noradrenergic system of the BLA in enhancing memory consolidation of inhibitory avoidance training. (a) Retention latencies of rats given posttraining intra-BLA infusions of the cannabinoid agonist WIN55,212-2 (10, 30, or 100 ng in 0.2 μl) alone or together with the β-adrenoceptor antagonist propranolol (0.5 μg). **P<0.01 vs vehicle; ♦♦P<0.01 vs WIN55,212-2 alone (n=9–12/group). (b) Retention latencies of rats given posttraining intra-BLA infusions of the β-adrenoceptor agonist clenbuterol (1, 10, or 100 ng in 0.2 μl) alone or together with GR antagonist RU 38486 (1 ng) and a non-enhancing dose of WIN55,212-2 (1 ng). **P<0.01 vs vehicle (n=10–14/group). (c) Retention latencies of rats given posttraining intra-BLA infusions of clenbuterol (1, 10, or 100 ng in 0.2 μl) alone or together with AM251 (0.14 ng). *P<0.05 vs vehicle; ♦P<0.05; ♦♦P<0.01 vs clenbuterol alone (n=9–12/group). Data depict 48-h retention latencies (mean+SEM) in seconds(s).

Figure 4

The endocannabinoid system enables glucocorticoid effects on the CRF system of the BLA in enhancing memory consolidation of inhibitory avoidance training. (a) Retention latencies of rats given posttraining intra-BLA infusions of the CRF-binding protein inhibitor CRF_6–33 (0.1 μg in 0.2 μl) alone or together with GR antagonist RU 38486 (1 ng) and cannabinoid agonist WIN55,212-2 (1 ng). *P<0.01, **P<0.01 vs vehicle; ♦♦P<0.01 vs CRF_6–33 alone (n=9–13/group). (b) Retention latencies of rats given posttraining intra-BLA infusions of CRF_6–33 (0.01, 0.1, or 1 μg in 0.2 μl) alone or together with the CB1 receptor antagonist AM251 (0.14 ng). **P<0.01 vs vehicle; ♦♦P<0.01 vs CRF_6–33 alone (n=10–13/group). Data depict 48-h retention latencies (mean+SEM) in seconds(s).

Figure 5

A model illustrating the role of the endocannabinoid system within the BLA in integrating the effects of glucocorticoids and the norepinephrine and CRF systems on memory consolidation. Corticosterone (CORT) is released during emotionally arousing events and binds to a membrane-bound GR, which activates the intracellular cAMP/PKA signaling pathway to induce endocannabinoid (eCB) synthesis. Endocannabinoids are then released into the synapse where they bind to CB1 receptors on GABAergic terminals and thereby inhibit the release of GABA. This suppression of GABA release subsequently disinhibits norepinephrine (NE) release, and increases the excitability of BLA pyramidal neurons, rendering them more sensitive to the effects of norepinephrine and CRF. Together, these effects will result in an increased activation of the cAMP/PKA pathway and phosphorylation of cAMP response element binding (CREB) protein. These stress hormone effects in the BLA are required for enhancing memory consolidation of emotionally arousing experiences.