Fast feedback inhibition of the HPA axis by glucocorticoids is mediated by endocannabinoid signaling

Abstract

Glucocorticoid hormones are secreted in response to stimuli that activate the hypothalamo-pituitary-adrenocortical (HPA) axis and self-regulate through negative feedback. Negative feedback that occurs on a rapid time scale is thought to act through nongenomic mechanisms. In these studies, we investigated fast feedback inhibition of HPA axis stress responses by direct glucocorticoid action at the paraventricular nucleus of the hypothalamus (PVN). Local infusion of dexamethasone or a membrane-impermeant dexamethasone-BSA conjugate into the PVN rapidly inhibits restraint-induced ACTH and corticosterone release in a manner consistent with feedback actions at the cell membrane. The dexamethasone fast feedback response is blocked by the cannabinoid CB1 receptor antagonist AM-251, suggesting that fast feedback requires local release of endocannabinoids. Hypothalamic tissue content of the endocannabinoid 2-arachidonoyl glycerol is elevated by restraint stress, consistent with endocannabinoid action on feedback processes. These data support the hypothesis that glucocorticoid-induced fast feedback inhibition of the HPA axis is mediated by a nongenomic signaling mechanism that involves endocannabinoid signaling at the level of the PVN.

Figure 1

Bilateral cannula placement in the PVN. Cannula placement was verified using Nissl staining. The injection locations are outlined by dashed lines, and the end of the guide cannulas are indicated with asterisks. Scale bar, 100 μm.

Figure 2

Intra-PVN administration of dexamethasone causes rapid inhibition of the HPA axis response to restraint. Animals were given bilateral intra-PVN injections of dexamethasone (10 ng per side) or an equimolar amount of dex:BSA (1.25 μg per side), and plasma hormone responses to 25 min of restraint stress were measured. A, Time course of ACTH response to restraint. Dexamethasone and dex:BSA treatment both attenuated the ACTH response to restraint within 15 min of injection. B, Integrated ACTH response to restraint stress (AUC). The magnitude of the integrated ACTH response to restraint was not significantly decreased by either dexamethasone or dex:BSA. C, Time course of corticosterone (CORT) response to restraint. Dexamethasone and dex:BSA both significantly attenuated the corticosterone response to restraint within 60 min of the injection. D, Integrated corticosterone response to restraint. Dexamethasone and dex:BSA both decreased the magnitude of the integrated corticosterone response to restraint. *, P < 0.05 vs. vehicle-treated animals.

Figure 3

Dexamethasone-induced fast feedback does not alter c-Fos expression in the PVN. To test whether dexamethasone treatment was altering the activation of paraventricular neurons, the expression of c-Fos protein was assessed by immunohistochemistry. A–C, Representative photomicrographs are shown from animals treated with vehicle (A), dexamethasone (B), and dex:BSA (C). Scale bar, 100 μm. D, There was no significant difference in the number of c-Fos-immunoreactive cells (fos IR) in the PVN among the three treatment groups.

Figure 4

BSA immunohistochemistry of brains of dex:BSA-injected animals. Immunohistochemistry was performed on brain slices from animals treated with dex:BSA to determine the extent of diffusion of the injection. A, Low-power photomicrograph showing the spread of BSA immunoreactivity in the region of the PVN; B, photomicrograph from the area of the median eminence, showing a lack of immunoreactivity either along the edge of the third ventricle or at the median eminence. These data suggest that injections at the PVN covered the PVN but did not diffuse to distant areas, including the pituitary gland. In addition, the immunoreactivity against BSA shown does not appear to be inside the cell bodies. This supports the model for fast feedback effects occurring at the membrane, rather than at receptors inside the cell body. Scale bars, 100 μm.

Figure 5

Restraint stress leads to elevated 2-AG but not AEA content of hypothalamic tissue. To determine whether restraint per se increases endocannabinoid synthesis in the PVN, AEA, and 2-AG levels were measured in the hypothalamus of rats subjected to 30 min restraint stress. A, AEA levels in control, restrained, and recovered animal groups; B, 2-AG levels. 2-AG levels were significantly elevated in restrained and recovery animals compared with unstressed controls. AEA levels were not altered. *, P < 0.05 vs. basal levels.

Figure 6

CB1 receptor signaling is necessary for fast feedback. Animals were treated with dexamethasone, the CB1 receptor antagonist AM-251, or both and then subjected to 30 min restraint. Plasma ACTH and corticosterone responses were measured for 60 min after the injection. A, Plasma ACTH response to restraint. Dexamethasone treatment rapidly reduced the magnitude of the ACTH response, whereas AM-251 alone did not affect the stress-induced elevation of ACTH. AM-251 also completely reversed the dexamethasone-induced inhibition of HPA axis activity. B, Total magnitude of ACTH response, expressed as AUC. The magnitude of the integrated ACTH response was lower in dexamethasone-treated animals compared with dexamethasone and AM-251 cotreatment. C, Plasma corticosterone (CORT) response to restraint. Again, dexamethasone decreased the corticosterone response to restraint at 60 min after injection, whereas AM-251 treatment had no effect on the response. AM-251 also completely reversed the dexamethasone-induced inhibition of the corticosterone response. D, AUC of corticosterone secretion. Dexamethasone treatment decreased the corticosterone response relative to all other groups. *, P < 0.05 vs. vehicle; #, P < 0.05 vs. dexamethasone.

Figure 7

Model for cannabinoid-mediated actions of the PVN on the HPA axis. The results of these studies are consistent with the model for fast feedback actions at the PVN proposed by Di et al. (10). A, Glucocorticoids in the vicinity of the parvocellular neuron bind to a putative membrane-localized glucocorticoid receptor. Binding of glucocorticoid to this receptor leads to synthesis of endocannabinoids, which are released into the synapse. After diffusion in a retrograde manner across the synapse, the endocannabinoids initiate CB1 receptor signaling, which leads to decreased glutamate release onto the CRH-containing neuron. This leads to decreased CRH release from the cell. B, Blocking CB1 receptors leads to normalization of the glutamate release and, thus, normalization of the CRH output of the cells. Parvo, Parvocellular neuron; PLC, phospholipase C; DAG, diacyl glycerol. Adapted from Di et al. (10).