Termination of HPA axis activation following exposure to stress is essential to limit the duration of glucocorticoid secretion and prevent the deleterious effects of persistently elevated levels of glucocorticoid hormones on cardiovascular, immune, metabolic and neural systems. Our data demonstrate that stress, via activation of GR, increases 2-AG levels within the mPFC, and that eCB signaling contributes to the appropriate termination of glucocorticoid secretion following cessation of stress. Immunohistochemical and EM data indicate that CB1
R are expressed almost entirely by GABAergic terminals in layer V of the prelimbic region of the mPFC, particularly on axons which synapse onto the soma of principal neurons. Functional studies support a role for CB1
R to inhibit GABA release within this same neuronal population. Our data also demonstrate that incubation of slices from the mPFC with corticosterone induces eCB-mediated inhibition of GABA release onto principal neurons. Collectively, these data support the novel hypothesis that glucocorticoid hormones released following stress exposure activate eCB/CB1
R signaling within the mPFC, inhibiting GABA release onto layer V pyramidal neurons in the prelimbic cortex, and promoting the termination of corticosterone secretion. Consistent with this model, pharmacological disruption of GABAA
receptor signaling within the mPFC also decreases stress-induced activation of the HPA axis (Weinberg et al., 2010
). Taken together, these data indicate that prefrontal cortical eCB signaling links glucocorticoids and neuronal activation within the mPFC and contributes to the long negative feedback loop to inhibit corticosterone secretion following cessation of stress.
The present data are consistent with the established role of the mPFC, and particularly the prelimbic region of the mPFC, in the regulation of the HPA axis and termination of the stress response. Lesion studies have demonstrated that selective ablation of the prelimbic region of the PFC does not alter the magnitude, but rather the duration, of corticosterone secretion following exposure to a psychogenic stressor (Diorio et al., 1993
; Radley et al., 2006
). In the present study, both genetic deletion of the CB1
R, and local antagonism of CB1
R signaling within the mPFC prolonged the elevation in stress-induced levels of circulating corticosterone. Our histological and electrophysiological experiments demonstrate the presence of CB1
R on GABAergic terminals impinging upon pyramidal neurons within layer V of the prelimbic region of the mPFC. These findings support the hypothesis that activation of CB1
R signaling in this brain region could result in disinhibition of excitatory projections from the prelimbic mPFC to other brain regions.
The circuit by which the efferent projection neurons from the prelimbic region of the PFC inhibit the HPA axis involves a secondary activation of inhibitory GABAergic neurons within subregions of the bed nucleus of the stria terminalis (BNST; Spencer et al., 2005
; Radley et al., 2009
) or the peri-PVN region (Herman et al., 2005
). Activation of either of these inhibitory circuits feeding into the PVN, dampens neuronal activation of the CRH secreting cells of the PVN (Herman et al., 2005
). Layer V of the prelimbic cortex is the primary site for projection neurons which extend to subcortical limbic structures (Gabbott et al., 2005
), such as the BNST; thus the identification of eCB-mediated regulation of neuronal excitability within layer V neurons in the prelimbic region of the mPFC provides a neurochemical and functional mechanism which compliments the previously established neuroanatomical networks involved in prefrontocortical regulation of HPA axis activity. Recent data indicate that excitatory afferents arising from the ventral subiculum activate the same inhibitory relays within the BNST as are activated by excitatory afferents originating from the mPFC (Radley and Sawchenko, 2010
). These data suggest that both hippocampal and mPFC projections are involved in the long glucocorticoid-mediated negative feedback loop via inputs into the BNST. In the current study, disruption of eCB signaling within the mPFC attenuated, but did not completely prevent, the return of circulating glucocorticoids to baseline concentrations. It is possible that the projections from the ventral subiculum to the BNST, which would not be affected by CB1
receptor blockade in the mPFC, are responsible for the ultimate return of circulating corticosterone concentrations to baseline.
It is possible that disruption of CB1
R within the mPFC regulates corticosterone secretion through an HPA axis-independent pathway. For example, ventral regions of the mPFC can regulate autonomic outflow during conditions of stress (Neafsey, 1990
) and it has recently been demonstrated that subregions of the BNST are capable of modulating corticosterone secretion independent of ACTH levels (Choi et al., 2007
). As such, it is plausible that CB1
R within the mPFC could contribute to higher order regulation of autonomic outflow, and that the ability of prefrontal cortical CB1
R to regulate corticosterone secretion involves the activity of the sympatho-medullary arm of the stress response (Bornstein et al., 2008
). Further studies are required to differentiate between these possibilities.
Within the context of HPA axis regulation, however, the activation of efferent projections from the mPFC has been shown to function as a prominent pathway in glucocorticoid mediated negative feedback. Local activation of glucocorticoid receptors within the mPFC accelerates the post-stress decline in circulating levels of corticosterone following exposure to stress (Diorio et al., 1993
) and downregulation of glucocorticoid receptors within the mPFC following chronic stress or in aging is associated with impaired glucocorticoid negative feedback regulation (Mizoguchi et al., 2003
). Unlike the local glucocorticoid effects in the PVN that rapidly decrease activation of CRH neurosecretory cells governing HPA axis output and promote fast-feedback inhibition on the HPA axis (Di et al., 2003
; Evanson et al., 2010
), disruption of the mPFC to PVN circuit prolongs the recovery to normal circulating glucocorticoids levels following stress exposure (Diorio et al., 1993
; Radley et al., 2006
). As such, these data demonstrate that glucocorticoid-mediated negative feedback possesses both short-loop (locally within the PVN) and long-loop (distally within the mPFC and ventral subiculum) components. Moreover, there is evidence that eCB signaling contributes to both of these phases of glucocorticoid feedback. The current data create an argument for a role of prefrontal cortical eCB signaling in the long-loop phase of glucocorticoid feedback. And, it has recently been reported that local antagonism of the CB1
R within the PVN impairs fast-feedback inhibition of HPA axis activity by glucocorticoids (Evanson et al., 2010
). In the present study, we report that mice globally deficient in CB1
R exhibit a larger peak in corticosterone secretion following stress, which is consistent with these mice lacking fast feedback inhibition due to the absence of CB1
R signaling within the PVN.
Taking these data together, we propose the following model for the integration of eCB signaling into the temporal phases of glucocorticoid feedback. Glucocorticoid hormones are released into the circulation in response to stress. In the PVN, glucocorticoids evoke a rapid induction of eCB release through a non-genomic pathway, which results in a rapid suppression of glutamatergic inputs to CRH neurosecretory cells and decreases the excitatory drive to the HPA axis (Di et al., 2003
; Evanson et al., 2010
). In the mPFC, glucocorticoids produce a time-delayed increase in 2-AG, which, via CB1
R activation, suppresses GABAergic inputs to principal neurons. This suppression of GABAergic inputs to principal neurons could act to increase the outflow of these projection neurons to inhibitory relays within the BNST, and thus contribute to the long-loop of glucocorticoid negative feedback. In sum, our model proposes a temporally- and structurally-specific role of eCB signaling in distinct phases of glucocorticoid feedback.
The mechanism by which glucocorticoids increase eCB signaling within the mPFC was not elucidated in the current study, but appears to be distinct from the process which occurs within the PVN. In the PVN, glucocorticoid regulation of eCB signalling is not blocked by an antagonist of the nuclear GR and is driven by glucocorticoid-induced G-protein signaling (Di et al., 2003
). Furthermore, administration of glucocorticoids in the absence of stress can rapidly (~10 min) increase eCB content within the hypothalamus but not the mPFC (Hill et al., 2010a
). Thus, we hypothesize that the actions of glucocorticoids on eCB content within the mPFC require coincident increases in neuronal activation which occur following exposure to stress to produce a detectable increase in 2-AG content using bulk tissue measurements.
Termination of HPA axis activation following exposure to stress is an essential process for maintaining optimal health in the face of persistent stress. The data presented herein suggest an important role of the eCB system within the mPFC in the termination of glucocorticoid release following exposure to stress. Increased 2-AG within the mPFC following exposure to stress provides a mechanism of coincidence detection that can fine tune the excitability of pyramidal neurons within the prelimbic region of the mPFC and contribute to termination of corticosterone secretion following cessation of stress exposure. These data contribute to our general understanding of the mechanisms subserving glucocorticoid-mediated negative feedback and stress recovery. Furthermore, they provide a mechanism that could underlie modulation by glucocorticoids of neuronal sensitivity in extrahypothalamic structures that contribute to feedback and recovery.