Nongenomic glucocorticoid inhibition via endocannabinoid release in the hypothalamus: a fast feedback mechanism

Abstract

Glucocorticoid negative feedback in the brain controls stress, feeding, and neural-immune interactions by regulating the hypothalamic-pituitary-adrenal axis, but the mechanisms of inhibition of hypothalamic neurosecretory cells have never been elucidated. Using whole-cell patch-clamp recordings in an acute hypothalamic slice preparation, we demonstrate a rapid suppression of excitatory glutamatergic synaptic inputs to parvocellular neurosecretory neurons of the hypothalamic paraventricular nucleus (PVN) by the glucocorticoids dexamethasone and corticosterone. The effect was maintained with dexamethasone conjugated to bovine serum albumin and was not seen with direct intracellular glucocorticoid perfusion via the patch pipette, suggesting actions at a membrane receptor. The presynaptic inhibition of glutamate release by glucocorticoids was blocked by postsynaptic inhibition of G-protein activity with intracellular GDP-beta-S application, implicating a postsynaptic G-protein-coupled receptor and the release of a retrograde messenger. The glucocorticoid effect was not blocked by the nitric oxide synthesis antagonist N(G)-nitro-L-arginine methyl ester hydrochloride or by hemoglobin but was blocked completely by the CB1 cannabinoid receptor antagonists AM251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] and AM281 [1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide] and mimicked and occluded by the cannabinoid receptor agonist WIN55,212-2 [(beta)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate], indicating that it was mediated by retrograde endocannabinoid release. Several peptidergic subtypes of parvocellular neuron, identified by single-cell reverse transcripton-PCR analysis, were subject to rapid inhibitory glucocorticoid regulation, including corticotropin-releasing hormone-, thyrotropin-releasing hormone-, vasopressin-, and oxytocin-expressing neurons. Therefore, our findings reveal a mechanism of rapid glucocorticoid feedback inhibition of hypothalamic hormone secretion via endocannabinoid release in the PVN and provide a link between the actions of glucocorticoids and cannabinoids in the hypothalamus that regulate stress and energy homeostasis.

Figure 1.

Glucocorticoids inhibited glutamate release onto PVN parvocellular neurons. a, Bath application of DEX (1 μm) elicited a decrease in the frequency of mEPSCs. b, Cumulative frequency plots of mEPSC interval and amplitude distributions from the same cell showed a significant reduction in mEPSC frequency (p < 0.01) with no change in mEPSC amplitude (p = 0.28). c, Mean changes in the average mEPSC frequency, amplitude, and decay time in dexamethasone (1 μm) in 18 PVN parvocellular neurons. d, The dexamethasone-induced decrease in mEPSC frequency was dose dependent. e, Corticosterone (CORT, 1 μm) caused a similar decrease in the mean frequency of mEPSCs, whereas the corticosteroid precursor cholesterol (5 μm) and the physiologically inactive steroid isopregnanolone (5 μm) had no effect on mEPSC frequency. Numbers in parentheses represent numbers of cells analyzed in each condition in this and in the following figures. ^*p < 0.05; ^**p < 0.01.

Figure 2.

The glucocorticoid effect was mediated by a postsynaptic, G-protein-coupled membrane receptor. a, Bath application of membrane-impermeant DEX—BSA maintained the inhibitory effect, whereas intracellular application of dexamethasone (1 μm) had no effect on the frequency of mEPSCs. The intracellular glucocorticoid receptor antagonist RU486 (10 μm) and the intracellular mineralocorticoid receptor antagonist spironolactone (10 μm) failed to block the inhibitory effect of dexamethasone on mEPSCs.b, G-protein and protein-kinase blockers abolished the effect of glucocorticoid on mEPSCs. Postsynaptic G-protein blockade with intracellular GDP-β-S application (500 μm) blocked the effect of dexamethasone on mEPSCs, implicating a postsynaptic G-protein-coupled mechanism. Kinase inhibition with bath application of staurosporine (0.5 μm) or a PKC blocker, GF109203X (0.5 μm), also abolished the inhibitory effect of dexamethasone on mEPSCs, suggesting a PKC-dependent mechanism that is either presynaptic or postsynaptic. ^**p < 0.01.

Figure 3.

Glucocorticoid inhibition of glutamate release was not mediated by NO or CO. a, The dexamethasone-induced suppression of mEPSCs was not blocked by the previous bath application of the NOS inhibitor l-NAME (10 μm). b, Cumulative frequency plots showed a significant reduction in mEPSC frequency (p < 0.05) with no change in mEPSC amplitude (p = 0.24) in the presence of l-NAME and DEX. c, The NO—CO scavenger hemoglobin (10 μm) also failed to block the inhibitory effect of dexamethasone on the frequency of mEPSCs. ^*p < 0.05; ^**p < 0.01.

Figure 4.

The effect of glucocorticoid on glutamate release was mediated by an endocannabinoid. a, The dexamethasone-induced suppression of mEPSCs was prevented by previous bath application of the CB₁ receptor antagonist AM251 (1 μm). b, There was no effect of dexamethasone on the cumulative mEPSC interevent interval or amplitude distribution in the presence of AM251. c, Mean changes in average mEPSC frequency in the presence of cannabinoid receptor antagonists. The CB₁ receptor antagonist AM251 blocked the effect of dexamethasone on mEPSC frequency. The CB₂ receptor antagonist AM630 failed to block the DEX effect at a 1 μm concentration but blocked the DEX effect at the higher, nonselective 10 μm concentration. d, A cannabinoid receptor agonist, WIN55,212-2 (1 μm), mimicked the glucocorticoid effect, causing a selective reduction in the frequency of mEPSCs. e, Cumulative frequency plots showed a significant increase in mEPSC interval (p < 0.01) but no change in mEPSC amplitude (p = 0.42) in WIN55,212-2. f, Mean changes in the average mEPSC frequency in the presence of glucocorticoid and the cannabinoid receptor agonist. The effect of WIN55,212-2 was similar to that of dexamethasone, but these effects were not additive, suggesting converging mechanisms. ^*p < 0.05; ^**p < 0.01.

Figure 5.

RT-PCR analysis in individual parvocellular neurons. a, CRH mRNA expression in six individual PVN parvocellular neurons. The expected CRH PCR product is 326 bp. b, TRH, VP, OT, and GAPDH mRNA expression in individual PVN parvocellular neurons. The expected PCR product weights are 276 bp (TRH), 440 bp (VP), 463 bp (OT) and 441 bp (GAPDH). The cell identification numbers are shown above each lane. A DNA ladder ranging from 300 to 500 bp is shown in the left lane of each panel.

Figure 6.

A model of the rapid glucocorticoid actions in PVN parvocellular neurons. The proposed mechanisms include glucocorticoid (CORT) binding to a G-protein-coupled glucocorticoid receptor (GR_mb) and activation of an intracellular signaling pathway (dashed arrow) that leads to endocannabinoid synthesis (+). Endocannabinoid (CB) is released from the PVN neuron and binds to a G-protein-coupled CB₁ cannabinoid receptor (CBR) on presynaptic glutamate terminals, activating a signaling cascade (dashed arrow) that leads to the inhibition of glutamate (GLU) release onto the PVN neuron-leading to decreased PVN neuronal activity and hormone secretion.