Nongenomic Glucocorticoid Suppression of a Postsynaptic Potassium Current via Emergent Autocrine Endocannabinoid Signaling in Hypothalamic Neuroendocrine Cells following Chronic Dehydration

Dexamethasone had no rapid effect on I_A activation and inactivation in magnocellular neurons from untreated rats. A, Voltage-clamp protocol for isolating I_K and I_A. Combined I_K and I_A currents were evoked by stepping from a holding potential (V_h) of −115 mV to test steps of −75 to 35 mV in 10 mV increments (left: voltage steps below, current responses above). I_K was evoked by the same stepping protocol, except from a V_h of −45 mV (middle: voltage steps below, current responses above). Subtraction of the currents activated from a V_h of −45 mV from those activated from a V_h of −115 mV yielded the isolated low voltage-activated I_A (right current traces). B, Representative traces showing voltage-dependent activation of I_A in PVN magnocellular neurons from untreated rats before and at the end of a 10 min Dex application (1 µm). Inset, Activation voltage-clamp protocol. C, Plots of the mean current amplitude of the I_A vs the test step potential, showing no effect of Dex on the I_A current amplitude. D, Plots of the mean current density of the I_A (peak I_A/capacitance) vs the test step potential, showing no effect of Dex on the I_A current density. E, Plots of the normalized chord conductance of the I_A vs the test step potential, showing no effect of Dex on the voltage dependence of activation of the I_A. F, Plots of the mean I_A 10–90% rise time vs test step potential; the Dex effect on the activation rate of I_A was not significant. G, Representative traces showing voltage-dependent inactivation of I_A in response to voltage steps to −15 mV from 200 ms conditioning steps between −135 and −25 mV before and after 10 min of Dex application (1 μm). Dexamethasone had no effect on the inactivation voltage dependence of the I_A. Inset, Inactivation voltage-clamp protocol. H, Plots of the mean normalized current amplitude vs the conditioning step potential, showing no effect of DEX on the voltage dependence of I_A inactivation. All recordings were in PVN magnocellular neurons in slices from untreated rats.

Rapid glucocorticoid modulation of I_A in PVN magnocellular neurons from salt-loaded rats. A, Representative traces showing voltage-dependent activation of I_A in PVN magnocellular neurons from salt-loaded rats before and at the end of a 10 min Dex application (1 µm). Inset, Activation voltage-clamp protocol. B, Plots of the mean peak amplitude of the I_A vs the test step potential; Dex caused a significant reduction in the I_A current amplitude. C, Plots of the mean current density (peak current/capacitance) of the I_A vs the test step potential; Dex caused a significant reduction in the I_A current density. D, Plots of the normalized chord conductance of the I_A vs the test step potential; Dex caused a significant shift to the right in the voltage dependence of activation of the I_A. E, Plots of the mean 10–90% I_A rise time vs the test step potential; Dex did not change the I_A activation kinetics. F, Representative traces showing voltage-dependent inactivation of I_A in response to voltage steps to −15 mV from 200 ms conditioning steps between −135 and −25 mV before and at the end of a 10 min Dex application (1 μm). Inset, Inactivation voltage-clamp protocol. G, Plots of the mean normalized current amplitude vs the conditioning potential. Dexamethasone had no effect on the voltage dependence of I_A inactivation. All recordings were in PVN magnocellular neurons in slices from salt-loaded rats. *p < 0.05; **p < 0.01 with ANOVA.

The rapid glucocorticoid effect on I_A in magnocellular neurons from salt-loaded rats was mediated by a membrane-associated receptor. A–F, Plots of the mean current amplitude of the I_A (A, C, E) and the I_A voltage dependence of activation (normalized chord conductance; B, D, F) vs the test step potential before and at the end of a 10 min application of the endogenous glucocorticoid corticosterone (Cort), the membrane-impermeant glucocorticoid Dex–BSA, and Dex in the presence of the protein translation inhibitor CHX. The endogenous glucocorticoid Cort (A, B), and the membrane-impermeant glucocorticoid Dex–BSA (C, D) reduced the I_A peak amplitude and shifted the I_A voltage dependence to the right. Blocking protein synthesis (E, F) failed to block the glucocorticoid modulation of the I_A. *p < 0.05; **p < 0.01 with ANOVA in A, C, E and with Student’s paired t test in B, D, F.

CB1 receptor agonists mimicked the glucocorticoid modulation of I_A. A, B, Bath application of the endocannabinoid 2-AG (1 μm) elicited a significant decrease in the I_A mean peak amplitude (A) and a positive shift in the I_A activation voltage dependence (normalized chord conductance; B) in magnocellular neurons from untreated rats. C, D, The CB1 receptor inverse agonist SR141716 (rimonabant, 1 μm) blocked the decrease in mean I_A peak amplitude (C) and the rightward shift in the chord conductance curve (D). E, F, AEA elicited a decrease in the I_A mean peak amplitude (E) and a positive shift in the I_A activation voltage dependence (chord conductance; F) in the presence of the TRPV1 receptor antagonist capsazepine (1 μm). G, The TRPV1 agonist capsaicin (1 μm) had no effect on the I_A voltage dependence of activation (chord conductance). *p < 0.05; **p < 0.01 with ANOVA in A, C, E and with Student’s paired t test in B, D, F, G.

CB1 receptor analogs blocked the glucocorticoid modulation of I_A. A, B, The CB1 receptor inverse agonist SR141716 (1 μm) blocked the decrease in I_A peak amplitude (A) and the rightward shift in the voltage dependence of activation (normalized chord conductance; B) induced by Dex (1 μm) in PVN magnocellular neurons in slices from salt-loaded rats. C, D, The CB1 receptor inverse agonist am251 also blocked the Dex-induced decrease in I_A peak amplitude (C) and the positive shift in activation voltage dependence (normalized chord conductance; D) in PVN magnocellular neurons from salt-loaded rats. E, F, 2-AG (1 μm; E) and AEA (1 μm; F) caused a positive shift in the I_A voltage-dependence curve (normalized chord conductance) from the control value, but subsequent application of Dex (1 μm) failed to further shift the curve, suggesting occlusion of the Dex effect by prior CB1 receptor activation. *p < 0.05 with ANOVA. Student’s paired t test.