P/Q-type voltage-gated calcium channels mediate the ethanol and CRF sensitivity of central amygdala GABAergic synapses

Abstract

The central amygdala (CeA) GABAergic system is hypothesized to drive the development of alcohol dependence, due to its pivotal roles in the reinforcing actions of alcohol and the expression of negative emotion, anxiety and stress. Recent work has also identified an important role for the CeA corticotropin-releasing factor (CRF) system in the interaction between anxiety/stress and alcohol dependence. We have previously shown that acute alcohol and CRF each increase action potential-independent GABA release in the CeA via their actions at presynaptic CRF type 1 receptors (CRF₁s); however, the shared mechanism employed by these two compounds requires further investigation. Here we report that acute alcohol interacts with the CRF/CRF₁ system, such that CRF and alcohol act via presynaptic CRF₁s and P/Q-type voltage-gated calcium channels to promote vesicular GABA release and that both compounds occlude the effects of each other at these synapses. Chronic alcohol exposure does not alter P/Q-type voltage-gated calcium channel membrane abundance or this CRF₁/P/Q-type voltage-gated calcium channel mechanism of acute alcohol-induced GABA release, indicating that alcohol engages this molecular mechanism at CeA GABAergic synapses throughout the transition to dependence. Thus, P/Q-type voltage-gated calcium channels, like CRF₁s, are key regulators of the effects of alcohol on GABAergic signaling in the CeA.

Keywords: Alcohol dependence; Alcohol/ethanol; Central amygdala; Corticotropin-releasing factor (CRF); Corticotropin-releasing factor type 1 receptor (CRF(1)); GABA; P/Q-type voltage-gated calcium channel.

Fig. 1

Acute alcohol increased GABA release in the naïve rat CeA via P/Q-type voltage-gated calcium channel activity. A: (Left) Representative mIPSC traces from a naïve rat CeA neuron in baseline conditions and during acute alcohol (44 mM EtOH) superfusion. (Right) EtOH significantly increased the mIPSC frequency, but had no effect on the mIPSC amplitude or kinetics (14 cells from 10 rats). B: EtOH significantly increased the mIPSC frequency in CeA neurons that were pre-loaded with 10 mM BAPTA (9 cells from 3 rats). C: (Left) Representative mIPSCs in low (0.5 mM) and high (5 mM) Ca²⁺ aCSF and during subsequent EtOH superfusion. (Right) EtOH significantly increased the mIPSC frequency in CeA neurons exposed to high Ca²⁺ aCSF, but not low Ca²⁺ aCSF (normalized to pre-alcohol baseline). For these extracellular calcium experiments, 6–7 cells from a minimum of 5 rats were used for each experimental group. D: (Left) Representative mIPSCs in the P/Q-type voltage-gated calcium channel blocker ω-Agatoxin TK (500 nM Aga) and during subsequent EtOH superfusion. (Right) EtOH’s enhancement of the mIPSC frequency was blocked in the presence of Aga (10 cells from 4 rats), but was unchanged by the L-type calcium channel blocker Nifedipine (10 μM Nif; 6 cells from 4 rats) or the N-type calcium channel blocker ω-Conotoxin GVIA (1 μM Cono; 6 cells from 5 rats).

Fig. 2

CRF increased GABA release in the naïve rat CeA via P/Q-type voltage-gated calcium channel activity. A: (Left) Representative mIPSCs from a naïve rat CeA neuron in baseline conditions and during CRF (200 nM) superfusion. (Right) CRF significantly increased the mIPSC frequency, but had no effect on the mIPSC amplitude or kinetics (8 cells from 5 rats). B: CRF significantly increased the mIPSC frequency in CeA neurons that were pre-loaded with 10 mM BAPTA (6 cells from 3 rats). C: (Left) Representative mIPSCs in low (0.5 mM) and high (5 mM) Ca²⁺ aCSF and during subsequent CRF superfusion. (Right) CRF significantly increased the mIPSC frequency in CeA neurons exposed to high Ca²⁺ aCSF, but not low Ca²⁺ aCSF (normalized to pre-CRF baseline). For these extracellular calcium experiments, 6–7 cells from a minimum of 4 rats were used for each experimental group. D: (Left) Representative mIPSCs in the P/Q-type voltage-gated calcium channel blocker ω-Agatoxin TK (500 nM Aga) and during subsequent CRF superfusion. (Right) CRF’s enhancement of the mIPSC frequency was blocked in the presence of Aga (5 cells from 3 rats).

Fig. 3

Acute alcohol interacts with the CRF/CRF₁ system to enhance GABA release. A: (Left) Representative mIPSCs from a naïve rat CeA neuron in baseline conditions, during CRF (200 nM) superfusion and following acute alcohol (44 mM EtOH) co-application in the continued presence of CRF. (Right) CRF significantly increased the mIPSC frequency, and EtOH (in CRF) had no further effect (6 cells from 4 rats). B: (Left) Representative mIPSCs in baseline conditions, during EtOH superfusion and following CRF + EtOH co-application. (Right) EtOH significantly increased the mIPSC frequency, and CRF (in EtOH) had no further effect (9 cells from 4 rats). C: (Left) Representative mIPSCs in the CRF₁ antagonist R121919 (1 μM) and during subsequent EtOH superfusion. (Right) EtOH’s enhancement of the mIPSC frequency was blocked in the presence R121919 (7 cells from 3 rats).

Fig. 4

Alcohol dependence does not alter the effects of acute alcohol at CeA GABAergic synapses. A: (Left) Representative mIPSCs from a CIE rat CeA neuron in baseline conditions and during acute alcohol (44 mM EtOH) superfusion. (Right) EtOH significantly increased the mIPSC frequency, but had no effect on the mIPSC amplitude or kinetics (6 cells from 5 rats). B: EtOH significantly increased the mIPSC frequency in CeA neurons that were pre-loaded with 10 mM BAPTA (6 cells from 3 rats). C: (Left) Representative mIPSCs in low (0.5 mM) and high (5 mM) Ca²⁺ aCSF and during subsequent EtOH superfusion. (Right) EtOH significantly increased the mIPSC frequency in CIE CeA neurons exposed to high Ca²⁺ aCSF, but not low Ca²⁺ aCSF (normalized to pre-alcohol baseline). For these extracellular calcium experiments, 6 cells from a minimum of 4 rats were used for each experimental group. D: (Left) Representative mIPSCs in the P/Q-type voltage-gated calcium channel blocker ω-Agatoxin TK (500 nM Aga) and during subsequent EtOH superfusion. (Right) EtOH’s enhancement of the mIPSC frequency was blocked in the presence of Aga (5 cells from 3 rats). E: (Top) Representative western blot image of P/Q-type voltage-gated calcium channel (Ca_v2.1) membrane abundance from the CeA of naïve and CIE rats. (Bottom) Quantification revealed no difference in CeA Ca_v1.2 membrane expression in CIE vs. naïve rats (6 rats were used for each experimental group). F: (Left) Representative mIPSCs in the CRF₁ antagonist R121919 (1 μM) and during subsequent EtOH superfusion. (Right) EtOH’s enhancement of the mIPSC frequency was blocked in the presence of R121919 (6 cells from 3 rats).