Search

Research on the actions of ethanol at the GABAergic synapse has traditionally focused on postsynaptic mechanisms, but recent data demonstrate that ethanol also increases both evoked and spontaneous GABA release in many brain regions. Using whole-cell voltage-clamp recordings, we previously showed that ethanol increases spontaneous GABA release at the rat interneuron-Purkinje cell synapse. This presynaptic ethanol effect is dependent on calcium release from internal stores, possibly through activation of inositol 1,4,5-trisphosphate receptors (IP(3)Rs). After confirming that ethanol targets vesicular GABA release, in the present study we used electron microscopic immunohistochemistry to demonstrate that IP(3)Rs are located in presynaptic terminals of cerebellar interneurons. Activation of IP(3)Rs requires binding of IP(3), generated through activation of phospholipase C (PLC). We find that the PLC antagonist edelfosine prevents ethanol from increasing spontaneous GABA release. Diacylglycerol generated by PLC and calcium released by activation of the IP(3)R activate protein kinase C (PKC). Ethanol-enhanced GABA release was blocked by two PKC antagonists, chelerythrine and calphostin C. When a membrane impermeable PKC antagonist, PKC (19-36), was delivered intracellularly to the postsynaptic neuron, ethanol continued to increase spontaneous GABA release. Overall, these results suggest that activation of the PLC/IP(3)R/PKC pathway is necessary for ethanol to increase spontaneous GABA release from presynaptic terminals onto Purkinje cells.

While research on the actions of ethanol at the GABAergic synapse has focused on postsynaptic mechanisms, recent data have demonstrated that ethanol also facilitates GABA release from presynaptic terminals in many, but not all, brain regions. The ability of ethanol to increase GABA release can be regulated by different G protein-coupled receptors (GPCRs), such as the cannabinoid-1 receptor, corticotropin-releasing factor 1 receptor, GABAB receptor, and the 5-hydroxytryptamine 2C receptor. The intracellular messengers linked to these GPCRs, including the calcium that is released from internal stores, also play a role in ethanol-enhanced GABA release. Hypotheses are proposed to explain how ethanol interacts with the GPCR pathways to increase GABA release and how this interaction contributes to the brain region specificity of ethanol-enhanced GABA release. Defining the mechanism of ethanol-facilitated GABA release will further our understanding of the GABAergic profile of ethanol and increase our knowledge of how GABAergic neurotransmission may contribute to the intoxicating effects of alcohol and to alcohol dependence. Research Highlights ► Ethanol facilitates GABA release in some, but not all, brain regions. ► Different GPCRs regulate ethanol-enhanced GABA release. ► Intracellular messengers alter the ability of ethanol to increase GABA release.

Ethanol increases miniature inhibitory postsynaptic current frequency and decreases the paired-pulse ratio, which suggests that ethanol increases both spontaneous and evoked GABA release, respectively. We have shown previously that ethanol increases GABA release at the rat interneuron–Purkinje cell synapse and that this ethanol effect involves calcium release from internal stores; however, further exploration of the mechanism responsible for ethanol-enhanced GABA release was needed. We found that a cannabinoid receptor 1 (CB1) agonist, WIN-55212, and a GABABreceptor agonist, baclofen, decreased baseline spontaneous GABA release and prevented ethanol from increasing spontaneous GABA release. The CB1 receptor and GABABreceptor are Gα i–linked G protein–coupled receptors with common downstream messengers that include adenylate cyclase and protein kinase A (PKA). Adenylate cyclase and PKA antagonists blocked ethanol from increasing spontaneous GABA release, whereas a PKA antagonist limited to the postsynaptic neuron did not block ethanol from increasing spontaneous GABA release. These results suggest that presynaptic PKA plays an essential role in ethanol-enhanced spontaneous GABA release. Similar to ethanol, we found that the mechanism of the cannabinoid-mediated decrease in spontaneous GABA release involves internal calcium stores and PKA. A PKA antagonist decreased baseline spontaneous GABA release. This effect was reduced after incubating the slice with a calcium chelator, BAPTA-AM, but was unaffected when BAPTA was limited to the postsynaptic neuron. This suggests that the PKA antagonist is acting through a presynaptic, calcium-dependent mechanism to decrease spontaneous GABA release. Overall, these results suggest that PKA activation is necessary for ethanol to increase spontaneous GABA release.

Whereas ethanol has behavioral actions consistent with increased GABAergic function, attempts to demonstrate a direct enhancement of GABA-gated currents by ethanol have produced mixed results. Recent work has suggested that a part of the GABAergic profile of ethanol may result from enhanced GABA release from presynaptic terminals. The present study examines the effect of ethanol on GABA release in several brain regions to assess the regional nature of ethanol-induced GABA release. Whole-cell voltage clamp recording of spontaneous inhibitory postsynaptic currents (sIPSCs) from mechanically dissociated neurons and miniature inhibitory postsynaptic currents (mIPSCs) and paired-pulse ratio (PPR) from a slice preparation were used to quantify GABA release. Ethanol produced a concentration-dependent increase in the frequency of sIPSCs recorded from mechanically dissociated cerebellar Purkinje neurons and mIPSCs from substantia nigra neurons without having an effect on sIPSCs recorded from lateral septal or cerebrocortical neurons. This regional difference in the effect of ethanol on GABA release was confirmed with PPR recording from brain slices. These data indicate that ethanol can act on presynaptic terminals to increase GABA release in some brain regions while having little or no effect on GABA release in others. This regional difference is consistent with earlier in vivo studies in which ethanol affected neural activity and sensitivity to GABA in some, but not all, brain sites.

Recent data have demonstrated that ethanol increases gamma-aminobutyric acid (GABA) release in many brain regions, but little is known about the mechanism responsible for this action. Consistent with previous results, ethanol increased miniature inhibitory postsynaptic current (mIPSC) frequency at the interneuron-Purkinje cell synapse in the slice and in mechanically dissociated neurons. These data suggest that ethanol is increasing spontaneous GABA release at this synapse. It is generally accepted that ethanol increases levels of intracellular calcium and that changes in intracellular calcium can alter neurotransmitter release. Therefore, we examined the contribution of calcium-dependent pathways to the effect of ethanol on spontaneous GABA release at the interneuron-Purkinje cell synapse. Ethanol continued to increase mIPSC frequency in a nominally calcium-free extracellular solution and in the presence of a voltage-dependent calcium channel inhibitor, cadmium chloride. These data suggest that influx of extracellular calcium does not play a critical role in the mechanism of ethanol-enhanced spontaneous GABA release. However, a sarco/endoplasmic-reticulum calcium ATPase pump inhibitor (thapsigargin), an inositol 1,4,5-trisphosphate receptor antagonist (2-aminoethoxydiphenylborate) and a ryanodine receptor antagonist (ryanodine) significantly reduced the ability of ethanol to increase mIPSC frequency. In addition, ethanol was still able to increase mIPSC frequency in the presence of intracellular 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and a cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM-251); thus, retrograde messengers are not involved in ethanol-enhanced spontaneous GABA release. Overall, these data suggest that calcium release from presynaptic internal stores plays a vital role in the mechanism of ethanol-enhanced spontaneous GABA release at the interneuron-Purkinje cell synapse.