Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599-7365, USA.
Neuropharmacology. 2010 Jun;58(7):1179-86. doi: 10.1016/j.neuropharm.2010.02.018. Epub 2010 Mar 4.
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.
乙醇在 GABA 能突触的作用的研究传统上集中于突触后机制,但最近的数据表明,乙醇还增加了许多脑区的诱发和自发 GABA 释放。我们以前使用全细胞膜片钳记录表明,乙醇增加了大鼠中间神经元-浦肯野细胞突触的自发 GABA 释放。这种突触前乙醇效应依赖于内质网钙释放,可能通过激活肌醇 1,4,5-三磷酸受体 (IP3Rs)。在确认乙醇靶向囊泡 GABA 释放后,在本研究中我们使用电子显微镜免疫组织化学证明 IP3Rs 位于小脑中间神经元的突触前末梢。IP3Rs 的激活需要 IP3 的结合,通过激活磷脂酶 C (PLC)产生。我们发现 PLC 拮抗剂埃达福林可防止乙醇增加自发 GABA 释放。PLC 产生的二酰基甘油和 IP3R 激活释放的钙激活蛋白激酶 C (PKC)。两种 PKC 拮抗剂Chelerythrine 和 calphostin C 阻断了乙醇增强的 GABA 释放。当膜不可渗透的 PKC 拮抗剂 PKC(19-36)被递送到突触后神经元的细胞内时,乙醇继续增加自发 GABA 释放。总的来说,这些结果表明 PLC/IP3R/PKC 通路的激活是乙醇增加从突触前末梢到浦肯野细胞的自发 GABA 释放所必需的。