Dissection of Ethanol Actions on Cholinergic Nerve Endings

描述（由申请人提供）：本提案的长期目标是阐明酒精影响神经递质分泌的分子机制。对于这种应用，我们将研究乙醇对乙酰胆碱分泌的影响。乙醇对CNS中乙酰胆碱释放具有双相浓度依赖性作用。初步实验表明，乙醇对乙酰胆碱释放的双相浓度依赖性影响非常相似，在实验上更容易接近哺乳动物突触，骨骼神经肌肉接头。哺乳动物运动神经末梢对酒精非常敏感，在低毫摩尔浓度的乙醇中观察到神经递质释放增加。具体目标1：确定乙醇影响神经递质乙酰胆碱（Ach）释放的总体神经末梢靶点。我们的初步数据表明，乙醇的一些影响是由于直接对分泌机制的行动。对于诱发的乙酰胆碱释放，我们将询问乙醇对乙酰胆碱释放的影响是由于突触前离子通道或膜离子通道下游的神经递质释放机制的作用。为此，我们将同时进行突触前离子电流和诱发神经递质释放的电生理测量。具体目的2：研究乙醇对突触前分泌器相关蛋白改变的影响。对于缺失研究，起点将是Rab3A敲除小鼠。初步实验表明，Rab3A基因删除后，乙醇的自发乙酰胆碱释放的敏感性增加，这种效应发生在膜离子通道的下游。对于切割研究，我们将切割的战略核心蛋白的分泌器在特定的残基使用不同馏分的肉毒杆菌毒素，并确定是否乙酰胆碱释放引起独立的切割的蛋白质受到乙醇的影响。我们对肉毒杆菌毒素的初步研究表明，突触囊泡蛋白synaptobrevin是乙醇作用的重要靶点。

DESCRIPTION (provided by applicant): The LONG TERM OBJECTIVE of this proposal is to elucidate the molecular mechanisms by which alcohol affects neurotransmitter secretion. For this application, we will study the effects of ethanol on the secretion of acetylcholine. Ethanol has biphasic concentration dependent effects on acetylcholine release in the CNS. Preliminary experiments show very similar biphasic concentration dependent effects of ethanol on acetylcholine release at the more experimentally accessible mammalian synapse, the skeletal neuromuscular junction. Mammalian motor nerve endings are very sensitive to alcohols, with increases in neurotransmitter release being observed at low millimolar concentrations of ethanol. There are 2 SPECIFIC AIMS underlying the overall objectives as follows: SPECIFIC AIM 1: To determine the global nerve terminal targets by which ethanol affects the release of the neurotransmitter acetylcholine (Ach). Our preliminary data show that some of the effects of ethanol are due to actions directly on the secretory machinery. For evoked Ach release, we will inquire which of the effects of ethanol on Ach release are due to an action on presynaptic ionic channels or on the neurotransmitter release machinery downstream of membrane ionic channels. To this end, we will make simultaneous electrophysiological measurements of presynaptic ionic currents and evoked neurotransmitter release. SPECIFIC AIM 2: To examine the effect of ethanol after alteration of presynaptic proteins associated with the secretory apparatus. For the deletion studies, the starting point will be the Rab3A knockout mouse. Preliminary experiments reveal an increased sensitivity to ethanol of spontaneous Ach release after deletion of the Rab3A gene; this effect occurs downstream of membrane ionic channels. For the cleavage studies, we will cleave the strategic core proteins of the secretory apparatus at specific residues using various fractions of botulinum toxins, and determine if Ach release elicited independently of the cleaved proteins is affected by ethanol. Our preliminary data with botulinums toxins demonstrate that the synaptic vesicle protein synaptobrevin is an important target for the effects of ethanol.