Dissection of Ethanol Actions on Cholinergic Nerve Endings

乙醇对胆碱能神经末梢作用的剖析

基本信息

批准号：
7465500
负责人：
Timothy Searl
金额：
$ 17.93万
依托单位：
NORTHWESTERN UNIVERSITY AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-07-10 至 2010-06-30
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：该提案的长期目标是阐明酒精会影响神经递质分泌的分子机制。对于此应用，我们将研究乙醇对乙酰胆碱分泌的影响。乙醇对CNS中乙酰胆碱释放的双相浓度依赖性影响。初步实验显示，在更实验可访问的哺乳动物突触（骨骼神经肌肉连接）上，乙醇对乙醇对乙醇释放的双相浓度依赖性效应非常相似。哺乳动物的运动神经末端对醇非常敏感，在低毫米浓度的乙醇中观察到神经递质释放的增加。总体目标的基础有2个特定目标：特定目的1：确定乙醇影响神经递质乙酰乙酰胆碱（ACH）的释放的全球神经末端目标。我们的初步数据表明，乙醇的某些影响是由于直接对分泌机械的作用引起的。对于诱发的ACH释放，我们将询问乙醇对ACH释放的哪些影响是由于对突触前离子通道的作用或对膜离子离子通道下游的神经递质释放机械的作用。为此，我们将对突触前离子电流的同时进行电生理测量，并唤起神经递质释放。具体目的2：检查与分泌仪相关的突触前蛋白改变后乙醇的作用。对于删除研究，起点将是Rab3a敲除鼠标。初步实验表明，Rab3a基因缺失后，对自发ACH释放的乙醇的敏感性提高。这种影响发生在膜离子通道的下游。对于裂解研究，我们将使用肉毒杆菌毒素的各种馏分在特定残基上切割分泌仪的策略核心蛋白，并确定是否独立于切割蛋白的ACh释放受到乙醇的影响。我们使用肉毒杆菌毒素的初步数据表明，突触囊泡蛋白突触纤维蛋白是乙醇作用的重要靶标。