Dissection of Ethanol Actions on Cholinergic Nerve Endings

描述(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。对于这一应用，我们将研究乙醇对乙酰胆碱分泌的影响。乙醇对中枢神经系统中乙酰胆碱的释放具有浓度依赖性的双相效应。初步实验显示，乙醇对更容易通过实验获得的哺乳动物突触--骨骼神经肌肉接头--的乙酰胆碱释放具有非常相似的两相浓度依赖效应。哺乳动物的运动神经末梢对酒精非常敏感，在低毫摩尔浓度的乙醇下，神经递质释放增加。总体目标有两个具体目标如下：特定目标1：确定乙醇影响神经递质乙酰胆碱(Ach)释放的全局神经末梢靶点。我们的初步数据显示，乙醇的一些影响是由于直接作用于分泌机器。对于诱发的Ach释放，我们将探讨乙醇对Ach释放的影响是由于作用于突触前离子通道还是作用于膜离子通道下游的神经递质释放机制。为此，我们将对突触前离子电流和诱发的神经递质释放进行同步电生理测量。特异性目的2：观察乙醇对突触前分泌器官相关蛋白改变的影响。对于缺失研究，起点将是Rab3A基因敲除小鼠。初步实验表明，在Rab3A基因缺失后，Ach的自发释放对乙醇的敏感性增加；这种影响发生在膜离子通道的下游。在切割研究中，我们将使用肉毒杆菌毒素的不同部分来切割特定残基上的分泌机构的战略核心蛋白，并确定乙醇是否影响不依赖于被切割的蛋白而引起的Ach释放。我们用肉毒杆菌毒素的初步数据表明，突触小泡蛋白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.