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. [unreadable] [unreadable] [unreadable] [unreadable]