Intracellular Study of Ethanol Effects on Brain Neurons

腹侧被盖区（VTA）的多巴胺能（DA）神经元提供对该核的DA神经支配 这种中脑边缘DA通路对于乙醇的奖励性质很重要。乙醇直接激发 急性分离的DA VTA神经元，在没有来自周围细胞的输入的情况下。DA VTA的乙醇激发 神经元与动作电位后超极化（AHP）的减少有关，这表明这是由于 降低钾（K）电流，这有助于AHP。乙醇激发被完全阻断， 奎尼丁，但不被其他K通道阻滞剂apamin，四乙铵，钡或铯。全电池电压 钳夹实验中，乙醇可降低去极化电压阶跃诱发的持续外向电流 电位为-40 mV（至mA电流）。这些数据表明，乙醇兴奋DA VTA神经元，通过减少 延迟整流型非失活或缓慢失活、奎尼丁敏感的K电流。的具体目标 本申请是双重的。1)这种乙醇的电生理学和药理学特性- DA VTA神经元中敏感的K电流，以及确定乙醇兴奋和减少 乙醇敏感性钾电流由较长链长的醇。2)通过分子生物学研究试图找出 乙醇敏感性钾电流的克隆已知结构的钾通道。已选择候选通道 根据他们的电生理学和药理学的相似性，以天然通道。混合神经元的RT-PCR 单细胞RT-PCR将用于确定哪些候选通道mRNA在DA VTA神经元中表达。 然后，我们将用荧光化学方法来观察哪些通道蛋白实际上存在于索马和树突上 DA VTA神经元最后，最可能的候选通道将在非洲爪蟾卵母细胞中表达， 将确定这些K电流的电生理学特性和较长链醇的截止值，并进行比较 DA VTA神经元中天然乙醇敏感性K电流的性质。这些数据将提供重要的 乙醇直接兴奋DA VTA奖励神经元的机制的信息。总的来说， 电生理学和分子生物学数据应有助于确定DA乙醇敏感的天然通道 腹侧被盖区奖励神经元，因此指向负责其表达的基因。这样的发现可能会对 理解乙醇对中脑边缘奖赏通路的影响的遗传差异及其变化 在慢性酒精消耗过程中，DA VTA神经元的反应导致酒精渴望和成瘾。

Dopaminergic (DA) neurons in the ventral tegmental area (VTA) provide the DA innervation of the nucleus accumbens; this mesolimbic DA pathway is important for the rewarding properties of ethanol. Ethanol directly excites acutely dissociated DA VTA neurons, in the absence of input from surrounding cells. Ethanol excitation of DA VTA neurons is associated with a reduction in the action potential afterhyperpolarization (AHP) suggesting that it is due to a decrease in a potassium (K) current which contributes to the AHP. Ethanol excitation is completely blocked by quinidine, but not by the other K channel blockers apamin, tetraethylammonium, barium or cesium. In whole cell voltage clamp experiments, ethanol reduced the sustained outward current evoked by depolarizing voltage steps from aholding potential of-40 mV (to inactivate A-current). These data suggest that ethanol excites DA VTA neurons by reducing a non- or slowly inactivating, quinidine-sensitive, K current of the delayed rectifier type. The specific aims of the present application are two-fold. 1) Electrophysiological and pharmacological characterization of this ethanol- sensitive K current in DA VTA neurons, and determination of the cutoff for ethanol excitation and for reduction of the ethanol-sensitive K current by longer chain length alcohols. 2) Molecular biological studies to try to identify the native ethanol-sensitive K current in terms of cloned K channels of known structure. Candidate channels have been selected according to their electrophysiological and pharmacological similarityto the native channel. RT-PCR on pooled neurons and single cell RT-PCR will be used to determine which candidate channel mRNAs are expressed in DA VTA neurons. Then immunohistochemistrywill be used to see which channel proteins are actually present on the soma and dendrites of DA VTA neurons. Finally, the most likely candidate channels will be expressed in Xenopus oocytes and the electrophysiological properties and cutoff for longer chain alcohols will be determined for these K currents and compared to the properties of the native ethanol-sensitive K current in DA VTA neurons. These data should give important information on the mechanism by which ethanol directly excites DA VTA reward neurons. Taken together, the electrophysiological and molecular biological data should help to identify the ethanol-sensitive native channel on DA VTA reward neurons and therefore point to a gene responsible for its expression. Such a discovery could have major implications for understanding genetic differences in ethanol effects on the mesolimbic reward pathway and how changes in the response of DA VTA neurons during chronic ethanol consumption leads to alcohol craving and addiction.