Intracellular Study of Ethanol Effects on Brain Neurons

腹侧被盖区(VTA)中的多巴胺(DA)能神经元为核内的DA神经支配提供神经 这种中脑边缘DA通路对于乙醇的奖赏特性很重要。乙醇直接刺激 在没有周围细胞输入的情况下，DA VTA神经元急剧分离。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 对AHP有贡献的钾(K)电流减少。乙醇的兴奋被完全阻断 奎尼丁，但不受其他钾通道阻滞剂阿帕明、四乙基铵、钡或铯的影响。在整个电池电压 在钳制实验中，乙醇降低了由保持的去极化电压阶跃引起的持续外向电流 电位为-40 mV(以使A电流失活)。这些数据表明，乙醇通过使多巴胺VTA神经元兴奋 延迟整流型的非失活或缓慢失活的奎尼丁敏感的K电流。《公约》的具体目标 目前的应用有两个方面。1)该乙醇的电生理和药理特性- 多巴胺VTA神经元的敏感性钾电流及乙醇兴奋的截止和降低 较长链长的醇类对乙醇敏感的K电流。2)分子生物学研究，试图识别原住民 乙醇敏感性钾电流是通过克隆的已知结构的钾通道实现的。已选择候选频道 根据它们与天然通道的电生理和药理相似性。汇聚神经元的RT-PCR检测 单细胞RT-PCR将被用来确定在DA VTA神经元中表达哪些候选通道mRNAs。 然后，将使用免疫组织化学来查看在胞体和树突上实际存在哪些通道蛋白 多巴胺VTA神经元。最后，最有可能的候选通道将在非洲爪哇卵母细胞中表达， 将确定这些K电流的较长链醇的电生理特性和截止值并进行比较 对多巴胺VTA神经元的天然乙醇敏感性钾电流的特性有影响。这些数据应该会给出重要的 关于乙醇直接兴奋DA VTA感觉神经元的机制的信息。总而言之， 电生理和分子生物学数据应有助于识别DA上对乙醇敏感的天然通道 VTA奖励神经元，因此指向一个负责其表达的基因。这样的发现可能会对 了解乙醇对中脑边缘奖赏通路的遗传差异及其变化的意义 在DA的反应中，VTA神经元在长期饮酒时会导致酒精渴求和成瘾。

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.