Intracellular Study of Ethanol Effects on Brain Neurons

超出所提供的空间。腹侧被盖区（VTA）的多巴胺能（DA）神经元提供伏隔核的DA神经支配；这条中脑边缘DA通路对乙醇的奖赏特性很重要。在没有周围细胞输入的情况下，乙醇直接刺激急性解离的DA - VTA神经元。乙醇刺激的DA - VTA神经元与超极化后动作电位（AHP）的减少有关，这表明这是由于钾(K)电流的减少导致了AHP。乙醇的激发完全被奎尼丁阻断，但不被其他K通道阻滞剂apamin， tetra乙基铵，钡或铯阻断。在全细胞电压箝位实验中，乙醇降低了由去极化电压步骤引起的持续向外电流，从保持电位40 mV（灭活a电流）。这些数据表明，乙醇通过减少非或缓慢失活的、奎尼丁敏感的延迟整流型K电流来刺激DA - VTA神经元。本应用程序的具体目的有两个方面。1) DA - VTA神经元中乙醇敏感K电流的电生理和药理学特征，以及乙醇兴奋的截止点和较长链醇对乙醇敏感K电流的还原的确定。2)通过分子生物学研究，尝试从已知结构的克隆K通道中鉴定天然乙醇敏感K电流。候选通道是根据它们与天然通道的电生理和药理相似性来选择的。聚合神经元RT-PCR和单细胞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，免疫组织化学将被用来观察哪些通道蛋白实际上存在于DA - VTA神经元的体细胞和树突上。最后，最可能的候选通道将在爪蟾卵母细胞中表达，并将确定这些K电流的电生理特性和长链醇的切断，并将其与DA VTA神经元中天然乙醇敏感K电流的特性进行比较。这些数据应该为乙醇直接刺激DA - VTA奖励神经元的机制提供重要信息。综上所述，电生理学和分子生物学数据应该有助于识别DA - VTA奖励神经元上乙醇敏感的天然通道，从而指出负责其表达的基因。这一发现可能对理解乙醇对中脑边缘奖赏通路影响的遗传差异，以及慢性酒精摄入过程中DA - VTA神经元的反应变化如何导致酒精渴望和成瘾具有重要意义。网站性能 ======================================== 节结束 ===========================================

EXCEED THE SPACE PROVIDED. 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. PERFORMANCE SITE ========================================Section End===========================================