CHRONIC ALCOHOL ON CHOLINERGIC SIGNALING PATHWAY AND NOS

该项目旨在发起一项研究合作， PI和外国研究者的实验室。 PI的父母 项目重点关注聚磷酸肌醇（poly-PI）信号传导 慢性饮酒后神经元损伤的通路。 的 合作者的实验室积极研究脑损伤介导的 一氧化氮合酶（NOS）。本项目的目标是测试 假设慢性乙醇消耗改变了毒蕈碱 胆碱能受体介导的多聚PI信号活性和NOS 大脑中的活动。 该项目将使用大脑切片 准备. 第一个目的是检查神经NOS活性的影响， C57 BL/6 J小鼠不同脑区蛋白质和mRNA的表达及其相关性 这些变化的IP 3水平，IP 3R结合，和IP 3R mRNA在相同的 大脑区域。 此外，将检查nNOS活性的变化 在乙醇提取后的不同时间。 这些研究旨在 提供目前缺乏的关于影响的基本信息， 慢性乙醇对脑内NOS活性的影响。 小鼠将被给予 乙醇饮食的一段时间的几个星期，并测试的发展， 耐受性使用睡眠时间和翻正反射后ip注射 乙醇（这是与W。Kostowski in Warsaw）. 将对小鼠实施安乐死，并将脑组织（包括其中 胆碱能途径得到很好的代表）将进行评价， 测定nNOS活性、蛋白质（Western印迹）和mRNA（原位 杂交）; IP 3水平，结合，蛋白质和mRNA。 这些 参数将在乙醇耐受小鼠中进行评价，并在4、8和 退出后24小时。 联合实验室拥有专业知识 所有程序的资源。 第二个目的是验证nNOS的改变 慢性乙醇给药后的活性与毒蕈碱 胆碱能受体介导的多聚PI信号通路。 先前 研究人员的研究表明，慢性乙醇影响 小鼠大脑皮层和海马中的多聚PI信号，表明 胆碱能毒蕈碱神经元中的nNOS活性（很好地代表 在皮质）可能受到影响。 胆碱能激动剂的能力， 将研究刺激NOS活性，这些发现扩展到 研究小鼠脑切片暴露于慢性乙醇， 不同的时间后退出。

The project is aimed at initiating a research collaboration between the laboratories of the PI and foreign investigator. The PI's parent project places emphasis on the poly-phosphoinositide (poly-PI) signaling pathway in neuronal damage after chronic alcohol consumption. The collaborator's laboratory is active in studying cerebral injury mediated by nitric oxide synthase (NOS). The goal for this project is to test the hypothesis that chronic ethanol consumption alters the muscarinic cholinergic receptor-mediated poly-PI signaling activity and NOS activity in the brain. The project will use the brain slice preparation. The first aim is to examine the effects of neural NOS activity, its protein and mRNA in different brain regions of C57BL/6J mice and relate these changes to IP3 level, IP3R binding, and IP3R mRNA in the same brain regions. In addition, changes in nNOS activity will be examined at different times after ethanol withdrawal. These studies are designed to provide basic information, lacking at present, about the effect of chronic ethanol on NOS activity in the brain. Mice will be given an ethanol diet for a period of weeks and tested for the development of tolerance using sleep-time and righting reflex after ip injection of ethanol (this in collaboration with Professor W. Kostowski in Warsaw). Mice will be euthanized and brain tissue (including areas where cholinergic pathways are well represented) will be evaluated for determination of nNOS activity, protein (Western blot) and mRNA (in situ hybridization); IP3 levels, binding, proteins and mRNA. These parameters will be evaluated in ethanol tolerant mice and at 4, 8, and 24 hours after withdrawal. The combined laboratories have the expertise and resources for all procedures. The second aim is to test the hypothesis that alteration of nNOS activity upon chronic ethanol administration is linked to the muscarinic cholinergic receptor-mediated poly-PI signaling pathway. Previous studies by the investigators have shown that chronic ethanol affects poly-PI signaling in mouse cerebral cortex and hippocampus, suggesting that nNOS activity in cholinergic muscarinic neurons (well represented in cortex) may be affected. The ability of cholinergic agonists to stimulate NOS activity will be studied and these findings extended to the study of brain slices in mice exposed to chronic ethanol at different times after withdrawal.