CHRONIC ALCOHOL ON CHOLINERGIC SIGNALING PATHWAY AND NOS

该项目旨在启动两国之间的研究合作 PI和外国调查员的实验室。PI的父级 该项目将重点放在聚磷脂酰肌醇(Poly-PI)信号传递上 慢性饮酒后神经元损伤的通路。这个 合作者的实验室积极研究由脑损伤引起的 通过一氧化氮合酶(NOS)。该项目的目标是测试 慢性酒精摄入改变毒鼠强的假说 胆碱能受体介导的Poly-PI信号活性与一氧化氮合酶 大脑的活动。该项目将使用大脑切片 准备工作。 第一个目标是检查神经一氧化氮合酶活性的影响，其 C57BL/6J小鼠不同脑区蛋白质和mRNA的表达及其相互关系 这些变化对IP3水平、IP3R结合和IP3R mRNA的影响相同 大脑区域。此外，还将检查nNOS活性的变化 在酒精戒断后的不同时间。这些研究是专门设计的 提供目前缺乏的关于 慢性酒精对大脑中一氧化氮合酶活性的影响。老鼠将被给予一种 进行为期数周的酒精饮食，并测试其发育情况 IP注射后睡眠时间和翻正反射的耐受性 乙醇(这是与华沙的W.Kostowski教授合作)。 小鼠将被安乐死，脑组织(包括 胆碱能途径被很好地描述)将被评估 NNOS活性、蛋白(Western印迹)和mRNA(原位)的测定 杂交)；IP3水平、结合、蛋白质和mRNA。这些 参数将在酒精耐受小鼠中进行评估，并在4、8和 停药后24小时。合并后的实验室拥有专业知识 以及所有程序的资源。 第二个目的是检验nNOS改变的假设 长期服用乙醇的活动与毒扁豆碱有关 胆碱能受体介导的Poly-PI信号通路。上一首 研究人员的研究表明，慢性乙醇会影响 小鼠大脑皮层和海马区的Poly-PI信号，提示 胆碱能毒碱能神经元中的nNOS活性(很好地代表了 在大脑皮层)可能受到影响。胆碱能激动剂的能力 将对刺激一氧化氮合酶活性进行研究，这些发现扩展到 慢性乙醇染毒小鼠脑片的研究 停药后的不同时间。

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.