Cholinergic Signaling in the Brain

描述(由申请人提供)：中枢胆碱能通路在各种认知行为中的作用及其在神经退行性疾病中的作用已有很好的文献记载。它还被认为尼古丁对大脑的作用涉及破坏内源性胆碱能信号。尽管这一信号通路很重要，但在细胞水平上对胆碱能传递的特性几乎一无所知。这项建议将建立一个模型系统来研究胆碱能传递，并阐明尼古丁在大脑中传递的一些基本特性。将产生在胆碱乙酰转移酶启动子(ChAT)控制下表达GFP或GFP-tau蛋白的转基因小鼠。转基因针对大脑中胆碱能神经元的选择性靶点将通过使用抗ChAT和GFP抗体的免疫细胞化学技术来确定。使用ChAT GFP-tau小鼠，将通过使用贴片电极刺激隔区-海马片中的胆碱能纤维来检测在海马放射层的中间神经元上诱发的nAChR反应。将检测乙酰胆碱酯酶对nAChR反应的影响，以表征递质扩散及其作用于非突触受体的能力。数量的内容，发布的概率，和网站的数量将确定。利用海马区CA3区锥体细胞的全细胞电压钳记录，研究突触释放的乙酰胆碱对苔藓纤维终末释放谷氨酸的调节作用。直接监测从突触前突触释放ACh将使用苯乙酰染料和活细胞成像从急性切片以及从培养的隔区神经元。这些研究将是对中枢神经系统胆碱能传递的第一次详细检查，也将为研究人员研究胆碱能过程提供宝贵的工具。该提案还将开始研究中枢胆碱能系统在调节尼古丁成瘾中的作用。

DESCRIPTION (provided by applicant): The role of central cholinergic pathways in a variety of cognitive behaviors and its role in neurodegenerative diseases has been well documented. It is also recognized nicotine's actions on the brain involves disruption of endogenous cholinergic signaling. In spite of the importance of this signaling pathway, almost nothing is known about the properties of cholinergic transmission at a cellular level. This proposal will establish a model system for the examination of cholinergic transmission and elucidade some fundamental properties of nicotinic transmission in the brain. Transgenic mice expressing GFP or GFP-tau protein under the control of the choline acetyltransferase promoter (ChAT) will be generated. The selective targetting of the transgene to cholinergic neurons in the brain will be determined by immunocytochemical techniques using antibodies against ChAT and GFP. Using the ChAT GFP-tau mice, evoked nAChR responses on interneurons at the stratum radiatum of the hippocampus will be examined by stimulating cholinergic fibers in septo-hippocampal slices using a patch electrode. The effects of acetylcholine esterases on nAChR responses will be examined in order to characterize transmitter diffusion and its ability to act on non-synaptic receptors. Quantal content, probability of release, and number of sites will be determined. Using whole cell voltage-clamp recordings from CA3 pyramidal cells of the hippocampus, the modulation of glutamate release from mossy fiber terminals by synaptically released acetylcholine will be examined. Direct monitoring of ACh release from presynaptic boutons will be monitored using styryl dyes and live cell imaging from acute slices as well as from septal neurons in culture. These studies will be the first detailed examination of cholinergic transmission in the central nervous system and will also provide an invaluable tool to researchers for studying cholinergic processes. The proposal will also begin examining the role of central cholinergic system in mediating nicotine addiction.