MOLECULAR AND CELLULAR NEUROPHYSIOLOGY FOR MENTAL HEALTH

本研究建议的目的是研究监管 大脑神经元的机制，涉及主要的精神 紊乱 建议的实验将主要通过使用 原代培养黑质多巴胺能神经元， 大鼠大脑的腹侧被盖区。 方法将是 电生理技术结合分子生物学 方法. 第一个项目是关于这些DA的激发 神经元 神经降压素产生DA神经元的缓慢兴奋， 激活非选择性阳离子通道。 离子分析 机制表明，这种非选择性通道不同于 众所周知的非选择性离子通道，例如CAN通道（ 钙激活的非特异性阳离子通道）或环 核苷酸门控通道 拟议中的实验将阐明 神经降压素激活的信号转导机制 这个非选择性的频道。G蛋白的作用将是 严格评估，G蛋白的身份将是 决定。 另一个项目涉及DA的缓慢抑制 多巴胺本身的神经元。 这种自我抑制是由一种 G蛋白β-γ激活内向整流钾通道， 亚单位。 该项目将调查的机制， β-γ亚单位和内向整流分子的相互作用。 本文旨在寻找一种简便的原代培养方法 DA神经元与质粒cDNA。 然后是DA神经元（也是HEK 293细胞） 将用编码G蛋白β亚基的CDNA转染， γ亚基 β基因定点突变的影响 将对子单元进行评估。 调查将显示哪些地区 β-亚基分子负责与 和激活内向整流分子。

The objective of this research proposal is to study the regulatory mechanisms of brain neurons that are implicated in major mental disorders. The proposed experiments will be done mainly by using primary cultured dopaminergic (DA) neurons from the substantia nigra and the ventral tegmental area of the rat brain. The methods will be electrophysiological techniques combined with molecular biological methods. The first project is concerned with excitation of these DA neurons. Neurotensin produces a slow excitation of DA neurons by activating a non-selective cation channel. The analysis of the ionic mechanism has revealed that this non-selective channel is different from the well-known non-selective ion channels such as the CAN channels (the calcium-activated non-specific cation channels) or the cyclic nucleotide-gated channels. The proposed experiments will shed light on the signal transduction mechanisms of the neurotensin-induced activation of this non-selective channel. The role played by G proteins will be critically evaluated, and the identity of the G protein will be determine. The other project deals with a slow inhibition of the DA neurons by dopamine itself. This self-inhibition results from an activation of an inward rectifier K+ channel by the G protein betagamma- subunits. The project will investigate the mechanisms of the interaction of the betagamma-subunits and the inward rectifier molecule. It is proposed to find a convenient way to transfect primary cultured DA neurons with plasmid cDNA. Then the DA neurons (also HEK293 cells) will be transfected with CDNAs encoding the G protein beta-subunit and the gamma-subunit. The effects of site-directed mutations of the beta- subunit will be evaluated. The investigation will show which regions of the beta-subunit molecule are responsible for the association with and activation of the inward rectifier molecule.