ANALYSIS OF DOPAMINE NERVE TERMINALS BY FLOW CYTOMETRY

多巴胺(DA)神经元一直是人们关注的焦点 因为他们可能参与了精神病学和 神经紊乱。然而，与生俱来的 DA神经元的特性可以被限制在 当前可用的大多数实验的细胞复杂性 准备工作。我们建议开发分析和分析方法 基于Flow的DA神经末梢(突触体)的分离 细胞学。首先，纹状体突触体将被 固定以使免疫球蛋白能够穿透。有选择性的 多巴胺突触体的标记将通过间接的方式完成 使用单抗的免疫荧光技术 针对酪氨酸羟基酶(TH)和荧光素- 标记的二抗。DA和非DA神经末梢将 然后用荧光激活细胞分选法进行分离。控制 实验将确定标记的突触体和 使用此方法排序的内容是否丰富，以及是否 黑质纹状体DA损伤后标记减少 终点站。纯种群DA神经末梢的制备 这种方法将用于蛋白质的二维凝胶分析。 用电泳法确定盘状蛋白是否 相对于非DA终端的浓缩或枯竭。隔离DA 也可以使用流式细胞仪和/或 蛋白质印迹技术，用于特定蛋白质的存在 它们已被提议参与DA的调节 活动。具体地说，我们将调查鸟嘌呤的存在 纯化的DA神经中的核苷酸调节蛋白(G蛋白) 终点站。已知G蛋白在许多情况下起偶联作用 大脑中的受体-效应器系统，被认为与DA有关 感受器占领到几秒的产生 信使。流式细胞术可以定量测定 特定G蛋白是否与DA神经相关 终端或DA终端的亚群。这些数据 可以为合理的模型提供参与的基础 G蛋白参与突触前DA受体的功能。

Dopamine (DA) neurons have been the focus of intensive interest because of their possible involvement in psychiatric and neurological disorders. However, the extent to which the inherent properties of DA neurons can be characterized is limited by the cellular complexity of most currently available experimental preparations. We propose to develop methods for the analysis and isolation of DA nerve terminals (synaptosomes) based upon flow cytometry. First, striatal synaptosomes will be permeabilized by fixation to enable penetration by immunoglobulins. Selective labeling of DA synaptosomes will be accomplished by indirect immunofluorescence techniques usng a monoclonal antibody directed against tyrosine hydroxylase (TH) and a fluorescein- labeled secondary antibody. DA and non-DA nerve terminals will then be separated by fluorescence-activated cell sorting. Control experiments will determine whether synaptosomes labeled and sorted using this method are enriched for TH content and whether labeling is decreased in response to lesions of nigrostriatal DA terminals. Pure populations of DA nerve terminals prepared in this manner will be used for protein analysis by 2 dimensional gel electrophoresis to determine whether discete proteins are enriched or depleted relative to non-DA terminals. Isolated DA terminals can also be examined, using flow cytometric and/or Western blot techniques, for the presence of particular proteins which have been proposed to be involved in the regulation of DA activity. Specifically, we will investigate the presence of guanine nucleotide regulatory proteins (G proteins) in purified DA nerve terminals. G proteins are known to play a coupling role in many receptor-effector systems in brain, and are thought to link DA receptor occupation to the generation of several second messengers. Flow cytometry can determine in a quantitative manner whether specific G proteins are associated with DA nerve terminals or with subpopulations of DA terminals. These data could provide the basis for rational models of the involvement of G proteins in the function of presynaptic DA receptors.