ANALYSIS OF DOPAMINE NERVE TERMINALS BY FLOW CYTOMETRY

多巴胺（DA）神经元是近年来研究的热点 因为他们可能参与精神病治疗， 神经系统疾病 然而，内在的程度 DA神经元的特性可以被表征是有限的， 细胞复杂性的最目前可用的实验 准备工作 我们建议开发分析方法， 基于流动的DA神经末梢（突触体）的分离 细胞仪 首先，纹状体突触体将被 固定以使免疫球蛋白能够穿透。 选择性 DA突触体的标记将通过间接的 使用单克隆抗体的免疫荧光技术 针对酪氨酸羟化酶（TH）和荧光素- 标记的第二抗体。 DA和非DA神经末梢将 然后通过荧光激活细胞分选进行分离。 控制 实验将确定是否标记的突触体， 使用该方法分选的样品富集TH含量， 黑质纹状体DA损伤后标记减少 terminals. 制备的DA神经末梢的纯群体， 这种方式将用于通过二维凝胶进行蛋白质分析 电泳，以确定是否discete蛋白质， 相对于非DA末端富集或耗尽。 孤立DA 还可以使用流式细胞术和/或 蛋白质印迹技术，用于特定蛋白质的存在 建议参与DA的监管 活动 具体来说，我们将研究鸟嘌呤的存在 纯化的DA神经中的核苷酸调节蛋白（G蛋白） terminals. 已知G蛋白在许多细胞中起偶联作用。 大脑中的受体效应器系统，并被认为是连接DA 受体占据数秒 使者 流式细胞术可以定量测定 特异性G蛋白是否与DA神经相关 终末或DA终末亚群。 这些数据 可以为参与的合理模式提供基础， G蛋白在突触前DA受体功能中的作用

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.