ANALYSIS OF DOPAMINE NERVE TERMINALS BY FLOW CYTOMETRY

流式细胞术分析多巴胺神经末梢

• 批准号: 3428771
• 负责人: GREGORY KAPATOS
• 金额: $ 3.49万
• 依托单位: SINAI HOSPITAL OF DETROIT
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-03-01 至 1989-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3428771
• 关键词: G protein; corpus striatum; dopamine receptor; flow cytometry; gel electrophoresis; immunochemistry; immunofluorescence technique; laboratory rat; monoclonal antibody; substantia nigra; synaptosomes; tyrosine 3 monooxygenase

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.

多巴胺（DA）神经元一直是人们密切关注的焦点 因为他们可能涉及精神科和 神经系统疾病。 然而，固有的程度 DA 神经元的特性可以表征为有限的 目前大多数实验的细胞复杂性 准备工作。 我们建议开发分析方法和 基于流量分离 DA 神经末梢（突触体） 细胞计数术。 首先，纹状体突触体将被透化 固定以使免疫球蛋白能够渗透。 选择性 DA突触体的标记将通过间接完成 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- 标记的二抗。 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 实验将确定突触体是否被标记和 使用此方法排序丰富了 TH 内容以及是否 黑质纹状体 DA 损伤后标记减少 终端。 制备的 DA 神经末梢纯群体 这种方式将用于二维凝胶的蛋白质分析 电泳以确定离散蛋白质是否 相对于非 DA 终端富集或耗尽。 隔离DA 也可以使用流式细胞仪和/或检查终端 蛋白质印迹技术，用于检测特定蛋白质的存在 已提议参与 DA 监管 活动。 具体来说，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） 终端。 众所周知，G 蛋白在许多方面发挥着偶联作用。 大脑中的受体效应系统，被认为与 DA 相关 受体占据到几秒的产生 使者。 流式细胞仪可以定量测定 特定G蛋白是否与DA神经相关的方式 终端或 DA 终端子群。 这些数据 可以为合理的参与模式提供基础 G 蛋白在突触前 DA 受体的功能中。