Nigrostriatal co-cultures with dopaminergic neurons carrying a fluorescent tag

项目概要/摘要 多巴胺能（DA）神经元在帕金森病（PD）中退化，并且在滥用成瘾性药物时退化。 兴奋剂、甲基安非他明（METH）和有关安非他明类兴奋剂（ATS）。的 DA神经元变性的分子机制尚不清楚，无论是在PD还是在ATS中 神经毒性为了开发新的治疗方法，防止DA神经变性， 需要更好地理解各种机制。为了研究这些机制，进行了几项体外实验 已经开发了模型。然而，这些模型都没有具有轴突的DA神经元 终末，支配其适当的目标，纹状体神经元。纹状体神经元的存在是 因为这些神经元产生化学吸引线索，不仅指导DA的生长， 而且还促进DA轴突末梢中的多巴胺转运体（DAT）表达。如果 纹状体线索不可用，DA神经元似乎表达DAT不当，在体细胞质 膜的值得注意的是，体细胞DAT表现异常-它在暴露于 METH.这种DAT内化在体内未在支配纹状体的DA轴突终末中观察到。 神经元本课题的目的是建立一种改良的体外培养DA细胞模型， 纹状体神经元将由表达DAT的轴突末梢支配。在该模型中，主要 腹侧中脑（VM）和纹状体神经元的培养物将在同一培养物上邻近铺板。 底物，使得VM培养物中存在的多巴胺能神经元可以支配纹状体 神经元VM和纹状体神经元将从HA-DAT小鼠获得。这些老鼠 插入DAT中的血凝素（HA）标签。HA标签不影响DAT运输和活性 但允许荧光可视化活细胞中的DAT。预期在培养期间， 将促进DAT的表达和活动的VM神经突支配的纹状体神经元 纹状体神经元该项目有两个目标。目标1：培养共同文化。目标2：描述DAT 在共培养物中的表达和活性。一旦开发和验证，拟议的模型将 用于研究DAT参与MET诱导的DA神经毒性的机制， PD中DA细胞变性的机制。

Project Summary/Abstract Dopaminergic (DA) neurons degenerate in Parkinson’s disease (PD) and upon abuse of the addictive stimulant, methamphetamine (METH) and related amphetamine-type stimulants (ATS). The molecular mechanisms involved in DA neuron degeneration are not yet clear, either in PD or ATS neurotoxicity. To develop novel treatments that prevent DA neural degeneration, underlying mechanisms need to be better understood. To study these mechanisms, several in vitro experimental models have been developed. However, none of these models features DA neurons with axon terminals that innervate their proper target, striatal neurons. The presence of striatal neurons is essential because these neurons produce chemo-attractive cues that not only direct the growth of DA axons and but also promote dopamine transporter (DAT) expression in the DA axon terminals. If striatal cues are not available, DA neurons appear to express DAT improperly, in the somatic plasma membrane. Notably, the somatic DAT behaves abnormally – it is internalized upon an exposure to METH. Such DAT internalization is not observed in vivo in the DA axon terminals innervating striatal neurons. The goal of this project is to develop an improved in vitro cultured DA cell model in which striatal neurons will be innervated by DAT-expressing axon terminals. In the proposed model, primary cultures of ventral mesencephalon (VM) and striatal neurons will be plated nearby on the same substrate, such that the dopaminergic neurons present in the VM cultures can innervate the striatal neurons. The VM and striatal neurons will be obtained from HA-DAT mice. These mice have a hemagglutinin (HA) tag inserted into the DAT. The HA tag does not affect DAT trafficking and activity but allows to fluorescently visualize DAT in live cells. It is expected that during culturing, the cues from the striatal neurons will promote DAT expression and activity in the VM neurites innervating the striatal neurons. The project has two aims. Aim 1: Develop the co-cultures. Aim 2: Characterize DAT expression and activity in the co-cultures. Once developed and validated, the proposed model will be used to study the mechanisms underlying DAT involvement in METH-induced DA neurotoxicity and the mechanisms underlying DA cell degeneration in PD.