使用 DAT-pHluorin 对多巴胺转运体的基础表面部分和动态可用性进行活体神经元成像分析的方案。

IF 1.3 Q4 BIOCHEMICAL RESEARCH METHODS

STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-10-04 DOI:10.1016/j.xpro.2024.103358

Elnaz Khezerlou, Jacqueline Saenz, Sanjana Surya Prakash, Ping-Yue Pan

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引用次数: 0

摘要

多巴胺（DA）转运体（DAT）的表面可用性对DA的传递有着至关重要的影响。在此，我们介绍一种方案，该方案描述了小鼠腹侧中脑神经元的制备、新型光学传感器 DAT-pHluorin 的表达，以及利用该传感器通过荧光显微镜分析活体神经元中 DAT 的表面可用性。这种方法可以定量测量相关遗传背景下的基础表面 DAT 部分，以及 DAT 在精神活性物质作用下的活体贩运情况。有关该方案使用和执行的完整细节，请参阅 Saenz 等人的文章1。

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Protocol for live neuron imaging analysis of basal surface fraction and dynamic availability of the dopamine transporter using DAT-pHluorin.

Surface availability of the dopamine (DA) transporter (DAT) critically influences DA transmission. Here, we present a protocol that describes the preparation of mouse ventral midbrain neurons, the expression of a new optical sensor, DAT-pHluorin, and the utilization of this sensor to analyze the surface availability of DAT in live neurons via fluorescent microscopy. This approach allows quantitative measures of basal surface DAT fraction under genetic backgrounds of interest and live trafficking of DAT in response to psychoactive substances. For complete details on the use and execution of this protocol, please refer to Saenz et al.¹.

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