使用多巴胺转运体pH荧光蛋白对多巴胺转运体基底表面分数和动态可用性进行活神经元成像分析的方案。

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

作者信息

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

机构信息

Department of Neuroscience and Cell Biology, Rutgers University Robert Wood Johnson Medical School, 675 Hoes Lane West, Piscataway, NJ 08854, USA; Rutgers Addiction Research Center, Rutgers Brain Health Institute, 683 Hoes Lane West, Piscataway, NJ 08854, USA.

Department of Neuroscience and Cell Biology, Rutgers University Robert Wood Johnson Medical School, 675 Hoes Lane West, Piscataway, NJ 08854, USA; Rutgers School of Graduate Studies, Molecular Biosciences Graduate Program, 25 Bishop Place, New Brunswick, NJ 08901-1178, USA; Rutgers Addiction Research Center, Rutgers Brain Health Institute, 683 Hoes Lane West, Piscataway, NJ 08854, USA.

出版信息

STAR Protoc. 2024 Dec 20;5(4):103358. doi: 10.1016/j.xpro.2024.103358. Epub 2024 Oct 4.

DOI:10.1016/j.xpro.2024.103358

PMID:39368094

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11490699/

Abstract

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..

摘要

多巴胺（DA）转运体（DAT）的表面可用性对DA传递有至关重要的影响。在此，我们展示了一种实验方案，该方案描述了小鼠腹侧中脑神经元的制备、一种新型光学传感器DAT-pHluorin的表达，以及利用该传感器通过荧光显微镜分析活神经元中DAT的表面可用性。这种方法能够在感兴趣的基因背景下对基础表面DAT比例进行定量测量，并检测DAT在响应精神活性物质时的实时转运情况。有关此实验方案使用和执行的完整详细信息，请参考Saenz等人的研究。