Delgado-Peraza Francheska, Nogueras-Ortiz Carlos, Acevedo Canabal Agnes M, Roman-Vendrell Cristina, Yudowski Guillermo A
Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus, San Juan, PR, USA; Department of Anatomy and Neurobiology, School of Medicine, University of Puerto Rico, San Juan, PR, USA.
Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus, San Juan, PR, USA.
Methods Cell Biol. 2016;132:25-33. doi: 10.1016/bs.mcb.2015.10.002. Epub 2015 Dec 24.
Total internal reflection fluorescence (TIRF) microscopy allows probing the cellular events occurring close and at the plasma membrane. Over the last decade, we have seen a significant increase in the number of publications applying TIRF microscopy to unravel some of the fundamental biological questions regarding G protein-coupled receptors (GPCRs) function such as the mechanisms controlling receptor trafficking, quaternary structure, and signaling among others. Most of the published work has been performed in heterologous systems such as HEK293 and CHO cells, where the imaging surface available is higher and smoother when compared with the narrow processes or the smaller cell bodies of neurons. However, some publications have expanded our understanding of these events to primary cell cultures, mostly rat hippocampal and striatal neuronal cultures. Results from these cells provide a bona fide model of the complex events controlling GPCR function in living cells. We believe more work needs to be performed in primary cultures and eventually in intact tissue to complement the knowledge obtained from heterologous cell models. Here, we described a step-by-step protocol to investigate the surface trafficking and signaling from GPCRs in rat hippocampal and striatal primary cultures.
全内反射荧光（TIRF）显微镜技术能够探测发生在细胞膜附近及细胞膜上的细胞活动。在过去十年中，我们看到应用TIRF显微镜技术来阐明一些关于G蛋白偶联受体（GPCRs）功能的基本生物学问题（如控制受体运输、四级结构和信号传导等机制）的出版物数量显著增加。大多数已发表的研究工作是在异源系统（如HEK293和CHO细胞）中进行的，与神经元狭窄的突起或较小的细胞体相比，这些系统中可用的成像表面更大且更平滑。然而，一些出版物已将我们对这些事件的理解扩展到原代细胞培养，主要是大鼠海马和纹状体神经元培养。来自这些细胞的结果提供了一个真实的模型，用于研究活细胞中控制GPCR功能的复杂事件。我们认为，需要在原代培养物中并最终在完整组织中开展更多工作，以补充从异源细胞模型中获得的知识。在此，我们描述了一个逐步的实验方案，用于研究大鼠海马和纹状体原代培养物中GPCRs的表面运输和信号传导。
