Dept. of Biomedical Engineering, Center for Systems Neuroscience, Neurophotonics Center, Boston University, Boston, MA, 02215, United States.
Dept. of Biomedical Engineering, Center for Systems Neuroscience, Neurophotonics Center, Boston University, Boston, MA, 02215, United States.
J Neurosci Methods. 2021 Mar 1;351:109064. doi: 10.1016/j.jneumeth.2020.109064. Epub 2020 Dec 30.
Fluorescence imaging is a widely used technique that permits for cell-type-specific recording from hundreds of neurons simultaneously. Often, to obtain cell-type-specific recordings from more than one cell type, researchers add an additional fluorescent protein to mark a second neuronal subpopulation. Currently, however, no consensus exists on the best expression method for multiple fluorescent proteins.
We optimized the coexpression of two fluorescent proteins across multiple brain regions and mouse lines.
The single-virus method, a viral injection in a double transgenic reporter mouse, results in limited fluorescent coexpression. In contrast the double-virus method, injecting a mixture of two viruses in a Cre driver mouse, results in up to 70 % coexpression of the fluorescent markers in vitro. Using the double-virus method allows for population activity recording and neuronal subpopulation determination.
The standard for expressing two fluorescent proteins is to use a double transgenic reporter mouse with a single viral injection. Injecting two viruses into a Cre driver mouse resulted in significantly higher coexpression compared to the standard method. This result generalized to multiple brain regions and mouse lines in vitro, as well as in vivo.
Efficiently coexpressing multiple fluorescent proteins provides population activity while identifying a neuronal subpopulation of interest. The improved coexpression is applicable to a wide breadth of experiments, ranging from engram investigation to voltage imaging.
荧光成像是一种广泛应用的技术，可实现同时对数百个神经元进行细胞类型特异性记录。通常，为了从超过一种细胞类型获得细胞类型特异性记录，研究人员会添加另一种荧光蛋白来标记第二个神经元亚群。然而，目前对于多种荧光蛋白的最佳表达方法尚无共识。
我们优化了两种荧光蛋白在多个脑区和小鼠品系中的共表达。
单病毒法（在双转基因报告小鼠中进行病毒注射）导致荧光共表达有限。相比之下，双病毒法（在 Cre 驱动小鼠中注射两种病毒的混合物）可使体外荧光标记物的共表达率高达 70%。使用双病毒法可以进行群体活动记录和神经元亚群的确定。
表达两种荧光蛋白的标准方法是使用带有单病毒注射的双转基因报告小鼠。与标准方法相比，将两种病毒注射到 Cre 驱动小鼠中可导致显著更高的共表达率。这一结果在体外、体内以及多个脑区和小鼠品系中均得到了验证。
高效共表达多种荧光蛋白可提供群体活动记录，并鉴定出感兴趣的神经元亚群。改进的共表达方法适用于广泛的实验，包括印记调查和电压成像。
