Background

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.

New Method

We optimized the coexpression of two fluorescent proteins across multiple brain regions and mouse lines.

Results

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.

Comparison with Existing Method

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.

Conclusion

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.

中文翻译：

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确定双色成像的最佳表达方法

背景

荧光成像是一种广泛使用的技术，它允许同时从数百个神经元进行细胞类型特异性记录。通常，为了从一种以上的细胞类型中获得细胞类型特异性记录，研究人员会添加一种额外的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。

新方法

我们优化了两种荧光蛋白在多个大脑区域和小鼠系中的共表达。

结果

单病毒方法，在双转基因报告小鼠中进行病毒注射，导致有限的荧光共表达。与双病毒方法相比，在 Cre 驱动小鼠中注射两种病毒的混合物，可导致高达 70% 的荧光标记在体外共表达。使用双病毒方法可以进行种群活动记录和神经元亚群测定。

与现有方法的比较

表达两种荧光蛋白的标准是使用单次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 驱动鼠标中导致显着更高的共表达。这一结果在体外和体内推广到多个大脑区域和小鼠系。

结论

有效地共表达多种荧光蛋白可提供种群活动，同时识别感兴趣的神经元亚群。改进的共表达适用于广泛的实验，从印迹调查到电压成像。