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Summary: Here, we present a comprehensive protocol to analyze the roles of disease-related genes in synaptic transmission. We have developed a pipeline of electrophysiological techniques and combined these with optogenetics in the medial prefrontal cortex of mice. This methodology provides a cost-effective, faster, and easier screening approach to elucidate functional aspects of single genes in several regions in the mouse brain such as a specific layer of the mPFC.For complete details on the use and execution of this protocol, please refer to Nagahama et al. (2020) and Sacai et al. (2020).