Quantification of Sparse Neuronal Projections

The mice were transcardially perfused with phosphate-buffered saline (PBS) followed by 4% PFA-PBS solution. The brains were post-fixed in 4% PFA-PBS for overnight at 4 °C and placed in 30% sucrose PBS solution for 2 days. Coronal sections were obtained using a cryostat with a thickness of 40 μm. The sections were analyzed using a fluorescence microscope (KEYENCE BZ-X810) with a 10X, 20X or 40X objective. For AAV2-retro experiments, the sections including the primary cortical sensory area (S1) or the nucleus accumbens (NAc) were imaged. The fluorescence intensity of the cells and the number of cells were automatically measured with a software (KEYENCE Image Analyzer). Since retrograde projections are sparse in the above-mentioned circuits, we quantified GFP signal to evaluate the transduction efficiency as conducted in the other studies [23 (link)–25 (link)]. For AAV1 experiments, fifty consecutive sections including the superior colliculus (SC) were imaged per a mouse. The number of cells was automatically measured with a software (KEYENCE Image Analyzer). During the whole imaging and analysis processing, the information of samples (the original virus or the mutant virus) was blinded to the experimenter.