Chen, T.; Deng, Q.; Lin, K.; Zheng, X.; Wang, X.; Zhong, Y.; Ning, X.; Li, Y.; Xu, F.; Du, J.; Du, X.

The larval zebrafish is a vertebrate model for in vivo monitoring and manipulation of whole-brain neuronal activities. Tracing its neural circuits still remains challenging. Here we report an applicable methodology tailored for larval zebrafish to achieve efficient retrograde trans-monosynaptic tracing from genetically defined neurons via EnvA-pseudotyped glycoprotein-deleted rabies viruses. By combinatorially optimizing multiple factors involved, we identified the CVS strain trans-complemented with advanced expression of N2cG at 36 degrees C as the optimal combination. It yielded a tracing efficiency of up to 20 inputs per starter cell. Its low cytotoxicity enabled the viable labeling and calcium imaging of infected neurons 10 days post-infection, spanning larval ages commonly used for functional examination. Cre-dependent labeling was further developed to enable input cell-type-specific tracing and circuit reconstruction. We mapped cerebellar circuits and uncovered the ipsilateral preference and subtype specificity of granule cell-to-Purkinje cell connections. Our method offers an efficient way for tracing neural circuits in larval zebrafish