Cell Type-specific Anterograde Circuit Mapping and Functional Control by Optimizing YFV-17D Transneuronal Systems Summary To elucidate the functional organization of brain circuitry we need to delineate neuronal connectivity and control the activity of neurons with specific connectivity. Both tasks have increasingly relied on transneuronal viral vectors. Anterograde transneuronal viral vectors, which spread from presynaptic neurons to the postsynaptic neurons, can reveal the neuronal projections and selectively target postsynaptic neurons. Due to the lack of ideal anterograde transneuronal viral vectors, we have spent the last few years developing a new anterograde system based on yellow fever vaccine—YFV-17D. We have successfully constructed two major anterograde tools: the packaging defective YFV∆CME for mapping neuronal projectomes, and the replication defective YFV∆NS1 (or YFV∆CMENS1) for transneuronal control of gene expression in postsynaptic neurons, which can be used for functional observation or manipulation. Neuronal toxicity can be avoided in the case of YFV∆NS1 and YFV∆CMENS1, but not in YFV∆CME. These new systems provide benefits of transneuronal efficacy, diverse applications, and ease of engineering, but they still have limitations for many experimental scenarios. Here we propose to further improve these viral systems to make them broadly applicable, powerful and safe tools for functional dissection of the brain circuits. Firstly, we will construct self- constrained YFV-17D to further minimize neuronal toxicity incurred by viral replication in neurons, which will make these vectors more useful for both research and potential clinical applications. Secondly, we will test multiple strategies to target this system to specific neurons for cell-type specific tracing or functional control in both local circuits and long-range projections. Some of the cell type specific tools will not rely on the availability of genetically modified mouse lines and can be applied to broad species. Thirdly, we will incorporate commonly used tags, sensors or effectors in the optimized transneuronal viral vectors for versatile applications. We will also apply the improved versions to delineate a selected neuronal circuit that is of great interest to neuroscientists. Therefore, this project will yield a set of highly powerful tools widely applicable to neuroscience research, and will reveal the projectomes of multiple classical neuronal types.