SCALABLE CIRCUIT MAPPING IN THE MAMMALIAN BRAIN USING A BARCODED RABIES VIRUS

Single-cell transcriptomics has revolutionized our understanding of neuronal heterogeneity, revealing hundreds of molecularly defined neuronal cell types which are postulated to serve unique roles in brain function. However, a comprehensive map of circuit connectivity between molecularly defined cell types in the mammalian brain remains elusive. Barcoded viral tools present an opportunity to multiplex neuronal tracing experiments to study many neurons in parallel by delivering a unique molecular barcode to each mapped neuron. We developed a high-complexity barcoded rabies virus (RV) which enables us to map the inputs to thousands of single neurons in parallel. In addition, we developed a strategy to target RV-encoded barcodes to the nuclear membrane, allowing them to be read out using single-nucleus RNA-sequencing (snRNA-seq). We demonstrate the utility of barcoded RV to map neural circuits in the mouse primary visual cortex (V1) in vivo, as well as in organotypic slices of the developing human cerebral cortex. In addition to snRNA-seq, RVΔG-encoded barcodes can be read out using BarSeq2-like in situ sequencing. Together, these technological advances provide a framework for scalable, sequencing-based connectomics across brain regions and species. By reducing the problem of synaptic connectivity into a problem of barcode sequencing, our approach has the potential to dramatically increase throughput, decrease costs and provide a direct link to the transcriptome of each mapped pre- and post-synaptic cell.