Cell type-specific barcoding reveals the projectional architecture of the mouse midbrain dopaminergic system

Brain-wide neural circuits are formed by the diverse axonal branching patterns of many individual neurons. Here we introduce POlNTseq (projections of interest by sequencing), a high-throughput and user-friendly barcoded connectomics method that uses cell type specific barcoding and sequencing to rapidly map single-cell projections of a cell type of interest for thousands of neurons per animal. POlNTseq leverages pseudotyping of Sindbis virus and a specific alphavirus- cellular receptor pair to make Sindbis infections cell type specific. lt thus integrates MAPseq-style high-throughput barcoded projection mapping with the established viral-genetic neural circuit analysis toolbox. We validated POlNTseq by mapping genetically and projection-defined cell populations in the mouse motor cortex. We then applied POlNTseq to midbrain dopaminergic neurons and reconstructed the brain-wide single-cell projections of 3,813 dopaminergic neurons in ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). We define over 30 connectomic cell types, vastly exceeding the known diversity of dopaminergic cell types, and identify stereotyped projection motifs that may mediate parallel dopamine signaling. This data constitutes the anatomical substrate on which the diverse functions of dopamine in the brain are built. HIGHLIGHTSO_LIWe develop POlNTseq, which uses pseudotyped Sindbis virus and cell type-specific expression of a viral receptor for cell type-specific barcoding. C_LIO_LIPOlNTseq enables massively multiplexed single-cell projection mapping of cell types of interest. C_LIO_LIWe map the brain-wide projections of 3,813 individual VTA and SNc dopaminergic neurons. C_LIO_LIVTA and SNc dopaminergic neurons form over 30 connectomic cell types. C_LIO_LIProjections organize into stereotyped motifs that may mediate parallel dopamine signalling. C_LI