Highly multiplexed circuit mapping using barcoded rabies viruses and in situ sequencing.

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ABSTRACT Understanding the function of neural circuits requires thorough investigation of two circuit elements: cell types and connectivity. The combination of axonal tracing with high-throughput DNA sequencing of genetically barcoded neurons has enabled the simultaneous characterization of anatomical and molecular identities of neurons and their projection fields. However, no high- throughput tools currently exist that allow us to map connections between presynaptic and postsynaptic neurons while identifying the cell types of mapped neurons. Here we propose to apply a family of novel sequencing-based approaches using barcoded rabies viruses to achieve two main goals: first, to allow characterization of projection neurons everywhere in the brain that project to multiple target regions; and second, to allow simultaneous mapping of the inputs to transcriptomically-distinct subtypes of neurons. We will combine our well- established single-cell RNA-seq (scRNA-seq) pipeline with retrograde labeling (Retro-seq) for rabies barcode detection in order to conduct highly multiplexed cell type characterization of neurons projecting to each of the many target areas across the brain. Furthermore, we will adapt our barcoded anatomy resolved by sequencing (BARseq) pipeline to characterize the spatial distribution and cell types of those projection neurons (Retro-BARseq). We will further establish the methodology to investigate the anatomical and transcriptomic features of neural circuitry revealed by rabies-based retrograde trans-synaptic circuit tracing using scRNA-seq/BARseq (TransR-seq and TransR-BARseq). Finally, we will strive to reach single-neuron-level connectome tracing and sequencing in wild-type mice by applying the optimized barcoded rabies viruses to achieve unique barcoding of individual neurons. Success of the project will result in a set of high-throughput, high-resolution methods for sequencing the connectome by combining barcoded rabies viruses with omics, therefore revealing both the connectivity and transcriptomic identities of neurons within a given neural circuit. These techniques, the majority of which should be applicable to any mammalian species, will provide orders of magnitude increases in both throughput and resolution of neuronal circuit analysis, with a transformative impact on many branches of neuroscience.