High-throughput sequencing of synaptic partnerships and gene expression at single-cell resolution in vivo

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PROJECT SUMMARY Brain function depends on forming and maintaining synaptic connections between neurons of specific types, yet systematic descriptions of cell-type connectivity and the molecules that instruct these relationships remain challenging because we lack some necessary tools. Traditional approaches for measuring synaptic connections and networks – such as whole-cell electrophysiology and anatomical reconstructions – sample only a few cells or small tissue volumes, do not readily scale to many animals or genotypes, and do not ascertain the molecular type and state of each cell. To bridge this gap, I have developed a barcoded rabies virus-based method called SBARRO which stores synaptic partnership data in each cells' RNA, allowing synaptic networks and gene expression to be measured simultaneously in hundreds of thousands of cells using high-throughput single-cell RNA sequencing. SBARRO experiments in vitro have demonstrated that quantitative models of cell type-specific connectivity can be systematically generated and used to discover gene expression signatures associated with connectivity properties. Yet technical limitations related to cell sampling, recombinant adeno-associated “helper” viruses (rAAVs) and rabies virus biology have precluded analyses of intact brain tissue. Here, I propose to address these limitations and advance SBARRO to generate quantitative models of synaptic networks in vivo based on the following aims: 1) create an anatomically-informed version of SBARRO based on spatially-resolved single-cell RNA sequencing (called “Slide-SBARRO”); 2) develop a new class of CRE recombinase-sensitive rAAVs which deliver transgenes while reporting the recombination state of expressed RNA; and 3) evaluate the synapse-selectivity of rabies virus transmission from postsynaptic to presynaptic cells. I will focus on mouse striatum because: 1) striatal cell populations are now well-characterized by me and others; 2) little is known about the synaptic organization of intrinsic striatal cell types and 3) extensive mouse genetic tools enable Slide-SBARRO connectivity models to be carefully tested through cell-type-specific anatomy and electrophysiology.