Simultaneous acquisition of single-cell mRNA and neuroanatomy

Abstract

The accurate characterization of neurons and their connectivity is crucial for understanding higher neural functions in both daily life as well as disease. However, the characterization of synapses and their connectivity has been limited to bulk observations or more granular but slower methods. Furthermore, no technology exists to link synaptic or neuroanatomical information with single cell gene expression, used to infer neuronal identity. We introduce a novel technology called Synapse-seq, which enables the labeling of subcellular targets with an RNA cell barcode via synthetic viral constructs. In particular, this dissertation concentrates on the presynaptic Synapse-seq, where we label the presynaptic terminals of virally infected neurons with distinct RNA cell barcodes. The barcodes associated with these labeled presynapses are then examined through spatial transcriptomics (Slide-seq) or bulk sequencing of ~1mm resolution dissections. Furthermore, we sequence the barcodes associated with the labeled cells using single-nuclei RNA sequencing (snRNA-seq). This enables us to establish connections between spatial projections and the transcriptomic cellular identity at regional, pseudo-cellular, and cellular levels. We validated presynaptic Synapse-seq by applying this technology to the primary visual cortex (VISP) of mice, achieving unmatched resolution, specificity, and throughput. This groundbreaking approach emerges as a potent tool for exploring synaptic biology and connectomics in mammalian models.