Brain-wide monosynaptic single-neuron connectivity with ROInet-seq

Viral projection tracing strategies help establish regional connectomes of mammalian brains. Monosynaptic connectivity tracing with G-deleted rabies virus (RV) establishes synaptic input connectivity, but cannot distinguish networks at cell resolution. We implemented a barcoded ΔG rabies virus to introduce unique molecular tags - and make network tracing amenable to readout by RNA-sequencing. First, we optimized and characterized library complexity and uniformity, such that detection of specific barcodes can reliably distinguish the individual monosynaptic input networks of multiple infected neurons in parallel. To deploy the method at scale; to hundreds of cells and full-brain volume per experiment, we developed regions-of-interest network sequencing (ROInet-seq); an accessible, scalable and low-cost spatial assay. ROInet-seq combines routine fluorescent imaging and processing of fixed tissue sections with a simple molecular biology workflow to capture barcode sequences in relevant regions-of-interest, and ultimately describes single-neuron networks brain-wide. In cortical brain areas the assay revealed preserved regional network motives, including co-inputs to single cortical neurons from distant and local sites. Towards improved spatial resolution and simultaneous detection of transcriptomes and networks we finally sampled barcoded ΔG rabies virus-infected hippocampus on commercial spatial transcriptomics assays and reveal details of the regions’ neurons local network architecture.