Brain-wide monosynaptic connectivity mapping with ROInet-seq

Viral projection tracing strategies help establish regional connectomes of mammalian brains. Monosynaptic connectivity tracing with G-deleted rabies virus (RV) establishes retrograde synaptic connectivity, but cannot distinguish networks at cell resolution. We developed ROInet-seq, an accessible spatial method using barcoded ΔG rabies virus, amenable to readout by RNA-sequencing. We optimized library complexity and uniformity, such that detection of specific barcodes can reliably distinguish multiple infected neurons, and their individual monosynaptic input networks. In cortical brain areas we found preserved regional network motives, including co-inputs to single cortical neurons from distant and local sites. Towards improved spatial resolution and simultaneous detection of the single cells’ transcriptomes and networks, we finally sampled barcoded ΔG rabies virus infected hippocampus on a commercial spatial transcriptomics assay and reveal details of the regions’ neurons local network architecture.