Barcoded Rabies In Situ Connectomics for high-throughput reconstruction of neural circuits

Sequencing of oligonucleotide barcodes holds promise as a high-throughput approach for reconstructing synaptic connectivity at scale (1). Rabies viruses can act as a vehicle for barcode transmission, thanks to their ability to spread between synaptically connected cells (2, 3). However, applying barcoded rabies viruses to map synaptic connections in vivo has proved challenging (4-7). Here, we develop Barcoded Rabies In Situ Connectomics (BRISC) for high-throughput connectivity mapping in the mouse brain. To ensure that the majority of post-synaptic "starter" neurons are uniquely labeled with distinct barcode sequences, we first generated libraries of rabies viruses with sufficient diversity to label >1000 neurons uniquely. To minimize the probability of barcode transmission between starter neurons, we developed a strategy to tightly control their density. We then applied BRISC to map inputs of single neurons in the primary visual cortex (V1). Using in situ sequencing, we read out the expression of viral barcodes in rabies-infected neurons, while preserving spatial information. We then matched barcode sequences between starter and presynaptic neurons, mapping the inputs of 385 neurons and identifying 7,814 putative synaptic connections. The resulting connectivity matrix revealed layer- and cell-type-specific local connectivity rules and topographic organization of long-range inputs to V1. These results show that BRISC can simultaneously resolve the synaptic connectivity of hundreds of neurons while preserving spatial information, enabling reconstruction of neural circuits at an unprecedented scale.