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

Mechanistic understanding of how networks of neurons carry out neural computations requires knowledge of their underlying synaptic connectivity. However, current methods for reconstructing synaptic connections are extremely laborious and typically limited to mapping connections between nearby neurons. Here we developed Barcoded Rabies In Situ Connectomics (BRISC), which uses rabies viruses carrying random molecular barcodes to map both local and long-range monosynaptic inputs of hundreds of neurons in parallel. To ensure that the majority of post-synaptic starter neurons are uniquely labeled with distinct barcode sequences, we generated libraries of rabies viruses with sufficient diversity to label >1000 neurons and controlled the probability of barcode transmission between starter neurons by limiting their density. We applied BRISC to map inputs of single neurons in the mouse primary visual cortex. We read out the expression of viral barcodes in rabies-infected neurons in situ, 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, revealing layer- and cell-type-specific local connectivity rules and topographic organization of long-range inputs. These results show that BRISC can simultaneously resolve the synaptic connectivity of hundreds of neurons, enabling reconstruction of neural circuits at an unprecedented scale.