SUMMARY

Mapping neuronal connectivity is essential for understanding the structure and function of neural circuits. While high-throughput, cost-effective methods using barcoded rabies viruses provide valuable cellular-level insights, they are limited by the spatial resolution of barcode sequencing. To provide a fluorescent in situ hybridization (FISH)-compatible alternative for spatial barcode readout, we developed the CASS (combination of artificial short sequences) barcode, an error-robust and in situ-hybridization-detectable tool. By combining CASS barcoding with rabies virus monosynaptic tracing, we enable in situ connectome mapping through FISH-based barcode decoding. Using this approach, we identified 1,532 synaptic pairs connected to neurons in the primary visual cortex with spatial precision across three mice, demonstrating its efficiency and scalability. This method provides a FISH-based approach for identifying neuronal connectivity with spatial context and may facilitate future integration of connectivity mapping with molecular profiling, thereby advancing our understanding of neural circuits.