High-Throughput Mapping of Synaptic Connectome with Single-Cell Spatial Resolution

Mapping neural connectivity requires spatial precision unattainable by conventional barcode sequencing. We developed CASS (Combination of Artificial Short Sequences) barcodes—error-resistant molecular tags optimized for in situ hybridization—and integrated them with rabies virus monosynaptic tracing. This synergy enables histological connectome mapping via FISH-based decoding, preserving anatomical context while achieving single-cell resolution. Applied to the primary visual cortex, we identified 1,532 spatially resolved synaptic pairs across three mice, revealing: 1) layer-specific intracortical connectivity patterns, and 2) topographically organized thalamocortical projections. The platform's throughput capacity allows parallel tracing of multiple neural populations, overcoming spatial disassociation limitations of sequencing-based methods. By combining viral tracing scalability with histological validation, our framework supports quantitative analysis of connectivity motifs in intact tissues at micron resolution. This advance provides a critical tool for investigating structure-function relationships in neural circuits, particularly where spatial organization underpins computational functions.