Spatial organization of projection neurons in the mouse auditory cortex identified by in situ barcode sequencing

Understanding neural circuits requires deciphering the interactions of myriad cell types defined by anatomy, spatial organization, gene expression, and functional properties. Resolving these cell types requires both single neuron resolution and high-throughput, a combination that is challenging to achieve with conventional anatomical methods. Here we introduce BARseq, a method for mapping the projections of thousands of spatially resolved neurons by combining the high throughput of DNA sequencing with the high spatial resolution of microscopy. We used BARseq to determine the projections of 1309 neurons in mouse auditory cortex to 11 targets. We observed 264 distinct projection patterns. Hierarchical clustering confirmed the major classical classes of projection neurons, segregated across cortical laminae. Further analysis revealed 25 subclasses, largely intermingled across laminae. Unlike cell types defined by gene expression, projection subclasses beyond the major classes were rarely enriched in specific laminae, raising the possibility that the organization of projection patterns in mature neurons is orthogonal to that of gene expression. In this way, downstream brain areas could receive information from multiple cell types through parallel pathways. By sequencing in situ, BARseq has the potential to bridge anatomical, transcriptomic, functional, and other approaches at single neuron resolution with high throughput, and thereby offer unprecedented insight of the structure and function of a neural circuit.