Extraction of Distinct Neuronal Cell Types from within a Genetically Continuous Population

Single-cell transcriptomics of neocortical neurons have revealed more than 100 clusters corresponding to putative cell types. For inhibitory and subcortical projection neurons (SCPNs), there is a strong concordance between clusters and anatomical descriptions of cell types. In contrast, cortico-cortical projection neurons (CCPNs) separate into surprisingly few transcriptomic clusters, despite their diverse anatomical projection types. We used projection-dependent single-cell transcriptomic analyses and monosynaptic rabies tracing to compare mouse primary visual cortex CCPNs projecting to different higher visual areas. We find that layer 2/3 CCPNs with different anatomical projections differ systematically in their gene expressions, despite forming only a single genetic cluster. Furthermore, these neurons receive feedback selectively from the same areas to which they project. These findings demonstrate that gene-expression analysis in isolation is insufficient to identify neuron types and have important implications for understanding the functional role of cortical feedback circuits.