Single-cell atlas uncovers layer 4 heterogeneity and functional gradients in rhesus macaque visual cortex

Non-human primates like rhesus macaques are pivotal models for decoding human visual cortex physiology and disorders. However, there is a lack of high-resolution transcriptomic atlases comparing distinct areas of the visual cortex in macaque. Here, we developed BrainSPACE, an innovative pipeline for rapid and precise brain tissue banking, to sample visual cortex areas: V1, V2, V4, MT, and TEO. We applied single-nucleus RNA-sequencing (snRNA-seq) to unravel neuronal diversity in BrainSPACE-banked V1 and V4 areas ( ~ 100,000 nuclei). We uncovered conserved GABAergic neuron profiles but stark area-specific diversity in principal neurons, featuring seven unique layer 4 subtypes in V1 and one in V4. Complementary smFISH validated transcriptional gradients across these areas, aligning with ventral and dorsal stream hierarchies. Gene ontology analyses highlighted plasticity-related pathways in unique layer 4 subtypes, with genes like NTNG1 and NLGN1 linked to neurodevelopmental disorders such as autism and schizophrenia. Our insights bridge molecular architecture to visual processing, offering an interactive atlas for community use. By revealing how layer 4 heterogeneity drives hierarchical specialization, our work advances primate brain mapping and informs therapeutic strategies for vision-related pathologies.