Higher-order cortical and thalamic pathways shape visual processing streams in the mouse cortex

Mammalian visual functions rely on distributed processing across interconnected cortical and subcortical regions. In higher-order visual areas (HVAs), visual features are processed in specialized streams that integrate feedforward and higher-order inputs from intracortical and thalamocortical pathways. However, the precise circuit organization responsible for HVA specialization remains unclear. We investigated the cellular architecture of primary visual cortex (V1) and higher-order visual pathways in the mouse, focusing on their roles in shaping visual representations. Using in vivo functional imaging and neural circuit tracing, we found that HVAs preferentially receive inputs from both V1 and higher-order pathways tuned to similar spatiotemporal properties, with the strongest selectivity seen in layer 2/3 neurons. These neurons exhibit target-specific tuning and sublaminar specificity in their projections, reflecting cell-type-specific visual information flow. In contrast, HVA layer 5 pathways nonspecifically broadcast visual signals across cortical areas, suggesting a role in distributing HVA outputs. Additionally, thalamocortical pathways from the lateral posterior thalamic nucleus (LP) provide highly specific, nearly non-overlapping visual inputs to HVAs, complementing intracortical inputs and contributing to input functional diversity. Our findings suggest that the convergence of laminar and cell-type-specific pathways V1 and higher-order intracortical and thalamocortical pathways plays a key role in shaping the functional specialization and diversity of HVAs.