State-Dependent Organization of Microscale Functional Circuitry in Visual Cortex.

Brain state modulates sensory processing across visual cortex, yet how it relates to the organization of functional circuitry at the level of individual neurons and cell types remains largely unknown. To address this, we constructed one of the largest microscale directed functional circuit maps in mouse visual cortex from calcium imaging of more than 57,000 neurons across four visual areas and five cortical layers. Using a time-aware causal inference framework, we found that intra-areal connections dominate across arousal states, consistent with experimental findings on the local bias of cortical anatomy. Among intra-areal connections, anterolateral area (AL) had the highest density, and among inter-areal connections, the AL↔rostrolateral area (RL) axis formed the strongest pathway. Laminar circuit organization was dominated by layer 6 recurrence within-layer, while the most prominent between-layer pathway was layer 5-to-layer 6 in low arousal and layer 4-to-layer 5 in high arousal. Spatial extent was selectively greater for excitatory-to-inhibitory connections in high arousal, but not for excitatory-to-excitatory connections. Across 6,597 electron-microscopy reconstructions of neuron pairs, synapse count predicted functional connection strength in both arousal states, but structure-function coupling was weaker in high arousal. In stimulus-driven response prediction, neuron pairs with stronger functional connections exhibited more similar predictive performance in both states, with performance varying by layer and cell type. Overall, our findings map, at single-neuron resolution, the multi-scale organization of directed functional circuitry in mouse visual cortex across brain states.