Linking functional activity and connectivity of neuronal circuits via fast cross-layer all-optical physiology

Interrogating neural circuits in vivo is crucial for understanding brain organization during diverse behaviors. However, causal connectivity mapping across cross-layer circuits is rarely studied. Here, we develop a cross-layer all-optical physiology (CLAOP) system that enables simultaneous recording and manipulation of single-neuron activities in multiple neuronal layers at high temporal resolution. Using synchronized spatiotemporal and spectral multiplexing, CLAOP achieves all-optical analysis with a minimal time delay in inter-layer imaging and photostimulation. Combined with behavioral inputs, CLAOP can perturb the activity response of inter-layer primary visual cortex (V1) neurons according to recorded functional signatures. CLAOP further reveals activity-connectivity coupling in the V1 and primary somatosensory cortex (S1), which provides all-optical evidence of the presence of widely observed like-to-like connectivity in the V1 and extends it to cross-layer levels and another cortex. CLAOP provides a high-throughput approach for mapping inter-layer causal connectivity during behavior and interpreting circuit mechanisms of diverse brain functions.