In vivo two-photon voltage-sensitive dye imaging reveals top-down control of cortical layers 1 and 2 during wakefulness

Conventional methods of imaging membrane potential changes have limited spatial resolution, particularly a long the axis perpendicular to the cortical surface. The laminar organization of the cortex suggests, however, that the distribution of activity in depth is not uniform. We developed a technique to resolve network activity of different cortical layers in vivo using two-photon microscopy of the voltage-sensitive dye (VSD) ANNINE-6. We imaged spontaneous voltage changes in the barrel field of the somatosensory cortex of head-restrained mice and analyzed their spatio-temporal correlations during anesthesia and wakefulness. EEG recordings always correlated more strongly with VSD signals in layer (L) 2 than in L1. Nearby (<200 mu m) cortical areas were correlated with one another during anesthesia. Waking the mouse strongly desynchronized neighboring cortical areas in L1 in the 4- to 10-Hz frequency band. Wakefulness also slightly increased synchrony of neighboring territories in L2 in the 0.5- to 4.0-Hz range. Our observations are consistent with the idea that, in the awake animal, long-range inputs to L1 of the sensory cortex from various cortical and thalamic areas exert top-down control on sensory processing.