Many mammalian species, including mice, switch between lit and dark environments during normal exploratory, foraging, and predator evasion behaviors by moving in and out of dark spaces. How neuronal activity encodes visual features in the primary visual cortex (V1) in lit environments and under various luminance levels has been well-studied in many mammalian species. However, how the functionally distinct superficial and deep cortical layers of the V1 encode abrupt and sustained changes in light conditions in freely exploring animals remains unclear. To address this question, we recorded neuronal activity from populations of V1 neurons located across the different layers (L2-L6) in freely exploring mice using our recently developed head-mounted 3-photon microscope that allows imaging in light or dark conditions. Using different mouse lines (Scnn1a-cre, Ntsr1-cre, and wild-type), the activity from V1 layers 2/3, 4, 5, and 6 were recorded from neurons labeled with the genetically encoded calcium indicator GCaMP7f, for up to 3 months, while mice were free to explore a linear track (0.8 x 0.1 m) under different sustained lighting conditions ranging from bright light (1.1 cd/m²), to complete darkness (>0.001 cd/m²). Initial analysis indicates that lighting conditions are differentially encoded across cortical layers, with sub-populations located in deep (L5 and 6) but not superficial (L2/3 and 4) layers displaying sustained activity during dark conditions. Additionally, for a subpopulation of dark-responsive neurons, the activity was sustained for the entire duration of darkness (>7 min) and was not dependent on the animal’s speed.