3-PHOTON CALCIUM IMAGING OF L6 NEURONS IN MOUSE PRIMARY VISUAL CORTEX DURING HEAD-FIXED SLEEP AND WAKEFULNESS

Current understanding of sleep dynamics in cortical networks suggests that state transitions may be coordinated by neuromodulatory systems engaging local cortical regulatory mechanisms. While the most superficial layer of the cortical column has long been considered a nexus for neuromodulatory integration, recent work has identified another convergence point in the deepest layer (L6). We present neuropixel probe recordings acquired in conjunction with electroencephalogram and electromyogram signals from head-fixed mice drifting between sleep and wakefulness. In concordance with literature acquired from freely moving mice, our sleep-stage segmented data shows the firing rates of deep-layer neurons to be more positively modulated by wake periods in comparison with superficial-layer neurons. By implementing our recently developed dual-plane three-photon calcium imaging method in the primary visual cortex of mice injected with AAV2/1-hSyn-jGCaMP8s, we find a small number of neurons at the base of cortex which increase firing rate immediately prior to waking, matching the sparsity of orexin-sensitive neurons expected from in vitro literature. Since Drd1a-Cre+ neurons which reside sparsely at the base of L6 are unique amongst cortical neurons in their sensitivity to the wake-promoting neuromodulator orexin, as well as in their targeted excitation of higher-order cortico-thalamo-cortical loops, they appear ideal candidate targets for the induction of sleep-wake state transitions. We will present preliminary data from dual-plane imaging of Drd1a-Cre+ neurons transduced with AAV2/1-hSyn-FLEX-jGCaMP8s along with more superficial Drd1a-Cre- neurons transduced with AAV2/1-hSyn-jGCaMP8s, experiments which are necessary to establish whether Drd1a-Cre+ neurons act as drivers of wake-like activity in cortex.