2-P imaging of mouse visual cortex layer 6 corticothalamic feedback during different behavior states

Layer 6 (L6), the deepest lamina of cerebral cortex, is one of the key structures regulating behavior state related information processing within the cortex and various subcortical areas. However, very little is known about the functional significance of different L6 circuits in vivo. L6 experiments in the behaving animals still remain challenging due to hard access of the recording / imaging site, complexity of neuronal circuits and heterogeneity in neuron morphology. Here, we focus on primary visual cortex L6 feedback projections to visual thalamus (lateral geniculate nucleus) which regulate visual signal transmission from retina to cortex. We developed a method to “dissect” and study these L6 corticothalamic feedback projections in vivo with 2P microscopy. With this method, we can reliably image retrogradely labeled corticothalamic and other excitatory L6 neurons throughout full layer, down to about 850 μm below dura in a head-fixed mouse. Up to a few hundred individual neurons can be recorded simultaneously for several hours and/or repeatedly recorded during different days, while monitoring mouse behavior state. This allows us to study the cortical feedback to the primary visual thalamus during different behavior states, ranging from full alertness to sleep.