The organization of functional subtypes of neurons shapes the basic structure of neocortical circuits. Our previous analysis revealed that subcerebral projection neurons (SCPNs), a major pyramidal neuron subtype in mouse neocortical layer 5 , form columnar clusters termed microcolumns. c-Fos expression induced by visual stimulation was strongly correlated among SCPNs in a single microcolumn, suggesting related functions (Maruoka et al., 2011). This analysis, however, had a limited temporal resolution because c-Fos expression was induced by one to two hours of stimulation.
In order to improve the temporal resolution for a better characterization of the function of microcolumns, we developed a method of in vivo two-photon calcium imaging of layer 5 excitatory neurons in awake mice. A genetically encoded calcium indicator was expressed in layer 5 excitatory neurons in the binocular visual cortex. A cranial window and a headplate were installed on the skull over the binocular visual cortex. The mouse was placed under a two-photon microscope and allowed to run on a freely rotating wheel during imaging.
The optimized method enabled imaging of sensory responses and spontaneous activity of layer 5 excitatory neurons in awake mice. Neuronal activity of layer 5 excitatory neurons differed from that in anesthetized mice, in accordance with similar observations in layer 2/3. Visual response properties of SCPNs in microcolumns will be discussed.