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.   Significance:  Cellular layering is a hallmark of the mammalian neocortex with layer and cell type-specific connections within the cortical mantle and subcortical connections. A key challenge in studying circuit function within the neocortex is to understand the spatial and temporal patterns of information flow between different columns and layers.   Aim:  We aimed to investigate the three-dimensional (3D) layer- and area-specific interactions in mouse cortex  in vivo .   Approach:  We applied a new promising neuroimaging method—fluorescence laminar optical tomography in combination with voltage-sensitive dye imaging (VSDi). VSDi is a powerful technique for interrogating membrane potential dynamics in assemblies of cortical neurons, but it is traditionally used for two-dimensional (2D) imaging. Our mesoscopic technique allows visualization of neuronal activity in a 3D manner with high temporal resolution.   Results:  We first demonstrated the depth-resolved capability of 3D mesoscopic imaging technology in Thy1-ChR2-YFP transgenic mice. Next, we recorded the long-range functional projections between sensory cortex (S1) and motor cortex (M1) in mice,  in vivo , following single whisker deflection.   Conclusions:  The results show that mesoscopic imaging technique has the potential to investigate the layer-specific neural connectivity in the mouse cortex  in vivo . Combination of mesoscopic imaging technique with optogenetic control strategy is a promising platform for determining depth-resolved interactions between cortical circuit elements.

In vivo voltage-sensitive dye imaging of mouse cortical activity with mesoscopic optical tomography