Lorenzo, D. A.; Gallero-Salas, Y.; Panzeri, M.; Wahl, A.-S.; Gilad, A.; Lewis, C. M.; Helmchen, F.

The mammalian neocortex is highly organized in local microcircuits and through long-range projection patterns between different regions. Since various features of local and long-range connectivity are determined by the cortical layer in which the respective neurons reside, understanding the flow of cortical information within and across layers is essential. Wide-field calcium imaging enables mesoscale functional mapping of genetically identified neurons across cortical areas. However, it has been applied primarily to superficial cortical layers, and systematic comparisons of wide-field signals across cortical layers are scarce. Here, we apply wide-field calcium imaging to different cortical layers using transgenic mouse lines with selective expression of GCaMP6f in layers 2/3, 5, and 6. We address several challenges of layer-specific wide-field imaging and provide possible solutions. First, to improve the registration of functional data to standard atlases, we demonstrate the benefit of layer-specific registration maps that are warped on the basis of the depth-dependent surface projections of the labeled cell populations. These maps help to assign the imaged calcium signals to the specific regions from which they originate. Second, we measure the depth-dependent blurring of wide-field fluorescence signals induced by light scattering and reveal stronger blurring in deep vs. superficial layers, in line with previous theoretical predictions from simulations. We used measured point spread functions to deconvolve single-whisker-evoked calcium signals in the barrel cortex and demonstrate improved signal confinement to individual barrel columns across layers. Finally, we investigate cross-regional functional connectivity during awake resting state periods for distinct layers. We find that mesoscopic functional connectivity is largely conserved between the cortical layers, with subtle differences for key regions of the default mode network (retrosplenial cortex and medial prefrontal cortex). Our approaches facilitate the comprehensive characterization of layer-specific cortico-cortical interactions, expanding wide-field calcium imaging as a powerful tool to investigate the layered organization of distributed brain dynamics.