Fig. 3. Simultaneous imaging reveals functional connectivity of single neurons with large-scale cortical networks.

a, Example average two-photon field-of-view showing pyramidal neurons in a P17 mouse during simultaneous imaging. Colored circles highlight cells for panels (b-f). Scale bar is 20 μm. b, Example mesoscopic ΔF/F images with simultaneous ΔF/F trace and deconvolved spike probability for cell 3 from (a). c, Schematized procedure for calculating cell-centered networks (CCNs) and significance maps. d, Left: example CCNs for the three cells indicated in (a). Middle left: corresponding significance maps. Middle right: significance maps overlaid with an anatomical parcellation based on the Allen CCFv3. Right: significance maps overlaid with a functional parcellation calculated for that mouse. e, Illustration of the functional parcellation with regions labelled based on correspondence with the anatomical parcellation. Plot below shows the conditional entropy of significance maps given the anatomical or functional parcellation for three mice. Lower values indicate better fit. Mean±SEM: Allen CCFv3: H=0.54±0.01, 0.56±0.01, 0.53±0.02; functional: H=0.41±0.01, 0.39±0.01, 0.42±0.02; p < 0.001, paired two-tailed t-test for each mouse, n = 238, 64, 41 significance maps. f, Activity index calculated from all significance maps for a single animal using the functional parcellation. Higher values indicate a large number of pixels that are significantly co-active with each cell. Cells are clustered into three groups (see Methods). Arrows on the left indicate rows corresponding to the cells in (a). g, Averages of the three clusters in (f) with parcels colored by their activity index. h, Schematized two-photon field-of-view, same as in (a), with pixels colored to indicate membership of individual cells in the three clusters shown in (g). Scale bar is 20 μm.