The sensory cortices of the brain exhibit large-scale functional topographic organization, such as the tonotopic organization of the primary auditory cortex (A1) according to sound frequency. However, at the level of individual neurons, layer 2/3 (L2/3) A1 appears functionally heterogeneous. To identify if there exists a higher-order functional organization of meso-scale neuronal networks within L2/3 that bridges order and disorder, we used in vivo two-photon calcium imaging of pyramidal neurons to identify networks in three-dimensional volumes of L2/3 A1 in awake mice. Using tonal stimuli, we found diverse receptive fields with measurable colocalization of similarly tuned neurons across depth but less so across L2/3 sublayers. These results indicate a fractured microcolumnar organization with a column radius of ∼50 µm, with a more random organization of the receptive field over larger radii. We further characterized the functional networks formed within L2/3 by analyzing the spatial distribution of signal correlations (SCs). Networks show evidence of Rentian scaling in physical space, suggesting effective spatial embedding of subnetworks. Indeed, functional networks have characteristics of small-world topology, implying that there are clusters of functionally similar neurons with sparse connections between differently tuned neurons. These results indicate that underlying the regularity of the tonotopic map on large scales in L2/3 is significant tuning diversity arranged in a hybrid organization with microcolumnar structures and efficient network topologies.
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