Macroscopic (millimeter scale) functional clustering is a hallmark characteristic of motor cortex spatial organization in awake behaving mammals; however, almost no information is known about the functional micro-organization (∼100 μm scale). Here, we optically recorded intracellular calcium transients of layer 2/3 neurons with cellular resolution over ∼200-μm-diameter fields in the forelimb motor cortex of mobile, head-restrained mice during two distinct movements (running and grooming). We showed that the temporal correlation between neurons was statistically larger the closer the neurons were to each other. We further explored this correlation by using two separate methods to spatially segment the neurons within each imaging field: K-means clustering and correlations between single neuron activity and mouse movements. The two methods segmented the neurons similarly and led to the conclusion that the origin of the inverse relationship between correlation and distance seen statistically was twofold: clusters of highly temporally correlated neurons were often spatially distinct from one another, and (even when the clusters were spatially intermingled) within the clusters, the more correlated the neurons were to each other, the shorter the distance between them. Our results represent a direct observation of functional clustering within the microcircuitry of the awake mouse motor cortex.
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