The recordings were analyzed offline using either ImageJ (National Institutes of Health) or NETCAL (36). For analyses on ImageJ, 60 neurons (15 per module) were randomly selected, and their somas were manually defined as regions of interest (ROIs). Relative fluorescence intensity of cell i, (Fi(t) − F0)/F0 = fi(t), with Fi and F0 being the fluorescence intensity and the background fluorescence, respectively, was then analyzed as described previously (14). Alternatively, the recordings were analyzed using the NETCAL software. For this method, the image of the average fluorescence of the recording was first created and the background (areas with no change in fluorescence) was removed. Neuronal somas were next automatically detected and ascribed as ROIs. An average of about 90 neurons was identified per network, which were uniformly distributed across modules. The average fluorescence in each ROI along the recording was then extracted, and fi(t) was finally obtained as described above.

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