At 10 DIV, networks bearing 70 to 130 neurons (20 to 30 neurons) were randomly selected from each coverslip for the single-bond, triple-bond, and merged (no-bond) patterns. Cultured neurons were loaded with a fluorescence calcium indicator Cal-520 AM (AAT Bioquest) by first rinsing the cells in Hepes-buffered saline (HBS) containing 128 mM NaCl, 4 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM d-glucose, 10 mM Hepes, and 45 mM sucrose and subsequently incubating in HBS containing 2 μM Cal-520 AM and 0.01% Pluronic F-127 for 30 min at 37°C. The cells were then rinsed in fresh HBS and incubated for an additional 10 min to complete the deesterification of the loaded AM dyes. The samples were imaged on an inverted microscope (IX83, Olympus) equipped with a 20× objective lens (numerical aperture, 0.75), a light-emitting diode light source (Lambda HPX, Sutter Instrument), a scientific complementary metal-oxide semiconductor camera (Zyla 4.2, Andor), and an incubation chamber (Tokai Hit). All recordings were performed at 37°C. Three networks were selected from a coverslip, and for each network, fluorescence imaging was performed for 20 min at 10 frames/s on the Solis software (Andor). The thickness of the neurite bundle that interconnects the modules was estimated from phase-contrast micrographs based on the brightness profile.

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