Structure-dependent spontaneous calcium dynamics in cultured neuronal networks on microcontact-printed substrates

Microcontact printing (μCP) is widely used in neuroscience research. However, μCP yields reduced cell-substrate adhesion compared with directly coating cell adhesion molecules. Here, we demonstrate that the reduced cell-substrate adhesion caused by μCP, high seeding density, and the local restriction would separately contribute to more aggregated (neurons closer to each other in separate clusters) neuronal networks. Calcium recordings revealed that more aggregated networks presented fewer spontaneous calcium activity patterns, and were more likely dominated by synchronized network-wide calcium oscillation (network bursts). First, on a uniform microcontact-printed substrate, densely seeded neurons were reaggregated into a Petri dish-wide network consisting of small clusters, of which the calcium dynamics were dominated by network bursts. Next, further analysis revealed this dominance was maintained since its appearance, and the initiation and propagation of bursts in the small-cluster network shared a similar mechanism with that of homogeneous networks. Then, sparsely seeded neurons formed several networks with different aggregation degrees, in which the less clustered ones presented abundant time-varying subnetwork burst patterns. Finally, by printing locally restricted patterns, highly clustered networks formed, where dominant network bursts reappeared. These findings demonstrate the existence of structure-dependent spontaneous calcium dynamics in cultured networks on microcontact-printed substrates, which provide important insights into designing cultured networks by using μCP, and into deciphering the onset and evolution of network bursts in developmental nerve systems. ### Competing Interest Statement The authors have declared no competing interest.