Subsequently, we investigated the capability of the neurons to generate spontaneous activity in these networks by monitoring Ca2+ transients. We loaded the neurons with Fluo-5-AM and imaged Ca2+ transients in regions of interest (ROIs) on somas and neurites. We observed abundant spontaneous network activity, which was blocked by TTX (Fig 4G, 4I, 4C and S4A Fig) and re-established after wash out, except for a few ROIs (17% for both hiPSC- and hESC-derived neurons). Addition of bicuculline triggered a change in spontaneous activity (Fig 4H, 4J, 4D and S4B Fig) with some neurons increasing (>60% increase in 15% of hiPSC- and in 25% of hESC-derived neurons) and others decreasing frequency of events (>60% reduction in 50% of hiPSC- and in 40% of hESC-derived neurons). Subsequent addition of AP5 and DNQX blocked spontaneous calcium transients in all hiPSC-derived neurons, but remained in 10% of the hESC-derived neurons, suggestive of intrinsic pacemaker activity in a small group of neurons. The network also responded to electrical field stimulation with increased activity/amplitude correlated to the number of pulses given (Fig 4K and S4E Fig). These
