In order to explore the nature of the recorded calcium oscillations, we performed additional experiments. First, treatment of co-cultures with tetrodotoxin (TTX), an inhibitor of voltage-gated sodium channels, completely blocked all calcium oscillations (Fig. 4a), suggesting that calcium oscillations are a secondary effect to voltage-gated sodium channel mediated action potentials. Moreover, this observation excludes astrocyte-induced calcium oscillations in our model39. Furthermore, we measured spontaneous postsynaptic currents (sPSCs, Fig. 4b) in co-cultures compared to monocultures. The spontaneous electrical activity represented synaptic events because the burst firing was completely abolished in 5 out of 7 cells by combined application of the competitive AMPA/kainate receptor antagonist CNQX (20 μM) and competitive NMDA receptor antagonist DAP5 (50 μM) in the recording buffer during 5 minutes (data not shown). Co-cultures displayed more bursts of postsynaptic events (86.4% of cells) than monocultures (7.9% of cells) 5 weeks after final plating (Fig. 4c). More specifically, monocultures were mainly inactive (47.4% of cells compared to only 4.5% in co-cultures) or only showed sparse activity (44.7% of cells compared to 9.1% in co-cultures). These patch clamp experiments confirm that
