We found that the majority (88.8%) of neurons exhibited spontaneous synaptic activity (n = 27 neurons from hCSs differentiated from two hiPSC clones). This activity was completely abolished by the application of the AMPA-receptor antagonist NBQX (25 μM) and the NMDA-receptor antagonist D-AP5 (50 μM) (P = 0.001), suggesting that synaptic currents are due solely to the activation of glutamate receptors (Fig. 5c; Supplementary Fig. 7a). Further characterization of the synaptic activity revealed that TTX (1 μM) reduced the amplitude (n = 10 cells, P < 0.0001) and frequency of excitatory postsynaptic currents (EPSCs; n = 10, P = 0.002) by approximately 50% (Fig. 5d,e, Supplementary Fig. 7; hCSs differentiated from two hiPSC clones), suggesting that half of the events observed were evoked by action potential–dependent vesicle release and the remainder were due to spontaneous vesicle release.
