To understand the excitability of the differentiated neurons (direct contact), a voltage step protocol was applied to record sodium and potassium currents (Fig 4A). Quantification of the peak size of these currents showed that hiPSC-derived neurons on average had larger currents than hESC-derived neurons (Fig 4B). The presence of voltage-gated Na+ and K+ channels suggests that these neurons can fire action potentials. Indeed, the majority of cells generated action potentials in response to step current injections (Fig 4C; >90% of hiPSC-derived neurons and 70% of hESC-derived neurons). Cells unable to generate action potentials showed smaller sodium responses and responded to a smaller voltage range (-40 to -10 mV) compared to the cells that could generate action potentials (-30 to +50 mV). 30% of hESC-derived neurons and 38% of hiPSC-derived neurons spontaneously elicited action potentials in current clamp (Fig 4D). To confirm the presence of functional synapses, we assessed spontaneous activity in the presence of the voltage-gated sodium channel antagonist TTX (Fig 4E). We observed that a proportion of the spontaneous events were blocked by the GABAA receptor antagonist bicuculline. Subsequent
