A reproducible balance in excitatory and inhibitory activity will facilitate the study of cortical networks and neuronal communication. We characterized and quantified the number of pre- and post-synaptic terminals. End-stage direct cultures demonstrated the presence of 50–55% GABAergic (VGAT) and 50–67% glutamatergic (VGLUT1) pre-synaptic terminals and 17–27% of neurons expressed GAD65/67, which is representative of human cortical development [46]. A subset of the glutamatergic post-synaptic HOMER1-positive puncta appeared VGLUT1-negative and may express other glutamatergic transporters like VGLUT2 [47, 48]. Since indirect cultures did not receive AraC treatment, we suspect that the implementation of AraC treatments increases maturity levels of these cultures. The hPSC-derived neuronal cultures manifested functional neurons by presence of evoked and spontaneous action potentials as well as miPSCs and mEPSCs mediated by GABA and NMDA/AMPA receptors, respectively in both co-cultures. The peak size of sodium currents showed that hiPSC-derived neurons had on average larger currents than hESC-derived neurons, suggestive of a higher maturation state, as reported previously [49]. When spontaneous calcium transients were measured, few ROIs increased activity upon TTX wash-out, likely due to a rebound effect of
