the presence of synchronized neuron cluster inside the hMGEOs (Figure 6F). Treating hMGEOs with bicuculline, an antagonist of GABAA receptors, robustly enhanced synchronization of calcium surges in the areas of the organoids, suggesting that GABAergic inhibition controls neuronal synchronization (Figure 6G, n=2 hMGEOs). Furthermore, histological analysis of hMGEOs revealed the presence of an extensive network of vesicular GABA transporter (vGAT) puncta, marking presynaptic structures of GABAergic synapses. Gephyrin, the postsynaptic scaffold protein essential for inhibitory synapses, showed similar subcellular distributions adjacent to vGAT puncta (Figure 6H), confirming inhibitory synaptogenesis in hMGEOs. We also examine the electrophysiological properties of cells in hMGEOs by whole-cell patch-clamp recording (Figure 6I, see STAR methods for details) that revealed that 8 of 14 recorded cells exhibited APs (Figure 6J). Increasing injection current resulted in an increase in firing frequency of the recorded neurons (Figure 6K; from 4.2 ± 1.5 Hz, +5 pA to 16.8 ± 2.9 Hz, +25 pA, n=7 cells). TTX completely abolished APs in all tested cells (Figure 6L, n=3). The neuronal morphologies of recorded cells were confirmed by injecting biocytin into the cells during the patch-clamp recordings (Figure 6I).
