To examine whether our organoid culture system produced functionally active neurons, we utilized the genetically encoded calcium indicator GCaMP6s under human synapsin I promoter (Figure 6A). Neurons inside hMGEOs differentiated for 40∼50 days already displayed calcium transient, which was blocked by adding Na+ channel blocker tetrodotoxin (TTX) (Figure 6B; Movie S1), indicating these calcium oscillations were neuronal activity-dependent. Importantly, when we performed calcium imaging over larger fields of view, we found that hMGEOs showed areas with highly synchronized calcium surges (Figure 6C, n=3 hMGEOs, with 1 to 2 synchronized areas observed for each hMGEO), representing the functional neuronal networks. The synchronized pattern of neuronal activity within the organoids was further investigated by performing higher magnification imaging where individual neurons displayed calcium surges simultaneously (Figure 6D and 6E). Synchronization matrix analysis based on calcium spike patterns of individual neurons further revealed 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
