To examine the neuronal activity of migrating interneurons, we generated hfMCOs by fusing hsyn-GCamp6s-labeled hMGEO with hsyn -RFP-labeled hCO, and measured the calcium transient at the hCO side of hfMCOs. A robust calcium transient at hCO side was observed with 80.40 ± 7.80% of detected cells (n=6 hfMCOs, mean ± SD, 85 cells in total) and with active calcium surges as early as 12 dpf (Figure 7M), indicating that migrating interneurons developed a normal neuronal activity. Furthermore, neurons from hMGEO maintained the GABAergic identity during migration, as revealed by the expression of GABAergic marker vGAT (Figure 7N and Figure S7E). Importantly, migrated interneurons displayed a normal neuronal morphology and formed excitatory postsynaptic densities both at the soma and neurite areas (Figure 7O), further confirming their integration into local neuronal network of hCOs. Together, our data demonstrate that hfMCOs serves as a model for human interneuron migration with a 3-D cellular environment.
