Efficient Functional Maturation of hMGEOs(A) Schematic view of the methods for calcium imaging of intact hMGEOs.(B) Representative image showing cells expressing hsyn-GCaMP6s in intact 42 day old hMGEOs. The single cell tracings of calcium transient (region of interest (ROI) indicated on top) are shown, which are blocked by application of TTX (1 μM). Scale bar, 25 μm.(C) Representative image of area-scale calcium imaging in intact 45 day old hMGEOs. The synchronized calcium surges are indicated with arrows. Time is shown in min:sec. Scale bar, 100 μm.(D and E) Calcium imaging of synchronized area (C) at single cell level. ROIs are indicated (D) and tracings of single cell calcium surges are shown (E). Time is shown as min: sec. Scale bar, 25 μm.(F) Synchronization matrix of calcium surges from recorded single neurons in intact 45 day old hMGEOs.(G) Area-scale calcium imaging reveals bicuculline disinhibition enhanced area synchronization of calcium surges in intact hMGEOs (47 day old). Arrows in bicuculline treated group indicate the synchronized calcium surge, while there is no synchronization in the same area before bicuculline treatment. Time is shown as min:sec. Scale bar, 100 μm.(H) Immunostaining for pre-synaptic protein vGAT and post-synaptic protein gephyrin in 47 day old hMGEOs section. Scale bar, 5 μm.(I) Diagram showing slice patch-clamp and identification of the neuronal morphology of the recorded cell by filling with biocytin. Scale bar, 25 μm.(J) Representative voltage traces of current-clamp recordings of a cell in hMGEO slice in response to current steps (-5 pA, +5 pA, and +25 pA from -60 mV, 1 s).(K) Graph depicting the firing frequency of the recorded cells from hMGEOs plotted against injected current (n=7 cells). Mean ± SE are shown.(L) Representative image of APs of a cell in hMGEO slice before and during application of TTX (1 μM).See also Figure S5; Movie S1-S3.
