Characterization of neural bundle-spheroid network function. (a) Fluorescence image of neuronal spheroids. Green and blue indicate neurites (β-tubulin) and nuclei (DAPI), respectively. (b) Bundle formation in neurospheroid networks; each neural bundle is composed of several neurite fibers. (c) Schematic depiction of the method used to assess signal transmission within a neurospheroid network. One neuronal spheroid was electrically stimulated and the electrical signal was detected by imaging Ca2+ in another spheroid. Red rectangle indicates the region of interest (ROI). (d) An optical microscope image of an electric stimulator on the nerve-like bundle and the ROI at which Ca2+ signals were recorded. Black arrowheads indicate a neural bundle. (e) A fluorescence microscope image of Ca2+ signals (dotted circles) detected in the ROI shown in (d). The white and black arrowheads indicate satellite spheroid and bundle formation in deep HCWN (f) Electrical transmission through the nerve-like bundle without (left) and with (right) TTX. Signals were detected at all sites prior to TTX treatment, and all signals were eliminated by TTX.