Fig. 4: Characterization of electrophysiological activity and axonal outgrowth in hMNS exposed to IH.

a Timeline for hMNS under normoxia (Ctrl) and intermittent hypoxia (IH), detailing the IH exposure phases. b Schematic representation of optogenetic control in an MEA system using ChR2-transfected hMNS to activate ion channels under 473 nm optical stimulation. Created in BioRender. Zhang, W. (2025) https://BioRender.com/z81fuip. c Plate maps showing optically evoked spikes per min, with each node representing a well and color intensity indicating standardized spike events. d–f Quantification of activity parameters, including mean firing rate (d), number of bursts per min (e), and synchrony index (f) from 10 wells (n = 3 hMNS per well). g Activity heat maps showing optically evoked spike amplitude (µV), with each map representing a well. h Plate map illustrating the time of first optically evoked spike latency, with each node representing a well and color indicating latency duration. i Representative images of hMNS axon growth (TUJ1, gray; nuclei, blue) in Ctrl and IH groups. After formation on D28, including 4 days of IH exposure, hMNS were transferred to a Matrigel-coated dish and cultured for an additional 6 days to visualize neurite elongation. Three independent experiments was repeated independently with similar results. j and k Representative image (j) of reactive oxygen species (ROS) intensity in hMNS exposed to IH, with quantification (k) of ROS distribution radially across the spheroids. Data are presented as median with interquartile range. Statistical analysis: d, e, two-tailed Mann-Whitney test. f Two-tailed Student’s t test. ***p  <  0.001, **p <  0.01, *p  <  0.05. Source data are provided as a Source Data file.