Fig. 3

Effect of the optogenetic stimulation of hMNs on the myotubes Ca²⁺ dynamic. a ReaChR was transduced into hMNs progenitors to enable ion channel activation by light allowing for control of neuronal activity by red light stimulation (590 nm). Optogenetic activation was confirmed by reading the Ca²⁺ response generated in myoblasts transduced with GCaMP6f by quantifying fluorescent intensity. b AAV2-hSyn-ReaChR-citrine expression in hMNs for optogenetic control and AAV2-CMV-GCaMP6f-WPRE-SV40pA expression in myotubes. Scale bars: 50 µm. c Representative schema of the optical stimulation protocol at 590 nm: 20 red light pulses, each pulse was 20 ms long. Optogenetics activation was confirmed by GCaMP6f in myotubes by quantifying fluorescent intensity. Scale bars: 50 µm. d-d’ Representative traces of normalized GCaMP6f fluorescence before and after red light stimulation (red bar) and quantification of Ca²⁺ oscillations. Data are represented as mean ± SEM (N = 3 independent experiments, 3 myofibers/well, 3 wells/condition). ANOVA with Sidak’s post hoc tests (****p < 0.0001; ns, not significant), with the recording times 60–120 and 120–180 compared to the time 0–60. e-e’ Effect of the addition of 150 µM tubocurarine before and after red light stimulation (red bar) and quantification of Ca²⁺ oscillations. Data are represented as mean ± SEM (N = 3 independent experiments, 3 myofibers/well, 3 wells/condition). ANOVA with Sidak’s post hoc tests (****p < 0.0001), with the recording times 60–120 and 120–180 compared to the time 0–60. f–f’ Effect of the addition of 10–50 µM range of glutamate before and after red light stimulation (red bar) and quantification of Ca²⁺ oscillations. Data are represented as mean ± SEM (N = 3 independent experiments, 3 myofibers/well, 3 wells/condition). ANOVA with Sidak’s post hoc tests (****p < 0.0001), with the recording times 60–120 and 120–180 compared to the time 0–60