Fig. 4: Functional characterization of the soNMJ model.

a The neuromuscular cultures were incubated with Fluo8-AM at day 75 for visualizing calcium transients with a spinning disk confocal microscope. Representative frames are shown for both spontaneous and 10 µM curare conditions. Individual neurons and muscle cells were identified by their morphology, and their calcium transients were plotted separately. Administration of 10 μM curare blocked the calcium activity in the skeletal muscle fibers but not in the neurons, supporting that the muscle contraction was driven by the neurons through the NMJs. Scale bars: 50 µm. H1: N = 2; XM001: N = 1. Source data are provided as a Source Data file. b Current clamp recording from a neuron transduced with an AAV encoding hSYN:NLS-GFP to label neuronal nuclei. The static current injection caused spontaneous action potential firing, while 10 Hz stimulation with brief square pulses revealed the potential for the repetitive firing of the neurons. Inset shows the first action potential. c Current clamp recording from a neuron transduced with an AAV encoding hSYN:ChR2(H134R)-GFP. The neuron reliably fired action potentials in response to 0.5 ms blue light (470 nm) flashes delivered at 2 Hz. Inset shows the first light-evoked action potential. d schematic representation of the optogenetic experiments. Neurons transduced with an AAV encoding hSYN:ChR2(H134R)-GFP were stimulated with blue light to drive the contraction of the skeletal muscle cells. e Immunofluorescence image of day 75 soNMJ culture model showing neurons expressing ChR2-GFP 3 weeks post-transduction with an AAV encoding hSYN:ChR2(H134R)-GFP. Scale bars: 100 µm. H1: N = 1; XM001: N = 2. Source data are provided as a Source Data file. f Optogenetic analysis of the soNMJ model at day 75. Every stimulation of ChR2-GFP⁺ neurons by 470 nm light pulse resulted in the concomitant contraction of the skeletal muscle cells. Exposure to 10 μM curare blocked the neural transmission at the NMJs resulting in the inhibition of muscle contraction even upon optogenetic stimulation. The muscle contraction response to blue light stimulation of the neurons was restored by washing out curare.