Fig. 1

Generation of an in vitro functional muscle-nerve coculture system at DIV 15. a Representative images of the muscle-nerve coculture where hMNs neurites made contacts with myotubes (black triangle in the phase contrast image). b hMNs express Islet and Tuj1. c, d/d’ Myotubes express MF20 and SAA. e/e’–g The coculture system expresses SYN (right and lower panels represent cross sections of myotubes in the orthogonal view), SMI32, SNAP25 and presents AChR clusters. a–g Scale bars: 100 µm. h Quantification of Islet1⁺ hMNs and quantification of fusion index in myotubes. Data are represented as mean ± SD (N = 3 independent experiments, each performed in triplicate n = 3). Mann–Whitney test (ns., not significant). i Disruption of myotubes contractions after treatment with 5 µM TTX and 150 µM tubocurarine compared to control condition with no treatment. Each drug was added at DIV 15, the recording of myotubes contractions was performed before treatment (baseline) and 30 min after treatment. j Disruption of Ca²⁺ oscillations in myotubes after treatment with 5 µM TTX and 150 µM tubocurarine compared to control condition with no treatment. For Ca²⁺, measurement cells were stained with 2 µM Cal520 dye the day of the recording. Each drug was added at DIV 15, the recording of Ca²⁺ oscillations was performed before treatment (baseline) and 30 min after treatment. i, j Data are represented as mean ± SEM (N = 3 independent experiments, each performed in triplicate n = 3). ANOVA with Sidak’s post hoc tests (****p < 0.0001)