Electrical stimulation was delivered via platinum electrodes positioned 3 mm away from each other across the neuromuscular tissue. They were controlled by an Arduino circuit delivering 12-V square inputs. To activate the MNs, they were stimulated by adding glutamic acid (0.1 mM) to the culture medium. For the excitotoxicity experiment, excess glutamic acid (5 mM) was added to the culture medium for 7 days. Before the measurement of the muscle contraction force, this high concentration of glutamic acid was rinsed away with phosphate-buffered saline (PBS) and then replaced with a low concentration of glutamic acid (0.1 mM). For simulation of a neurotoxin added to a human NMJ model, TTX (final concentration, 1 μM) was added to the culture medium and the muscle contraction force was measured. Then, the TTX was rinsed away with PBS and replaced with a low concentration of glutamic acid (0.1 mM).

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