Development of a novel technique for the measurement of neuromuscular junction functionality in isotonic conditions

Introduction: The neuromuscular junction (NMJ) is a chemical synapse responsible for converting electrical pulses generated by the motor neuron into electrical activity in muscle fibers, and is severely impaired in various diseases, such as Amyotrophic Lateral Sclerosis (ALS). Here, we proposed a novel technique to measure, for the first time, NMJ functionality in isotonic conditions, which better reflect muscle physiological activity. Methods: We employed the in-situ testing technique, studied a proper placing of two pairs of wire electrodes for nerve and muscle stimulation, developed an extensive testing protocol, and proposed a novel parameter, the Isotonic Neurotransmission Failure (INF), to properly capture the impairments in neurotransmission during isotonic fatigue. We employed wild-type mice to assess the feasibility of the proposed technique, and the ALS model SOD1G93A mice to demonstrate the validity of the INF. Results: Results confirmed the measurement accuracy in term of average value and coefficient of variation of the parameters measured through nerve stimulation in comparison with the corresponding values obtained for membrane stimulation. The INF values computed for the SOD1G93A tibialis anterior muscles pointed out an impairment of ALS mice during the isotonic fatigue test, whereas, as expected, their resistance to fatigue was higher. Conclusions: In this work we devised a novel technique and a new parameter for a deep assessment of NMJ functionality in isotonic conditions, including fatigue, which is the most crucial condition for the neuronal signal transmission. This technique may be applied to other animal models, to unravel the mechanisms behind muscle-nerve impairments in other neurodegenerative pathologies.