An Optimized Ex Vivo Protocol for Quantitative Electrophysiological Assessment of Neuromuscular Junctions and...

The neuromuscular junction (NMJ) is critical for muscle function, and its dysfunction underlies conditions such as sarcopenia and motor neuron diseases. Current protocols for assessing NMJ function often lack standardized stimulation parameters, limiting reproducibility. This study presents an optimized ex vivo method to evaluate skeletal muscle and NMJ function using the Aurora Scientific system, incorporating validated stimulation protocols for both nerve and muscle to ensure consistency. Key steps include tissue preparation in a low-calcium, high-magnesium solution to preserve NMJ integrity, determination of optimal muscle length, and sequential stimulation protocols to quantify neurotransmission failure and intratetanic fatigue. By integrating rigorous standardization, this approach enhances reproducibility and precision, providing a robust framework for investigating NMJ pathophysiology in aging and disease models. Key features • Dual stimulation modes enable direct muscle and indirect nerve stimulation to isolate NMJ-specific dysfunction. • Optimized stimulation parameters for nerve (5 mA, 0.8 ms pulse width) and muscle (300 mA, 0.2 ms pulse width) on mouse model. • Preservation of NMJ integrity through dissection in low-calcium, high-magnesium artificial cerebrospinal fluid (aCSF) and synthetic interstitial fluid (SIF). • Quantitative analysis of neurotransmission failure and intratetanic fatigue using standardized equations. This protocol is used in: Ageing Cell (2024), DOI: 10.1111/acel.14156.