Background

The neuromuscular junction (NMJ), a specialized synapse bridging motor neurons and skeletal muscle fibers, is indispensable for voluntary movement and autonomic function. Its dysfunction underlies a spectrum of pathologies, including sarcopenia, amyotrophic lateral sclerosis (ALS), and myasthenic syndromes, which affect millions globally [1–3]. Traditional methodologies for assessing NMJ integrity and function have relied on in vivo electrophysiology, histological analyses, and electron microscopy (EM), which provide critical insights into synaptic ultrastructure [4,5]. Ex vivo systems, such as the Aurora Scientific apparatus, have emerged as powerful tools for isolating NMJ physiology by enabling direct muscle and indirect nerve stimulation [6]. Despite their utility, existing protocols exhibit critical limitations, notably inconsistent stimulation parameters (e.g., pulse width, frequency) and inadequate preservation of NMJ structure during tissue preparation, leading to interlaboratory variability and diminished reproducibility [7].

Recent studies employing ex vivo systems have underscored the importance of standardized parameters in quantifying neurotransmission failure (NF) and intratetanic fatigue (IF), key metrics of NMJ resilience. For instance, Rizzuto et al. (2015) demonstrated that precise stimulation protocols are essential for detecting NMJ dysfunction in the SOD1(G93A) mouse model of amyotrophic lateral sclerosis (ALS), highlighting how variable pulse widths and frequencies can obscure synaptic deficits [7]. Similarly, Pratt et al. (2015) reported that inconsistent stimulation currents in dystrophic (mdx) mice led to misinterpretations of NMJ fatigue, emphasizing the need for protocol standardization [8]. Moreover, the use of non-physiological dissection buffers often compromises NMJ morphology [9]. Franco et al. (2014) revealed that conventional saline solutions accelerate synaptic vesicle depletion, compromising NMJ integrity [10]. This issue is particularly relevant in aging models, where NMJ morphology is already vulnerable. Recent work by Wang et al. (2023) further demonstrated that low-calcium, high-magnesium dissection buffers mitigate synaptic transmission inhibition, preserving NMJ architecture in murine sarcopenia models [11]. These technical shortcomings could impede translational research, leading to conflicting outcomes in therapeutic trials for neuromuscular disorders [12,13].

To address these challenges, this protocol introduces an optimized ex vivo framework that integrates (1) validated stimulation parameters for nerve (5 mA, 0.8 ms pulse width) and muscle (300 mA, 0.2 ms pulse width) to maximize activation fidelity; (2) a low-calcium, high-magnesium dissection buffer to preserve NMJ ultrastructure by inhibiting spontaneous neurotransmitter release [10]; and (3) standardized equations for quantifying NF and IF, enabling direct comparisons across experimental models. By coupling these advancements with the Aurora Scientific system’s dual-mode lever and real-time data acquisition, this approach enhances precision in evaluating NMJ pathophysiology, as demonstrated in murine models of sarcopenia [14–16]. The protocol’s reproducibility and translational relevance are further evidenced by its application in identifying NMJ-specific deficits in aging mice [17].

This methodological advancement bridges a critical gap in neuromuscular research and provides a platform for mechanistic studies of NMJ-targeted therapies. It could accelerate the development of interventions for conditions where synaptic failure precedes muscle atrophy, such as early-stage ALS or chemotherapy-induced neuropathy in the murine model [18].