Background

Neurotransmission is the physiological process by which neurons transfer information to target cells on a rapid time scale (usually < 1 msec). The structure mediating this communication is the synapse, a specialized structure formed either between neurons (a pre- and a post-synaptic neuron) or between a neuron (presynaptic neuron) and an effector cell (post-synaptic cell). Neuromuscular junction (NMJ) is the chemical synapse enabling communication between motor neuron and skeletal muscle fiber. This is the best characterized synapse and most of the knowledge on maturation, structure and function of synapses derives from its study (Li et al., 2016). At the NMJ, the action potential running along the motor axon invades the nerve terminal (presynaptic bouton) and induces the opening of voltage-gated calcium channels. The ensuing Ca²⁺ influx in presynaptic nerve terminal triggers (approximately in 0.3 µsec (Kuffler et al., 1984)) the fusion with the presynaptic membrane of about 100 synaptic vesicles from a ready to release pool (10-20% of all vesicles) (Del Castillo and Katz, 1954; Denker and Rizzoli, 2010). Around 1,000 acetylcholine (ACh) molecules per vesicle diffuse in the synaptic cleft (Kuffler and Yoshikami, 1975) and, in about 0.5 msec, bind to the nicotinic ACh Receptors (nAChRs) on the postsynaptic muscle fiber membrane. nAChRs are ionotropic ligand-gated Na⁺/K⁺ channels which open upon ACh binding and cause a local depolarizing potential of the postsynaptic membrane (end-plate) by mediating a large inward flux of Na⁺ (and a smaller outward flow of K⁺). This local depolarization is named evoked End-Plate Potential (eEPP) (or Evoked Junction Potential). In mice, the resting membrane potential of a skeletal muscle fiber lies around -75 mV and the eEPP has an amplitude of ~15-30 mV (depending on muscle type). When the eEPP amplitude is sufficiently high to reach or overcome action potential threshold, voltage-gated Na⁺ channels open thus triggering an action potential into the muscle fiber, which ultimately spreads out along the sarcolemma and invades muscle fiber T-tubules. Here, an excitation-contraction molecular machinery transduces this electric signal into the cytosolic release of Ca²⁺ from the sarcoplasmic reticulum, leading to muscle fiber contraction (Figure 1).


Figure 1. Mechanism of muscle fiber contraction. Acetylcholine (ACh), released by synaptic vesicle (SVs) fusing with the motor axon terminal membrane, binds to post synaptic nicotinic ACh Receptors, ionotropic cation channels that upon binding allow the leakage of cations (Na⁺ inward, K⁺ outward), leading to a local depolarization of the sarcolemmatic membrane (eEPP). When depolarization is sufficiently large to overcome the voltage threshold (red dotted line), voltage gated Na⁺ channels get open and trigger a post synaptic action potential (AP) spreading along the sarcolemma and invading the T-tubules (invagination of the sarcolemma within the muscle fiber). Dihydropyridine (DHP) receptors sense this membrane depolarization and stimulate the opening of Ryanodine Receptors (RyR) on the sarcoplasmic reticulum which release Ca⁺⁺ into the cytosol eliciting muscle contraction. µ-conotoxin inhibits voltage gated Na⁺ channels thus allowing to record membrane depolarization due to the opening of the sole nicotinic AChR, i.e., the eEPP.

Random fusion of synaptic vesicles also takes place in the absence of a presynaptic action potential thereby inducing a very small (~0.4 mV) depolarization of the end-plate, which is not sufficient to trigger muscle contraction. This spontaneous activity is called ‘miniature End-Plate Potential’ (mEPP) and, according to the quantal hypothesis, it is generated by the release of a single synaptic vesicle (Katz, 2003).

NMJ is easily accessible to many kinds of experimental manipulation. Since the ‘50s’ electrophysiology applied at the NMJ provided seminal discoveries on basic aspects of synaptic transmission (Augustine and Kasai, 2007). Thereafter, a continuous development in technics and animal models paved the way to sophisticate investigation of pathological alterations occurring at the NMJ in neuromuscular diseases, including myasthenic syndromes and peripheral neuropathies, as well as neuroparalytic syndromes caused by animal (Duchen et al., 1981; Duregotti et al., 2015a) and bacterial toxins (Colasante et al., 2013, Pirazzini et al., 2014).

We describe here a detailed protocol to evaluate NMJ functionality in murine muscle-nerve preparations. The method is based on the intracellular recording of spontaneous mEPPs and nerve-evoked EPPs in muscle fibers of soleus nerve-muscle preparations, thus allowing accurate investigation of NMJ functionality at a single synapse resolution (Tremblay et al., 2017). We have recently used this method to test engineered botulinum neurotoxins and to assay the efficacy of novel putative antitoxins (Pirazzini et al., 2014; Zanetti et al., 2017). In addition, we successfully employed this technique to study NMJ nerve degeneration and to test molecules promoting its regeneration (Duregotti et al., 2015b; Negro et al., 2017; Rigoni and Montecucco, 2017).

This procedure represents a basic technique that can be easily adopted to investigate NMJ activity in mouse models of any neuromuscular diseases, including peripheral neuropathies, motor neuron disorders and myasthenic syndromes.