Background

The neuromuscular junction (NMJ) is the chemical synapse enabling communication between motor neurons and skeletal muscle fibers. 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., 2017). At the NMJ, the action potential running along the motor axon invades the nerve terminal (presynaptic bouton) and induces the fusion of synaptic vesicles with the presynaptic membrane. This triggers the release of acetylcholine (ACh), the neurotransmitter that binds the nicotinic ionotropic ACh receptors (nAChRs) on the postsynaptic muscle fiber. Upon ACh binding to nAChRs, a postsynaptic action potential spreads out along the muscle fiber causing Ca²⁺ release from the sarcoplasmic reticulum into the cytosol, thereby inducing muscle contraction. At variance from central synapses, NMJs are not protected by anatomical barriers, like the blood brain barrier or the blood nerve barrier, and are exposed to the action of various pathogenic molecules, including chemicals, toxins from plant, animal and bacteria as well as autoantibodies raised against NMJ antigens. Depending on the way they act, these agents produce distinct kinds of injury to the NMJ, eventually leading to impairment of muscle contraction.

The ‘mouse phrenic nerve hemidiaphragm assay’ (MPN) is an established model to study ex vivo NMJ function and offers a valuable tool to investigate the mechanism of action of toxins and molecules that produce NMJ alteration. In addition, MPN can be used to evaluate skeletal muscle contractility and measure diaphragmatic muscle force elicited by electrical stimulation of its phrenic nerve, so providing a model which recapitulates the complexity of the neuromuscular system in a more accessible and isolated environment.

The MPN provided fundamental insights to tackle the mechanism of action of Botulinum Neurotoxins (BoNTs), and it is currently used in many laboratories worldwide to perform qualitative/quantitative analysis of BoNTs. Its employment allows a significant refinement and reduction in the use of animals and results are in good agreement with the classical mouse lethality bioassay (Rasetti-Escargueil et al., 2011; Bigalke and Rummel, 2015).

For this, and because the hemidiaphragm-phrenic nerve intoxicated by BoNTs closely mimics the failure of respiratory muscles occurring in vivo, the MPN is presently listed in the European Pharmacopeia as an alternative method to the mouse bioassay for assaying BoNT/A lots for human use (https://ntp.niehs.nih.gov/iccvam/suppdocs/feddocs/eur/eph_botat-508.pdf). This system is also a valuable tool to test new BoNTs (Zanetti et al., 2017) and the neutralizing potency of antibodies and inhibitors (Rasetti-Escargueil et al., 2011; Azarnia Tehran et al., 2015; Beske et al., 2017).

Besides BoNTs, the MPN can be used to study many other bioactive substances, comprising toxins like phospholipases, myotoxins and complete venoms, neuroactive molecules like peptides, lipids and drugs (Rigoni et al., 2005; Caccin et al., 2006; Bercsenyi et al., 2014; Yan et al., 2014; Caccin et al., 2015), or to measure muscle force in diaphragms from mice pre-treated with autoantibodies involved in myasthenic syndromes (Klooster et al., 2012) or from animal models of neuromuscular diseases (Nascimento et al., 2014).

Here, we describe a very detailed protocol to successfully dissect the mouse diaphragmatic muscle with both the phrenic nerves completely functional. This is a remarkable advantage as it allows obtaining two autonomously innervated hemidiaphragms to be independently used, either as an internal control or to increment the number of experimental data. The small volume of muscle bath-chambers, the possibility of finely control bath concentration of substances used, the easy manipulability of experimental conditions (temperature, washes, etc.) and the possibility to use the muscles for further analysis (immunofluorescence, Western blot, etc.), represent significant advantages as well.