Abstract
The neuromuscular junction (NMJ) is the highly specialised peripheral synapse formed between lower motor neuron terminals and muscle fibres. Post‐synaptic acetylcholine receptors (AChRs), which are found in high density in the muscle membrane, bind to acetylcholine released into the synaptic cleft of the NMJ, thereby enabling the conversion of motor action potentials to muscle contractions. NMJs have been studied for many years as a general model for synapse formation, development and function, and are known to be early sites of pathological changes in many neuromuscular diseases. However, information is limited on the diversity of NMJs in different muscles, how synaptic morphology changes during development, and the relevance of these parameters to neuropathology. Here, this crucial gap was addressed using a robust and standardised semi‐automated workflow called NMJ‐morph to quantify features of pre‐ and post‐synaptic NMJ architecture in an unbiased manner. Five wholemount muscles from wild‐type mice were dissected and compared at immature (post‐natal day, P7) and early adult (P31−32) timepoints. The inter‐muscular variability was greater in mature post‐synaptic AChR morphology than that of the pre‐synaptic motor neuron terminal. Moreover, the developing NMJ showed greater differences across muscles than the mature synapse, perhaps due to the observed distinctions in synaptic growth between muscles. Nevertheless, the amount of nerve to muscle contact was consistent, suggesting that pathological denervation can be reliably compared across different muscles in mouse models of neurodegeneration. Additionally, mature post‐synaptic endplate diameters correlated with fibre type, independently of muscle fibre diameter. Altogether, this work provides detailed information on healthy pre‐ and post‐synaptic NMJ morphology from five anatomically and functionally distinct mouse muscles, delivering useful reference data for future comparison with neuromuscular disease models.
Highlights
The neuromuscular junction (NMJ) is a peripheral synapse formed between lower motor neurons and skeletal muscle fibres that has been studied in many different species, including humans (Nyström, 1968; Kuno et al, 1971; Cramer and Van Essen, 1995; Prakash et al, 1996; Campbell and Ganetzky, 2012; Sakowski et al, 2012; Hammond et al, 2016; Jones et al, 2017; Grice et al, 2018)
It is well known that NMJ size and morphology are predictive of function, and that muscle fibre types differ in their functional requirements of neurotransmission (Kuno et al, 1971; Harris and Ribchester, 1979; Waerhaug and Lømo, 1994; Ribchester et al, 2004; Jones et al, 2016)
NMJ morphologies at both timepoints were correlated with fast twitch muscle fibre percentages
Summary
The neuromuscular junction (NMJ) is a peripheral synapse formed between lower motor neurons and skeletal muscle fibres that has been studied in many different species, including humans (Nyström, 1968; Kuno et al, 1971; Cramer and Van Essen, 1995; Prakash et al, 1996; Campbell and Ganetzky, 2012; Sakowski et al, 2012; Hammond et al, 2016; Jones et al, 2017; Grice et al, 2018). This process is thought to refine peripheral nerve architecture, and is driven by activity, inter-nerve competition and reciprocal nerve−muscle−glia signalling (Favero et al, 2012; Turney and Lichtman, 2012; Smith et al, 2013b) This is accompanied by significant changes in the post-synapse to maintain the close proximity of motor terminals to AChRs. For example, the density and number of AChRs increase, and the morphology of AChR clusters transforms from a simple, plaquelike shape to a complex, multi-perforated, pretzel-like structure (Marques et al, 2000). Performing analyses in five different wholemount muscles with varying fibre type populations, this novel morphometric approach was used to assess the influence of post-natal maturation, fibre type and fibre diameter on pre- and post-synaptic NMJ morphology, as well as post-natal synaptic development/growth
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