Comparative anatomy of the mammalian neuromuscular junction

Abstract

The neuromuscular junction (NMJ)-a synapse formed between lower motor neuron and skeletal muscle fibre-represents a major focus of both basic neuroscience research and clinical neuroscience research. Although the NMJ is known to play an important role in many neurodegenerative conditions affecting humans, the vast majority of anatomical and physiological data concerning the NMJ come from lower mammalian (e.g. rodent) animal models. However, recent findings have demonstrated major differences between the cellular anatomy and molecular anatomy of human and rodent NMJs. Therefore, we undertook a comparative morphometric analysis of the NMJ across several larger mammalian species in order to generate baseline inter-species anatomical reference data for the NMJ and to identify animal models that better represent the morphology of the human NMJ in vivo. Using a standardized morphometric platform ('NMJ-morph'), we analysed 5,385 individual NMJs from lower/pelvic limb muscles (EDL, soleus and peronei) of 6 mammalian species (mouse, cat, dog, sheep, pig and human). There was marked heterogeneity of NMJ morphology both within and between species, with no overall relationship found between NMJ morphology and muscle fibre diameter or body size. Mice had the largest NMJs on the smallest muscle fibres; cats had the smallest NMJs on the largest muscle fibres. Of all the species examined, the sheep NMJ had the most closely matched morphology to that found in humans. Taken together, we present a series of comprehensive baseline morphometric data for the mammalian NMJ and suggest that ovine models are likely to best represent the human NMJ in health and disease.

Keywords: NMJ-morph; comparative anatomy; mammalian; neuromuscular junction; synapse.

FIGURE 1

Gross anatomy of hindlimb muscles in cat, dog, sheep and pig. Representative photographs illustrating gross muscle morphology in each species. The proximal end of the limb is on the right‐hand side of the image. A block of tissue containing full‐length muscle fibres (from origin to insertion) was sampled from each of the selected muscles. Note the species‐specific absence of certain muscles—dog lacks soleus, and sheep and pig lack peroneus brevis. EDL, Extensor Digitorum Longus; PL, Peroneus Longus; PB, Peroneus Brevis; SOL, Soleus. Scale bar = 2 cm

FIGURE 2

Heterogeneity of mammalian NMJs. Representative confocal micrographs of ‘average’ NMJs from each species (ranked according to body mass). The selected NMJ images most closely represent the ‘average’ morphology (size, shape) across the limb muscles sampled (EDL, soleus, peronei). Mouse NMJs are typically ‘pretzel’‐shaped; human NMJs have a ‘nummular’ morphology. Of the species represented, sheep and pig NMJs appear most similar to human NMJs. Images have been pseudo‐coloured for display purposes; all analysis has been performed on grayscale images. α‐BTX (α‐bungarotoxin), acetylcholine receptors (magenta); SV2/2H3 = synaptic vesicle and neurofilament (green). Scale bar = 20 µm (across all images)

FIGURE 3

NMJ‐morph analysis reveals inter‐species variations in NMJ morphology. Comparison of ‘average’ NMJ morphology in each species for a range of pre‐ and post‐synaptic variables. Data for each morphological variable are pooled by muscle identity (EDL, PL, PB, S) for each species, and statistical comparison is made with the human NMJ. Boxes contain the mean (+) and median (line) values for each NMJ variable and extend from the 25th to 75th percentiles, with the whiskers representing the maximum and minimum values. Compared to humans, mouse and dog NMJs are significantly larger, with equivalent differences in axon calibre. In contrast, sheep and pig NMJs are the most similar to humans, with the majority of NMJ variables showing no significant differences between the species (see also Table S1). In total, 5,385 individual NMJs were analysed [cat: N = 3 animals, n = 12 muscles, 465 NMJs; dog: N = 3, n = 9, 341 NMJs; sheep: N = 3, n = 9, 313 NMJs; pig: N = 3, n = 9, 446 NMJs; mouse: N = 3, n = 24, 960 NMJs; human: N = 21, n = 72, 2860 NMJs. Mouse and human data from Jones et al., 2017]. One‐way ANOVA with Dunnett's post hoc analysis (for parametric variables) and Kruskal–Wallis test with Dunn's post hoc analysis (for non‐parametric variables) *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

FIGURE 4

NMJ‐morph analysis of inter‐muscle variations in NMJ morphology. The pooled data in Figure 3 have been segregated to demonstrate ‘average’ NMJ morphology for individual muscles (EDL, PL, PB, S). Compared with the marked inter‐species variation, the differences between individual muscles are much less pronounced

FIGURE 5

No significant relationship between NMJ morphology and muscle fibre diameter. Scatterplots demonstrating the correlation between size‐related NMJ variables and muscle fibre diameter. Each data point represents an individual muscle (mean of 40 NMJs/40 muscle fibres). Despite modest correlations for some variables, there is no significant relationship between NMJ size and muscle fibre diameter across the species investigated. Mouse = purple circles; cat = green squares; dog = blue triangles; sheep = orange diamonds; pig = black stars; Human = grey circles. Correlation coefficients (Pearson) are for individual species (within the box) and all species pooled (above the box). *p < 0.05; **p < 0.01; ***p < 0.001

FIGURE 6

Schematic overview of NMJ morphology, muscle fibre diameter and body size. Schematic diagram illustrating the relationship between NMJ size, muscle fibre diameter and body weight. For each species, the mean values for AChR area and muscle fibre diameter are depicted (Table 1) to provide an accurate visual representation of inter‐species differences/similarities. Of the species investigated, the starkest contrast is between the two smallest animals; the mouse has the largest NMJs on the smallest muscle fibres, and the cat has the smallest NMJs on the largest muscle fibres. Sheep and pig are most similar to human, with sheep and human bearing the closest resemblance. Overall, there is no relationship between NMJ size, muscle fibre diameter and body mass; the ratio between NMJ size and muscle fibre diameter is therefore unique to each species. Scale bar = 20 µm