A method to identify, dissect and stain equine neuromuscular junctions for morphological analysis.

Morphological study of the neuromuscular junction (NMJ), a specialised peripheral synapse formed between a lower motor neuron and skeletal muscle fibre, has significantly contributed to the understanding of synaptic biology and neuromuscular disease pathogenesis. Rodent NMJs are readily accessible, and research into conditions such as amyotrophic lateral sclerosis (ALS), Charcot-Marie-Tooth disease (CMT), and spinal muscular atrophy (SMA) has relied heavily on experimental work in these small mammals. However, given that nerve length dependency is an important feature of many peripheral neuropathies, these rodent models have clear shortcomings; large animal models might be preferable, but their size presents novel anatomical challenges. Overcoming these constraints to study the NMJ morphology of large mammalian distal limb muscles is of prime importance to increase cross-species translational neuromuscular research potential, particularly in the study of long motor units. In the past, NMJ phenotype analysis of large muscle bodies within the equine distal pelvic limb, such as the tibialis cranialis, or within muscles of high fibrous content, such as the soleus, has posed a distinct experimental hurdle. We optimised a technique for NMJ location and dissection from equine pelvic limb muscles. Using a quantification method validated in smaller species, we demonstrate their morphology and show that equine NMJs can be reliably dissected, stained and analysed. We reveal that the NMJs within the equine soleus have distinctly different morphologies when compared to the extensor digitorum longus and tibialis cranialis muscles. Overall, we demonstrate that equine distal pelvic limb muscles can be regionally dissected, with samples whole-mounted and their innervation patterns visualised. These methods will allow the localisation and analysis of neuromuscular junctions within the muscle bodies of large mammals to identify neuroanatomical and neuropathological features.

Comparative anatomy of the mammalian neuromuscular junction.

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.

Morphological variability is greater at developing than mature mouse neuromuscular junctions.

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.

Terminal Schwann cells at the human neuromuscular junction.

Terminal Schwann cells are non-myelinating glial cells localized to the neuromuscular junction. They play an important role in regulating many aspects of neuromuscular junction form and function, in health and during disease. However, almost all previous studies of mammalian terminal Schwann cells have used rodent models. Despite a growing awareness of differences in the cellular and molecular anatomy of rodent and human neuromuscular junctions, it remains unclear as to whether these differences also extend to the terminal Schwann cells. Here, we have adapted immunohistochemical protocols to facilitate visualization and comparative morphometric analyses of terminal Schwann cells at the human and mouse neuromuscular junction. We labelled terminal Schwann cells in the peroneus brevis muscle in six adult mice and five humans with antibodies against S100 protein. All human neuromuscular junctions were associated with at least one terminal Schwann cell, consistent with findings from other species, with an average of ∼1.7 terminal Schwann cells per neuromuscular junction in both humans and mice. In contrast, human terminal Schwann cells were significantly smaller than those of mice (P ≤ 0.01), in keeping with differences in overall synaptic size. Human terminal Schwann cell cytoplasm extended significantly beyond the synaptic boundaries of the neuromuscular junction, whereas terminal Schwann cells in mice were largely restricted to the synapse. Moreover, there was a significant difference in the location of terminal Schwann cell nuclei (P ≤ 0.01), with human terminal Schwann cells having their nuclear compartment located beyond the perimeter of the synapse more than the mouse. Taken together, these findings demonstrate that terminal Schwann cells at the human neuromuscular junction have notable differences in their morphology and synaptic relationships compared to mice. These fundamental differences need to be considered when translating the findings of both neuromuscular junction biology and pathology from rodents to humans.

aNMJ-morph: a simple macro for rapid analysis of neuromuscular junction morphology.

Large-scale data analysis of synaptic morphology is becoming increasingly important to the field of neurobiological research (e.g. 'connectomics'). In particular, a detailed knowledge of neuromuscular junction (NMJ) morphology has proven to be important for understanding the form and function of synapses in both health and disease. The recent introduction of a standardized approach to the morphometric analysis of the NMJ-'NMJ-morph'-has provided the first common software platform with which to analyse and integrate NMJ data from different research laboratories. Here, we describe the design and development of a novel macro-'automated NMJ-morph' or 'aNMJ-morph'-to update and streamline the original NMJ-morph methodology. ImageJ macro language was used to encode the complete NMJ-morph workflow into seven navigation windows that generate robust data for 19 individual pre-/post-synaptic variables. The aNMJ-morph scripting was first validated against reference data generated by the parent workflow to confirm data reproducibility. aNMJ-morph was then compared with the parent workflow in large-scale data analysis of original NMJ images (240 NMJs) by multiple independent investigators. aNMJ-morph conferred a fourfold increase in data acquisition rate compared with the parent workflow, with average analysis times reduced to approximately 1 min per NMJ. Strong concordance was demonstrated between the two approaches for all 19 morphological variables, confirming the robust nature of aNMJ-morph. aNMJ-morph is a freely available and easy-to-use macro for the rapid and robust analysis of NMJ morphology and offers significant improvements in data acquisition and learning curve compared to the original NMJ-morph workflow.

NMJ-morph reveals principal components of synaptic morphology influencing structure-function relationships at the neuromuscular junction.

The ability to form synapses is one of the fundamental properties required by the mammalian nervous system to generate network connectivity. Structural and functional diversity among synaptic populations is a key hallmark of network diversity, and yet we know comparatively little about the morphological principles that govern variability in the size, shape and strength of synapses. Using the mouse neuromuscular junction (NMJ) as an experimentally accessible model synapse, we report on the development of a robust, standardized methodology to facilitate comparative morphometric analysis of synapses ('NMJ-morph'). We used NMJ-morph to generate baseline morphological reference data for 21 separate pre- and post-synaptic variables from 2160 individual NMJs belonging to nine anatomically distinct populations of synapses, revealing systematic differences in NMJ morphology between defined synaptic populations. Principal components analysis revealed that overall NMJ size and the degree of synaptic fragmentation, alongside pre-synaptic axon diameter, were the most critical parameters in defining synaptic morphology. 'Average' synaptic morphology was remarkably conserved between comparable synapses from the left and right sides of the body. Systematic differences in synaptic morphology predicted corresponding differences in synaptic function that were supported by physiological recordings, confirming the robust relationship between synaptic size and strength.