Morphological analysis of neuromuscular junction development and degeneration in rodent lumbrical muscles

BACKGROUND The neuromuscular junction (NMJ) is a specialised synapse formed between a lower motor neuron and a skeletal muscle fibre, and is an early pathological target in numerous nervous system disorders, including amyotrophic lateral sclerosis (ALS), Charcot-Marie-Tooth disease (CMT), and spinal muscular atrophy (SMA). Being able to accurately visualise and quantitatively characterise the NMJ in rodent models of neurological conditions, particularly during the early stages of disease, is thus of clear importance. NEW METHOD We present a method for dissection of rodent deep lumbrical muscles located in the hind-paw, and describe how to perform immunofluorescent morphological analysis of their NMJs. RESULTS These techniques allow the temporal assessment of a number of developmental and pathological NMJ phenotypes in lumbrical muscles. COMPARISON WITH EXISTING METHODS Small muscles, such as the distal hind-limb lumbrical muscles, possess a major advantage over larger muscles, such as gastrocnemius, in that they can be whole-mounted and the entire innervation pattern visualised. This reduces preparation time and ambiguity when evaluating important neuromuscular phenotypes. CONCLUSIONS Together, these methods will allow the reader to perform a detailed and accurate analysis of the neuromuscular system in rodent models of disease in order to identify pertinent features of neuropathology.

[1]  R. Ruiz,et al.  SMN Requirement for Synaptic Vesicle, Active Zone and Microtubule Postnatal Organization in Motor Nerve Terminals , 2011, PloS one.

[2]  R. Burgess,et al.  Neuromuscular disease models and analysis. , 2010, Methods in molecular biology.

[3]  K. Talbot,et al.  Transgenics, toxicity and therapeutics in rodent models of mutant SOD1-mediated familial ALS , 2008, Progress in Neurobiology.

[4]  J. Lichtman,et al.  In vivo observations of pre- and postsynaptic changes during the transition from multiple to single innervation at developing neuromuscular junctions , 1993, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[5]  J. Duncan,et al.  An adaptive coding model of neural function in prefrontal cortex , 2001, Nature Reviews Neuroscience.

[6]  R. Kothary,et al.  Defects in neuromuscular junction remodelling in the Smn 2B/− mouse model of spinal muscular atrophy , 2013, Neurobiology of Disease.

[7]  E. Tizzano,et al.  Synaptic defects in type I spinal muscular atrophy in human development , 2013, The Journal of pathology.

[8]  R. Burgess,et al.  An Active Dominant Mutation of Glycyl-tRNA Synthetase Causes Neuropathy in a Charcot-Marie-Tooth 2D Mouse Model , 2006, Neuron.

[9]  J. McArdle,et al.  Advantages of the triangularis sterni muscle of the mouse for investigations of synaptic phenomena , 1981, Journal of Neuroscience Methods.

[10]  J. Caldwell,et al.  The effects of partial denervation at birth on the development of muscle fibres and motor units in rat lumbrical muscle. , 1980, The Journal of physiology.

[11]  R. Ribchester,et al.  Compartmental neurodegeneration and synaptic plasticity in the Wlds mutant mouse , 2001, The Journal of physiology.

[12]  R. Burgess,et al.  A MusD Retrotransposon Insertion in the Mouse Slc6a5 Gene Causes Alterations in Neuromuscular Junction Maturation and Behavioral Phenotypes , 2012, PloS one.

[13]  J. Sanes,et al.  Induction, assembly, maturation and maintenance of a postsynaptic apparatus , 2001, Nature reviews. Neuroscience.

[14]  J. Sanes,et al.  Watching the neuromuscular junction , 2003, Journal of neurocytology.

[15]  J. Caldwell,et al.  Sprouting of active nerve terminals in partially inactive muscles of the rat. , 1980, The Journal of physiology.

[16]  I. Jirmanová.  Ultrastructure of motor end-plates during pharmacologically-induced degeneration and subsequent regeneration of skeletal muscle , 1975, Journal of neurocytology.

[17]  J. Sanes,et al.  Roles of Neurotransmitter in Synapse Formation Development of Neuromuscular Junctions Lacking Choline Acetyltransferase , 2002, Neuron.

[18]  P. Caroni,et al.  Assembly, plasticity and selective vulnerability to disease of mouse neuromuscular junctions , 2003, Journal of neurocytology.

[19]  G. Feng,et al.  Genetic evidence that relative synaptic efficacy biases the outcome of synaptic competition , 2003, Nature.

[20]  R. Kothary,et al.  Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis , 2014, Journal of visualized experiments : JoVE.

[21]  C. Lorson,et al.  Development and characterization of an SMN2-based intermediate mouse model of Spinal Muscular Atrophy. , 2013, Human molecular genetics.

[22]  Ju Lu,et al.  The Interscutularis Muscle Connectome , 2009, PLoS biology.

[23]  S. Nagataki,et al.  The motor end‐plate fine structure and ultrastructural localization of acetylcholine receptors in amyotrophic lateral sclerosis , 1984, Muscle & nerve.

[24]  K. Talbot,et al.  Selective vulnerability of motor neurons and dissociation of pre- and post-synaptic pathology at the neuromuscular junction in mouse models of spinal muscular atrophy. , 2008, Human molecular genetics.

[25]  K. Talbot,et al.  The contribution of mouse models to understanding the pathogenesis of spinal muscular atrophy , 2011, Disease Models & Mechanisms.

[26]  J. Glass,et al.  Amyotrophic lateral sclerosis is a distal axonopathy: evidence in mice and man , 2004, Experimental Neurology.

[27]  R. Burgess,et al.  An assessment of mechanisms underlying peripheral axonal degeneration caused by aminoacyl-tRNA synthetase mutations , 2011, Molecular and Cellular Neuroscience.

[28]  D. Kullmann,et al.  Myasthenia and related disorders of the neuromuscular junction , 2010, Journal of Neurology, Neurosurgery & Psychiatry.

[29]  R. Burgess,et al.  A valid mouse model of AGRIN-associated congenital myasthenic syndrome. , 2011, Human molecular genetics.

[30]  J. Sanes,et al.  Development of the vertebrate neuromuscular junction. , 1999, Annual review of neuroscience.

[31]  J. Lichtman,et al.  In Vivo Time-Lapse Imaging of Synaptic Takeover Associated with Naturally Occurring Synapse Elimination , 2003, Neuron.

[32]  W. Robberecht,et al.  HDAC6 inhibitors reverse axonal loss in a mouse model of mutant HSPB1–induced Charcot-Marie-Tooth disease , 2011, Nature Medicine.

[33]  T. Gillingwater,et al.  Using mouse cranial muscles to investigate neuromuscular pathology in vivo , 2010, Neuromuscular Disorders.

[34]  J. Lichtman,et al.  From Plaque to Pretzel: Fold Formation and Acetylcholine Receptor Loss at the Developing Neuromuscular Junction , 2000, The Journal of Neuroscience.

[35]  M. Z. Cader,et al.  Neuromuscular junction maturation defects precede impaired lower motor neuron connectivity in Charcot-Marie-Tooth type 2D mice. , 2014, Human molecular genetics.

[36]  K. Talbot,et al.  Pre-symptomatic development of lower motor neuron connectivity in a mouse model of severe spinal muscular atrophy. , 2010, Human molecular genetics.

[37]  J. Wokke,et al.  Morphological changes in the human end plate with age , 1990, Journal of the Neurological Sciences.

[38]  T. Gillingwater,et al.  Review: Neuromuscular synaptic vulnerability in motor neurone disease: amyotrophic lateral sclerosis and spinal muscular atrophy , 2010, Neuropathology and applied neurobiology.

[39]  R. Burgess,et al.  Loss of the E3 ubiquitin ligase LRSAM1 sensitizes peripheral axons to degeneration in a mouse model of Charcot-Marie-Tooth disease , 2013, Disease Models & Mechanisms.

[40]  M. Caleo,et al.  Central effects of tetanus and botulinum neurotoxins. , 2009, Toxicon : official journal of the International Society on Toxinology.

[41]  L. Mei,et al.  Morphological analysis of neuromuscular junctions by immunofluorescent staining of whole-mount mouse diaphragms. , 2013, Methods in molecular biology.

[42]  M. Rich,et al.  Impaired Synaptic Vesicle Release and Immaturity of Neuromuscular Junctions in Spinal Muscular Atrophy Mice , 2009, The Journal of Neuroscience.

[43]  T. Mizushige,et al.  Active zone density is conserved during synaptic growth but impaired in aged mice , 2012, The Journal of comparative neurology.

[44]  R. Ribchester,et al.  ApoE isoform-specific regulation of regeneration in the peripheral nervous system. , 2011, Human molecular genetics.

[45]  R. Dieler,et al.  Scanning electron microscopic study of denervated and reinnervated intrafusal muscle fibers in rats , 1992, Muscle and Nerve.

[46]  G. Hamilton,et al.  Morphological Characteristics of Motor Neurons Do Not Determine Their Relative Susceptibility to Degeneration in a Mouse Model of Severe Spinal Muscular Atrophy , 2012, PloS one.

[47]  T. Gillingwater,et al.  Loss of translation elongation factor (eEF1A2) expression in vivo differentiates between Wallerian degeneration and dying‐back neuronal pathology , 2008, Journal of anatomy.

[48]  Hyuno Kang,et al.  Attenuation of age-related changes in mouse neuromuscular synapses by caloric restriction and exercise , 2010, Proceedings of the National Academy of Sciences.

[49]  Zhihua Feng,et al.  Severe neuromuscular denervation of clinically relevant muscles in a mouse model of spinal muscular atrophy. , 2012, Human molecular genetics.

[50]  E. Costanzo,et al.  Co‐regulation of synaptic efficacy at stable polyneuronally innervated neuromuscular junctions in reinnervated rat muscle , 1999, The Journal of physiology.

[51]  L. Piedrafita,et al.  Defective Neuromuscular Junction Organization and Postnatal Myogenesis in Mice With Severe Spinal Muscular Atrophy , 2011, Journal of neuropathology and experimental neurology.