Imaging Analysis of the Neuromuscular Junction in Dystrophic Muscle

  • Stephen J. P. Pratt
  • Shama R. Iyer
  • Sameer B. Shah
  • Richard M. Lovering
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1687)

Abstract

Duchenne muscular dystrophy (DMD), caused by the absence of the protein dystrophin, is characterized as a neuromuscular disease in which muscle weakness, increased susceptibility to muscle injury, and inadequate repair appear to underlie the pathology. Considerable attention has been dedicated to studying muscle fiber damage, but there is little information to determine if damage from contraction-induced injury also occurs at or near the nerve terminal axon. Interestingly, both human patients and the mouse model for DMD (the mdx mouse) present fragmented neuromuscular junction (NMJ) morphology. Studies of mdx mice have revealed presynaptic and postsynaptic abnormalities, nerve terminal discontinuity, as well as increased susceptibility of the NMJ to contraction-induced injury with corresponding functional changes in neuromuscular transmission and nerve-evoked electromyography. Focusing on the NMJ as a contributor to functional deficits in the muscle represents a paradigm shift from the more prevalent myocentric perspectives. Further studies are needed to determine the extent to which the nerve-muscle interaction is disrupted in DMD and the role of the NMJ in the dystrophic progression. This chapter lists the tools needed for nerve terminal and NMJ structural analysis using fluorescence imaging, and provides a step-by-step outline for how to stain, image, and analyze the NMJ in skeletal muscle, with specific attention to mdx muscle.

Key words

Skeletal muscle Neuromuscular junction NMJ structure NMJ occupancy NMJ area Duchenne muscular dystrophy Bungarotoxin mdx 

Notes

Acknowledgements

This work was supported by grants from the National Institutes of Health by grants to S.R.I. (AR07592-20), and to R.M.L. (R01-AR059179 and R21-AR067872-01).

References

  1. 1.
    Grady RM, Zhou H, Cunningham JM, Henry MD, Campbell KP, Sanes JR (2000) Maturation and maintenance of the neuromuscular synapse: genetic evidence for roles of the dystrophin – glycoprotein complex. Neuron 25:279–293CrossRefPubMedGoogle Scholar
  2. 2.
    Wilson MH, Deschenes MR (2005) The neuromuscular junction: anatomical features and adaptations to various forms of increased, or decreased neuromuscular activity. Int J Neurosci 115:803–828CrossRefPubMedGoogle Scholar
  3. 3.
    Wood SJ, Slater CR (2001) Safety factor at the neuromuscular junction. Prog Neurobiol 64:393–429CrossRefPubMedGoogle Scholar
  4. 4.
    Sanes JR, Lichtman JW (1999) Development of the vertebrate neuromuscular junction. Annu Rev Neurosci 22:389–442CrossRefPubMedGoogle Scholar
  5. 5.
    Wood SJ, Slater CR (1997) The contribution of postsynaptic folds to the safety factor for neuromuscular transmission in rat fast- and slow-twitch muscles. J Physiol 500(Pt 1):165–176CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Grinnell AD (1995) Dynamics of nerve-muscle interaction in developing and mature neuromuscular junctions. Physiol Rev 75:789–834CrossRefPubMedGoogle Scholar
  7. 7.
    Sieck DC, Zhan WZ, Fang YH, Ermilov LG, Sieck GC, Mantilla CB (2012) Structure-activity relationships in rodent diaphragm muscle fibers vs. neuromuscular junctions. Respir Physiol Neurobiol 180:88–96CrossRefPubMedGoogle Scholar
  8. 8.
    Jang YC, Van Remmen H (2011) Age-associated alterations of the neuromuscular junction. Exp Gerontol 46:193–198CrossRefPubMedGoogle Scholar
  9. 9.
    Kawabuchi M, Tan H, Wang S (2011) Age affects reciprocal cellular interactions in neuromuscular synapses following peripheral nerve injury. Ageing Res Rev 10:43–53CrossRefPubMedGoogle Scholar
  10. 10.
    Adams ME, Kramarcy N, Krall SP, Rossi SG, Rotundo RL, Sealock R, Froehner SC (2000) Absence of alpha-syntrophin leads to structurally aberrant neuromuscular synapses deficient in utrophin. J Cell Biol 150:1385–1398CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Banks GB, Chamberlain JS, Froehner SC (2009) Truncated dystrophins can influence neuromuscular synapse structure. Mol Cell Neurosci 40:433–441CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Chipman PH, Franz CK, Nelson A, Schachner M, Rafuse VF (2010) Neural cell adhesion molecule is required for stability of reinnervated neuromuscular junctions. Eur J Neurosci 31:238–249CrossRefPubMedGoogle Scholar
  13. 13.
    Kulakowski SA, Parker SD, Personius KE (2011) Reduced TrkB expression results in precocious age-like changes in neuromuscular structure, neurotransmission, and muscle function. J Appl Physiol 111:844–852CrossRefPubMedGoogle Scholar
  14. 14.
    Kong J, Anderson JE (1999) Dystrophin is required for organizing large acetylcholine receptor aggregates. Brain Res 839:298–304CrossRefPubMedGoogle Scholar
  15. 15.
    Chamberlain JS, Metzger J, Reyes M, Townsend D, Faulkner JA (2007) Dystrophin-deficient mdx mice display a reduced life span and are susceptible to spontaneous rhabdomyosarcoma. FASEB J 21:2195–2204CrossRefPubMedGoogle Scholar
  16. 16.
    Li Y, Lee Y, Thompson WJ (2011) Changes in aging mouse neuromuscular junctions are explained by degeneration and regeneration of muscle fiber segments at the synapse. J Neurosci 31:14910–14919CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Li Y, Thompson WJ (2011) Nerve terminal growth remodels neuromuscular synapses in mice following regeneration of the postsynaptic muscle fiber. J Neurosci 31:13191–13203CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Minatel E, Santo NH, Marques MJ (2001) Acetylcholine receptors and neuronal nitric oxide synthase distribution at the neuromuscular junction of regenerated muscle fibers. Muscle Nerve 24:410–416CrossRefPubMedGoogle Scholar
  19. 19.
    Kong J, Yang L, Li Q, Cao J, Yang J, Chen F, Wang Y, Zhang C (2012) The absence of dystrophin rather than muscle degeneration causes acetylcholine receptor cluster defects in dystrophic muscle. Neuroreport 23:82–87CrossRefPubMedGoogle Scholar
  20. 20.
    Kuromi H, Kidokoro Y (1984) Denervation disperses acetylcholine receptor clusters at the neuromuscular junction in Xenopus cultures. Dev Biol 104:421–427CrossRefPubMedGoogle Scholar
  21. 21.
    Apel PJ, Alton T, Northam C, Ma J, Callahan M, Sonntag WE, Li Z (2009) How age impairs the response of the neuromuscular junction to nerve transection and repair: an experimental study in rats. J Orthop Res 27:385–393CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Valdez G, Tapia JC, Kang H, Clemenson GD Jr, Gage FH, Lichtman JW, Sanes JR (2010) Attenuation of age-related changes in mouse neuromuscular synapses by caloric restriction and exercise. Proc Natl Acad Sci U S A 107:14863–14868CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Marques MJ, Taniguti AP, Minatel E, Neto HS (2007) Nerve terminal contributes to acetylcholine receptor organization at the dystrophic neuromuscular junction of mdx mice. Anat Rec (Hoboken) 290:181–187CrossRefGoogle Scholar
  24. 24.
    Hoffman EP, Brown RH Jr, Kunkel LM (1987) Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 51:919–928CrossRefPubMedGoogle Scholar
  25. 25.
    Lovering RM, Michaelson L, Ward CW (2009) Malformed mdx myofibers have normal cytoskeletal architecture yet altered EC coupling and stress-induced Ca2+ signaling. Am J Physiol Cell Physiol 297:C571–C580CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Pratt SJ, Shah SB, Ward CW, Inacio MP, Stains JP, Lovering RM (2013) Effects of in vivo injury on the neuromuscular junction in healthy and dystrophic muscles. J Physiol 591:559–570CrossRefPubMedGoogle Scholar
  27. 27.
    Pratt SJ, Shah SB, Ward CW, Kerr JP, Stains JP, Lovering RM (2015) Recovery of altered neuromuscular junction morphology and muscle function in mdx mice after injury. Cell Mol Life Sci 72:153CrossRefPubMedGoogle Scholar
  28. 28.
    Deschenes MR, Roby MA, Eason MK, Harris MB (2010) Remodeling of the neuromuscular junction precedes sarcopenia related alterations in myofibers. Exp Gerontol 45:389–393CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Dachs E, Hereu M, Piedrafita L, Casanovas A, Caldero J, Esquerda JE (2011) Defective neuromuscular junction organization and postnatal myogenesis in mice with severe spinal muscular atrophy. J Neuropathol Exp Neurol 70:444–461CrossRefPubMedGoogle Scholar
  30. 30.
    Sleigh JN, Burgess RW, Gillingwater TH, Cader MZ (2014) Morphological analysis of neuromuscular junction development and degeneration in rodent lumbrical muscles. J Neurosci Methods 227:159–165CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Stephen J. P. Pratt
    • 1
  • Shama R. Iyer
    • 2
  • Sameer B. Shah
    • 3
    • 5
  • Richard M. Lovering
    • 2
    • 4
  1. 1.Department of Biochemistry and Molecular BiologyUniversity of Maryland, Baltimore School of MedicineBaltimoreUSA
  2. 2.Department of OrthopaedicsUniversity of Maryland, Baltimore School of MedicineBaltimoreUSA
  3. 3.Departments of Orthopaedic Surgery and BioengineeringUniversity of California San DiegoLa JollaUSA
  4. 4.Department of PhysiologyUniversity of Maryland, Baltimore School of MedicineBaltimoreUSA
  5. 5.Research DivisionVeterans Administration San Diego Healthcare SystemSan DiegoUSA

Personalised recommendations