Skip to main content
SpringerLink
Log in

Imaging Analysis of the Neuromuscular Junction in Dystrophic Muscle

  • Protocol
  • First Online:
Duchenne Muscular Dystrophy

Part of the book series: Methods in Molecular Biology ((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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Institutional subscriptions

References

  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 25:279–293

    Article  CAS  PubMed  Google Scholar 

  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 115:803–828

    Article  PubMed  Google Scholar 

  3. Wood SJ, Slater CR (2001) Safety factor at the neuromuscular junction. Prog Neurobiol 64:393–429

    Article  CAS  PubMed  Google Scholar 

  4. Sanes JR, Lichtman JW (1999) Development of the vertebrate neuromuscular junction. Annu Rev Neurosci 22:389–442

    Article  CAS  PubMed  Google Scholar 

  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):165–176

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Grinnell AD (1995) Dynamics of nerve-muscle interaction in developing and mature neuromuscular junctions. Physiol Rev 75:789–834

    Article  CAS  PubMed  Google Scholar 

  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 180:88–96

    Article  PubMed  Google Scholar 

  8. Jang YC, Van Remmen H (2011) Age-associated alterations of the neuromuscular junction. Exp Gerontol 46:193–198

    Article  CAS  PubMed  Google Scholar 

  9. Kawabuchi M, Tan H, Wang S (2011) Age affects reciprocal cellular interactions in neuromuscular synapses following peripheral nerve injury. Ageing Res Rev 10:43–53

    Article  CAS  PubMed  Google Scholar 

  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 150:1385–1398

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Banks GB, Chamberlain JS, Froehner SC (2009) Truncated dystrophins can influence neuromuscular synapse structure. Mol Cell Neurosci 40:433–441

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 31:238–249

    Article  PubMed  Google Scholar 

  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 111:844–852

    Article  CAS  PubMed  Google Scholar 

  14. Kong J, Anderson JE (1999) Dystrophin is required for organizing large acetylcholine receptor aggregates. Brain Res 839:298–304

    Article  CAS  PubMed  Google Scholar 

  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 21:2195–2204

    Article  CAS  PubMed  Google Scholar 

  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 31:14910–14919

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Li Y, Thompson WJ (2011) Nerve terminal growth remodels neuromuscular synapses in mice following regeneration of the postsynaptic muscle fiber. J Neurosci 31:13191–13203

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 24:410–416

    Article  CAS  PubMed  Google Scholar 

  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 23:82–87

    Article  CAS  PubMed  Google Scholar 

  20. Kuromi H, Kidokoro Y (1984) Denervation disperses acetylcholine receptor clusters at the neuromuscular junction in Xenopus cultures. Dev Biol 104:421–427

    Article  CAS  PubMed  Google Scholar 

  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 27:385–393

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 107:14863–14868

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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) 290:181–187

    Article  CAS  Google Scholar 

  24. Hoffman EP, Brown RH Jr, Kunkel LM (1987) Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 51:919–928

    Article  CAS  PubMed  Google Scholar 

  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–C580

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 591:559–570

    Article  CAS  PubMed  Google Scholar 

  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 72:153

    Article  CAS  PubMed  Google Scholar 

  28. Deschenes MR, Roby MA, Eason MK, Harris MB (2010) Remodeling of the neuromuscular junction precedes sarcopenia related alterations in myofibers. Exp Gerontol 45:389–393

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 70:444–461

    Article  PubMed  Google Scholar 

  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 227:159–165

    Article  PubMed  PubMed Central  Google Scholar 

Download references

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).

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Maryland, Baltimore School of Medicine, Baltimore, MD, USA

    Stephen J. P. Pratt

  2. Department of Orthopaedics, University of Maryland, Baltimore School of Medicine, 100 Penn St., AHB, Room 540, Baltimore, MD, 21201, USA

    Shama R. Iyer & Richard M. Lovering

  3. Departments of Orthopaedic Surgery and Bioengineering, University of California San Diego, La Jolla, CA, 92093, USA

    Sameer B. Shah

  4. Department of Physiology, University of Maryland, Baltimore School of Medicine, Baltimore, MD, USA

    Richard M. Lovering

  5. Research Division, Veterans Administration San Diego Healthcare System, San Diego, CA, 92121, USA

    Sameer B. Shah

Authors
  1. Stephen J. P. Pratt
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shama R. Iyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sameer B. Shah
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Richard M. Lovering
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Richard M. Lovering .

Editor information

Editors and Affiliations

  1. Istituto di Anatomia Umana e Biologia Cellulare, Università Cattolica del Sacro Cuore, Roma, Italy

    Camilla Bernardini

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Pratt, S.J.P., Iyer, S.R., Shah, S.B., Lovering, R.M. (2018). Imaging Analysis of the Neuromuscular Junction in Dystrophic Muscle. In: Bernardini, C. (eds) Duchenne Muscular Dystrophy. Methods in Molecular Biology, vol 1687. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7374-3_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7374-3_5

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7373-6

  • Online ISBN: 978-1-4939-7374-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation