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Axon Growth and Regeneration pp 43–56Cite as

Neurite Outgrowth and Growth Cone Collapse Assays to Assess Neuronal Responses to Extracellular Cues

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1162))

Abstract

The identification of molecular processes involved in regulating neurite outgrowth is an active area of interest for investigators studying neural development and regeneration. In vitro assays designed to measure growth cone morphology and neurite length are frequently used to assess neuronal responses to developmental guidance cues and inhibitory cues that exist in the adult CNS. Here, we describe the procedures to assess morphological responses of cultured dorsal root ganglion neurons to attractive and repellent cues, with a focus on repellents found in the injured adult CNS. The chapter describes methods to culture the DRGs, apply inhibitory ligands, and assess morphological responses. These assays provide biological readouts to assess the capacity of a molecule to act as an inhibitory or growth promoting cue. The readouts can be used as screening tools to aid in the identification of novel targets or drugs for promoting nerve regeneration.

Andrew Kaplan, Ricardo Sanz, Gino B. Ferraro, and Ricardo Alchini contributed equally to the manuscript.

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References

  1. Kaech S, Banker G (2006) Culturing hippocampal neurons. Nat Protoc 1(5):2406–2415. doi:10.1038/nprot.2006.356

    Article  CAS  PubMed  Google Scholar 

  2. Yiu G, He Z (2006) Glial inhibition of CNS axon regeneration. Nat Rev Neurosci 7(8):617–627. doi:10.1038/nrn1956

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  3. Chen MS, Huber AB, van der Haar ME, Frank M, Schnell L, Spillmann AA, Christ F, Schwab ME (2000) Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1. Nature 403(6768):434–439. doi:10.1038/35000219

    Article  CAS  PubMed  Google Scholar 

  4. Koprivica V, Cho KS, Park JB, Yiu G, Atwal J, Gore B, Kim JA, Lin E, Tessier-Lavigne M, Chen DF, He Z (2005) EGFR activation mediates inhibition of axon regeneration by myelin and chondroitin sulfate proteoglycans. Science 310(5745):106–110. doi:10.1126/science.1115462

    Article  CAS  PubMed  Google Scholar 

  5. Prinjha R, Moore SE, Vinson M, Blake S, Morrow R, Christie G, Michalovich D, Simmons DL, Walsh FS (2000) Inhibitor of neurite outgrowth in humans. Nature 403(6768):383–384. doi:10.1038/35000287

    Article  CAS  PubMed  Google Scholar 

  6. Raper JA, Kapfhammer JP (1990) The enrichment of a neuronal growth cone collapsing activity from embryonic chick brain. Neuron 4(1):21–29

    Article  CAS  PubMed  Google Scholar 

  7. Cai D, Qiu J, Cao Z, McAtee M, Bregman BS, Filbin MT (2001) Neuronal cyclic AMP controls the developmental loss in ability of axons to regenerate. J Neurosci 21(13):4731–4739

    CAS  PubMed  Google Scholar 

  8. Norton WT, Poduslo SE (1973) Myelination in rat brain: method of myelin isolation. J Neurochem 21(4):749–757

    Article  CAS  PubMed  Google Scholar 

  9. Nash M, Pribiag H, Fournier AE, Jacobson C (2009) Central nervous system regeneration inhibitors and their intracellular substrates. Mol Neurobiol 40(3):224–235. doi:10.1007/s12035-009-8083-y

    Article  CAS  PubMed  Google Scholar 

  10. Busch SA, Silver J (2007) The role of extracellular matrix in CNS regeneration. Curr Opin Neurobiol 17(1):120–127. doi:10.1016/j.conb.2006.09.004

    Article  CAS  PubMed  Google Scholar 

  11. Kwok JC, Afshari F, Garcia-Alias G, Fawcett JW (2008) Proteoglycans in the central nervous system: plasticity, regeneration and their stimulation with chondroitinase ABC. Restor Neurol Neurosci 26(2–3):131–145

    PubMed  Google Scholar 

  12. Sharma K, Selzer ME, Li S (2012) Scar-mediated inhibition and CSPG receptors in the CNS. Exp Neurol 237(2):370–378. doi:10.1016/j.expneurol.2012.07.009

    Article  CAS  PubMed  Google Scholar 

  13. Ahmed Z, Mazibrada G, Seabright RJ, Dent RG, Berry M, Logan A (2006) TACE-induced cleavage of NgR and p75NTR in dorsal root ganglion cultures disinhibits outgrowth and promotes branching of neurites in the presence of inhibitory CNS myelin. FASEB J 20(11):1939–1941. doi:10.1096/fj.05-5339fje

    Article  CAS  PubMed  Google Scholar 

  14. Alabed YZ, Pool M, Ong Tone S, Fournier AE (2007) Identification of CRMP4 as a convergent regulator of axon outgrowth inhibition. J Neurosci 27(7):1702–1711. doi:10.1523/JNEUROSCI.5055-06.2007

    Article  CAS  PubMed  Google Scholar 

  15. Huebner EA, Kim BG, Duffy PJ, Brown RH, Strittmatter SM (2011) A multi-domain fragment of Nogo-A protein is a potent inhibitor of cortical axon regeneration via Nogo receptor 1. J Biol Chem 286(20):18026–18036. doi:10.1074/jbc.M110.208108

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. GrandPre T, Nakamura F, Vartanian T, Strittmatter SM (2000) Identification of the Nogo inhibitor of axon regeneration as a Reticulon protein. Nature 403(6768):439–444. doi:10.1038/35000226

    Article  CAS  PubMed  Google Scholar 

  17. Hsieh SH, Ferraro GB, Fournier AE (2006) Myelin-associated inhibitors regulate cofilin phosphorylation and neuronal inhibition through LIM kinase and Slingshot phosphatase. J Neurosci 26(3):1006–1015. doi:10.1523/JNEUROSCI.2806-05.2006

    Article  CAS  PubMed  Google Scholar 

  18. Domeniconi M, Zampieri N, Spencer T, Hilaire M, Mellado W, Chao MV, Filbin MT (2005) MAG induces regulated intramembrane proteolysis of the p75 neurotrophin receptor to inhibit neurite outgrowth. Neuron 46(6): 849–855. doi:10.1016/j.neuron.2005.05.029

    Article  CAS  PubMed  Google Scholar 

  19. Ferraro GB, Morrison CJ, Overall CM, Strittmatter SM, Fournier AE (2011) Membrane-type matrix metalloproteinase-3 regulates neuronal responsiveness to myelin through Nogo-66 receptor 1 cleavage. J Biol Chem 286(36):31418–31424. doi:10.1074/jbc.M111.249169

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Wulf E, Deboben A, Bautz FA, Faulstich H, Wieland T (1979) Fluorescent phallotoxin, a tool for the visualization of cellular actin. Proc Natl Acad Sci U S A 76(9):4498–4502

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. Pool M, Thiemann J, Bar-Or A, Fournier AE (2008) NeuriteTracer: a novel ImageJ plugin for automated quantification of neurite outgrowth. J Neurosci Methods 168(1):134–139. doi:10.1016/j.jneumeth.2007.08.029

    Article  PubMed  Google Scholar 

  22. Neve RL, Lim F (2013) Generation of high-titer defective HSV-1 vectors. Curr Protoc Neurosci Chapter 4:Unit 4 13. doi:10.1002/0471142301.ns0413s62. Editorial board, Jacqueline N Crawley [et al.]

  23. Alabed YZ, Pool M, Ong Tone S, Sutherland C, Fournier AE (2010) GSK3 beta regulates myelin-dependent axon outgrowth inhibition through CRMP4. J Neurosci 30(16):5635–5643. doi:10.1523/JNEUROSCI.6154-09.2010

    Article  CAS  PubMed  Google Scholar 

  24. Saijilafu, Zhou FQ (2012) Genetic study of axon regeneration with cultured adult dorsal root ganglion neurons. J Vis Exp (66). doi:10.3791/4141

  25. Albuquerque C, Joseph DJ, Choudhury P, MacDermott AB (2009) Dissection, plating, and maintenance of dorsal root ganglion neurons for monoculture and for coculture with dorsal horn neurons. Cold Spring Harb Protoc 2009(8):pdb.prot5275. doi:10.1101/pdb.prot5275

    PubMed  Google Scholar 

  26. Pool M, Niino M, Rambaldi I, Robson K, Bar-Or A, Fournier AE (2009) Myelin regulates immune cell adhesion and motility. Exp Neurol 217(2):371–377. doi:10.1016/j.expneurol.2009.03.014

    Article  CAS  PubMed  Google Scholar 

  27. Fournier AE, Gould GC, Liu BP, Strittmatter SM (2002) Truncated soluble Nogo receptor binds Nogo-66 and blocks inhibition of axon growth by myelin. J Neurosci 22(20): 8876–8883

    CAS  PubMed  Google Scholar 

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Acknowledgments

A.E.F. is a Canada Research Chair and R.A. is supported by a Lloyd Carr-Harris Studentship. This work is supported by the McGill Program in Neuroengineering and by grants from the Canadian Institutes of Health Research and the MS Society of Canada.

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Authors and Affiliations

  1. Department of Neurology and Neurosurgery, Montreal Neurological Institute, BT-109, 3801 Rue University, Montreal, QC, Canada, H3A 2B4

    Andrew Kaplan, Ricardo Sanz, Ricardo Alchini & Alyson E. Fournier

  2. Edwin L. Steele Laboratory of Tumor Biology, Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, 02114, USA

    Gino B. Ferraro

Authors
  1. Andrew Kaplan
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  2. Ricardo Sanz
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  3. Gino B. Ferraro
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  4. Ricardo Alchini
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  5. Alyson E. Fournier
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Corresponding author

Correspondence to Alyson E. Fournier .

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Editors and Affiliations

  1. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York, USA

    Andrew J. Murray

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Kaplan, A., Sanz, R., Ferraro, G.B., Alchini, R., Fournier, A.E. (2014). Neurite Outgrowth and Growth Cone Collapse Assays to Assess Neuronal Responses to Extracellular Cues. In: Murray, A. (eds) Axon Growth and Regeneration. Methods in Molecular Biology, vol 1162. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0777-9_4

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  • DOI: https://doi.org/10.1007/978-1-4939-0777-9_4

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0776-2

  • Online ISBN: 978-1-4939-0777-9

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