Mammalian Growth Cone Turning Assays Identify Distinct Cell Signalling Mechanisms That Underlie Axon Growth, Guidance and Regeneration

Part of the Methods in Molecular Biology book series (MIMB, volume 846)


The cell signalling mechanisms underlying mammalian central nervous system axon growth and guidance change during development, such that axons that establish appropriate connectivity in the embryo fail to regenerate after injury to the adult nervous system. The growth cone turning assay has been used in Xenopus neurons to elucidate mechanisms of axon guidance during development. Here, we describe how we have adapted this assay for rat dorsal root ganglion neurons to study the influence of extracellular secreted factors causing growth cone attraction and repulsion. Additionally, we describe how this method can be combined with small interfering RNA and cDNA transfections to manipulate protein expression in growth cones, and fluorescence resonance energy transfer to monitor the activity of signalling pathways in live neurons. This assay provides the unique ability to manipulate and visualise the internal status of growth cone signalling whilst challenged with extracellular chemotropic signalling molecules, and can be used to develop strategies to promote axon regeneration in the mature mammalian central nervous system.

Key words

Axon guidance Regeneration Growth cone Turning assay Fluorescence resonance energy transfer Cell signalling cAMP 


  1. 1.
    Filbin MT (2003) Myelin-associated inhibitors of axonal regeneration in the adult mammalian CNS. Nat Rev Neurosci 4, 703–713PubMedCrossRefGoogle Scholar
  2. 2.
    Lohof AM, Quillan M, Dan Y, Poo MM (1992) Asymmetric modulation of cAMP activity induces growth cone turning. J Neurosci 12, 1253–1261PubMedGoogle Scholar
  3. 3.
    Song H, Poo MM (1999) Signal transduction underlying growth cone responses to diffusible factors. Curr Opin Neurobiol 9, 355–363PubMedCrossRefGoogle Scholar
  4. 4.
    Shewan D, Dwivedy A, Anderson R, Holt CE (2002) Age related changes underlie switch in netrin-1 responsiveness as growth cones advance along the visual pathway. Nat Neurosci 5, 955–962PubMedCrossRefGoogle Scholar
  5. 5.
    Murray AJ, Shewan DA (2008) Epac medi-ates cAMP-dependent axon growth, guidance and regeneration. Mol Cell Neurosci 38, 578–588PubMedCrossRefGoogle Scholar
  6. 6.
    Murray AJ, Peace AG, Shewan DA (2009) cGMP promotes neurite outgrowth and growth cone turning and improves axon regeneration on spinal cord tissue in combination with cAMP. Brain Res 1294, 12–21PubMedCrossRefGoogle Scholar
  7. 7.
    Murray AJ, Tucker SJ, Shewan DA (2009) cAMP-dependent axon guidance is distinctly regulated by Epac and protein kinase A. J Neurosci 29, 15434–15444PubMedCrossRefGoogle Scholar
  8. 8.
    Golding J, Shewan D, Cohen J (1997) Maturation of the mammalian dorsal root entry zone—from entry to no entry. Trends Neurosci 20, 303–308PubMedCrossRefGoogle Scholar
  9. 9.
    Shipman C Jr (1969) Evaluation of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) as a tissue culture buffer. Proc Soc Exp Biol Med 130, 305–310PubMedGoogle Scholar
  10. 10.
    Erskine L, Reijntjes S, Pratt T, Denti L, Schwarz Q, Vieira JM, Alakakone B, Shewan D, Ruhrberg C (2011) VEGF signaling through neuropilin 1 guides commissural axon crossing at the optic chiasm. Neuron 70, 951–965Google Scholar
  11. 11.
    Wouters FS, Verveer PJ, Bastiaens PIH (2001) Imaging biochemistry inside cells. Trends Cell Biol 11, 203–211PubMedCrossRefGoogle Scholar
  12. 12.
    Tamada A, Kawase S, Murakami F, Kamiguchi H (2010). Autonomous right screw rotation of growth cone filopodia drives neurite turning. J Cell Biol 188, 429–441PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiophysicsColumbia UniversityNew YorkUSA
  2. 2.School of Medical Sciences, College of Life Sciences and MedicineUniversity of AberdeenAberdeenUK

Personalised recommendations