Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Single-cell axotomy of cultured hippocampal neurons integrated in neuronal circuits

Nature Protocols volume 9, pages 1028–1037 (2014)Cite this article

Subjects

Abstract

An understanding of the molecular mechanisms of axon regeneration after injury is key for the development of potential therapies. Single-cell axotomy of dissociated neurons enables the study of the intrinsic regenerative capacities of injured axons. This protocol describes how to perform single-cell axotomy on dissociated hippocampal neurons containing synapses. Furthermore, to axotomize hippocampal neurons integrated in neuronal circuits, we describe how to set up coculture with a few fluorescently labeled neurons. This approach allows axotomy of single cells in a complex neuronal network and the observation of morphological and molecular changes during axon regeneration. Thus, single-cell axotomy of mature neurons is a valuable tool for gaining insights into cell intrinsic axon regeneration and the plasticity of neuronal polarity of mature neurons. Dissociation of the hippocampus and plating of hippocampal neurons takes ∼2 h. Neurons are then left to grow for 2 weeks, during which time they integrate into neuronal circuits. Subsequent axotomy takes 10 min per neuron and further imaging takes 10 min per neuron.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Main steps of the coculture of dissociated WT and GFP-expressing neurons.
Figure 2: Development of single-labeled GFP neurons in coculture.
Figure 3: Axotomy setup.
Figure 4: Example of axotomy of a GFP neuron and axonal regrowth.
Figure 5: Analysis of axon growth after axotomy.
Figure 6: Analysis of synaptic markers after axotomy.

Similar content being viewed by others

References

  1. Bradke, F., Fawcett, J.W. & Spira, M.E. Assembly of a new growth cone after axotomy: the precursor to axon regeneration. Nat. Rev. Neurosci. 13, 183–193 (2012).

    Article  CAS  Google Scholar 

  2. Cafferty, W.B.J., McGee, A.W. & Strittmatter, S.M. Axonal growth therapeutics: regeneration or sprouting or plasticity? Trends Neurosci. 31, 215–220 (2008).

    Article  CAS  Google Scholar 

  3. Kwon, B.K., Oxland, T.R. & Tetzlaff, W. Animal models used in spinal cord regeneration research. Spine 27, 1504–1510 (2002).

    Article  Google Scholar 

  4. Rosenzweig, E.S. & McDonald, J.W. Rodent models for treatment of spinal cord injury: research trends and progress toward useful repair. Curr. Opin. Neurol. 17, 121–131 (2004).

    Article  Google Scholar 

  5. Tuszynski, M.H.M. & Steward, O.O. Concepts and methods for the study of axonal regeneration in the CNS. Neuron 74, 777–791 (2012).

    Article  CAS  Google Scholar 

  6. Hellal, F. et al. Microtubule stabilization reduces scarring and causes axon regeneration after spinal cord injury. Science 331, 928–931 (2011).

    Article  CAS  Google Scholar 

  7. Dimou, L. et al. Nogo-A–deficient mice reveal strain-dependent differences in axonal regeneration. J. Neurosci. 26, 5591–5603 (2006).

    Article  CAS  Google Scholar 

  8. Misgeld, T., Nikic, I. & Kerschensteiner, M. In vivo imaging of single axons in the mouse spinal cord. Nat. Protoc. 2, 263–268 (2007).

    Article  CAS  Google Scholar 

  9. Ertürk, A., Hellal, F., Enes, J. & Bradke, F. Disorganized microtubules underlie the formation of retraction bulbs and the failure of axonal regeneration. J. Neurosci. 27, 9169–9180 (2007).

    Article  Google Scholar 

  10. Laskowski, C.J. & Bradke, F. In vivo imaging: a dynamic imaging approach to study spinal cord regeneration. Exp. Neurol. 242, 11–17 (2013).

    Article  Google Scholar 

  11. Silver, J. & Miller, J.H. Regeneration beyond the glial scar. Nat. Rev. Neurosci. 5, 146–156 (2004).

    Article  CAS  Google Scholar 

  12. Ylera, B. et al. Chronically CNS-injured adult sensory neurons gain regenerative competence upon a lesion of their peripheral axon. Curr. Biol. 19, 930–936 (2009).

    Article  CAS  Google Scholar 

  13. Chuckowree, J.A. & Vickers, J.C. Cytoskeletal and morphological alterations underlying axonal sprouting after localized transection of cortical neuron axons in vitro. J. Neurosci. 23, 3715–3725 (2003).

    Article  CAS  Google Scholar 

  14. Chung, R.S. et al. Mild axonal stretch injury in vitro induces a progressive series of neurofilament alterations ultimately leading to delayed axotomy. J. Neurotrauma 22, 1081–1091 (2005).

    Article  Google Scholar 

  15. Smith, D.H., Wolf, J.A., Lusardi, T.A., Lee, V.M. & Meaney, D.F. High tolerance and delayed elastic response of cultured axons to dynamic stretch injury. J. Neurosci. 19, 4263–4269 (1999).

    Article  CAS  Google Scholar 

  16. Gomis-Rüth, S., Wierenga, C.J. & Bradke, F. Plasticity of polarization: changing dendrites into axons in neurons integrated in neuronal circuits. Curr. Biol. 18, 992–1000 (2008).

    Article  Google Scholar 

  17. Dotti, C.G.C. & Banker, G.A.G. Experimentally induced alteration in the polarity of developing neurons. Nature 330, 254–256 (1987).

    Article  CAS  Google Scholar 

  18. Bradke, F. & Dotti, C.G. Differentiated neurons retain the capacity to generate axons from dendrites. Curr. Biol. 10, 1467–1470 (2000).

    Article  CAS  Google Scholar 

  19. Goslin, K.K. & Banker, G.G. Experimental observations on the development of polarity by hippocampal neurons in culture. J. Cell Biol. 108, 1507–1516 (1989).

    Article  CAS  Google Scholar 

  20. Stiess, M. & Bradke, F. Neuronal polarization: the cytoskeleton leads the way. Dev Neurobiol 71, 430–444 (2011).

    Article  CAS  Google Scholar 

  21. Takahashi, D., Yu, W., Baas, P.W., Kawai-Hirai, R. & Hayashi, K. Rearrangement of microtubule polarity orientation during conversion of dendrites to axons in cultured pyramidal neurons. Cell Motil. Cytoskeleton 64, 347–359 (2007).

    Article  Google Scholar 

  22. Calderon de Anda, F., Gartner, A., Tsai, L.H. & Dotti, C.G. Pyramidal neuron polarity axis is defined at the bipolar stage. J. Cell Sci. 121, 178–185 (2008).

    Article  CAS  Google Scholar 

  23. Ohara, R. et al. Axotomy induces axonogenesis in hippocampal neurons by a mechanism dependent on importin β. Biochem. Biophys. Res. Commun. 405, 697–702 (2011).

    Article  CAS  Google Scholar 

  24. Cho, Y. & Cavalli, V. HDAC5 is a novel injury-regulated tubulin deacetylase controlling axon regeneration. EMBO J. 31, 3063–3078 (2012).

    Article  CAS  Google Scholar 

  25. Stiess, M. et al. Axon extension occurs independently of centrosomal microtubule nucleation. Science 327, 704–707 (2010).

    Article  CAS  Google Scholar 

  26. Busch, S.A., Horn, K.P., Silver, D.J. & Silver, J. Overcoming macrophage-mediated axonal dieback following CNS injury. J. Neurosci. 29, 9967–9976 (2009).

    Article  CAS  Google Scholar 

  27. Verma, P. et al. Axonal protein synthesis and degradation are necessary for efficient growth cone regeneration. J. Neurosci. 25, 331–342 (2005).

    Article  CAS  Google Scholar 

  28. Vogelaar, C.F. et al. Axonal mRNAs: characterisation and role in the growth and regeneration of dorsal root ganglion axons and growth cones. Mol. Cell. Neurosci. 42, 102–115 (2009).

    Article  CAS  Google Scholar 

  29. Rasband, M.N.M. The axon initial segment and the maintenance of neuronal polarity. Nat. Rev. Neurosci. 11, 552–562 (2010).

    Article  CAS  Google Scholar 

  30. Kaech, S. & Banker, G. Culturing hippocampal neurons. Nat. Protoc. 1, 2406–2415 (2007).

    Article  Google Scholar 

  31. Bradke, F. & Dotti, C.G. Neuronal polarity: vectorial cytoplasmic flow precedes axon formation. Neuron 19, 1175–1186 (1997).

    Article  CAS  Google Scholar 

  32. Okabe, M.M., Ikawa, M.M., Kominami, K.K., Nakanishi, T.T. & Nishimune, Y.Y. 'Green mice' as a source of ubiquitous green cells. FEBS Lett. 407, 7–7 (1997).

    Article  Google Scholar 

  33. Flynn, K.C. et al. ADF/Cofilin-mediated actin retrograde flow directs neurite formation in the developing brain. Neuron 76, 1091–1107 (2012).

    Article  CAS  Google Scholar 

  34. Garvalov, B.K. et al. Cdc42 regulates cofilin during the establishment of neuronal polarity. J. Neurosci. 27, 13117–13129 (2007).

    Article  CAS  Google Scholar 

  35. Nikonenko, I., Boda, B., Alberi, S. & Muller, D. Application of photoconversion technique for correlated confocal and ultrastructural studies in organotypic slice cultures. Microsc. Res. Tech. 68, 90–96 (2005).

    Article  Google Scholar 

  36. Grabenbauer, M. et al. Correlative microscopy and electron tomography of GFP through photooxidation. Nat. Methods 2, 857–862 (2005).

    Article  CAS  Google Scholar 

  37. Knott, G.W., Holtmaat, A., Trachtenberg, J.T., Svoboda, K. & Welker, E. A protocol for preparing GFP-labeled neurons previously imaged in vivo and in slice preparations for light and electron microscopic analysis. Nat. Protoc. 4, 1145–1156 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank B. Garvalov and K. Flynn for their suggestions and revisions to this manuscript. F.B. was supported by Wings for Life (WfL), the International Foundation for Research in Paraplegia (IRP) and the Deutsche Forschungsgemeinschaft (DFG). S.G.-R. was supported by a grant from Boehringer Ingelheim Fonds. M.S. was supported by a European Molecular Biology Organization (EMBO) long-term fellowship and is supported by the Human Frontier Science Program.

Author information

Author notes

  1. Michael Stiess

    Present address: Present address: Biozentrum, University of Basel, Basel, Switzerland.,

  2. Susana Gomis-Rüth and Michael Stiess: These authors contributed equally to this work.

Authors and Affiliations

  1. Axonal Growth and Regeneration, Max Planck Institute of Neurobiology, Martinsried, Germany

    Susana Gomis-Rüth, Michael Stiess, Liane Meyn & Frank Bradke

  2. Axon Growth and Regeneration, Deutsches Zentrum für Neurodegenerative Erkrankungen, Bonn, Germany

    Michael Stiess, Liane Meyn & Frank Bradke

  3. Division of Cell Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands

    Corette J Wierenga

Authors
  1. Susana Gomis-Rüth
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michael Stiess
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Corette J Wierenga
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Liane Meyn
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Frank Bradke
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.G.-R., L.M. and F.B. conceived and designed the protocol and experimental procedures. S.G.-R., M.S., L.M. and C.J.W. performed cultures and experiments. S.G.-R., M.S. and F.B. wrote the manuscript. F.B. supervised the project.

Corresponding author

Correspondence to Frank Bradke.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gomis-Rüth, S., Stiess, M., Wierenga, C. et al. Single-cell axotomy of cultured hippocampal neurons integrated in neuronal circuits. Nat Protoc 9, 1028–1037 (2014). https://doi.org/10.1038/nprot.2014.069

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2014.069

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing