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:

Production of compartmented cultures of rat sympathetic neurons

Nature Protocols volume 4, pages 1869–1887 (2009)Cite this article

Abstract

The compartmented culture, in which primary neurons plated in a proximal compartment send their axons under silicone grease barriers and into left and right distal compartments, has enhanced the experimental capabilities of neuronal cultures. Treatments can be applied separately to cell bodies/proximal axons or distal axons, and cell bodies/proximal axons and distal axons can be separately harvested and analyzed. Distal axons can be axotomized, and the neurons can be studied while their axons regenerate. Construction of the culture dishes requires 3 h for 48 cultures, and preparing the neurons also requires 3 h. Compartmented cultures provide enough cellular material for biochemical analyses such as immunoblotting. The uses of compartmented cultures have included studies of neurotrophic factor retrograde signaling, axonal transport, and axonal protein and lipid biosynthesis. Here we focus on sympathetic neurons cultured from neonatal rats and provide protocols for the production and some of the uses of compartmented cultures.

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: The compartmented culture system.
Figure 2: Assembly of three-compartment cultures.
Figure 3: Sympathetic neurons from neonatal rats after 1 and 5 d in culture.
Figure 4: Construction of the pin rake.
Figure 5: Specialty equipment used in constructing compartmented cultures.
Figure 6: Dissection of superior cervical ganglia from a neonatal rat.
Figure 7: Assembling a culture with an intermediate compartment using a three-compartment Teflon divider.

Similar content being viewed by others

References

  1. Campenot, R.B. Local control of neurite development by nerve growth factor. Proc. Natl. Acad. Sci. USA 74, 4516–4519 (1977).

    Article  CAS  PubMed  Google Scholar 

  2. Campenot, R.B. Development of sympathetic neurons in compartmentalized cultures. II. Local control of neurite survival by nerve growth factor. Dev. Biol. 93, 13–21 (1982).

    Article  CAS  PubMed  Google Scholar 

  3. Campenot, R.B. Development of sympathetic neurons in compartmentalized cultures. I. Local control of neurite growth by nerve growth factor. Dev. Biol. 93, 1–12 (1982).

    Article  CAS  PubMed  Google Scholar 

  4. MacInnis, B.L. & Campenot, R.B. Retrograde support of neuronal survival without retrograde transport of nerve growth factor. Science 295, 1536–1539 (2002).

    Article  CAS  PubMed  Google Scholar 

  5. Mok, S.A., Lund, K. & Campenot, R.B. A retrograde apoptotic signal originating in NGF-deprived distal axons of rat sympathetic neurons in compartmented cultures. Cell Res. 19, 546–560 (2009).

    Article  CAS  PubMed  Google Scholar 

  6. Senger, D.L. & Campenot, R.B. Rapid retrograde tyrosine phosphorylation of trkA and other proteins in rat sympathetic neurons in compartmented cultures. J. Cell. Biol. 138, 411–421 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. MacInnis, B.L., Senger, D.L. & Campenot, R.B. Spatial requirements for TrkA kinase activity in the support of neuronal survival and axon growth in rat sympathetic neurons. Neuropharmacology 45, 995–1010 (2003).

    Article  CAS  PubMed  Google Scholar 

  8. Mok, S.A. & Campenot, R.B. A nerve growth factor-induced retrograde survival signal mediated by mechanisms downstream of TrkA. Neuropharmacology 52, 270–278 (2007).

    Article  CAS  PubMed  Google Scholar 

  9. Eng, H., Lund, K. & Campenot, R.B. Synthesis of beta-tubulin, actin, and other proteins in axons of sympathetic neurons in compartmented cultures. J. Neurosci. 19, 1–9 (1999).

    Article  CAS  PubMed  Google Scholar 

  10. Vance, J.E., Pan, D., Campenot, R.B., Bussiere, M. & Vance, D.E. Evidence that the major membrane lipids, except cholesterol, are made in axons of cultured rat sympathetic neurons. J. Neurochem. 62, 329–337 (1994).

    Article  CAS  PubMed  Google Scholar 

  11. Campenot, R.B., Lund, K. & Senger, D.L. Delivery of newly synthesized tubulin to rapidly growing distal axons of sympathetic neurons in compartmented cultures. J. Cell. Biol. 135, 701–709 (1996).

    Article  CAS  PubMed  Google Scholar 

  12. Ure, D.R. & Campenot, R.B. Retrograde transport and steady-state distribution of 125I-nerve growth factor in rat sympathetic neurons in compartmented cultures. J. Neurosci. 17, 1282–1290 (1997).

    Article  CAS  PubMed  Google Scholar 

  13. Walicke, P.A., Campenot, R.B. & Patterson, P.H. Determination of transmitter function by neuronal activity. Proc. Natl. Acad. Sci. USA 74, 5767–5771 (1977).

    Article  CAS  PubMed  Google Scholar 

  14. Singh, K.K. & Miller, F.D. Activity regulates positive and negative neurotrophin-derived signals to determine axon competition. Neuron. 45, 837–845 (2005).

    Article  CAS  PubMed  Google Scholar 

  15. Kimpinski, K., Campenot, R.B. & Mearow, K. Effects of the neurotrophins nerve growth factor, neurotrophin-3, and brain-derived neurotrophic factor (BDNF) on neurite growth from adult sensory neurons in compartmented cultures. J. Neurobiol. 33, 395–410 (1997).

    Article  CAS  PubMed  Google Scholar 

  16. Watson, F.L. et al. Rapid nuclear responses to target-derived neurotrophins require retrograde transport of ligand–receptor complex. J. Neurosci. 19, 7889–7900 (1999).

    Article  CAS  PubMed  Google Scholar 

  17. Hayashi, H., Campenot, R.B., Vance, D.E. & Vance, J.E. Glial lipoproteins stimulate axon growth of central nervous system neurons in compartmented cultures. J. Biol. Chem. 279, 14009–14015 (2004).

    Article  CAS  PubMed  Google Scholar 

  18. Watson, F.L. et al. Neurotrophins use the Erk5 pathway to mediate a retrograde survival response. Nat. Neurosci. 4, 4 (2001).

    Article  Google Scholar 

  19. Ye, H., Kuruvilla, R., Zweifel, L.S. & Ginty, D.D. Evidence in support of signaling endosome-based retrograde survival of sympathetic neurons. Neuron. 39, 57–68 (2003).

    Article  CAS  PubMed  Google Scholar 

  20. Campenot, R.B. NGF uptake and retrograde signaling mechanisms in sympathetic neurons in compartmented cultures. in Results and Problems in Cell Differentiation, Vol. 48 Cell Biology of the Axon (ed. Koenig, E.) 141–158 (Springer, Berlin/Heidelberg, 2009).

  21. Zweifel, L.S., Kuruvilla, R. & Ginty, D.D. Functions and mechanisms of retrograde neurotrophin signalling. Nat. Rev. Neurosci. 6, 615–625 (2005).

    Article  CAS  PubMed  Google Scholar 

  22. Ginty, D.D. & Segal, R.A. Retrograde neurotrophin signaling: Trk-ing along the axon. Curr. Opin. Neurobiol. 12, 268–274 (2002).

    Article  CAS  PubMed  Google Scholar 

  23. Campenot, R.B. & MacInnis, B.L. Retrograde transport of neurotrophins: fact and function. J. Neurobiol. 58, 217–229 (2004).

    Article  CAS  PubMed  Google Scholar 

  24. Campenot, R.B. & Eng, H. Protein synthesis in axons and its possible functions. J. Neurocytol. 29, 793–798 (2000).

    Article  CAS  PubMed  Google Scholar 

  25. Vance, J.E., Campenot, R.B. & Vance, D.E. The synthesis and transport of lipids for axonal growth and nerve regeneration. Biochim. Biophys. Acta 1486, 84–96 (2000).

    Article  CAS  PubMed  Google Scholar 

  26. Bertrand, J., Winton, M.J., Rodriguez-Hernandez, N., Campenot, R.B. & McKerracher, L. Application of rho antagonist to neuronal cell bodies promotes neurite growth in compartmented cultures and regeneration of retinal ganglion cell axons in the optic nerve of adult rats. J. Neurosci. 25, 1113–1121 (2005).

    Article  CAS  PubMed  Google Scholar 

  27. Yang, X.M. et al. Autocrine hepatocyte growth factor provides a local mechanism for promoting axonal growth. J. Neurosci. 18, 8369–8381 (1998).

    Article  CAS  PubMed  Google Scholar 

  28. Chu, G.K. & Tator, C.H. Calcium influx is necessary for optimal regrowth of transected neurites of rat sympathetic ganglion neurons in vitro . Neuroscience 102, 945–957 (2001).

    Article  CAS  PubMed  Google Scholar 

  29. Song, M.S., Saavedra, L. & de Chaves, E.I. Apoptosis is secondary to non-apoptotic axonal degeneration in neurons exposed to Abeta in distal axons. Neurobiol. Aging 27, 1224–1238 (2006).

    Article  CAS  PubMed  Google Scholar 

  30. Ch′ng, T.H. & Enquist, L.W. Neuron-to-cell spread of pseudorabies virus in a compartmented neuronal culture system. J. Virol. 79, 10875–10889 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Ch′ng, T.H., Spear, P.G., Struyf, F. & Enquist, L.W. Glycoprotein D-independent spread of pseudorabies virus infection in cultured peripheral nervous system neurons in a compartmented system. J. Virol. 81, 10742–10757 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Manning, P.T., Johnson, E.M., Jr., Wilcox, C.L., Palmatier, M.A. & Russell, J.H. MHC-specific cytotoxic T lymphocyte killing of dissociated sympathetic neuronal cultures. Am. J. Pathol. 128, 395–409 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Pazyra-Murphy, M.F. & Segal, R.A. Preparation and maintenance of dorsal root ganglia neurons in compartmented cultures. J. Vis. Exp. published online, doi: 10.3791/951 (17 October 2008).

  34. Cui, B. et al. One at a time, live tracking of NGF axonal transport using quantum dots. Proc. Natl. Acad. Sci. USA 104, 13666–13671 (2007).

    Article  CAS  PubMed  Google Scholar 

  35. Heerssen, H.M. & Segal, R.A. Location, location, location: a spatial view of neurotrophin signal transduction. Trends Neurosci. 25, 160–165 (2002).

    Article  CAS  PubMed  Google Scholar 

  36. Heerssen, H.M., Pazyra, M.F. & Segal, R.A. Dynein motors transport activated Trks to promote survival of target-dependent neurons. Nat. Neurosci. (2004).

  37. Carlton, E. et al. Fusion of the tetanus toxin C fragment binding domain and Bcl-xL for protection of peripheral nerve neurons. Neurosurgery 63, 1175–1182; discussion 1182–1184 (2008).

    Article  PubMed  Google Scholar 

  38. Ng, B.K., Chen, L., Mandemakers, W., Cosgaya, J.M. & Chan, J.R. Anterograde transport and secretion of brain-derived neurotrophic factor along sensory axons promote Schwann cell myelination. J. Neurosci. 27, 7597–7603 (2007).

    Article  CAS  PubMed  Google Scholar 

  39. Bi, J., Tsai, N.P., Lin, Y.P., Loh, H.H. & Wei, L.N. Axonal mRNA transport and localized translational regulation of kappa-opioid receptor in primary neurons of dorsal root ganglia. Proc. Natl. Acad. Sci. USA 103, 19919–19924 (2006).

    Article  CAS  PubMed  Google Scholar 

  40. Bi, J., Tsai, N.P., Lu, H.Y., Loh, H.H. & Wei, L.N. Copb1-facilitated axonal transport and translation of kappa opioid-receptor mRNA. Proc. Natl. Acad. Sci. USA 104, 13810–13815 (2007).

    Article  CAS  PubMed  Google Scholar 

  41. Copray, S., Liem, R., Mantingh-Otter, I.J. & Brouwer, N. Coculture of rat embryonic proprioceptive sensory neurons and myotubes. Muscle Nerve 19, 1401–1412 (1996).

    Article  CAS  PubMed  Google Scholar 

  42. Silva, A., Wang, Q., Wang, M., Ravula, S.K. & Glass, J.D. Evidence for direct axonal toxicity in vincristine neuropathy. J. Peripher. Nerv. Syst. 11, 211–216 (2006).

    Article  CAS  PubMed  Google Scholar 

  43. Melli, G., Keswani, S.C., Fischer, A., Chen, W. & Hoke, A. Spatially distinct and functionally independent mechanisms of axonal degeneration in a model of HIV-associated sensory neuropathy. Brain 129, 1330–1338 (2006).

    Article  PubMed  Google Scholar 

  44. Guertin, A.D., Zhang, D.P., Mak, K.S., Alberta, J.A. & Kim, H.A. Microanatomy of axon/glial signaling during Wallerian degeneration. J. Neurosci. 25, 3478–3487 (2005).

    Article  CAS  PubMed  Google Scholar 

  45. Edstrom, A. & Ekstrom, P.A. Role of phosphatidylinositol 3-kinase in neuronal survival and axonal outgrowth of adult mouse dorsal root ganglia explants. J. Neurosci. Res. 74, 726–735 (2003).

    Article  PubMed  Google Scholar 

  46. Kimpinski, K., Jelinski, S. & Mearow, K. The anti-p75 antibody, MC192, and brain-derived neurotrophic factor inhibit nerve growth factor-dependent neurite growth from adult sensory neurons. Neuroscience 93, 253–263 (1999).

    Article  CAS  PubMed  Google Scholar 

  47. Tsiang, H., Ceccaldi, P.E. & Lycke, E. Rabies virus infection and transport in human sensory dorsal root ganglia neurons. J. Gen. Virol. 72 (Part 5): 1191–1194 (1991).

    Article  PubMed  Google Scholar 

  48. Lycke, E. & Tsiang, H. Rabies virus infection of cultured rat sensory neurons. J. Virol. 61, 2733–2741 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Ekstrom, P.A., Bergstrand, H. & Edstrom, A. Effects of protein kinase inhibitors on regeneration in vitro of adult frog sciatic sensory axons. J. Neurosci. Res. 31, 462–469 (1992).

    Article  CAS  PubMed  Google Scholar 

  50. Ekstrom, P.A. Insulin stimulates ganglionic protein synthesis and reduces thymidine incorporation in support cells of the in vitro regenerating adult frog sciatic sensory neurons. Neurosci. Lett. 132, 183–186 (1991).

    Article  CAS  PubMed  Google Scholar 

  51. Ivins, K.J., Bui, E.T. & Cotman, C.W. Beta-amyloid induces local neurite degeneration in cultured hippocampal neurons: evidence for neuritic apoptosis. Neurobiol. Dis. 5, 365–378 (1998).

    Article  CAS  PubMed  Google Scholar 

  52. Underhill, S.M. & Goldberg, M.P. Hypoxic injury of isolated axons is independent of ionotropic glutamate receptors. Neurobiol. Dis. 25, 284–290 (2007).

    Article  CAS  PubMed  Google Scholar 

  53. Hayashi, H. et al. Methods for the study of lipid metabolism in neurons. Anal. Biochem. 331, 1–16 (2004).

    Article  CAS  PubMed  Google Scholar 

  54. Hayashi, H., Campenot, R.B., Vance, D.E. & Vance, J.E. Apolipoprotein E-containing lipoproteins protect neurons from apoptosis via a signaling pathway involving low-density lipoprotein receptor-related protein-1. J. Neurosci. 27, 1933–1941 (2007).

    Article  CAS  PubMed  Google Scholar 

  55. Sonderegger, P. et al. A few axonal proteins distinguish ventral spinal cord neurons from dorsal root ganglion neurons. J. Cell. Biol. 98, 364–368 (1984).

    Article  CAS  PubMed  Google Scholar 

  56. Klostermann, S. & Bonhoeffer, F. Investigations of signaling pathways in axon growth and guidance. Perspect. Dev. Neurobiol. 4, 237–252 (1996).

    CAS  PubMed  Google Scholar 

  57. Brayfield, C.A., Marra, K.G., Leonard, J.P., Tracy Cui, X. & Gerlach, J.C. Excimer laser channel creation in polyethersulfone hollow fibers for compartmentalized in vitro neuronal cell culture scaffolds. Acta Biomater. 4, 244–255 (2008).

    Article  CAS  PubMed  Google Scholar 

  58. Bergen, J.M. & Pun, S.H. Analysis of the intracellular barriers encountered by nonviral gene carriers in a model of spatially controlled delivery to neurons. J. Gene Med. 10, 187–197 (2008).

    Article  CAS  PubMed  Google Scholar 

  59. Ravula, S.K., Wang, M.S., Asress, S.A., Glass, J.D. & Bruno Frazier, A. A compartmented neuronal culture system in microdevice format. J. Neurosci. Methods 159, 78–85 (2007).

    Article  PubMed  Google Scholar 

  60. Ravula, S.K., McClain, M.A., Wang, M.S., Glass, J.D. & Frazier, A.B. A multielectrode microcompartment culture platform for studying signal transduction in the nervous system. Lab. Chip 6, 1530–1536 (2006).

    Article  CAS  PubMed  Google Scholar 

  61. Liu, W.W., Goodhouse, J., Jeon, N.L. & Enquist, L.W. A microfluidic chamber for analysis of neuron-to-cell spread and axonal transport of an alpha-herpesvirus. PLoS ONE 3, e2382 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  62. Taylor, A.M., Rhee, S.W. & Jeon, N.L. Microfluidic chambers for cell migration and neuroscience research. Methods. Mol. Biol. 321, 167–177 (2006).

    CAS  PubMed  Google Scholar 

  63. Park, J.W., Vahidi, B., Taylor, A.M., Rhee, S.W. & Jeon, N.L. Microfluidic culture platform for neuroscience research. Nat. Protoc. 1, 2128–2136 (2006).

    Article  CAS  PubMed  Google Scholar 

  64. Campenot, R.B. Independent control of the local environment of somas and neurites. Methods Enzymol. 58, 302–307 (1979).

    Article  CAS  PubMed  Google Scholar 

  65. Campenot, R.B. Compartmented culture analysis of nerve growth. In Cell–Cell Interactions: A Practical Approach (eds. Stevenson, B.R., Gallin, W.J. & Paul, D.L.) 275–298 (IRL Press, Oxford, 1992).

  66. Campenot, R.B. & Martin, G. Construction and use of compartmented cultures for studies of cell biology of neurons. In Protocols for Neural Cell Culture (eds. Federoff, S. & Richardson, A.) 49–57 (Humana Press, Totowa, New Jersey, 2001).

  67. Hawrot, E. & Patterson, P.H. Long-term culture of dissociated sympathetic neurons. Methods Enzymol. 58, 574–584 (1979).

    Article  CAS  PubMed  Google Scholar 

  68. Mains, R.E. & Patterson, P.H. Primary cultures of dissociated sympathetic neurons. I. Establishment of long-term growth in culture and studies of differentiated properties. J. Cell. Biol. 59, 329–345 (1973).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Tsui-Pierchala, B.A. & Ginty, D.D. Characterization of an NGF-P-TrkA retrograde-signaling complex and age-dependent regulation of TrkA phosphorylation in sympathetic neurons. J. Neurosci. 19, 8207–8218 (1999).

    Article  CAS  PubMed  Google Scholar 

  70. Chun, L.L. & Patterson, P.H. Role of nerve growth factor in the development of rat sympathetic neurons in vitro. II. Developmental studies. J. Cell. Biol. 75, 705–711 (1977).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank the researchers who have worked in the laboratory and contributed improvements to our techniques over the years: Dr. Daren Ure, Dr. Donna Senger, Grace Martin, Russell Watts, Dr. Bronwyn MacInnis, Norma Jean Valli and Dr. Barbara Karten. Special thanks to Dr. Edwin Furshpan and Dr. David Potter in whose laboratory R.B.C. developed the compartmented culture during a postdoctoral fellowship in 1976–1978. Work contributing to the development and refinement of the techniques described here has been funded by The National Institutes of Health (USA), The Canadian Institutes of Health Research, The Alberta Heritage Foundation for Medical Research, The Alberta Paraplegic Foundation and the Rick Hansen Man in Motion Foundation.

Author information

Authors and Affiliations

  1. Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada.,

    Robert B Campenot, Karen Lund & Sue-Ann Mok

Authors
  1. Robert B Campenot
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Karen Lund
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sue-Ann Mok
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors contributed extensively to the development of this protocol and preparation of the paper. K.L. developed several of the procedures and prepared a laboratory manual upon which much of the detail in the protocol is based; S.-A.M. developed several of the procedures, created the graphic images and prepared part of the initial draft of the paper; R.B.C. originally developed the compartmented culture system, produced most of the photographs and wrote the final paper.

Corresponding author

Correspondence to Robert B Campenot.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Campenot, R., Lund, K. & Mok, SA. Production of compartmented cultures of rat sympathetic neurons. Nat Protoc 4, 1869–1887 (2009). https://doi.org/10.1038/nprot.2009.210

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2009.210

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