Methods for Culturing Primary Sympathetic Neurons and for Determining Neuronal Viability

  • Jonathan Whitfield
  • Stephen J. Neame
  • Jonathan Ham
Part of the Methods in Molecular Biology book series (MIMB, volume 282)

Abstract

Developing nerve growth factor (NGF)-dependent sympathetic neurons are one of the best-studied in vitro models of neuronal apoptosis and have been used to identify key components of the neuronal cell death pathway. This chapter describes how to prepare purified cultures of primary sympathetic neurons and how to induce apoptosis by NGF deprivation. In addition, a simple method for measuring neuronal viability based on the live/dead assay is also described. This can be used for assessing the effect of small molecule inhibitors of protein kinases, caspases and other enzymes, on NGF withdrawal-induced death.

Key Words

Sympathetic neuron nerve growth factor developmental neuronal death apoptosis live/dead cell viability assay 

References

  1. 1.
    Yuan, J. and Yankner, B. A. (2000) Apoptosis in the nervous system. Nature 407, 802–809.PubMedCrossRefGoogle Scholar
  2. 2.
    Wright, L. L., Cunningham, T. J., and Smolen, A. J. (1983) Developmental neuron death in the rat superior cervical sympathetic ganglion: cell counts and ultrastructure. J. Neurocytol. 12, 727–738.PubMedCrossRefGoogle Scholar
  3. 3.
    Martin, D. P., Schmidt, R. E., DiStefano, P. S., Lowry, O. H., Carter, J. G., and Johnson, E. M. (1988) Inhibitors of protein synthesis and RNA synthesis prevent neuronal Death caused by nerve growth factor deprivation. J. Cell. Biol. 106, 829–844.PubMedCrossRefGoogle Scholar
  4. 4.
    Deckwerth, T. L., and Johnson, E. M. (1993) Temporal analysis of events associated with programmed cell death (apoptosis) of sympathetic neurons deprived of nerve growth factor. J. Cell. Biol. 123, 1207–1222.PubMedCrossRefGoogle Scholar
  5. 5.
    Edwards, S. N. and Tolkovsky, A. M. (1994) Characterization of apoptosis in cultured rat sympathetic neurons after nerve growth factor withdrawal. J. Cell. Biol. 124, 537–546.PubMedCrossRefGoogle Scholar
  6. 6.
    Rimon, G., Bazenet, C. E., Philpott, K. L., and Rubin, L. L. (1997) Increased surface phosphatidylserine is an early marker of neuronal apoptosis. J. Neurosci. Res. 48, 563–570.PubMedCrossRefGoogle Scholar
  7. 7.
    Martinou, I., Fernandez, P.-A., Missotten, M., White, E., Allet, B., Sadoul, R., et al (1995) Viral proteins E1B19K and p35 protect sympathetic neurons from cell death induced by NGF deprivation. J. Cell. Biol. 128, 201–208.PubMedCrossRefGoogle Scholar
  8. 8.
    Deshmukh, M., Vasilakos, J., Deckwerth, T. L., Lampe, P. A., Shivers, B. D., and Johnson, E. M. (1996) Genetic and metabolic status of NGF-deprived sympathetic neurons saved by an inhibitor of ICE family proteases. J. Cell. Biol. 135, 1341–1354.PubMedCrossRefGoogle Scholar
  9. 9.
    McCarthy, M. J., Rubin, L. L., and Philpott, K. L. (1997) Involvement of caspases in sympathetic neuron apoptosis. J. Cell. Sci. 110, 2165–2173.PubMedGoogle Scholar
  10. 10.
    Neame, S. J., Rubin, L. L., and Philpott, K. L. (1998) Blocking cytochrome c activity within intact neurons inhibits apoptosis. J. Cell. Biol. 142, 1583–1593.PubMedCrossRefGoogle Scholar
  11. 11.
    Deshmukh, M. and Johnson, E. M. (1998) Evidence of a novel event during neuronal death: development of competence-to-die in response to cytoplasmic cytochrome c. Neuron 21, 695–705.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Jonathan Whitfield
    • 1
  • Stephen J. Neame
    • 1
  • Jonathan Ham
    • 2
  1. 1.Eisai London Research LaboratoriesUniversity College LondonLondonUK
  2. 2.Molecular Hematology and Cancer Biology Unit, Institute of Child HealthUniversity College LondonLondonUK

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