Journal of Neurocytology

, Volume 15, Issue 2, pp 169–176 | Cite as

Nerve growth factor immunohistochemistry and biological activity in the rat iris

  • P. J. Finn
  • I. A. Ferguson
  • F. J. Renton
  • R. A. Rush
Article

Summary

Nerve growth factor (NGF) production in the cultured rat iris was examined using immunohistochemistry and bioassay of irides and conditioned media. NGF immunoreactivity increased steadily with days in culture so that the intensity of staining was maximal after 6 days of culture. The localization was shown to be sensitive to the presence of cross-linking fixatives such as formaldehyde and glutaraldehyde and this effect was only partially alleviated by the use of very high concentrations of antibodies. NGF immunoreactivity was localized in Schwann cells and possibly nerve axons, but with no antigen detectable in smooth muscle fibres. Media conditioned over irides initially supported a high percentage of dissociated sympathetic neurons, but the number supported decreased with time in culture until day 4. Moreover, the use of antibodies to NGF allowed the detection of at least two types of neuronotropic activity, NGF accounting for at least 94% of the total trophic activity present after 4 days of culture. These findings provide support for the proposal that Schwann cells produce NGF and question the accepted hypothesis that the molecule is produced by smooth muscle fibres as a peripheral maintenance factor for sympathetic and sensory nerves. The results also suggest that two survival factors may be involved in the regulation of sympathetic function.

Keywords

Formaldehyde Smooth Muscle Biological Activity Glutaraldehyde Nerve Growth Factor 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. Abrahamson, I. K., Murdoch, S. Y. &Rush, R. A. (1984) The effect of culture on the neuronotrophic activity of avian smooth muscle.International Journal of Developmental Neuroscience 2, 229–39.Google Scholar
  2. Ayer-Lelievre, C. S., Ebendal, T., Olson, L. &Seiger, A. (1983) Localization of NGF-like immunoreactivity in rat nervous tissue.Medical Biology 61, 296–304.Google Scholar
  3. Barth, E. -M., Korsching, S. &Thoenen, H. (1984) Regulation of NGF synthesis and release in organ cultures of rat iris,journal of Cell Biology 99, 839–43.Google Scholar
  4. Belford, D. A. &Rush, R. A. (1983) A survival factor for sympathetic neurons from avian smooth muscle.Developmental Brain Research 6, 304–7.Google Scholar
  5. Benowitz, L. I. &Shashoua, V. E. (1979) Immunoreactive sites for nerve growth factor (N.G.F.) in the goldfish brain.Brain Research 172, 561–5.Google Scholar
  6. Bradshaw, R. A. (1978) Nerve Growth Factor.Annual Review of Biochemistry 47, 191–217.Google Scholar
  7. Ebendal, T., Olson, L. &Seiger, A. (1983) The level of Nerve Growth Factor as a function of innervation.Experimental Cell Research 148, 311–17.Google Scholar
  8. Ebendal, T., Olson, L., Seiger, A. &Hedlund, K. O. (1980) Nerve growth factors in the rat iris.Nature 286, 25–8.Google Scholar
  9. Harper, G. P., Al-Saaffar, A. M., Pearce, F. L. &Vernon, C. A. (1980) The production and storage of Nerve Growth Factor in vitro by tissues of the mouse, rat and embryonic chick.Developmental Biology 77, 379–90.Google Scholar
  10. Hazen-Martin, D. J. &Simson, J. A. V. (1984) Immunocytochemical localization of nerve growth factor.Journal of Histochemistry and Cytochemistry 32, 30–6.Google Scholar
  11. Henderson, C. E., Huchet, M. &Changeux, J. P. (1983) Denervation increases a neurite-promoting activity in extracts of skeletal muscle.Nature 302, 609–11.Google Scholar
  12. Heumann, R., Korsching, S., Scott, J. &Thoenen, H. (1984) Relationship between levels of NGF and its mRNA in sympathetic ganglia and peripheral target tissues.European Molecular Biology Organisation Journal 3, 3183–90.Google Scholar
  13. Hill, M. A. &Bennett, M. R. (1983) Cholinergic growth factor from skeletal muscle elevated following denervation.Neuroscience Letters 35, 31–5.Google Scholar
  14. Johnson, D. G., Silberstein, S. D., Hanbauer, I. &Kopin, I. J. (1972) The role of nerve growth factor in the ramification of sympathetic nerve fibres into the rat iris in organ culture.Journal of Neurochemistry 19, 2025–9.Google Scholar
  15. Kessler, J. A., Bell, W. O. &Black, I. B. (1983) Interactions between the sympathetic and sensory innervation of the iris.Journal of Neuroscience 3, 1301–7.Google Scholar
  16. Korsching, S. &Thoenen, H. (1983a) Nerve Growth Factor in sympathetic ganglia and corresponding target organs of the rat: correlation with density of sympathetic innervation.Proceedings of the National Academy of Sciences USA 80, 3513–6.Google Scholar
  17. Korsching, S. &Thoenen, H. (1983b) Quantitative demonstration of the retrograde axonal transport of endogenous nerve growth factor.Neuroscience Letters 9, 1–4.Google Scholar
  18. Lees, G., Chubb, I., Freeman, C., Geffen, L. &Rush, R. A. (1981) Effect of nerve activity on transport of NGF and dopamine-β-hydroxylase antibodies in sympathetic neurones.Brain Research 214, 186–9.Google Scholar
  19. Mobley, W. C., Schenenker, A. &Shooter, E. M. (1976) Characterization and isolation of proteolytically modified nerve growth factor.Biochemistry 15, 5543–52.Google Scholar
  20. Palmatier, M. A., Hartman, B. K. &Johnson, E. M. Jr. (1984) Demonstration of retrogradely transported endogenous NGF in axons of sympathetic neurons.Journal of Neuroscience 4, 751–7.Google Scholar
  21. Puchtler, H. &Meloan, S. N. (1985) On the chemistry of formaldehyde fixation and its effects on immunohistochemical reactions.Histochemistry 82, 201–4.Google Scholar
  22. Richardson, P. M. &Ebendal, T. (1982) Nerve growth activities in rat peripheral nerve.Brain Research 246, 57–64.Google Scholar
  23. Rush, R. A. (1983) Dopamine-β-hydroxylase (DBH) immunohistochemistry and immunocytochemistry. InImmunohistochemistry (edited byCuello, A. C.), pp. 139–58. New York: Wiley.Google Scholar
  24. Rush, R. A. (1984) Immunohistochemical localization of endogenous Nerve Growth Factor.Nature 312, 364–7.Google Scholar
  25. Rush, R. A. (1985) Tenotomy decreases sympathetic neuronal survival factors in avian smooth muscle.Developmental Brain Research,22, 93–7.Google Scholar
  26. Shelton, D. L. &Reichardt, L. F. (1984) Expression of the β-nerve growth factor gene correlates with the density of sympathetic innervation in effector organs.Proceedings of the National Academy of Sciences USA 81, 7951–5.Google Scholar
  27. Slack, J. R. &Pockett, S. (1982) Motor neurotrophic factor in denervated adult skeletal muscle.Brain Research 247, 138–40.Google Scholar
  28. Stefanini, M., De Martino, C. &Zamboni, L. (1967) Fixation of ejaculated spermatozoa for electron microscopy.Nature 26, 173–4.Google Scholar
  29. Stoeckel, K., Gagnon, K., Guroff, G. &Thoenen, H. (1976) Purification of nerve growth factor antibodies by affinity chromatography.Journal of Neurochemistry 26, 1207–11.Google Scholar
  30. Straznicky, C. &Rush, R. A. (1985) Nerve growth factor treatment does not prevent dorsal root ganglion cell death induced by target removal in chick embryos.Anatomy and Embryology 171, 357–63.Google Scholar
  31. Thoenen, H. &Barde, Y. A. (1980) Physiology of Nerve Growth Factor.Physiological Reviews 60, 1284–335.Google Scholar
  32. Wilson, P. A., Scott, J., Pennschow, J., Coghlan, J. &Rush, R. A. (1986) Identification and quantification of mRNA for nerve growth factor in histological preparations.Neuroscience Letters (in press).Google Scholar

Copyright information

© Chapman and Hall Ltd. 1986

Authors and Affiliations

  • P. J. Finn
    • 1
  • I. A. Ferguson
    • 1
  • F. J. Renton
    • 1
  • R. A. Rush
    • 1
  1. 1.Centre for Neuroscience and Unit of PhysiologyFlinders University of South AustraliaBedford ParkAustralia

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