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Localization of Neurotrophin Proteins Within the Central Nervous System by Immunohistochemistry

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Neurotrophin Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 169))

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Abstract

Neurotrophins are a family of growth factor proteins sharing a considerable degree of primary sequence and tertiary structure homology (1–4). Members of this protein family, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5), have been identified as important signals in the developing peripheral nervous system where they serve as survival-promoting and differentiation factors for various populations of neurons (5,6). The presence of each of these factors within the central nervous system (CNS) has also been confirmed (7–12), although their physiological role in the CNS has yet to be fully elucidated. Identifying where in the CNS these proteins are made, where they are stored, how they are transferred from one cell to another, what cells they are transferred to, and their final fate are some key steps toward reaching an understanding of endogenous neurotrophin function. One technique for addressing many of these issues is immunohistochemistry. The strength of the immunohistochemical approach lies with its ability to precisely define the distribution of a given protein within a tissue sample obtained under defined experimental conditions. By manipulating the conditions under which a specimen is collected, it is possible to gain a great deal of insight into the nature by which a protein interacts within its environment and to obtain clues regarding its functional role in the animal. Nevertheless, the immunohistochemical approach has distinct limitations that must be considered when interpreting results and drawing conclusions.

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References

  1. Berkemeier, L. R., Winslow, J. W., Kaplan, D. R., Nikolics, K., Goeddel, D. V., and Rosenthal, A. (1991) Neurotrophin-5: a novel neurotrophic factor that activatestrk and trkB. Neuron 7(5), 857–866.

    Article  PubMed  CAS  Google Scholar 

  2. Narhi, L. O., Rosenfeld, R., Talvenheimo, J., Prestrelski, S. J., Arakawa, T., Lary, J. W., et al. (1993) Comparison of the biophysical characteristics of human brainderived neurotrophic factor, neurotrophin-3 and nerve growth factor. J. Biol. Chem. 268(18), 13,309–13,317.

    PubMed  CAS  Google Scholar 

  3. Gotz, R., Koster, R., Winkler, C., Raulf, F., Lottspeich, F., Schartl, M., et al. (1994) Neurotrophin-6 is a new member of the nerve growth factor family. Nature 372(6503), 266–269.

    Article  PubMed  CAS  Google Scholar 

  4. Ibanez, C. F. (1994) Structure-function relationships in the neurotrophin family. J. Neurobiol. 25(11), 1349–1361.

    Article  PubMed  CAS  Google Scholar 

  5. Levi-Montalcini, R. and Angeletti, P. U. (1968) Nerve growth factor. Physiol. Rev. 48, 534–569.

    PubMed  CAS  Google Scholar 

  6. Thoenen, H. and Barde, Y.-A. (1980) Physiology of nerve growth factor. Physiol. Rev. 60, 1285–1335.

    Google Scholar 

  7. Leibrock, J., Lottspeich, F., Hohn, A., Hofer, M., Hengerer, B., Masiakowski, P., et al. (1989) Molecular cloning and expression of brain-derived neurotrophic factor. Nature 341(6238), 149–152.

    Article  PubMed  CAS  Google Scholar 

  8. Ernfors, P., Wetmore, C., Olson, L., and Persson, H. (1990) Identification of cellsin rat brain and peripheral tissues expressing mRNA for members of the nerve growth factor family. Neuron 5(4), 511–526.

    Article  PubMed  CAS  Google Scholar 

  9. Hofer, M., Pagliusi, S. R., Hohn, A., Leibrock, J., and Barde, Y-A. (1990) Regional distribution of brain-derived neurotrophic factor mRNA in the adultmouse brain. EMBO J. 9(8), 2459–2464.

    PubMed  CAS  Google Scholar 

  10. Jones, K. R. and Reichardt, L. F. (1990) Molecular cloning of a human gene that is a member of the nerve growth factor family. Proc. Natl. Acad. Sci. USA 87(20), 8060–8064.

    Article  PubMed  CAS  Google Scholar 

  11. Maisonpierre, P. C., Belluscio, L., Friedman, B., Alderson, R. F., Weigand, S. J., Furth, M. E., et al. (1990) NT-3, BDNF, and NGF in the developing rat nervous system: parallel as well as reciprocal patterns of expression. Neuron 5, 501–509.

    Article  PubMed  CAS  Google Scholar 

  12. Ip, N. Y., Ibanez, C. F., Nye, S. H., McClain, J., Jones, P. F., Gies, D. R., et al. (1992) Mammalian neurotrophin-4: structure, chromosomal localization, tissue distribution, and receptor specificity. Proc. Natl. Acad. Sci. USA 98(7), 3060–3064.

    Article  Google Scholar 

  13. Blochl, A. and Thoenen, H. (1996) Localization of cellular storage compartments and sites of constitutive and activity-dependent release of nerve growth factor (NGF) in primary cultures of hippocampal neurons. Mol. Cell. Neurosci. 7, 173–190.

    Article  PubMed  CAS  Google Scholar 

  14. Seidah, N. G., Benjannet, S., Pareek, S., Savaria, D., Hamelin, J., Goulet, B., et al. (1996) Cellular processing of the nerve growth factor precursor by the mammalian pro-protein convertases. Biochem. J. 314, 951–960.

    PubMed  CAS  Google Scholar 

  15. Seidah, N. G., Benjannet, S., Pareek, S., Chretien, M., and Murphy, R. A. (1996) Cellular processing of the neurotrophin precursors of NT-3 and BDNF by the mammalian proprotein convertases. FEBS Lett. 379, 247–250.

    Article  PubMed  CAS  Google Scholar 

  16. von Bartheld, C. S., Byers, M. R., Williams, R., and Bothwell, M. (1996) Anterograde transport of neurotrophins and axodendritic transfer in the developing visual system. Nature 379, 830–833.

    Article  Google Scholar 

  17. Goodman, L. J., Valverde, J., Lim, F., Geschwind, M. D., Federoff, H. J., Geller, A. I., et al. (1996) Regulated release and polarized localization of brain-derived neurotrophic factor in hippocampal neurons. Mol. Cell. Neurosci. 7, 222–238.

    Article  PubMed  CAS  Google Scholar 

  18. Conner, J. M., Lauterborn, J. C., Yan, Q., Gall, C. M., and Varon, S. (1997) Distribution of brain-derived neurotrophic factor (BDNF) protein and mRNA in the normal rat CNS: evidence for anterograde axonal transport. J. Neurosci. 17(7), 2295–2313.

    PubMed  CAS  Google Scholar 

  19. Holstege, J. C., Kennis, J. H. H., van Rooijen-Boot, A., Taal, W., Yan, Q., and Vecht, Ch. J. (1997) Light and electron microscopical identification of BDNF immunoreactivity in the rat spinal cord. Soc. Neurosci. Abstr. 23, 882.

    Google Scholar 

  20. Michael, G. J., Averill, S., Nitkunan, A., Rattray, M., Bennett, D. L. H., Yan, Q., et al. (1997) Nerve growth factor treatment increases brain-derived neurotrophic factor selectively in TrkA-expressing dorsal root ganglion cells and in their central terminations within the spinal cord. J. Neurosci. 17(21), 8467–8490.

    Google Scholar 

  21. Hoener, M. C., Hewitt, E., Conner, J. M., Costello, J. W., and Varon, S. (1996) Nerve growth factor (NGF) content in adult rat brain tissues is several-fold higher than generally reported and is largely associated with sedimentable fractions. Brain Res. 728, 47–56.

    Article  PubMed  CAS  Google Scholar 

  22. Schwab, M. E., Otten, U., Agid, Y., and Thoenen, H. (1979) Nerve growth factor (NGF) in the rat CNS: abscence of specific retrograde axonal transport and tyrosine hydroxylase induction in locus coeruleus and substantia nigra. Brain Res. 168, 473–483.

    Article  PubMed  CAS  Google Scholar 

  23. Seiler, M. and Schwab, M. E. (1984) Specific retrograde transport of nerve growth factor (NGF) from neocortex to nucleus basalis in the rat. Brain Res. 300, 33–39.

    Article  PubMed  CAS  Google Scholar 

  24. Conner, J. M., Muir, D., Varon, S., Hagg, T., and Manthorpe, M. (1992) The localization of nerve growth factor-like immunoreactivity in the adult rat basal forebrain and hippocampal formation. J. Comp. Neurol. 319, 454–462.

    Article  PubMed  CAS  Google Scholar 

  25. Hu, L., Ribeiro-Da Silva, A., Garofalo, L., and Cuello, A. C. (1994) Subcellular localization of NGF immunoreactivity in basal forebrain cholinergic neurons. Anultrastructural double-labeling study in the rat. Soc. Neurosci. Abstr. 20, 1305.

    Google Scholar 

  26. Wetmore, C., Cao, Y. H., Pettersson, R. F., and Olson, L. (1991) Brain-derived neurotrophic factor—subcellular compartmentalization and interneuronal transfer as visualized with anit-peptide antibodies. Proc. Natl. Acad. Sci. USA 88, 9843–9847.

    Article  PubMed  CAS  Google Scholar 

  27. Brandtzaeg, P. (1982) Tissue preparation methods for immunohistochemistry. In Techniques in Immunohistochemistry (Bullock, G. R., Perusz, P., eds.), Academic, London, pp. 1–75.

    Google Scholar 

  28. Hazin-Martin, D. J. and Simson, J. A. V. (1984) Immunocytochemical localization of nerve growth factor: effects of fixation. J. Histochem. Cytochem. 32 30–36.

    Google Scholar 

  29. Olson, L., Ayer-LeLievre, C., Ebendal, T., and Seiger, A. (1987) Nerve growth factor-like immunoreactivities in rodent salivary glands and testis. Cell Tissue Res. 248, 275–286.

    Article  PubMed  CAS  Google Scholar 

  30. Zhou, X. F., Zettler, C., and Rush, R. A. (1994) An improved procedure for the immunohistochemical localization of nerve growth factor-like immunoreactivity. J. Neurosci. Methods 54(1), 95–102.

    Article  PubMed  CAS  Google Scholar 

  31. Lee, T-H., Kato, H., Pan, L-H., Ryu, J. H., Kogure, K., and Itoyama, Y. (1998) Localization of nerve growth factor, TrkA and p75 immunoreactivity in the hippocampal formation and basal forebrain of adult rat. Neuroscience 83, 335–349.

    Article  PubMed  CAS  Google Scholar 

  32. Zhou, X. F. and Rush, R. A. (1994) Localization of neurotrophin-3-like immunoreactivity in the rat central nervous system. Brain Res. 643(1-2), 162–172.

    Article  PubMed  CAS  Google Scholar 

  33. Aloe, L., Alleva, E., and de Simone, R. (1990) Changes in NGF level in mouse hypothalamus following internal aggressive behavior: biological and immunohistochemical evidence. Behav. Brain Res. 39, 53–61.

    Article  PubMed  CAS  Google Scholar 

  34. Mufson, E. J., Conner, J. M., Varon, S., and Kordower, J. H. (1994) Nerve growth factor immunoreactive profiles in the primate basal forebrain and hippocampal formation. J. Comp. Neurol. 341, 507–519.

    Article  PubMed  CAS  Google Scholar 

  35. Ayer-LeLievre, C. S., Ebendal, T., Olson, L., and Seiger, A. (1983) Localization of nerve growth factor-like immunoreactivity in rat nervous tissue. Med. Biol. 61, 296–304.

    PubMed  CAS  Google Scholar 

  36. Whittemore, S. R., Ebendal, T., Larkfors, L., Olson, L., Seiger, A., Stromberg, I., et al. (1986) Developmental and regional expression of b nerve growth factor messanger RNA and protein in the rat central nervous system. Proc. Natl. Acad. Sci. USA 83, 817–821.

    Article  PubMed  CAS  Google Scholar 

  37. Conner, J. M. and Varon, S. (1992) Distribution of nerve growth factor-like immunoreactive neurons in the adult rat brain following colchicine treatment. J. Comp. Neurol. 326(3), 347–362.

    Article  PubMed  CAS  Google Scholar 

  38. Mufson, E. J., Conner, J. M., and Kordower, J. H. (1995) Nerve growth factor in Alzheimer′s disease-defective retrograde transport to nucleus basalis. Neuroreport 6, 1063–1066.

    Article  PubMed  CAS  Google Scholar 

  39. Acheson, A., Barker, P. A., Alderson, R. F., Miller, F. D., and Murphy, R. A. (1991) Detection of brain-derived neurotrophic factor-like activity in fibroblasts and Schwann cells: inhibition by antibodies to NGF. Neuron 7(2), 265–275.

    Article  PubMed  CAS  Google Scholar 

  40. Gloster, A. and Diamond, J. (1992) Sympathetic nerves in adult rats regenerate normally and restore pilomotor function during anti-NGF treatment that prevents their collateral sprouting. J. Comp. Neurol. 326(3), 363–374.

    Article  PubMed  CAS  Google Scholar 

  41. Murphy, R. A., Acheson, A., Hodges, R., Haskins, J., Richards, C., Reklow, E., et al. (1993) Immunological relationships of NGF, BDNF, and NT-3: recognition and functional inhibition by antibodies to NGF. J. Neurosci. 13(7), 2853–2862.

    PubMed  CAS  Google Scholar 

  42. Conner, J. M. and Varon, S. (1996) Characterization of β-NGF antibodies: assay dependent variability in the cross-reactivity with various neurotrophin family members. J. Neurosci. Methods 65, 93–99.

    Article  PubMed  CAS  Google Scholar 

  43. Cuello, A. C. (1983) Immunohistochemistry. Wiley, New York.

    Google Scholar 

  44. De Mey, J. and Moeremans, M. (1986) Raising and testing polyclonal antibodies for immunocytochemistry. In Immunocytochemistry: Modern Methods and Applications (Polak, J. M., Van Noorden, S., eds.), John Wright & Sons, Bristol, UK, pp. 3–12.

    Google Scholar 

  45. Ritter, M. A. (1986) Raising and testing monoclonal antibodies for immunocytochemistry. In Immunocytochemistry: Modern Methods and Applications (Polak, J. M., Van Noorden, S., eds.), John Wright & Sons, Bristol, UK, pp. 13–25.

    Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Neurosciences, University of California, San Diego, La Jolla, CA

    James M. Conner

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  1. James M. Conner
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Editors and Affiliations

  1. Department of Human Physiology, Flinders University, Adelaide, Australia

    Robert A. Rush

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© 2001 Humana Press Inc.

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Conner, J.M. (2001). Localization of Neurotrophin Proteins Within the Central Nervous System by Immunohistochemistry. In: Rush, R.A. (eds) Neurotrophin Protocols. Methods in Molecular Biology™, vol 169. Humana Press. https://doi.org/10.1385/1-59259-060-8:3

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  • DOI: https://doi.org/10.1385/1-59259-060-8:3

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-699-4

  • Online ISBN: 978-1-59259-060-5

  • eBook Packages: Springer Protocols

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