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Estrogen synthesis in the male brain triggers development of the avian song control pathway in vitro

Abstract

Sexual differentiation of the brain is determined in part by steroids such as estrogen, which are generally assumed to arise from the gonads. Here we show that estrogens are produced autonomously in cultured juvenile male zebra finch brain slices, and this brain-derived estrogen is both necessary and sufficient to trigger formation in vitro of a key male-specific synaptic connection in the telencephalic song control circuit. Male-like development was stimulated in female slices cultured with male slices or exposed to estrogen, and estrogen antagonists inhibited song circuit development in slices of either sex. These results reveal a new mode of sex-specific neural development, induced not by differential exposure to gonadal steroids, but rather by differential synthesis of steroids in the brain.

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Figure 1: Analysis of HVC–RA pathway development in cultured slices from 25-d zebra finches.
Figure 2: Quantitative analysis of HVC fiber terminal area at RA after various culture conditions.
Figure 3: Estrogen in conditioned medium from slice cultures.

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References

  1. Cooke, B., Hegstrom, C. D., Villeneuve, L. S. & Breedlove, S. M. Sexual differentiation of the vertebrate brain: principles and mechanisms. Front. Neuroendocrinol. 19, 323–362 (1998).

    Article  CAS  PubMed  Google Scholar 

  2. Mooney, R. Sensitive periods and circuits for learned birdsong. Curr. Opin. Neurobiol. 9, 121–127 (1999).

    Article  CAS  PubMed  Google Scholar 

  3. Schlinger, B. A. Sex steroids and their actions on the birdsong system. J. Neurobiol. 33, 619–631 (1997).

    Article  CAS  PubMed  Google Scholar 

  4. Brenowitz, E. A., Margoliash, D. & Nordeen, K. W. An introduction to birdsong and the avian song system. J. Neurobiol. 33, 495–500 (1997).

    Article  CAS  PubMed  Google Scholar 

  5. Bottjer, S. W. Building a bird brain—sculpting neural circuits for a learned behavior. Bioessays 19, 1109–1116 (1997).

    Article  Google Scholar 

  6. Nottebohm, F., Stokes, T. & Leonard, C. M. Central control of song in the canary. J. Comp. Neurol. 165, 457–486 (1976).

    Article  CAS  PubMed  Google Scholar 

  7. Mooney, R. & Rao, M. Waiting periods versus early innervation: the development of axonal connections in the zebra finch song system. J. Neurosci. 14, 6532–6543 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Konishi, M. & Akutagawa, E. Neuronal growth, atrophy and death in a sexually dimorphic song nucleus in the zebra finch. Nature 315, 145–147 (1985).

    Article  CAS  PubMed  Google Scholar 

  9. Arnold, A. P. Sexual differentiation of the zebra finch song system—positive evidence, negative evidence, null hypotheses, and a paradigm shift. J. Neurobiol. 33, 572–584 (1997).

    Article  CAS  PubMed  Google Scholar 

  10. Gahr, M. & Metzdorf, R. The sexually dimorphic expression of androgen receptors in the song nucleus hyperstriatalis ventrale pars caudale of the zebra finch develops independently of gonadal steroids. J. Neurosci. 19, 2628–2636 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Nordeen, E. J., Nordeen, K. W. & Arnold, A. P. Sexual differentiation of androgen accumulation within the zebra finch brain through selective cell loss and addition. J. Comp. Neurol. 259, 393–399 (1987).

    Article  CAS  PubMed  Google Scholar 

  12. Gahr, M. & Konishi, M. Developmental changes in estrogen-sensitive neurons in the forebrain of the zebra finch. Proc. Natl. Acad. Sci. USA 85, 7380–7383 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Jacobs, E. C., Arnold, A. P. & Campagnoni, A. T. Developmental regulation of the distribution of aromatase- and estrogen-receptor- mRNA-expressing cells in the zebra finch brain. Dev. Neurosci. 21, 453–472 (1999).

    Article  CAS  PubMed  Google Scholar 

  14. Gurney, M. & Konishi, M. Hormone induced sexual differentiation of brain and behavior in zebra finches. Science 208, 1380–1382 (1980).

    Article  CAS  PubMed  Google Scholar 

  15. Simpson, H. B. & Vicario, D. S. Early estrogen treatment of female zebra finches masculinizes the brain pathway for learned vocalizations. J. Neurobiol. 22, 777–793 (1991).

    Article  CAS  PubMed  Google Scholar 

  16. Simpson, H. B. & Vicario, D. S. Early estrogen treatment alone causes female zebra finches to produce learned, male-like vocalizations. J. Neurobiol. 22, 755–776 (1991).

    Article  CAS  PubMed  Google Scholar 

  17. Dittrich, F., Feng, Y., Metzdorf, R. & Gahr, M. Estrogen-inducible, sex-specific expression of brain-derived neurotrophic factor mRNA in a forebrain song control nucleus of the juvenile zebra finch. Proc. Natl. Acad. Sci. USA 96, 8241–8246 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Wade, J. & Arnold, A. Post-hatching inhibition of aromatase activity does not alter sexual differentiation of the zebra finch song system. Brain Res. 639, 347–350 (1994).

    Article  CAS  PubMed  Google Scholar 

  19. Mathews, G. A. & Arnold, A. P. Antiestrogens fail to prevent the masculine ontogeny of the zebra finch song system. Gen. Comp. Endocrinol. 80, 48–58 (1990).

    Article  CAS  PubMed  Google Scholar 

  20. Mathews, G. A. & Arnold, A. P. Tamoxifen's effects on the zebra finch song system are estrogenic, not antiestrogenic. J. Neurobiol. 22, 957–969 (1991).

    Article  CAS  PubMed  Google Scholar 

  21. Balthazart, J., Absil, P., Fiasse, V. & Ball, G. F. Effects of the aromatase inhibitor r76713 on sexual differentiation of brain and behavior in zebra finches. Behaviour 131, 225–260 (1994).

    Article  Google Scholar 

  22. Adkins-Regan, E., Yang, S. & Mansukhani, V. Behavior of male and female zebra finches treated with an estrogen synthesis inhibitor as nestlings. Behaviour 133, 847–862 (1996).

    Article  Google Scholar 

  23. Adkins-Regan, E., Abdelnabi, M., Mobarak, M. & Ottinger, M. A. Sex steroid levels in developing and adult male and female zebra finches (Poephila guttata). Gen. Comp. Endocrinol. 78, 93–109 (1990).

    Article  CAS  PubMed  Google Scholar 

  24. Schlinger, B. A. & Arnold, A. P. Plasma sex steroids and tissue aromatization in hatchling zebra finches: implications for the sexual differentiation of singing behavior. Endocrinology 130, 289–299 (1992).

    Article  CAS  PubMed  Google Scholar 

  25. Hutchison, J. B., Wingfield, J. C. & Hutchison, R. E. Sex differences in plasma concentrations of steroids during the sensitive period for brain differentiation in the zebra finch. J. Endocrinol. 103, 363–369 (1984).

    Article  CAS  PubMed  Google Scholar 

  26. Wade, J., Schlinger, B. A. & Arnold, A. P. Aromatase and 5 beta-reductase activity in cultures of developing zebra finch brain: an investigation of sex and regional differences. J. Neurobiol. 27, 240–251 (1995).

    Article  CAS  PubMed  Google Scholar 

  27. Vockel, A., Pröve, E. & Balthazart, J. Sex- and age-related differences in the activity of testosterone-metabolizing enzymes in microdissected nuclei of the zebra finch brain. Brain Res. 511, 291–302 (1990).

    Article  CAS  PubMed  Google Scholar 

  28. Distler, P. G. & Robertson, R. T. Development of AChE-positive neuronal projections from basal forebrain to cerebral cortex in organotypic tissue slice cultures. Brain Res. Dev. Brain Res. 67, 181–196 (1992).

    Article  CAS  PubMed  Google Scholar 

  29. Dailey, M. E. & Smith, S. J. The dynamics of dendritic structure in developing hippocampal slices. J. Neurosci. 16, 2983–2994 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Molnar, Z. & Blakemore, C. Development of signals influencing the growth and termination of thalamocortical axons in organotypic culture. Exp. Neurol. 156, 363–393 (1999).

    Article  CAS  PubMed  Google Scholar 

  31. Toran-Allerand, C. D. Organotypic culture of the developing cerebral cortex and hypothalamus: relevance to sexual differentiation. Psychoneuro endocrinology 16, 7–24 (1991).

    Article  CAS  Google Scholar 

  32. Wray, S., Castel, M. & Gainer, H. Characterization of the suprachiasmatic nucleus in organotypic slice explant cultures. Microsc. Res. Tech. 25, 46–60 (1993).

    Article  CAS  PubMed  Google Scholar 

  33. Stoppini, L., Buchs, P.-A. & Muller, D. A simple method for organotypic cultures of nervous tissue. J. Neurosci. Methods 37, 173–182 (1991).

    Article  CAS  PubMed  Google Scholar 

  34. Goldman, S. A., Zaremba, A. & Niedzwiecki, D. In vitro neurogenesis by neuronal precursor cells derived from the adult songbird brain. J. Neurosci. 12, 2532–2541 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Honig, M. G. & Hume, R. I. Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures. J. Cell Biol. 103, 171–187 (1986).

    Article  CAS  PubMed  Google Scholar 

  36. Niblock, M. M., Brunso-Bechtold, J. K. & Henkel, C. K. Fiber outgrowth and pathfinding in the developing auditory brainstem. Brain Res. Dev. Brain Res. 85, 288–292 (1995).

    Article  CAS  PubMed  Google Scholar 

  37. Foster, E. F. & Bottjer, S. W. Axonal connections of the high vocal center and surrounding cortical regions in juvenile and adult male zebra finches. J. Comp. Neurol. 397, 118–138 (1998).

    Article  CAS  PubMed  Google Scholar 

  38. Wade, J., Schlinger, B. A., Hodges, L. & Arnold, A. P. Fadrozole: a potent and specific inhibitor of aromatase in the zebra finch brain. Gen. Comp. Endocrinol. 94, 53–61 (1994).

    Article  CAS  PubMed  Google Scholar 

  39. MacGregor, J. I. & Jordan, V. C. Basic guide to the mechanisms of antiestrogen action. Pharmacol. Rev. 50, 151–196 (1998).

    CAS  PubMed  Google Scholar 

  40. Wade, J. & Arnold, A. P. Functional testicular tissue does not masculinize development of the zebra finch song system. Proc. Natl. Acad. Sci. USA 93, 5264–5268 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Toran-Allerand, C. D., Singh, M. & Setalo, G. Jr. Novel mechanisms of estrogen action in the brain: new players in an old story. Front. Neuroendocrinol. 20, 97–121 (1999).

    Article  CAS  PubMed  Google Scholar 

  42. MacLusky, N. J., Walters, M. J., Clark, A. S. & Toran-Allerand, C. D. Aromatase in the cerebral cortex, hippocampus, and mid-brain: ontogeny and developmental implications. Mol. Cell. Neurosci. 5, 691–698 (1994).

    Article  CAS  PubMed  Google Scholar 

  43. Schlinger, B. & Arnold, A. P. Circulating estrogens in a male songbird originate in the brain. Proc. Natl. Acad. Sci. USA 89, 7650–7653 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Schlinger, B. A. & Arnold, A. P. Brain is the major site of estrogen synthesis in a male songbird. Proc. Natl. Acad. Sci. USA 88, 4191–4194 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Vanson, A., Arnold, A. P. & Schlinger, B. A. 3-beta-hydroxysteroid dehydrogenase/isomerase and aromatase activity in primary cultures of developing zebra finch telencephalon—dehydroepiandrosterone as substrate for synthesis of androstenedione and estrogens. Gen. Comp. Endocrinol. 102, 342–350 (1996).

    Article  CAS  PubMed  Google Scholar 

  46. Schlinger, B. A., Amurumarjee, S., Shen, P., Campagnoni, A. T. & Arnold, A. P. Neuronal and non-neuronal aromatase in primary cultures of developing zebra finch telencephalon. J. Neurosci. 14, 7541–7552 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors thank E. Brenowitz, B. Schlinger, S. Fahrbach and R. Stripling for advice, and T. Mittelmeier for performing the blind anatomical analyses. Supported by NIH grants NS25742 and MH52086.

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Correspondence to David F. Clayton.

Supplementary information

Figure 1

(JPG 163.9 KB)

Fibers from HVC to RA in cultured adult male zebra finch slices. Confocal microscopic image of nucleus RA, visualized by diI labeling of fibers from HVC, in slices of adult male zebra finch brain after 6 weeks in culture. Labeled fiber bundles enter from upper left, and ramify within RA (visible as oval area of diffuse bright fluorescence in left panel). Right panel shows higher magnification of ramifying fibers within RA. A DiI crystal was placed on HVC at the initiation of the cultures.

Figure 2

(JPG 50.16 KB)

ingrowth in vitro (serial images) A single juvenile male slice, established at 25 days of age with diI label upon HVC, is imaged after 1, 2 & 3 weeks in culture. The fields shown are centered on the boundary of RA (indicated); dorsal is left.

Figure 3

(JPG 405.04 KB)

Fibers from HVC to RA develop during long-term culture of 25 day-old zebra finch brain slices Fiber terminals in nucleus RA of 25 day-old juvenile zebra finch brain slices (A) before, and (B) after 3 weeks in culture; visualized by Neurofilament immunoreactivity.

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Holloway, C., Clayton, D. Estrogen synthesis in the male brain triggers development of the avian song control pathway in vitro. Nat Neurosci 4, 170–175 (2001). https://doi.org/10.1038/84001

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