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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
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).
Mooney, R. Sensitive periods and circuits for learned birdsong. Curr. Opin. Neurobiol. 9, 121–127 (1999).
Schlinger, B. A. Sex steroids and their actions on the birdsong system. J. Neurobiol. 33, 619–631 (1997).
Brenowitz, E. A., Margoliash, D. & Nordeen, K. W. An introduction to birdsong and the avian song system. J. Neurobiol. 33, 495–500 (1997).
Bottjer, S. W. Building a bird brain—sculpting neural circuits for a learned behavior. Bioessays 19, 1109–1116 (1997).
Nottebohm, F., Stokes, T. & Leonard, C. M. Central control of song in the canary. J. Comp. Neurol. 165, 457–486 (1976).
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).
Konishi, M. & Akutagawa, E. Neuronal growth, atrophy and death in a sexually dimorphic song nucleus in the zebra finch. Nature 315, 145–147 (1985).
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).
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).
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).
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).
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).
Gurney, M. & Konishi, M. Hormone induced sexual differentiation of brain and behavior in zebra finches. Science 208, 1380–1382 (1980).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
Dailey, M. E. & Smith, S. J. The dynamics of dendritic structure in developing hippocampal slices. J. Neurosci. 16, 2983–2994 (1996).
Molnar, Z. & Blakemore, C. Development of signals influencing the growth and termination of thalamocortical axons in organotypic culture. Exp. Neurol. 156, 363–393 (1999).
Toran-Allerand, C. D. Organotypic culture of the developing cerebral cortex and hypothalamus: relevance to sexual differentiation. Psychoneuro endocrinology 16, 7–24 (1991).
Wray, S., Castel, M. & Gainer, H. Characterization of the suprachiasmatic nucleus in organotypic slice explant cultures. Microsc. Res. Tech. 25, 46–60 (1993).
Stoppini, L., Buchs, P.-A. & Muller, D. A simple method for organotypic cultures of nervous tissue. J. Neurosci. Methods 37, 173–182 (1991).
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).
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).
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).
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).
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).
MacGregor, J. I. & Jordan, V. C. Basic guide to the mechanisms of antiestrogen action. Pharmacol. Rev. 50, 151–196 (1998).
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).
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).
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).
Schlinger, B. & Arnold, A. P. Circulating estrogens in a male songbird originate in the brain. Proc. Natl. Acad. Sci. USA 89, 7650–7653 (1992).
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).
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).
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).
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.
Author information
Authors and Affiliations
Corresponding author
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.
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/84001
This article is cited by
-
Sex differences in song syntax and syllable diversity in testosterone-induced songs of adult male and female canaries
Biology of Sex Differences (2023)
-
Urotensin-related gene transcripts mark developmental emergence of the male forebrain vocal control system in songbirds
Scientific Reports (2019)
-
Tide-related Changes in mRNA Abundance of Aromatases and Estrogen Receptors in the Ovary and Brain of the Threespot Wrasse Halichoeres trimaculatus
Ocean Science Journal (2018)
-
The perinatal effects of maternal caffeine intake on fetal and neonatal brain levels of testosterone, estradiol, and dihydrotestosterone in rats
Naunyn-Schmiedeberg's Archives of Pharmacology (2017)
-
A genetically female brain is required for a regular reproductive cycle in chicken brain chimeras
Nature Communications (2013)