Influence of testosterone on cell proliferation in the telencephalic ventricle zone

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Image 1. Canaries. Source: “15 Canaries Stolen from Bird Breeder.” The Telegraph.

Introduction: 

Sexual selection is an important aspect in biology that allows scientist to decipher traits in animals which are being selected for among different species. Among the many species that follow this strategy are Canaries. Canaries, Serinus canaria domestica, are known for their loud and rhythmic singing. Most male canaries have adopted this trait through sexual selection in order to ensure their reproductive success. This is done by singing songs that attract their female opposites through the learning of songs from older members of the species as well as modifying vocal output to generate a memorized model. Early developmental factors play an important role in the expression of sexually selected traits. A better “song” is an indication of better genes. Testosterone levels in canaries reflect the type of songs the birds will sing, as well as the different impacts it will have on female/male reproduction. The song system in canaries is sexually dimorphic, in which the males have a more complex brain structure than females. Due to their ability to encode and produce song, canaries serve as model organisms in the field of scientific research in terms of understanding the underlying concepts that occur in the brain of vertebrates involving song production. Canaries are generally used as model organisms in terms of neurogenesis (the birth of new neurons), as well as understanding the functional components of vertebrate brain that control memory recollection and recalling motor movements. The song system of birds help with the understanding of brain circuits and their potential for rejuvenation as well as the repairing of brain variables,  involved in learning.

Neuroanatomy:

HVc: High vocal center in lateral caudal nidopallium

RA: robost nucleus of the arcopallium

Area X: Homologous to mammalian basal ganglia

LMAN: lateral part of the magnocellular nucleus of anterior neostriatum, also part of the avian basal ganglia

DLM: dorso-lateral division of the medial thalamus

PDP: Posterior descending pathway; homologous to the mammalian motor pathway starting in the cerebral cortex and leading to the brain stem

AFP: Anterior forebrain pathway; homologous to the mammalian cortical pathway through the basal ganglia and thalamus

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Figure 1. A schematic of the song system in the brain of song birds. Source: Nottebohm F (2005) The Neural Basis of Birdsong.

The production of learned song in the brain of song birds is created in a discrete area of the brain nuclei, the forebrain’s lateral ventricle. The system is composed of two main branches, the posterior descending pathway (PDP) and the anterior forebrain pathway (AFP). The PDP is needed for the acquisition and production of learned song, while the AFP is required for acquisition. Figure 1. is a schematic that outlines the song pathway. The HVc  is the starting point that feeds information for the two separate pathways. The neurons lead from the HVc to the RA  and then follow directly to the PDP pathway, and indirectly to sections such as Area X, DLM, and LMAN. The HVc to RA projection carries the learned song. LMAN to RA projection induces the variability in motor output. The male song birds have a larger nucleus, HVc, especially during breeding seasons in the Spring (usually during the month of March). The cells in control of the song nuclei are androgen and estrogen.

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Anatomically discrete sex differences and enhancement by testosterone of cell proliferation in the telencephalic ventricle zone of the adult canary brain by Jennifer M. Barker, Gregory G. Ball, and Jacques Balthazart published in the Journal of Chemical Neuroanatomy, Volume 55 in January 2014.

In songbirds, seasonal changes affect the number of new neurons in the song system nucleus known as the HVc. The birth of new neurons is part of a process that involves constant replacement while being added to the many regions of the adult avian telencephalon. New neurons are generated from the ventricular zone (VZ) around the lateral ventricles and follow throughout the telencephalon. The changes that occur in HVc volume and the amount of new neurons produced are predicted to be administered by the level of testosterone produced. The fluctuations in testosterone help control different aspects of neurogenesis in the adult brain, however the specific location where these effect occur are unclear. Previous work on songbirds have suggested that testosterone levels affect the number of neuronal recruitment and survival in HVc, but have no affect on neuronal proliferation in the ventricular zone. In order to determine the influence of testosterone on cellular proliferation in the VZ of both male and female adult songbirds, data was collected from the brains of birds with implanted testosterone.

Experimental Procedure:

1. Animals and in vivo treatment: Adult male and female canaries were purchased and capsules containing either 10 mm crystalline testosterone or an empty capsule were implanted under the skin between the shoulders. The level of testosterone administered were elevated concentrations that are typical of adult sexually mature birds. The birds were also injected with BrdU (DNA replication marker) five times, every two hours, at the start of the day to ensure the labeling. BrdU (bromodeoxyuridine) was used to quantify cellular proliferation in the VZ at different rostro-caudal levels, from where the song nucleus occurs (Area X) through the caudal extent of HVc. The birds were decapitated after the third injection and the brains were trimmed to a “Y” shape in order to focus on the dorsal part of the ventricle. The brain trimmings were then mounted with the rostral tip up . Twelve series of eleven sections were collected from the rostral section to the caudal end of the telencephalon (Figure 2).

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Figure 2. (Figure 1 from J.M Barker et al.) (A) Photomicrographs of stained sections from a control-treated canary brain illustrating the BrdU labeling of the ventricle wall from rostro-caudal levels going from left to right. Area X is represent from 1-3, the septum from 4-8, and the HVc from 9-11. The horizontal black bars show the separation between dorsal and ventral subregions of the lateral ventricles. The small figure in the lower right corner shows a schematic illustration (can also be seen in figure 1) of the 11 sections stained. (B) This smaller image is a higher magnification of sex differentiation at a rostral level in Area X. (C) This is a magnification of the septum. The influence of testosterone can be seen in comparison to the control.

2. Statistical Analysis: The sex and testosterone effects on proliferation were analyzed using repeated measures ANOVA. The factors being analyzed were the stained cells in the VZ, the sex of the canary, and the hormonal treatment (control versus testosterone). 

Results:

Morphology:
Testosterone treatment resulted in a slight increase of the oviduct in non-breeding females, but did not affect the ovarian weight in females nor the testes weight in males.

Ventricle length:
Ventricle length did not differ between sex or treatment group which can be seen in part A of figure 3. Part B and C of the figure show the variation of ventricle length between the dorsal and ventral extents, however, these changes were corrected by dividing the BrdU-stained area adjacent to the ventricle by the length of the ventricle in each section.

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Figure 3. (Figure 3 from J.M. Barker et al.) Figure shows ventricle length in the brain. (A). The overall ventricle length was similar in all four groups of birds (F: female; M: male; T: testosterone; C: control) (B & C). The ventricle length in the ventral region decreased from rostral to caudal levels, but increased in the dorsal region.

Main Effects:
BrdU-ir cells identified main effects of the endocrine treatment and of the position along the dorso-ventral and rostro-caudral axis. There was a significant interaction between treatment type (testosterone) and the dorso-ventral region of the brain. There was a significant influence of testosterone on proliferation in the VZ of adult birds only in the most caudal sections (4-11) which can be seen in figure 4. 

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Figure 4. (Figure 4 from J.M. Barker et al.) This figure shows the effects of testosterone and sex on BrdU labeling of the ventricular zone (VZ) in the ventral. A & C show BrdU labeling in the ventral part of the brain. B & D show BrdU labeling in the dorsal part of the brain. The panel in section B shows the overall effects of testosterone in the 4 experimental groups (Female vs male control and female vs. male testosterone). A: shows the results obtained from the control vs. testosterone. This helps shows that testosterone does help increase cell proliferation in comparison to B which does the effects of testosterone in the dorsal section.

Discussion:

Despite previous assumptions, testosterone increases cell proliferation in caudal regions of the VZ of an adult songbird. Neurogenesis in the songbirds helps demonstrate that the number of neurons in the HVc increase with testosterone treatment. The cell numbers in the regions outside of the HVc do not change, which means that testosterone does not affect the production of new cells, but either enhances the recruitment of migrating cells, or enhances the survival of cells that reach the HVc. Testosterone increases cell proliferation in caudal regions of the VZ in both male and female; suggesting VZ cells are divided into subpopulations, such as differential localization of the aromatase enzyme, oestrogen receptor alpha, androgen receptors, or steroid hormone receptors. The cells containing estrogen receptors tend to be localized around the ventral region of the lateral ventricle zone, which would be effected by testosterone, thus female adult song birds have more cell proliferation than males in limited parts of the rostral VZ near Area X.

Strengths versus Weakness:

The data obtained helps provide evidence that testosterone does in fact increase cell proliferation in the caudal regions of the VZ, and that female song birds have more cell proliferation than males. The weakness of the paper is that the results were not based on direct measurements of proliferation alone. The cells that were counted included both newly produced cells as well as cell that may have migrated or died, thus recruitment and survival of cells is not established only within the HVc and can also take place in the ventricle wall. Another weakness of the paper is that the destination of the cells dividing was not established, and therefore, the data does not demonstrate the the cells will end up as neurons in the HVc.

Importance:

  • Increase in testosterone in males, increases dominance, song activity, and song consistency
  • Hormones of maternal origins have been shown to significantly alter offspring phenotype (sexual selection)
  • Increase in testosterone, increased aggressive behavior leading to increase success in defending food sources
  • Testosterone controls regions of the forebrain the control song: testosterone regulates both the motivation to sing, as well as the quality of song produced

Citations:

Barker, Jennifer M., Gregory F. Ball, and Jacques Balthazart. “Anatomically Discrete Sex Differences and Enhancement by Testosterone of Cell Proliferation in the Telencephalic Ventricle Zone of the Adult Canary Brain.” Journal of Chemical Neuroanatomy 55 (2014)

Cynx, J. “Testosterone Facilitates Some Conspecific Song Discriminations in Castrated Zebra Finches (Taeniopygia Guttata).” Proceedings of the National Academy of Sciences 89.4 (1992): 1376-378

Nottebohm F (2005) The Neural Basis of Birdsong. PLoS Biol 3(5): e164. doi:10.1371/journal.pbio.0030164

Rutkowska, Joanna, Mariusz Cichoń, Marisa Puerta, and Diego Gil. “Negative Effects of Elevated Testosterone on Female Fecundity in Zebra Finches.” Hormones and Behavior 47.5 (2005): 585-91

Vergauwen, Jonas, Ton G.g. Groothuis, Marcel Eens, and Wendt Müller. “Testosterone Influences Song Behaviour and Social Dominance – But Independent of Prenatal Yolk Testosterone Exposure.” General and Comparative Endocrinology 195 (2014): 80-87

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