Age effect of deafening on stereotyped song maintenance in adult male bengalese finches Lonchura striata domestica

Birdsong is a complex learned vocal behavior that relies on auditory experience for development. However, it appears that among different species of close-ended songbirds, there are some variations in the necessity of auditory feedback for maintaining stereotyped adult song. In zebra finches, the deterioration of adult songs following deafness depends on the birds’ age. It is unknown whether this age effect is a general rule in other avian species as well. Therefore, we chose Bengalese finches, whose songs show more complexity and have much heavier dependency on auditory feedback than that of zebra finches, to compare the degree of song degradation after hearing loss in old (over 18 months old) and young adult birds (5–6 months old). We found that both syllable sequence and syllable phonology were much less severely affected by deafening in old adults than that in young ones. Moreover, young adults almost lost their capability to sing trills over 6 months following deafening, while old birds continued to sing plenty of trills and trilled syllables after the same period of deafening. Our results suggest that age plays an important role in affecting the dependency of adult song maintenance on auditory feedback in Bengalese finches. Furthermore, the age dependency may be a general phenomenon in different species of close-ended songbirds [Current Zoology 55(3):212–218, 2009]

Sexual Differences in Cell Loss during the Post-Hatch Development of Song Control Nuclei in the Bengalese Finch.

Birdsongs and the regions of their brain that control song exhibit obvious sexual differences. However, the mechanisms underlying these sexual dimorphisms remain unknown. To address this issue, we first examined apoptotic cells labeled with caspase-3 or TUNEL in Bengalese finch song control nuclei - the robust nucleus of the archopallium (RA), the lateral magnocellular nucleus of the anterior nidopallium (LMAN), the high vocal center (HVC) and Area X from post-hatch day (P) 15 to 120. Next, we investigated the expression dynamics of pro-apoptotic (Bid, Bad and Bax) and anti-apoptotic (Bcl-2 and Bcl-xL) genes in the aforementioned nuclei. Our results revealed that the female RA at P45 exhibited marked cell apoptosis, confirmed by low densities of Bcl-xL and Bcl-2. Both the male and female LMAN exhibited apoptotic peaks at P35 and P45, respectively, and the observed cell loss was more extensive in males. A corresponding sharp decrease in the density of Bcl-2 after P35 was observed in both sexes, and a greater density of Bid was noted at P45 in males. In addition, we observed that RA volume and the total number of BDNF-expressing cells decreased significantly after unilateral lesion of the LMAN or HVC (two areas that innervate the RA) and that greater numbers of RA-projecting cells were immunoreactive for BDNF in the LMAN than in the HVC. We reasoned that a decrease in the amount of BDNF transported via HVC afferent fibers might result in an increase in cell apoptosis in the female RA. Our data indicate that cell apoptosis resulting from different pro- and anti-apoptotic agents is involved in generating the differences between male and female song control nuclei

Sexual differences in cell proliferation in the ventricular zone, cell migration and differentiation in the HVC of juvenile Bengalese finch.

Song control nuclei have distinct sexual differences and thus are an ideal model to address how brain areas are sexually differentiated. Through a combination of histological analysis and electrical lesions, we first identified the ventricle site for HVC progenitor cells. We then found that there were significant sex differences in the cellular proliferation activity in the ventricular zone of the HVC, the number of migrating cells along the radial cells (positive immunoreactions to vimentin) and differentiation towards neurons. Through co-culturing of male and female slices containing the developing HVC in the same well, we found that the male slices could produce diffusible substances to masculinize the female HVC. By adding estrogen, an estrogen antagonist, brain-derived neurotrophic factor (BDNF) or its antibody into the culture medium, separately or in combination, we found that these diffusible substances may include estrogen and BDNF. Finally, we found that 1) estrogen-induced BDNF upregulation could be detected 48 hr after estrogen treatment and could not be blocked by a vascular endothelial growth factor (VEGF) receptor inhibitor and 2) the amount of VEGF mRNA expressed in the developing HVC and its adjacent area did not display any significant sex differences, as did the distribution of VEGF and laminin-expressing endothelial cells in the developing HVC. Because these findings are largely different from previous reports on the adult female HVC, it is suggested that our estrogen-induced BDNF up-regulation and the resultant sexual differentiation might not be mediated by VEGF and endothelial cells, but instead, may result from the direct effects of estrogen on BDNF

<i>In situ</i> hybridization for Bcl-2 mRNA in the RA, LMAN, HVC and Area X in the Bengalese finch.

<p>A1–D2: Labeled cells in the RA at post-hatching day (P) 45 (A1, A2), the LMAN at P35 (B1, B2), the HVC at P35 (C1, C2), and in Area X at P35 (D1, D2). E-H: Comparisons of the densities of Bcl-2 mRNA-positive cells in the RA (E), LMAN (F), HVC (G) and Area X (H) between males and females. Borders of the song nuclei (dashed lines) were determined with the help of another set of Nissl-stained sections. The Nissl-defined border of the female Area X was difficult to clearly identify, and the dashed lines in D2 indicate the approximate region corresponding to the male Area X. Dorsal is up and caudal is right. Scale bar = 200 μm in A1–C2 and 300 μm in D1–D2. The data are expressed as the mean ± SEM. **<i>P</i>< 0.01.</p

Immunohistochemistry for caspase-3 in the RA, LMAN, HVC and Area X in the Bengalese finch.

<p>A1–D4: Labeled cells are observed in the RA (A1–A4), LMAN (B1–B4), HVC (C1–C4) and Area X (D1–D4) in males at post-hatching days P15 (A1–D1) and P45 (A3–D3) and in females at P15 (A2–D2) and P45 (A4–D4). E-H: Comparison of the densities of caspase-3-positive cells in the RA (E), LMAN (F), HVC (G) and Area X (H) between males and females. Borders of the song nuclei (dashed lines) were determined with the help of another set of Nissl-stained sections. The Nissl-defined border of the female Area X was difficult to identify clearly, and the dashed lines in D2 and D4 indicate the approximate region that corresponds to the male Area X. Dorsal is up and caudal is right. Scale bar = 200 μm in A1–C4 and 300 μm in D1–D4. The data are expressed as the mean ± SEM. **<i>P</i>< 0.01.</p

Immunohistochemistry for Bcl-xL in the RA, LMAN, HVC and Area X of the Bengalese finch (visualized by nickel intensified DAB).

<p>A1–D4: Labeled cells in the RA at post-hatching day (P) 35 (A1, A2) and P45 (A3–A4), in the LMAN at P15 (B1, B2) and P45 (B3, B4), in the HVC at P45 (C1, C2) and in the adult (C3, C4), and in Area X at P15 (D1, D2) and P45 (D3, D4). E-H: Comparison of the densities of Bcl-xL-positive cells in the RA (E), LMAN (F), HVC (G) and Area X (H) between males and females. The borders of the song nuclei (dashed lines) were determined with the help of another set of Nissl-stained sections. The Nissl-defined border of the female Area X was difficult to clearly identify, and the dashed lines in D2 and D4 indicate the approximate region corresponding to the male Area X. Dorsal is up and caudal is right. Scale bar = 200 μm in A1–C4 and 300 μm in D1–D4. The data are expressed as the mean ± SEM. *<i>P</i>< 0.05, **<i>P</i>< 0.01.</p

<i>In situ</i> hybridization for Bid mRNA in the RA, LMAN, HVC and Area X of the Bengalese finch.

<p>A1–D2: Labeled cells in the RA at post-hatching day (P) 45 (A1, A2), the LMAN at P45 (B1, B2), the HVC at P35 (C1, C2), and in Area X at P15 (D1, D2). E-H: Comparisons of the densities of Bid mRNA-positive cells in the RA (E), LMAN (F), HVC (G) and Area X (H) between males and females. Borders of the song nuclei (dashed lines) were determined with the help of another set of Nissl-stained sections. The Nissl-defined border of the female Area X was difficult to clearly identify, and the dashed lines in D2 indicate the approximate region corresponding to the male Area X. Dorsal is up and caudal is right. Scale bar = 200 μm in A1–C2 and 300 μm in D1–D2. The data are expressed as the mean ± SEM. **<i>P</i>< 0.01.</p

BDNF immunoreactive cells and retrogradely labeled cells in the HVC and LMAN at post-hatching day (P) 45 after injection of fluorogold into the RA.

<p>A and B: BDNF-labeled positive cells in the HVC (A) and LMAN (B). C and D: Retrogradely labeled cells in the HVC (C) and LMAN (D) after injection of fluorogold into the RA. E and F: Merged images of A and C (E) and of B and D (F). The arrows in E and F indicate BDNF-positive cells, the curved arrows indicate fluorogold-positive cells, and the asterisks indicate double-labeled cells. Scale bar = 200 μm. G: The total numbers of BDNF- or TrkB-positive cells in the RA after unilateral HVC or LMAN lesion at P45. H: The total number of BDNF-positive cells projecting to the RA at P45 in the HVC or LMAN. The data are expressed as the mean ± SEM. *<i>P</i>< 0.05.</p

Nissl-defined volumetric changes of song control nuclei in the Bengalese finch from post-hatching day (P) 15 to 120.

<p>A: RA; B: LMAN; C: HVC; D: Area X; m: male; f: female. The mark “#” indicates that significant sexual differences are present in the marked groups towards adulthood. As the Nissl-defined borders of the female Area X in all studied age groups were difficult to clearly identify, the volume of the female Area X is not available in D.</p