Figure 1.
Photographs demonstrating the punches of song control nuclei collected for Western blot analyses.
All images are from females, and show typical collections from LMAN and the MSt (A), HVC (B), and RA (C). Black arrowheads point to the lateral ventricles. White arrowheads indicate the pallial-subpallial lamina (A) and dorsal arcopallial lamina (C). Scale bars = 500 µm.
Figure 2.
Top: Western blot analyses used total protein (30 µg) extracted from the whole telencephalon of two 25-day-old male zebra finches. The left image shows two bands (38 kDa proBDNF and 14 kDa mature mBDNF) recognized by BDNF primary antibody. The small box immediately below the proBDNF band indicates an example of where the background optical density would be quantified. The middle and right images document the absence of these bands on separate blots preadsorbed with 20-fold excess of the antigen and with the primary antibody omitted, respectively. Bottom: Immunohistochemistry showing distinct BDNF+ cells in RA with the primary antibody and a complete absence of labeling in neighboring section exposed to the antibody following preadsorption with 10-fold excess peptide. Scale bar = 30 µm for both photographs.
Table 1.
Number of animals from each group in which a distinct 14 kDa band representing the mature form of BDNF was detected in each song nucleus using Western blot analysis.
Figure 3.
proBDNF protein in song control nuclei.
Data and representative images are from the punches of LMAN (top) and RA (bottom). The ratio of proBDNF/actin was calculated from mean optical densities of bands in each individual. Values represent means+one standard error. Symbols indicate significant differences as follows: a = main effect of treatment; * = main effect of sex; # = control males greater than control females; + = E2 females greater than control females. Sample sizes are indicated at the bottom of each bar. Representative bands for each protein from the LMAN and RA of each group are shown above the histograms.
Figure 4.
Quantification of TrkB-T protein.
Data and representative bands from Western blots analyses of LMAN (top) and RA (bottom) punches are depicted. The ratio of TrkB-T/actin was calculated from mean optical densities of bands representing these proteins in each individual. Values indicate means+one standard error. Symbols represent trends as follows: ♦ = TrkB-T is greater in E2-treated than control males, p = 0.053; • = In E2-treated birds, TrkB-T is decreased in females compared with males; p = 0.052. The letter ‘a’ indicates a significant main effect of treatment. Sample sizes are indicated within the bars for each group. Representative bands for each protein from the LMAN and RA of each group are shown above the histograms.
Figure 5.
Western blot analyses of TrkB proteins in punches of HVC.
The full length receptor (TrkB-FL) is shown on the top, and the truncated form (TrkB-T) on the bottom. The ratio of each of these proteins relative to actin was calculated from mean optical densities for each individual. Values indicate means+one standard error. Symbols indicate significant effects as follows: a = main effect of treatment; * = main effect of sex; # = Control female>control male. Sample sizes are indicated in the bars. Representative bands for each protein from are shown above the histograms.
Figure 6.
Immunohistochemistry for BDNF in LMAN.
The top panels are photographs from a representative individual of each group. Arrow heads indicate the border of LMAN. Scale bar for all images (shown in top left photo) = 100 µm. On the bottom, the histogram shows means+one standard error for the estimated total number of BDNF+ cells for each group (bottom). * = significant main effect of sex; a = significant main effect of treatment. Sample sizes are indicated within each bar.
Figure 7.
Immunohistochemistry of BDNF in RA.
The top panels are photographs from a representative individual of each group. Arrow heads indicate the border of RA. Scale bar for all images (shown in top left photo) = 100 µm. On the bottom, the histogram shows means+one standard error for the estimated total number of BDNF+ cells for each group (bottom). * = significant main effect of sex; # = sex difference within control birds; + = significant effect of E2 within females. Sample sizes are indicated in the bars.
Figure 8.
Immunohistochemistry of BDNF in HVC.
The top panels are photographs from a representative individual of each group. Arrowheads indicate the ventral border of HVC. Scale bar for all images (shown in top left photo) = 100 µm. On the bottom, the histogram indicates means+one standard error for the estimated total number of BDNF+ cells (bottom). * = significant main effect of sex; # = sex difference within control animals; + = effect of estradiol within females. Sample sizes are indicated within the bars.
Figure 9.
Immunohistochemical labeling of BDNF in the control nucleus rotundus (RT).
The top panels are photographs from a representative individual of each group. Arrowheads depict the border of this brain region. Scale bar for all images (shown in top left photo) = 100 µm. On the bottom, the histogram indicates means+one standard error for the estimated total number of BDNF+ cells (bottom). No statistically significant effects of sex or treatment were detected, and no interaction between these variables existed.