Brain-derived neurotrophic factor signaling in the HVC is required for testosterone-induced song of female canaries

Abstract

Testosterone-induced singing in songbirds is thought to involve testosterone-dependent morphological changes that include angiogenesis and neuronal recruitment into the HVC, a central part of the song control circuit. Previous work showed that testosterone induces the production of vascular endothelial growth factor (VEGF) and its receptor (VEGFR2 tyrosine kinase), which in turn leads to an upregulation of brain-derived neurotrophic factor (BDNF) production in HVC endothelial cells. Here we report for the first time that systemic inhibition of the VEGFR2 tyrosine kinase is sufficient to block testosterone-induced song in adult female canaries, despite sustained androgen exposure and the persistence of the effects of testosterone on HVC morphology. Expression of exogenous BDNF in HVC, induced locally by in situ transfection, reversed the VEGFR2 inhibition-mediated blockade of song development, thereby restoring the behavioral phenotype associated with androgen-induced song. The VEGFR2-inhibited, BDNF-treated females developed elaborate male-like song that included large syllable repertoires and high syllable repetition rates, features known to attract females. Importantly, although functionally competent new neurons were recruited to HVC after testosterone treatment, the time course of neuronal addition appeared to follow BDNF-induced song development. These findings indicate that testosterone-associated VEGFR2 activity is required for androgen-induced song in adult songbirds and that the behavioral effects of VEGFR2 inhibition can be rescued by BDNF within the adult HVC.

Figure 1.

Experimental design (a, b) and BDNF expression of HVC (c, d). a, The experimental design (for details, see Materials and Methods, Experimental procedures). b, BDNF-expressing plasmid was transfected into nucleus HVC of the song control system, shown in a schematic sagittal view. The motor pathway (green arrows) is involved in song production and ultimately innervates the bird's sound-producing organ, the syrinx. The anterior forebrain pathway (yellow arrows) is an internal feedback system thought to be involved in song learning. Nuclei depicted in red express androgen receptors. HVC expresses both androgen and estrogen receptors (for additional details and abbreviations, see Bolhuis and Gahr, 2006). c, Local distribution of the BDNF plasmid in the HVC. Microphotographs of autoradiograms of sections that were obtained from males killed 4 and 26 d after in vivo transfection, respectively, and hybridized with a radioactive antisense probe for BDNF–eGFP. The black labeled areas are those that contain the BDNF–eGFP–mRNA. The dashed lines indicate the ventral border of HVC. d, Time course of expression of BDNF precursor protein (proBDNF) in the HVC as determined by Western blot analysis. The intensity of a major doublet band detectable at 29 kDa that is characteristic for proBDNF was relatively increased to controls at 4–14 d after in vivo transfection. Peak expression was between 6 and 10 d, in agreement with the rBDNF–GFP–mRNA expression kinetics shown in supplemental Figure S1 (available at www.jneurosci.org as supplemental material).

Figure 2.

Sonograms of typical songs produced by birds of different treatment groups. a, Depicted are the vocalizations of two females that were not treated with testosterone (null). These birds produce calls but never sing. b, Shown are the sonograms of two females that were implanted with testosterone and were injected with PBS (T+PBS). Most of these birds (84%) sing. Their songs vary from simple song (left) to more stereotyped male-like song (right). c, Depicted are the sonograms of two birds that were implanted with T and were injected with VEGFR2 inhibitor (T+VEGFR2-I). Only 19% of these females sing (right), but their songs are monotonous. Most such females (81%; left) never sing and produce only calls like null females. In d, typical sonograms are shown for two females treated with T, injected with VEGFR2-inhibitor, and infused with a BDNF-expressing vector into HVC (T+VEGFR2-I+BDNF). All of these birds sing in a male-like manner.

Figure 3.

BDNF facilitates the production of large song syllable repertoires (a) and high syllable repetition rates (b). a, Note that BDNF treatment (T+VEGFR2-I+BDNF) overcomes the inhibitory effect of VEGFR2-I (T+VEGFR2-I) on the development of syllable repertoires after testosterone treatment. Females not receiving testosterone (null) do not sing and differed from all other groups (p ≤ 0.0001). The length of each box shows the range of the central 50% of the syllable repertoire, with the borders of the box at the first and third quartiles, and the median value represented by a thick line. The whiskers show the range of values that fall within the inner fences. Values between the inner and outer fences are plotted with triangles, and values outside the outer fence are plotted with circles. *p ≤ 0.05; **p ≤ 0.001; ***p ≤ 0.0001, by Mann–Whitney U test. In b, we measured the percentage of tours that are produced with repetition rates above 10 Hz and above 17 Hz; the latter tours have the potential to be sexually salient (Vallet and Kreutzer, 1995). Testosterone-treated females that obtained BDNF (light boxes) are compared with those not treated with BDNF (dark boxes). The boxes represent the same characteristics as described in a. The values between the inner and outer fences are plotted with circles. *p ≤ 0.05; **p ≤ 0.001, by t test.

Figure 4.

Effect of testosterone on HVC volume (a) and HVC neuron numbers (b). a, Testosterone treatment increases the HVC volume compared with null females (no testosterone). This effect is not impaired by additional treatment with VEGFR2-I (T+VEGFR2-I) and/or BDNF (T+PBS+BDNF and T+VEGFR2-I+BDNF). b shows that testosterone increases the total number of HVC neurons compared with null females independent of additional treatments with BDNF and/or VEGFR2-I. ***p ≤ 0.0001, by one-way ANOVA.

Figure 5.

The recruitment of new functional HVC neurons is subsequent to BDNF-dependent singing activity. a, In the HVC of adult female canaries, we observed neurons triple labeled for the immediate early gene ZENK (red), the cell birth marker BrdU (green), and the DNA marker DAPI (blue). These neurons are newly born and functional, as indicated by ZENK expression after singing. Scale bar, 10 μm. b, After 40 d of testosterone treatment, the number of new functional neurons is smaller in females treated with testosterone and VEGFR2-I (T+VEGFR2-I) than in such females, in which we expressed exogenous BDNF locally in HVC (T+VEGFR2-I+BDNF). **p ≤ 0.0001, by one-way ANOVA. c, The number of new neurons of singing T+VEGFR2-I+BDNF females and of nonsinging T+VEGFR2-I females is similar at the beginning of song development, 9–13 d after testosterone treatment started.