Our experimental design is summarized in Figure 1 . Untreated females were first recorded for 2 weeks to ensure that they were not singing spontaneously. Afterward, between experimental days 1–7 (Fig. 1 ), birds were injected intraperitoneally twice daily with 50 μg of 5-bromo-2′-deoxyuridine (BrdU) per gram of body weight (Sigma) in 0.9% saline. On day 8, HVC was tranfected bilaterally with a BDNF plasmid expression vector (100 nl/hemisphere) under isoflurane anesthesia. Additional females were given BDNF plasmid injections bilaterally into the robust nucleus of the arcopallium (RA) or into the nidopallium adjacent to the HVC. To control for the surgery and the plasmid injection, other females underwent either a sham operation or were injected with an empty plasmid not containing the BDNF sequence into the HVC. After surgery, all birds were implanted with SILASTIC tubes (Dow Corning) containing either testosterone propionate (Sigma) or nothing (empty SILASTICs). Between days 8 and 34, females were injected intramuscularly with either VEGFR2-I (inhibitor of vascular endothelial growth factor receptor tyrosine kinase) (4-[(4′-chloro-2′-fluoro)phenylamino]-6,7-dimethoxy-quinazoline [Calbiochem]; 2.5 μg of VEGFR2-I per gram of body weight) dissolved in vehicle (DMSO/PBS, 2:1) or either the DMSO/PBS vehicle or PBS only. Because vehicle and PBS females did not differ in their measurements, we collectively refer to them as PBS-treated throughout this paper. At day 26, all SILASTIC implants were renewed. On day 44, the birds were killed by an overdose of isoflurane and perfused transcardially with 50 ml each of 0.9% saline and 4% formaldehyde solution. The brains were removed, postfixed in 4% formaldehyde for 1 week, and then cryoprotected in 10% sucrose for 5 d and 20% sucrose for 3 d. The right hemisphere was cut on a freezing microtome into 40 μm sagittal sections, and the left hemisphere was stored at −80°C.
An additional set of females (“BDNF only”) was injected with BrdU and given BDNF plasmid as above but was not treated with either testosterone or VEGFR2-I. Another set of females was treated as above, except for the date when the animals were killed (four “T+VEGFR2-I+BDNF” females and four “T+VEGFR2-I” females; the latter received an empty plasmid). Each of the latter was randomly assigned to one of the former, and each female was maintained single in a sound box. One day after start of singing of the T+VEGFR2-I+BDNF females, these females and their nonsinging matched control females were killed.
An additional set of females (“BDNF only”) was injected with BrdU and given BDNF plasmid as above but was not treated with either testosterone or VEGFR2-I. Another set of females was treated as above, except for the date when the animals were killed (four “T+VEGFR2-I+BDNF” females and four “T+VEGFR2-I” females; the latter received an empty plasmid). Each of the latter was randomly assigned to one of the former, and each female was maintained single in a sound box. One day after start of singing of the T+VEGFR2-I+BDNF females, these females and their nonsinging matched control females were killed.
