Histology

Immediately after the electrophysiological recordings, birds were deeply anesthetized by an overdose of pentobarbital (Vortech Pharmaceutical, Dearborn, MI), transcardially perfused with saline (0.9%, 40 ml) and paraformaldehyde (4%, 40 ml), and decapitated. The brains were extracted and postfixed with paraformaldehyde for at least 4 days, after which 50-µm sagittal sections were cut on a vibratome. Unstained sections were visualized under a fluorescence microscope, and grayscale digital images of the same sections were collected under 450–490/515-nm cut-on and 510–560/590-nm cut-on excitation/emission filters for anatomical markers and DiI, respectively. Two images from the same sections were superimposed to create composite images, and scaled drawings of the silicon probes were used to validate the recording sites that fell within the boundaries of NCM and HVC (Fig. 1, B and C).