Tract tracing by BDA and DiI

To reveal the efferent terminals from the MSt, we used biotinylated dextran amine (BDA, 0.1 μl per injection, 10% in distilled water, 10 kDa; {"type":"entrez-nucleotide","attrs":{"text":"D22910","term_id":"426835","term_text":"D22910"}}D22910, Molecular Probes®, Thermo Fisher Scientific Inc., USA) as an anterograde tracer. To reveal the MSt neurons projecting to the SN, we used 1,1′-dioctadecyl-3,3,3′3′-tetramethylindocarbocyanine perchlorate (DiI, 30 nl per injection, 7% solution in N,N-Dimethylformamide) as a retrograde tracer. We used a micro injection instrument (Nanoject II, Drummond Scientific Co., Broomall, Pennsylvania, USA) to inject the tracer into chicks aged approximately PH9. The injection was performed under ketamine/xylazine anesthesia, as described above. Either 7 days (BDA) or 11 days (DiI) after the operation, chicks were transcardially perfused with 4% PFA. Brains were dissected out and post-fixed in the same fixative at 4°C overnight (BDA) or for ≥ 3 days (DiI).

After 1 day of cryo-protection in PBS with 20% sucrose, the brains were frozen and stored at -30°C until sectioning. We used a sliding microtome with a freezing stage (TU-213, Yamato Kohki Industrial Co. Ltd., Saitama, Japan) to cut the brains into sagittal sections for single or double histochemical labeling.

For single labeling to visualize BDA, 60-μm-thick sections were cut and incubated in avidin-biotinylated horseradish peroxidase complex reagent (PK-6100, Vectastain® Elite ABC Kit, Vector Laboratories Co., USA) and DAB (SK-4100, DAB Peroxidase Substrate Kit, Vector Laboratories) as a chromogen. Sections were mounted on APS coated glass slides (S8441, Matsunami Glass Ind. LTD., Osaka, Japan), cover-slipped in Permount™ mounting medium (SP15-500, Thermo Fisher Scientific Inc., USA), and stored at room temperature.

For double labeling, 24-μm-thick sections were initially soaked in Alexa Fluor®488 streptavidin conjugate at room temperature for 1 h to visualize BDA (S32354, Molecular Probes®; dilution by 1:400). The sections were then processed with a primary antibody; rabbit anti-TH (1:1000, 4°C, overnight; AB152, Chemicon®, EMD Millipore Co., USA) or rabbit anti-GAD65 (1:1000, 4°C, 3 days; bs-0400R, Bioss Inc., USA). As the secondary antibody, we used goat anti-rabbit IgG - Alexa Fluor® 568 conjugate (1:400, A11011, Molecular Probes®) at room temperature for 1 h. Sections were then mounted on APS coated glass slides and cover-slipped in Prolong® Diamond antifade mountant with DAPI ({"type":"entrez-protein","attrs":{"text":"P36962","term_id":"547832","term_text":"P36962"}}P36962, Thermo Fisher Scientific Inc.) and stored at room temperature.

The fixated brains were embedded into yolk, post-fixated in 4% PFA for an additional ≥ 3 days, and cut into 50-μm-thick sections using a vibrating microtome (DTK-1000). Sections were collected in PBS, mounted onto APS coated glass slides, and cover-slipped in PBS. The cover glass was sealed with a transparent nail polish to prevent drying.

At low magnification, stained sections were photographed using a bright-field light microscope (Olympus BH-2) and fluorescence microscopes (Leica MZ16F and EVOS® FL Imaging System). We used a confocal microscope (Zeiss LSM 510) to examine the connectivity between BDA-positive terminals and tegmentum neurons at a high magnification. Scanned images were examined using Zeiss LSM 5 Image. Images of interest were edited using a free graphics editor GIMP2.8 (GNU Image Manipulation Program; URL: https://www.gimp.org/).