Sv120

After aforementioned deparaffinage, dehydration, and antigen recovery, mice brain slices were washed with PBS for 3 × 5 min and blocked in 5% donkey serum containing 0.5% Triton X-100 for 40 min. Next, slices were incubated with primary antibody (listed in Table 1) at 4°C for 24–48 h. After incubation with secondary antibody at 37°C for 1 h, slices were washed with PBS for 3 × 5 min and stained with DAPI reagent (G1012, Servicebio) for 10 min at room temperature. Slices were sealed with anti-fluorescence quencher (G1401, Servicebio) and imaged using a scanning microscope (SV120, OLYMPUS).

Paraffin-embedded brain sections (4-μm thick) were stained with Nissl staining solution (Servicebio, Wuhan, China) for 5 min and then washed with running water until they turned colourless. If the slice was hyperchromatic, 0.1% glacial acetic acid was used for differentiation. The sections were dried, mounted on coverslips using Permount TM Mounting Medium and examined under a scanning microscope (SV120, OLYMPUS, Japan).

TUNEL staining assay was performed using the One-Step TUNEL in situ Apoptosis Assay Kit (Elabscience, Wuhan, China) according to the manufacturer’s instructions. In brief, paraffin-embedded mouse brain slices (4 μm thick) were deparaffinised in xylene, rehydrated in gradient ethanol, incubated with TUNEL reagent mixture for 60 min, counterstained with DAPI for 5 min and examined under a fluorescence microscope (SV120, OLYMPUS, Japan). The TUNEL index (apoptosis rate) was calculated as the proportion (%) of TUNEL-positive neurons among DAPI-positive neurons.

Totally 150 nL of AAV9‐TH‐Cre, AAV9‐DIO‐TVA‐EYFP, and AAV9‐DIO‐G were mixed in 3:1:2 and injected into left SNc. Once the helper virus reach the expression peak, the retrograde virus RV‐EnVA‐ΔG‐dsRed were injected in the same site with a mass of 150 nL. After 10 days of expression, mice were sacrificed to remove the brains. All brains were fixed in 4% paraformaldehyde and dehydrated with 20% and 30% sucrose. Then, brains were sliced in 30 μm at nine representative coronal levels from bregma: +2.0, +1.5, +0.5, −0.5, −1.2, −2.0, −3.0, −4.0, −5.6, and −6.6 mm. Next, all the slices were imaged via an automatic scanning fluorescence microscope (Olympus, SV120). Finally, neurons marked by fluorescent proteins in corresponding upstream brain regions were calculated by Image J (Fiji). All brain regions we analyzed included the Primary motor area (MOp), Nucleus accumbens (ACB), Caudoputamen (CP), Substantia innominate (SI), External segment of Globus pallidus (GPe), Central amygdala nucleus (CeA), Paraventricular hypothalamic nucleus (PVH), Zona incerta (ZI), Reticular part of Substantia nigra (SNr), Periaqueductal gray (PAG), Motor related Superior colliculus (SCm), Inferior colliculus (IC), Parabrachial nucleus (PB), and Interposed nucleus (IP), which was aligned by the mouse brain map of Allen Brain Atlas.

Free-floating brain sections were washed with Tris-buffered saline (TBS) for 3 × 5 min, and then placed in 5% periodic acid followed by alkaline silver iodide solution and developer solution (#G1052, Servicebio, Wuhan, China). After washing with acetic acid and water, they were placed in 0.1% gold chloride, followed by sodium thiosulphate solution. The sections were placed in absolute ethanol for 3 × 5 min and in xylene for 2 × 5 min to become transparent. Images were taken by an automatic slice scanning system (SV120, Olympus, Tokyo, Japan) and analyzed at 100 × magnification.