A1 confocal

For histological analysis, mice were anesthetized with ketamine at the 5th week following TAM injection and perfused transcardially with 4% paraformaldehyde (PFA)^{15 (link)}. After dehydrated in 30% sucrose, the brain tissues were cut into 40 μm coronal sections using Leica cryostat (CM-1950S, Leica, Germany). They were incubated with blocking buffer (10% normal goat serum, 1% bovine serum albumin, 0.3% Triton X-100, PBS solution) overnight at 4 °C and were then incubated with the primary antibodies overnight at 4 °C (Table 1). For Ub staining, the sections were performed antigen repair by using citrate buffer (pH 6.0). The stained sections were imaged using a laser scanning confocal microscope (A1 confocal, Nikon instruments (Shanghai) Co., Ltd). The paired images in the figures were collected at the same gain and offset settings.

Based on the previous study (Lu and Yang, 2017 (link)), the AAV-infused mouse brains were sectioned at 60 μm of thickness. The sections were stained with GFP antibody (1:1000, ab6662; Abcam, Cambridge, UK) and Ctip2 antibody (1:500, ab18465, Abcam, Cambridge, UK). Afterward, the stained sections were imaged using a laser scanning confocal microscope (A1 confocal, Nikon Instruments [Shanghai]Co., Ltd.) under 40× objective lens. The MSNs were identified based on the positive staining of Ctip2. Neuronal structure reconstruction with neuTube (Feng et al., 2015 (link)) and Sholl analysis were performed with ImageJ (Longair et al., 2011 (link)).

Mice were anesthetized with ketamine and perfused transcardially with 40 mL PBS and then 60 mL 4% paraformaldehyde (PFA). After dehydrating 30% sucrose for 72 hours, the brain tissues were cut into 40 μm coronal sections using a Leica cryostat (CM-1950S, Leica, Germany). The slides were incubated with IFC blocking buffer (10% normal goat serum, 1% bovine serum albumin, 0.3% Triton X-100, PBS solution) for 2 hours at room temperature and were then incubated with the primary antibodies overnight at 4 °C (a complete list of primary antibodies information in Table 1). For Ub staining, the sections were subjected to antigen repair using citrate buffer (pH 6.0). The stained sections were visualized and photographed directly with a laser scanning confocal microscope (A1 confocal, Nikon Instruments (Shanghai) Co., Ltd). The paired images in the figures were collected at the same gain and offset settings. TH-positive cells in the SNc and VTA were calculated in every three sections from bregma −2.80 to −3.64 mm, and the data were collected from 9 slices per animal. The outline of the SNc and VTA was determined according to anatomical landmarks. The analysis of IFC staining on the number of the puncta, axon density, and the mean number of enlarged axon terminals was quantified using ImageJ software.

Cells were fixed at 12-14DIV, after co-culture describe above. Fixation was performed as described above. Subsequently, cells were incubated with 1 hr in blocking buffer (10% goat serum, 0.3% Triton-X in PBS). Glast and Gfap immunohistochemistry was performed as described above. Following primary antibody incubations, AlexaFlour 488 and 647 were used to visualize the primary antibodies with 1 hr incubations at 1:500. Prior to image acquisition, the experimenter was blinded to experimental conditions. Confocal images were acquired on Nikon A1 confocal with 40x objective and two digital zoom. Astrocyte complexity was assessed by Sholl analysis plugin in FIJI software according to Stogsdill et al. (2017) (link). Briefly, for each individual astrocyte, a line was drawn from the soma out to the furthest cellular process. Sholl radii were set at starting 10 µm from the start line with 1 µm increasing increments. Subsequently, the data were binned at 10 µm intervals in Excel.