Mice and MPTP treatment
Littermate D3 receptor-deficient (D3−/−) male mice (Ensemble number: ENSMUSG00000022705) and wild-type mice (provided by Dr. Xu M., University of Chicago), weighing 30–40 g and 2–3 months old were used in this study. The mice were developed by gene-targeting techniques, maintained on a 129/C57 mixed background, as described previously , and derived from heterozygous matings. DNA was isolated from mouse tail biopsies, and the D3−/− knockout genotype was confirmed by PCR using the forward primer (NeoF: 5′CATTCTGCACGCTTCAAAAGCG3′) and reverse primer (NeoR: 5′TTTCTCGGCAGGAGCAAGGTG3′). The PCR protocol was as follows: after denaturing at 94°C for 2 min, all reactions were followed by 30 cycles (94°C for 30 s, 60°C for 30 s, and 72°C for 30 s) using the Basic PCR mix (Biovisualab, Shanghai, China). Mice were individually housed on arrival in a stress-minimized specific pathogen-free facility with free access to food and water. After adaptation to a standard 12-h light/dark cycle (lights on from 6:00 am to 6:00 pm) for 3–4 weeks, the animals were randomly assigned to four groups: wild-type mice injected with saline, wild-type mice injected with MPTP, D3−/− mice injected with saline, and D3−/− mice injected with MPTP.
To establish an MPTP-induced acute mouse model for PD, animals received nine intraperitoneal injections of MPTP (5 or 15 mg/kg/day; Sigma, St. Louis, MO) or an equal volume of saline as a control . All animal experiments were conducted in accordance with internationally recognized guidelines for animal experiments (“Animal Research: Reporting In Vivo Experiments” (ARRIVE) guidelines) and were approved by the Animal Ethics Committee of Shanghai Jiao Tong University School of Medicine (reference number 2010–0018).
Locomotor activity was assessed by the following four experiments: the open-field test, rotarod test, pole test, and beam test. All animals were trained twice before each recorded test. All experiments were performed between 13:00 and 17:00, and mice were acclimated to the experimental environment for 30 min prior to training or testing.
The open-field test was performed in a plastic case (length×width×height: 30 × 20 × 20 cm) lit by a 40 W lamp. The base of the case was marked into six grids, and all mice were placed in the same corner to start the test. The case was thoroughly cleaned before each test. Each mouse was tested either three or four times during the experiment. Each test lasted 15 min, and locomotion was recorded at the 5-, 10-, and 15-min time points. Each grid over which the mouse stepped was recorded as one horizontal motion, and each time point at which the mouse’s forelegs were uplifted was recorded as one vertical motion.
The rotarod test was used to further quantify the locomotor activity and degree of motor impairment. Mice were trained at several different rod-rotation speeds. For testing, a 60 mm-diameter rotarod was set to rotate at 10 rpm. The duration from when the mouse was put on the rotarod until the mouse dropped off was recorded; the upper time limit was 180 s. All participants were tested three times, at 30-min intervals.
The pole test was performed according to the method established by Ogawa et al. . Briefly, animals were positioned with their head upward near the top of a rough-surfaced iron pole (1 cm in diameter and 50 cm high). The time taken until they turned completely downward (defined as a “T-turn”) and arrived on the floor was recorded. The maximum time allowed was 120 s.
The challenging beam test was performed according to previous studies using Parkinsonian genetic mouse models. A 2 cm-wide and 50 cm-long beam was set up in a bright laboratory. The mice were placed onto a hanging terminal, and the time required for the mice to traverse through the beam to a platform terminal was recorded.
Histology and immunohistochemistry
At the end of the behavioral testing period, brain segments were subjected to histopathologic analysis. The number of damaged neurons was calculated as the average number of cells counted per field. The general criteria to score damaged cells included hyperchromatic nuclei and cytoplasmic vacuolation. The number of damaged neurons was visually estimated on three sections from each animal for each experimental group. Coronal sections of the SNpc were cut on a vibratome, stained with anti-tyrosine hydroxylase (anti-TH) antibody (polyclonal rabbit anti-TH, 1:1000; Abcam, Cambridge, UK), followed by biotinylated secondary antibody (goat anti-rabbit IgG; Abcam, Cambridge, UK), and avidin-horseradish peroxidase conjugate (ABC, Vector, Peterborough, UK) according to the manufacturer’s instructions. The antibody binding was visualized with 3,3′-diaminobenzidine (Sigma, Milan, Italy) as the chromogen. TH-positive neurons were enumerated on three serial sections per animal. TH-labeled neurons were scored as positive only if their cell bodies included well-defined nuclear counterstaining. An independent pathologist who was blind to the specifics of the experiment determined the number of TH-positive neurons.
Because the behavioral data obtained in the locomotor activity tests were not normally distributed, comparisons of dose–response effects were conducted with Kruskal–Wallis one-way analysis of variance (ANOVA). Comparisons between the two groups of mice at each dose were conducted with the Mann–Whitney test. The remaining observations were compared with the Mann–Whitney test. p < 0.05 was considered statistically significant.