Animals and the TD model
The SPF-grade SD rats (male, 6 weeks old, weight 200±10 g) were purchased from Changzhou Cavens Experimental Animal Co., Ltd. All experimental animals were adapted to feed for a week before the formal experiment. First, we conducted a pilot experiment to validate the IDPN induced TD model. Rats in the TD group were intraperitoneally injected with iminodipropionitrile (IDPN, 200 mg/kg, Sigma Chemical Co., St. Louis, MO, USA, at 9:00-11:00, for 7 consecutive days). The control group was injected with the same dose of saline (5 ml/kg/day). In our previous investigation, the stereotypic behavior was triggered by IDPN injection (including biting, head twitching, shaking claws, continuous rotation, etc., and the stereotypic behavior score was significantly elevated).
For each batch of animals, after the behavioral observation, animals were sacrificed, and brains were sampled. A part of samples was fixed in 4% paraformaldehyde; for the other part of samples, the striatum tissue was separated for each animal and homogenized, the proteins and RNAs were extracted and stored at -80℃.
Primary culture of striatal dopaminergic neurons
Two SPF-grade SD pregnant mice were purchased from the Changzhou Cavens Laboratory Animal Co., Ltd., and at least 10 pups were obtained for primary culture of dopaminergic neurons. The 3-to-5-day-old SD rats were soaked in 75% ethanol for 30 min. Each rat was transferred to an ultra-clean table; under the aseptic conditions, the brain tissue was taken out, rinsed with pre-cooled PBS, and placed in a petri dish. Under a microscope, the striatal nucleus was exposed. The striatum was picked off and placed in a sterile EP tube, and PBS was added to rinse the tissue. Next, 0.25% pancreatin was added. After a 10-min digestion, the tissue was pipetted and transferred to a new EP tube. An appropriate amount of H-DMEM complete medium (90% H-DMEM medium + 10% FBS + 1% penicillin/streptomycin) was added, the tissue suspension was pipetted and centrifuged (1000 rpm for 5 min). The supernatant was removed, an appropriate amount of Neurobasal complete medium (90% Neurobasal medium + 10% FBS + 1% penicillin/streptomycin) was added and the single-cell suspension was generated by pipetting. All cells then passed through a 70-µm cell sieve. The filtered suspension was added to the cell culture flask, an appropriate amount of Neurobasal complete medium was supplemented. The plate was placed in a culture incubator (37°C, 5% CO2). One day after the cells were extracted, the medium was changed. Then, the medium was refreshed every 3 days. The striatal dopaminergic neurons were identified by the tyrosine hydroxylase (TH) staining.
Total RNA was extracted from the striatum region of the brain using TRIZOL (Takara, 9108), and cDNAs were synthesized using a TAKARA PrimeScript™ RT reagent Kit (Takara, RR047A). PCR amplification reactions were conducted using a SYBR Green Supermix system (Takara, RR820A) in a 20 µl reaction containing 0.8 µl primers (0.4 µl of the 10 µM Forward primer and 0.4 µl of the 10 µM Reverse primer, respectively) and 2 µl cDNA. The cycling program was as follow: (1) denaturation at 95°C for 1 minute, (2) 40 cycles of (95°C for 5 s + 55°C for 30 s + 72°C for 30 s), (3) melting curve. All measurements were performed in triplicates. The primers pairs (forward and reverse, respectively) used in amplification were as follow.
β-actin: Forward GGGCTTTATTGACAAGATTGCT; Reverse TCGGTAGTCTGACTGAGTGGC.
STX1A: Forward ACCACAGCTGAGAGGGAAATCG; Reverse AGAGGTCTTTACGGATGTCAACG.
For each well, the cell supernatant was collected and centrifuged at 1000×g for 20 min. Then, the supernatant was stored at 2 to 8°C. The above samples were used to detect the concentration of dopamine in the cell culture supernatant. In addition, the cells in each well were gently washed with pre-cooled PBS for 3 times, 200 µl of lysis buffer was added to each well, cells were lysed by pipetting and the lysis solution centrifuged at 10000×g for 5 min. Next, this supernatant was also stored at 2 to 8°C. The above samples were used to detect the concentration of intracellular dopamine. The DA ELISA Kit (Wuhan Fine Biotech Co., Ltd, Product code EU0392) was used for measurement of the supernatant dopamine content and intracellular dopamine content of the primary dopaminergic neurons. All ELISA operations followed the official instructions.
The cell samples or rat striatum tissues were homogenized in a lysis buffer with protein inhibitor and PMSF (1 mM). The lysates were centrifuged at 1000 rpm for 5 min, and the supernatant was collected. An amount of 20 µg protein samples were separated using 10% SDS-PAGE gel, followed by being transferred onto PVDF membranes. The blots were first blocked in TBST-milk and then incubated with the primary antibody overnight at 4°C. Next, the secondary antibodies conjugated with HRP (1: 1000) were used for 2 h of incubation. The expression of β-actin was used as an endogenous reference. The Tanon ECL Kit was used for chemiluminescence, and the Tanon 5200 chemiluminescence imager was used for image capture.
The primary antibodies applied in this study were as follow: Rabbit monoclonal [EPR19695] to Dopamine Transporter (Abcam, UK, ab184451), Rabbit monoclonal [EPR23457-15] to STXA (Abcam, UK, ab272736), Anti-beta-actin rabbit polyclonal antibody (Abcam, UK, ab8227). Goat anti-rabbit HRP (Abcam, UK, ab6721) was used as the secondary antibody.
For each protein sample, 500 µg protein was collected, and the STX1A antibody (1:30) or DAT antibody (1:30) was added. The rabbit IgG was used as a negative control. Each tube of mixture was slightly shacked overnight at 4°C. The remaining total protein was directly used in Western Blot as an Input control. Next, 40 µl of protein A/G agarose beads was added, and the mixture was shacked at 4°C for 4 h to couple the antibody to the agarose beads. Then, the suspension was centrifuged at 4°C for 3 min (1000 g), the supernatant was carefully aspirated off, and the precipitate was collected for analysis. Subsequently, 500 µl of RIPA lysate was added to the precipitate, and the system was homogenized. The lysates were centrifuged at 4°C for 3 min (1000 g), the supernatant was collected, and this step was repeated 3 times. The collected proteins were further added 5×SDS loading buffer and boiled for 5 min. Next, routine Western blotting analysis was performed.
Three pairs of Adenovirus were produced by Shanghai Genechem Co., Ltd., including the STX1A-overexpression adenovirus and the corresponding negative control virus (namely, STX1A vs NC), STX1A interfering shRNA adenovirus and corresponding scramble RNA control virus (namely, sh-STX1A vs sh-Control), DAT overexpression adenovirus and corresponding negative control virus (namely, DAT vs NC).
Intra-striatal virus injection
Each rat was fixed on a stereotaxic instrument, a micro-injector was used to inject the Adenovirus into the bilateral striatum (5 µl each side, the virus amount for each rat was 10^10 units). The injection site was as follow: AP + 1.0 mm, ML ± 2.5 mm, DV -3.8 mm. TD modeling was performed on the 3rd day after injection. After the behavioral observation, animals were sacrificed, and brain tissue samples were collected. Brains were fixed in 4% paraformaldehyde, and the reporter gene (GFP) staining was conducted to confirm the accurate injection of the virus.
The rats were transcardially perfused with 0.01M PBS followed by 50 ml of 4% paraformaldehyde in 0.1 M PBS (pH 7.4). The brain tissues were quickly separated and post-fixed in 4% paraformaldehyde overnight, then the brain was dehydrated in 20% sucrose (0.1 M PBS) for 24 h at 4°C and further dehydrated in 30% sucrose (0.1 M PBS) for 24 h at 4°C. The sections were cut into 15 µm sections on a cryostat. The sections were rinsed in 0.01 M PBS and blocked for 2 h with donkey serum (in 0.3% Tween-20 and 0.01 M PBS) and then incubated with rabbit anti STX1A antibody and mouse anti TH antibody (to label dopaminergic neurons) at 4°C overnight (1:500, Proteintech). Subsequently, sections were washed 3 times in 0.01 M PBS for 5 min and incubated with donkey anti-rabbit IgG conjugated with FITC (1:200) and donkey anti-mouse IgG conjugated with CY3 (1:200). Next, sections were incubated with DAPI for nucleus staining for 15 min and washed 3 times for 5 min each. Finally, sections were cover slipped, and images were captured under a fluorescence microscope.
Stereotyped behavior test
The stereotyped behavior test was conducted at 9:00- 11:00. The light condition during the observation period was consistent throughout the experiment. Each rat was placed in the observation box (diameter = 40 cm, height = 30 cm), and the 5-min recording for bite time, head shaking time, and rotation time were conducted by two observers. Finally, the stereotypy score was assessed using the following criterion: No stereotypy or normal activity (score = 0), Discontinuous circling behavior or occasional head twitching (score = 1), Occasionally vertical dyskinetic head and neck movements, occasional sniffing, licking, and biting (score = 2), Continuous circling behavior, increased body raising, increased sniffing, repetitive grooming (such as paw-to-mouth movements) (score = 3), Increased lateral and vertical dyskinetic head and neck movements (score =4).
Results were expressed as means ± standard error. For two-group comparison, Student’s t-test was used after the normal distribution test. A P value < 0.05 was considered as statistically significant.