Animals
C57BL/6 male mice at 6 weeks old were supplied from DaeHan Bio-Link Co., Ltd (Eumsung, Korea) and maintained in a humidity- and temperature-controlled environment (50 ± 5 %, 22 ± 1°C) under a 12 h light/dark cycle with free access to water and standard rodent food. Experimental procedures concerning animals were approved by the Ethics Committee of School of Medicine, Keimyung University (Daegu, Korea). All animal care and experimental procedures were accomplished with the National Institutes of Health guideline for laboratory animal care and use.
Reagents
METH was supplied from Ministry of Food and Drug Safety (Cheongju, Korea) and was dissolved in 0.9% sterile saline. Anti-actin antibody and other chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Anti-TH antibody was obtained from Millipore Co. (Temecula, CA, USA). Anti-D2R antibody was supplied by Biorbyt LLC. (Wobum, MA, USA) and Bioss Antibodies Inc. (Woburn, MA, USA), respectively. Anti-phospho-nuclear factor-erythroid 2- related factor 2 (Nrf2), 4-hydoxynonenal (4-HNE), and Iba-1 antibodies were obtained from Abcam (Waltham, MA, USA). Anti-Nrf2, manganese superoxide dismutase (MnSOD), glutamylcysteine synthase (GCS), Bax, and Bcl-2 antibodies were the products from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).
Procedures of drug treatment
After the adaptation for a week, mice were randomly divided into four groups (5 mice per group) as follows: group 1 (control), group 2 (METH), group 3 (METH+3 mg/kg of ΑPN), and group 4 (METH+10 mg/kg of APN). All groups except for control were administered METH (1 mg/kg, i.p.) daily for 8 days. The control group received an equivoluminal 0.9% normal saline solution. In the APN group, APN (3 and 10 mg/kg, i.p.) was also administered for 8 days before METH injections; on the other hand, mice in the groups (control and METH only) received the same amount of normal saline.
CPP apparatus
Conventional CPP apparatus with two compartments of equal size was utilized. One compartment was black-colored, with stainless steel grids in the floor consisting with perpendicularly placed rods, and the other compartment was white-colored, with stainless steel mesh floor. The time spent in each compartment and movement of the animals were recorded using a computerized video tracking system (Smart 3.0 software, Panlab, Barcelona, Spain).
Measurement of METH-induced CPP
The CPP paradigm consisted of three phases as follows: pre-tracking, conditioning, and post-tracking (Fig. 1B). In the pre-tracking phase (day 3), the mice were allowed to freely access the two compartments for 20 min daily. The time spent in each compartment was recorded during this habituation period to exclude biased mice that spent more than 800 s in either compartment. In the conditioning phase (day 4-11), the mice received METH (1 mg/kg, i.p.) or saline alternately on every other day for 8 days (4 METH sessions and 4 saline session). Each animal received saline injections in their initially preferred compartment (saline-pared side) and METH injections in their initially non-preferred compartment (drug-paired side). The mice were then immediately confined in the appropriate drug-paired or saline-paired compartments for 60 min. To assess the effects of APN on the acquisition of METH-induced CPP, naive animals received a vehicle or APN (3 and 10 mg/kg, i.p., n=5 per group) 30 min prior to each METH or saline injection during the METH conditioning phase. In the post-tracking phase (day 12), the CPP test was conducted 24 h after the last injection, and there was no drug treatment on the test day. A preference score was calculated as the difference between the relative amount of time spent in the drug-paired, non-preferred side of the apparatus and the time spent in the preferred side during the preconditioning phase.
Western blot analysis
After the treatments, cortical and striatal regions from the mice were rapidly isolated. Brain tissues were homogenized and lysed with ice-cold RIPA buffer [150 mM NaCl, 1.0% Triton-X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Trizma base, pH 8.0]. Lysates were centrifuged at 14,000 g for 30 min, and supernatants obtained were used for western blot analysis. Protein concentration was determined by using a BCA protein assay reagent (Thermo Scientific, Rockford, IL, USA). Total protein lysates were separated by 10-12% SDS-polyacrylamide gels, were transferred into the polyvinylidene fluoride membranes, and were blocked in PBS buffer containing a 5% non-fat dry milk for 1 h. The membranes were further incubated with primary antibodies at 4°C overnight. The membranes were then washed with PBS and then reacted with secondary anti-rabbit antibody for 60 min at room temperature. After the second incubation, the blots were washed in PBS for 10 min 3 times and developed with the enhanced chemiluminescence system (Pierce, IL, USA) for visualization. Relative intensities of the bands were quantified by using an ImageQuant LAS 4000 Multi Gauge software (Fujifilm, Tokyo, Japan) and were then normalized to the intensity of Actin.
Statistical analysis
The data were expressed as the means ± SEM (standard error of the mean). Differences between groups were compared by using a one-way analysis of variance (ANOVA) followed by least significant difference (LSD) post hoc test for multiple comparisons. All of the statistical analyses were performed by using SPSS ver. 20.0 (IBM, Chicago, IL, USA).
