Male Wistar rats weighing 180–220 g (Academy of Military Medical Science, Beijing, China) at the beginning of the experiment were used. The rats were housed in groups of five in a room with constant temperature (25°C) and humidity (70%) and a 12 h light/12 h dark cycle (08:00–20:00 light on), with free access to food and water. Animals were maintained according to the international guidelines for the care and use of laboratory animals, and all experimental procedures involving animals were approved by the Ethics Committee of Beijing Normal University (BNU/EC/01/2011).
Drugs and reagents
AJN was provided by Taier Company (Hunan, China). Morphine hydrochloride was purchased from Qinghai Pharmaceutical Co. (China). (S)- N-((1-ethyl-2-pyrrolidinyl) methyl)-2-hydroxy-6-methoxy-3-(trimethylstannyl) benzamide (TBZM), and (1R,2S,3S,5S)-methyl 8-methyl-3-(4-(trimethylstannyl) phenyl)-8-azabicyclo [3.2.1] octane-2-carboxylate (trimethylstannyl-β-CT, or TMS-β-CT) were purchased from Huayi Isotope Co. (Toronto, ON, Canada). Na125I (specific activity > 2200 Ci/mmol) was purchased from Perkin Elmer (Boston, MA, USA). [125I]-IBZM and [125I]-β-CIT were prepared as described by Kung et al., and Toyama et al.[19, 20].
Morphine dependence model and AJN treatment
The rats were divided into six groups: control group, morphine group, AJN pre-treatment group (0.555 g/kg), and three AJN post-treatment groups: low dose (AJN-L, 0.185 g/kg), medium dose (AJN-M, 0.555 g/kg), and high dose (AJN-H, 1.851 g/kg). The rats in the morphine, AJN pre-treatment and AJN post-treatment groups were administered morphine via intraperitoneal injection twice daily (09:00 and 15:00) for eight days in a volume of 1 mL/kg body weight, with a gradually increasing dose (10, 10, 15, 15, 15, 20, 20, and 20 mg/kg per injection on each day)[21, 22]. The control animals received 0.9% saline in the same volume. AJN was dissolved in deionized water and intragastrically administered to the rats in the AJN pre-treatment group at a dose of 0.555 g/kg (1 mL/kg of body weight) 30 min before each morphine injection. After morphine administrations, the rats in the AJN post-treatment groups were administered AJN intragastrically once daily at a dose of 0.185, 0.555 or 1.851 g/kg (1 mL/kg of body weight) for 10 days, whereas the other groups received the same volume of vehicle (saline). The AJN dosages were converted from those used clinically.
At 10:00 a.m. on days one, five, and ten after the withdrawal of morphine, the animals were placed individually into Plexiglas cages and observed for signs of spontaneous withdrawal. Following a 5 min acclimation in the cages, the number of wet dog shakes and writhing episodes were monitored during a 30 min period. Three observers blind to the groups completed the observation and score independently. Scores were averaged for each behavior test.
After the final behavioral observation, all the rats were sacrificed by decapitation. Rat brains were rapidly removed and stored at -80°C until use. The brains were then cut into 18 μm coronal slices with a cryostat (CM1900, Leica, Germany) at -20°C.
Immunohistochemical staining to determine levels of DAT and D2R proteins and TH activity was conducted with striatal slices. To prepare for staining, the slices were prewashed in PBS (0.01 M, pH 7.4) three times for 5 min each, then sequentially treated with 0.2% Triton X-100 in PBS for 5 min and with 0.3% H2O2 in PBS for 10 min, and washed in PBS three times for 5 min each, all at room temperature. Slices were initially incubated with 10% normal goat serum for 10 min (or normal donkey serum for TH measurement), then incubated for 20 h at 4°C with the primary antibodies (D2R antibody AB5084P, Millipore, CA, USA, 1:200 dilution; DAT antibody MAB369, Millipore, CA, USA, 1:100 dilution; TH antibody T1299, Sigma, CA, USA, 1:10,000 dilution), washed three times in PBS, and incubated for 60 min at 37°C with the secondary antibodies (anti-rabbit antibody PV-6001, ZSGB-BIO, CA, USA; anti-rat antibody ZB-2307, ZSGB-BIO, CA, USA; anti-mouse antibody PK4002, Vector, CA, USA). Slices were then washed five times for 3 min each in PBS, and incubated in 100 μL of 3,3′-diaminobenzidine tertrahydrochloride (DAB) for 3 min. The slices immunostained for TH were incubated in the ABC (VECTASTAIN ABC kit, ZSGB-BIO, Beijing, China) reagent for 30 min at 37°C and washed five times for 3 min each in PBS prior to the DAB treatment. Final wash of the slices was done in distilled water.
The striatal slices were washed for 20 min at room temperature in 50 mM Tris buffer (pH 7.4, containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, and 1 mM MgCl2) for D2R autoradiography, and in 50 mM Tris buffer (pH 7.4, containing 120 mM NaCl and 5 mM KCl) for DAT autoradiography. The slices were then incubated for 60 min in the same buffer with 50 pM [125I]-IBZM for D2R labeling or 50 pM [125I]-β-CIT in the presence of 1 mM fluoxetine (serotonin antagonist, Sigma-Aldrich Co., USA) for DAT labeling. The nonspecific binding was determined in the adjacent slices in the presence of 10 μM sulpiride (D2R antagonist, Sigma-Aldrich Co., USA) for D2R or in the presence of 100 mM nomifensine (DAT antagonist, Sigma-Aldrich Co., USA) and 100 mM fluoxetine for DAT. After incubation, the slices were washed five times for 1 min each in ice-cold 50 mM Tris buffer (pH 7.4), rapidly dipped in deionized water and dried under a stream of cold, dry air.
The labeled slices were mounted and exposed to a super-sensitive phosphor screen (PerkinElmer, USA) at room temperature for 8 h. Densitometry determinations were performed using a Cyclone Plus phosphor imager (PerkinElmer, USA) and analyzed using the Opti-Quant software (PerkinElmer, USA). The specific binding detected in each structure was quantified by subtracting the non-specific binding image from the total binding image. The results are shown as the ratio of specific binding relative to the control group.
All of the statistical analyses were conducted using SPSS software (version 20.0) with a type I error rate of α = 0.05 (two-tailed). Data are expressed as the mean ± SD. The significance of the changes in the behavioral assays was determined using the Kruskal-Wallis test. If the difference was found to be significant using this test, the Mann–Whitney U-test was used to compare the control and experimental groups. To compare the values obtained from two groups, Student’s t-test was performed.