2. Materials and methods
2.1. Drugs and reagents . Bulleyaconitine A (BAA) was purchased from Zelang Bio-Pharmaceutical (Nanjing, China) with the purity no less than 98% determined by manufacturer with high performance liquid chromatography. Morphine hydrochloride, minocycline, and pentobarbital sodium were purchased from the Northeast Pharmaceuticals Group (Shenyang, China), Yuanye Biotech (Shanghai, China), and Sinopharm Chemical Reagent Co. (Shanghai, China), respectively. Both 5′-guanidinonaltrindole (GNTI) and naloxone hydrochloride were obtained from Sigma-Aldrich (St. Louis, Mo, USA). The rabbit polyclonal antiserum neutralizing dynorphin A was purchased from Phoenix Pharmaceuticals (Burlingame, CA, USA). The antiserum was specific to dynorphin A (100%), but not to dynorphin B (0%), β-endorphin (0%), α-neo-endorphin (0%) or leu-enkephalin (0%) according to the manufacturer′s data sheet. Its specificity was also validated by the antigen absorption test from other laboratories (Wakabayashi et al., 2010; Yamada et al., 2013). All the drugs and reagents were dissolved or diluted in 0.9% normal saline.
2.2. Experimental animals . Male adult Swiss mice (8-9 weeks, 20-25 g) were purchase from the Shanghai Experimental Animal Institute for Biological Sciences (Shanghai, China). The animals were maintained in a 12-hr light/dark cycle (light period 7:00 a.m.-7:00 p.m.) with free access to food and water at standard room temperature (22±2℃) in the Shanghai Jiao Tong University Experimental Animal Center (Shanghai, China). All mice were acclimatized to 3-5 days before the experiments. Mice (n=10-12 per group) were randomly assigned and the behavior tests were performed in a blind manner. All housing conditions and experimental procedures were approved by the Animal Care and Welfare Committee of Shanghai Jiao Tong University (Shanghai, China).
2.3. Induction of morphine physical dependence . The morphine administration paradigm for induction of physical dependence was performed as previously established (Goeldner et al., 2011; Bobzean et al., 2019). Briefly, morphine was administered in mice with escalating doses (5, 10, 20, 40, 80 and 100 mg/kg) by twice-daily subcutaneous injections at 10:00 a.m. and 4:00 p.m. for 6 consecutive days. On the seventh day, mice received a single subcutaneous injection of morphine (100 mg/kg) at 10:00 a.m. followed by an intraperitoneal injection of naloxone (5 mg/kg) 4 hours later. The withdrawal signs included shakes, jumps, genital licks and fecal excretion and loss of body weight, which were observed and recorded for 30 minutes after naloxone injection.
2.4. CPP apparatus and paradigm. CPP has a position preference for different chambers and is used to explore the effects of drug rewarding (Bahi et al., 2008; Shi et al., 2019). The apparatus for the CPP test consisted of three compartments. Two equal-sized chambers (25 × 25 × 40 cm) with a connecting white protruded chamber (null compartment, 25 × 5 × 40 cm) were separated by a removable door. To distinguish each other, one of the main chambers was decorated with black walls and a striped floor, while the other one was with black and white striped walls and a round dot floor. The environmental lighting was adjusted to exclude baseline preference. The apparatus was kept in a quiet room and dim 40 lx illumination (Marszalek-Grabska et al., 2018).
The 10-day schedule CPP paradigm included three distinct phases: preconditioning, conditioning and post-conditioning (Khaleghzadeh-Ahangar and Haghparast, 2015, 2017). The preconditioning phase started with a 3-day twice-daily (10:00 a.m. and 4:00 p.m.) mouse handling with the cupping open gloved hand method (Gouveia and Hurst). On Day 4, each mouse was placed into the null compartment with full access to the entire apparatus for 15 minutes. The time spent in each chamber was recorded by a 3CCD camera (Panasonic Inc, Japan) and analyzed using the EthoVision XT 8.0 (Noldus Information Technology Co., China) to determine baseline preference. Animals that spent more than 450 seconds in any of the three chambers were excluded from the study. During the conditioning phase, mice underwent 5 days of morphine (10 mg/kg) or saline (10 mL/kg) alternate subcutaneous injections, with a 6-hour interval (between 10:00 a.m. and 4:00 p.m.) and included ten 45-min sessions in a five-day schedule. On day 5, 7 and 9 of conditioning, mice were treated with morphine in the morning and immediately confined to the morphine-paired chamber for 45 minutes and received saline in the afternoon and put in the saline-paired chamber for 45 minutes. On day 6 and 8, the injection time of morphine and saline was changed (Meng et al., 2012; Shirazy et al., 2020). Morphine-induced CPP in mice was tested by being allowed with free access to all three compartments for 15 minutes in the post-conditioning phase (On Day 10). The time spent in each chamber was recorded by the 3CCD camera and analyzed using the EthoVision XT 8.0. The conditioning score was expressed by the time spent in the drug-paired chamber minus the time spent in the saline-paired chamber.
2.5.Intracerebroventricular catheterization and injection in mice . For intracerebroventricular injection, mice were anesthetized by intraperitoneal injection of 1.5% pentobarbital sodium and positioned in a stereotaxic instrument (Stoelting Company, Wood Dale, IL, USA). The surgical site was shaved and sterilized with 70% ethanol and a 1.5 cm incision was made to expose the skull. A 22-gauge stainless steel cannula was directed to 0.6 mm lateral and 1.0 mm caudal to bregma and inserted 3 mm deep. Dental cement was applied to adhere the cannula to the skull. The incision was sutured and the cap of cannula was covered. Animals were returned to their cages and allowed at least 3 days recovery. The drug was administrated slowly over 3 minutes in a volume of 6 μL through the planted cannula, using an insulin needle mated with a 10-μL microsyringe via a polyethylene tube (Hylden and Wilcox, 1980; Lenard and Roerig, 2005).
2.6. RNA extraction, reverse transcription, andreal-time quantitative polymerase chain reaction (PCR) . The total RNAs were isolated from nucleus accumbens (NAc) and hippocampus of mice using the TRIzol reagent (Invitrogen, Carlsbad, USA) and were reversely transcribed into cDNA using the ReverTraAce RT-qPCR kit (Toyobo Co., Osaka, Japan) according to the instructions provided by the manufacturers. Real-time quantitative PCR was performed with a Mastercycler ep realplex (Eppendorf, Hamburg, Germany) using the Realmaster Mix (SYBR Green I, Toyobo, Japan). The forward and reverse primer sequences were ATG ATG AGA CGC CAT CCT TC and TTA ATG AGG GCT GTG GGA AC for prodynorphin, which was designed by Premier 6 (version 6.0, Premier Biosoft, San Francisco, USA); and CCA AGG TCA TCC ATG ACG AC and TCC ACA GTC TTC TGA GTG GC for gapdh (Reiss et al., 2017). The fold change was calculated using the 2-△△Ct method after normalization to gapdh (Huang et al., 2017b).
2.7. Measurement of dynorphin A . NAc and hippocampus were obtained and immediately frozen in liquid nitrogen and stored an -80℃ until further measurement. Tissues were homogenized at 4000 rpm for 15 seconds with a homogenizer (Fluko Equipment Co, Shanghai, China) in 10 mM Tris-HCl (pH 7.4) and centrifuged at 1500 rpm at 4℃ for 15 minutes. The total protein concentrations in NAc and hippocampus were determined by standard bicinchoninic acid protein assay (Beyotime Institute of Biotechnology, Jiangsu, China) and dynorphin A was assayed using commercialized fluorescence enzyme-linked immunosorbent assay (ELISA) kit (Phoenix Pharmaceuticals, Burlingame, CA, USA) according to the operation manual (Leitermann et al., 2004; Nocjar et al., 2012).
2.8.Immunofluorescence staining . Double immunofluorescence labeling of dynorphin A and cellular biomarkers of microglia, astrocytes, and neurons in NAc and hippocampus was carried out using a TCS SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) according to the previously published method with minor modifications. Mice were deeply anesthetized by intraperitoneal 1.5% pentobarbital sodium (5 mL/kg), and intracardially perfusion with 20 mL of 0.9% saline, followed by 20 mL of 4% paraformaldehyde. The brain was dissected and fixed in the 4% paraformaldehyde for 12 hours at 4℃. Paraformaldehyde was then removed with PBS and the brain was dehydrated with gradient sucrose solution (10%, 20% and 30% diluted with PBS) at 4℃. The dehydrated brain was embedded in the optimal cutting temperature embedding agent (Lecia Microsystems) and cut into 30-μm-thick transverse sections with sliding microtome. The frozen sections were incubated in 10% goat serum (v/v) and 0.5% Trixon X-100 (v/v) for 1 hour at room temperature and then incubated at 4℃ for 24 hours with primary antibodies. The applied primary antibodies included dynorphin A antibody (1:100; rabbit polyclonal; Phoenix Pharmaceuticals) and cellular markers: Iba-1 (1:100; mouse monoclonal; Millipore, Darmstadt, Germany) for microglia, GFAP (1:100; mouse monoclonal) for astrocytes and NeuN (1:60; mouse polyclonal; Millipore) for neurons. After washing with PBS, the sections were incubated for 1 hour at 37℃ with the Alexa-555-conjugated goat anti-rabbit secondary antibody for dynorphin A and the Alexa-488-conjugated goat anti-mouse secondary antibody for microglia, astrocytes or neurons (Qi et al., 2018). Expression of dynorphin A, Iba-1, GFAP, and NeuN was visualized in the shell of nucleus accumbens (NAcSh) (from bregma 1.70 mm to 0.98 mm, according to the brain in stereotaxic coordinates) and hippocampal CA1 (from bregma -1.46 mm to -1.94 mm, according to the brain stereotaxic coordinates) under a confocal microscope. To quantify the relative intensity of dynorphin A in Iba-1-, GFAP- or NeuN-immunopositive cells in NAcSh and hippocampal CA1, the images were acquired at a 10× or 30× magnification. The background fluorescence was excluded and only immunofluorescent intensity from positively stained areas were included using the low and high thresholds. A colocalization analysis was performed using the ImageJ software with a co-localization finder to generate images in which co-localized pixels appeared as white. All surface areas in each group were measured following the same setup configurations at the same time. The averaged value of the immunolabeled surface area was recorded as the positive immunofluorescence area from three nonadjacent sections of NAcSh or hippocampal CA1. Data was calculated from six mice of each group.
2.9. Statistical analysis . For the dose-response curve analysis, the parameters, i.e., minimum effect, half-effective dose (ED50), Emax and Hill coefficient (n), were calculated by fitting nonlinear least-squares curves to the relation Y = a + bx, where x = [D]n/ (ED50n + [D]n). The values of ED50 and b (Emax) were projected by yielding a minimum residual sum of squares of deviations from the theoretical curve (Zhang et al., 2013).
The data were summarized as means ± standard error of the mean (S.E.M.). The statistical significance was evaluated by unpaired and two-tailed Student t-test or one-way analysis of variance (ANOVA) using Prism (version 7.00, GraphPad Software Inc., San Diego, CA, USA). The ANOVA analysis was undertaken based on the assumptions of normal distribution and variance consistency verified by residual plots and the post-hoc Student-Newman-Keuls test was performed when the effect of the drug (dose) was observed to be statistically significant. The probability values were two-tailed and the statistical significance criterion value was 0.05.