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.