Figure legends
Fig. 1. Inhibitory effects of subcutaneous injection of
bulleyaconitine A on naloxone-induced withdrawal signs, including shakes
(A ), jumps (B ), genital licks (C ), fecal
excretion (D ) and body weight loss (E ), in morphine
physical dependence mice. Mice were subjected to bi-daily subcutaneous
injections of normal saline (10 mL/kg), BAA (300 μg/kg) or morphine
(escalating doses of 5, 10, 20, 40, 80 and 100 mg/kg) for 6 days. On the
7th day, mice received intraperitoneal injection of naloxone (5 mg/kg) 4
hours post last injection of morphine (100 mg/kg) to induce physical
dependence, and their withdrawal signs were observed immediately for 30
minutes. For BAA inhibitory effects, mice received a single injection of
saline (10 mL/kg) or BAA (30, 100 or 300 μg/kg) 40 minutes prior to
naloxone intraperitoneal injection. Dose-response analysis was best
projected by the nonlinear least squares methods in A and E. The data
are presented as means ± S.E.M (n=10 per group). *, #P<0.05, compared with the saline control group and morphine
physical dependence group, respectively, by one-way ANOVA followed by
the post-hoc Student-Newman-Keuls test.
Fig. 2. Inhibitory effects of subcutaneous injection of
bulleyaconitine A (BAA, 300 μg/kg) on morphine-induced conditioned place
preference (CPP) acquisition in mice. Mice were subjected to alternate
daily subcutaneous injections of normal saline (10 mL/kg/day), BAA (300
μg/kg/day) or morphine (10 mg/kg/day) for 5 days followed by a single
subcutaneous injection of saline (10 mL/kg) or BAA (300 μg/kg). Results
are presented as means ± S.E.M. (n=12 per group). *,# P<0.05 compared to the saline control and
morphine CPP control groups, respectively, by one-way ANOVA followed by
the post-hoc Student-Newman-Keuls test.
Fig. 3. Stimulatory effects of subcutaneous injection of
bulleyaconitine A (BAA, 300 μg/kg) on gene (A , B ) and
protein (C , D ) expression of dynorphin A in nucleus
accumbens (NAc) and hippocampus in multiple daily morphine (10
mg/kg)-treated mice. The mRNA and protein expression of dynorphin A was
measured using quantitative real-time PCR and fluorescent ELISA kit,
respectively. Data are presented as mean ± S.E.M (n=8-12 per group). *
P<0.05 compared to the saline control group, by unpaired and
two-tailed Student t-test.
Fig. 4. Specific stimulatory effects of subcutaneous injection
of bulleyaconitine A (BAA, 300 μg/kg) on dynorphin A expression in
microglia, but not in astrocytes or neurons in the shell of nucleus
accumbens (NAcSh) in multiple daily morphine (10 mg/kg)-treated mice.
Frozen sections of NAcSh were obtained 1 hour after subcutaneous
injection of saline (10 mL/kg) or BAA (300 μg/kg). Immunofluorescence
was doubly stained with dynorphin A/microglial marker Iba-1 (A ,B, G , H ), dynorphin A/astrocytic marker GFAP
(C, D , I , J ) and dynorphin A/neuronal marker
NeuN (E, F , K , L ) under 10× magnification
(scale bar: 250 μm) and 30× magnification (scale bar: 50 μm),
respectively. Arrowheads indicate colocalization of dynorphin A with
microglia, astrocytes or neurons. Colocalized areas of dynorphin A/Iba-1
(M ), dynorphin A/GFAP (N ) and dynorphin A/NeuN
(O ) were quantified at 10× magnification using the ImageJ
software. Data are presented as mean ± S.E.M. (n=6 per group). *
P<0.05 compared to the saline control group, by unpaired and
two-tailed Student t-test.
Fig. 5. Specific stimulatory effects of subcutaneous injection
of bulleyaconitine A (BAA, 300 μg/kg) on dynorphin A expression in
microglia, but not in astrocytes or neurons in hippocampal CA1 in
multiple twice-daily morphine (10 mg/kg)-treated mice. Frozen sections
of hippocampal CA1 were obtained 1 hour after subcutaneous injection of
saline (10 mL/kg) or BAA (300 μg/kg). Immunofluorescence was doubly
stained with dynorphin A/microglial marker Iba-1 (A , B,
G , H ), dynorphin A/astrocytic marker GFAP (C, D ,I , J ) and dynorphin A/neuronal marker NeuN (E,
F , K , L ) under 10× magnification (scale bar: 250 μm,
DAPI was also colabeled with the nucleus in blue) and 30× magnification
(scale bar: 50 μm), respectively. Arrowheads indicate colocalization of
dynorphin A with microglia, astrocytes or neurons. Colocalized areas of
dynorphin A/Iba-1 (M ), dynorphin A/GFAP (N ) and
dynorphin A/NeuN (O ) were quantified at 10× magnification using
the ImageJ software. Data are presented as mean ± S.E.M. (n=6 per
group). * P<0.05 compared to the saline control group, by
unpaired and two-tailed Student t-test.
Fig. 6. Blockade effects of intracerebroventricular (icv)
injection of the microglial activation inhibitor minocycline
(A-E ), specific dynorphin A antiserum (F-J ) and
selective κ-opioid receptor antagonist GNTI (K-O ) on
subcutaneous (sc) injection of bulleyaconitine A (BAA)-attenuated
withdrawal signs in morphine physical dependence mice. Naloxone-induced
withdrawal signs included shakes (A, F, K), jumps (B, G, L), genital
licks (C, H, M), fecal excretion (D, I, N) and body weight loss (E, J,
O). The data are presented as means ± S.E.M. (n=10 per group). *,# P<0.05, compared to the saline control
group and BAA group, respectively, by one-way ANOVA followed by the
post-hoc Student-Newman-Keuls test.
Fig. 7. Blockade effects of intracerebroventricular (icv)
injection of the microglial activation inhibitor minocycline
(A ), specific dynorphin A antiserum (B ) and selective
κ-opioid receptor antagonist GNTI (C ) on subcutaneous (sc)
injection of bulleyaconitine A (BAA)-attenuated conditioned place
preference (CPP) acquisition in mice. The data are presented as means ±
S.E.M. (n=10 per group). *, # P<0.05,
compared to the saline control group and BAA group, respectively, by
one-way ANOVA followed by the post-hoc Student-Newman-Keuls test.