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.