3. Results
3.1.B AA
attenuated naloxone-induced withdrawal signs in morphine physical
dependence . Six groups of mice (n=10 per group) 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 7 days. On the 7th day, mice received intraperitoneal injection of
naloxone (5 mg/kg) 4 hours post the last injection of morphine (100
mg/kg) to induce physical dependence, and their withdrawal signs were
observed immediately for 30 minutes. For the BAA inhibitory effects, the
mice received a single injection of saline (10 mL/kg) or BAA (30, 100 or
300 μg/kg) 40 minutes prior to the intraperitoneal injection of
naloxone. As shown in Fig. 1, intraperitoneal injection of naloxone did
not induce any abnormal behaviors in bi-daily saline- or BAA-treated
mice. In contrast, naloxone in bi-daily morphine injected mice induced
profound withdrawal signs including shakes (Fig. 1A), jumps (Fig. 1B),
genital licks (Fig. 1C), fecal excretion (Fig. 1D) and body weight loss
(Fig. 1E) (P<0.05, by one-way ANOVA followed by the post-hoc
Student-Newman-Keuls test). Moreover, pretreatment with subcutaneous
injection of BAA (30, 100 and 300 μg/kg) dose-dependently attenuated
naloxone-induced withdrawal signs in bi-daily morphine-treated mice,
with maximal inhibition of around 70-100% in each different sign
(P<0.05, by one-way ANOVA followed by the post-hoc
Student-Newman-Keuls test). The dose-response analysis was performed in
shakes with an ED50 of 74.4 μg/kg which was yielded
after data transformation (Fig. 1A), and in the body weight loss with an
ED50 of 105.8 μg/kg after data transformation (Fig. 1E).
3.2.BAA
attenuated morphine-induced CPP acquisition . Four groups of mice (n=12
per group) were subjected to the preconditioning phase of three days and
the place preference test on the 4th day followed by
alternate daily subcutaneous injections of normal saline (10 mL/kg), BAA
(300 μg/kg) or morphine (10 mg/kg)
for 5 days. On the 10th day, mice received single
subcutaneous injection of saline (10 mL/kg) or BAA (300 μg/kg) 50
minutes prior to the last injection and the place preference test was
conducted immediately afterwards. As shown in Fig. 2, all four groups of
mice did not show any obvious CPP acquisition in the preconditioning
phase. Moreover, bi-daily subcutaneous injections of saline did not
exhibit significant CPP acquisition in the post-conditioning phase. In
contrast, bi-daily injections of morphine but not BAA showed remarkable
CPP acquisition. However, pretreatment with subcutaneous injection of
BAA completely attenuated morphine-induced CPP acquisition
(P<0.05, by one-way ANOVA followed by the post-hoc
Student-Newman-Keuls test).
3.3. BAA specifically
stimulated microglial dynorphin A expression in NAc andhippocampus in
morphine-multiply treated mice . Two groups of mice (n=10 per group)
daily treated with morphine (10 mg/kg) for 5 days received subcutaneous
injection of normal saline (10 mL/kg) or BAA (300 μg/kg). Mice were
sacrificed 1 hour after subcutaneous injection and NAc and hippocampus
were obtained for the prodynorphin mRNA detection using RT-PCR. As
shown, treatment with BAA significantly increased prodynorphin gene
expression by 1.9-fold in NAc (Fig. 3A) and 1.7-fold in hippocampus
(Fig. 3B), respectively (P<0.05, by unpaired and two-tailed
Student t-test). The stimulatory effects of BAA on dynorphin A protein
expression were also measured in NAc and hippocampus in the same mice
using the commercial fluorescent ELISA kit. As exhibited, subcutaneous
BAA significantly increased dynorphin A expression in NAc
(P<0.05, by unpaired and two-tailed Student t-test; Fig. 3C)
and hippocampus (P=0.07, by unpaired and two-tailed Student t-test; Fig.
3D).
Dynorphin A is known to be localized in neurons, astrocytes and
microglia in the central nervous system (Wahlert et al., 2013; Ayrout et
al., 2019). To verify cell types that specifically upregulate dynorphin
A expression in NAcSh and hippocampal CA1 following BAA treatment,
dynorphin A was immunofluorescent labeled with the microglial cellular
marker Iba-1, astrocytic cellular marker GFAP and neuronal cellular
marker NeuN. Two groups of mice (n=6 per group) daily treated with
morphine (10 mg/kg) for 5 days received subcutaneous injection of saline
(10 mL/kg) or BAA (300 μg/kg). Mice were sacrificed 1 hour after the
subcutaneous injection and NAcSh and hippocampal CA1 were obtained for
fluorescent immunostaining. As shown, dynorphin A was colocalized with
Iba-1, GFAP and NeuN in NAcSh of saline-treated mice (Fig. 4A-4F).
Subcutaneous BAA specifically increased co-labeling of dynorphin A/Iba-1
(Fig. G, H) but not dynorphin A/GFAP (Fig. I, J) or dynorphin A/NeuN
(Fig. K, L) at 10× and 30× magnification. In addition, the ImageJ
software was used to quantify immunofluorescence intensity of dynorphin
A with Iba-1, GFAP or NeuN at 10× magnification. Treatment with
subcutaneous BAA significantly increased dynorphin A/Iba-1 by 2.4-fold
(P<0.05, by unpaired and two-tailed Student t-test; Fig. 4M),
but not dynorphin A/GFAP (Fig. 4N) or dynorphin A/NeuN (Fig. 4O).
Furthermore, the same specific stimulatory effects of BAA on microglial
dynorphin A expression were observed in hippocampal CA1 from the same
mice as above (Fig. 5A-5L), with increased immunofluorescence intensity
of dynorphin A/Iba-1 by 1.9-fold (P<0.05, by unpaired and
two-tailed Student t-test; Fig. 5M), but not dynorphin A/GFAP (Fig. 5N)
or dynorphin A/NeuN (Fig. 5O).
3.4. Brain microglial expression of dynorphin A mediated
BBA-inhibited morphine dependence . To verify the causal relationship
between the microglial expression of dynorphin A in brain and
BAA-inhibited morphine physical dependence, the microglial activation
inhibitor minocycline (Wu et al., 2002; Kobayashi et al., 2013),
dynorphin A antiserum (Li et al., 2016) and κ-opioid receptor antagonist
GNTI (Zhang et al., 2007; Liu et al., 2013) were
intracerebroventricularly injected separately. Four groups of morphine
physical dependence mice (n=10 per group) received the first
intracerebroventricular injection followed by the second subcutaneous
injection of 1) saline (6 μL) + saline (10 mL/kg), 2) minocycline (10
μg) + saline (10 mL/kg), 3) saline (6 μL) + BAA (300 μg/kg), and 4)
minocycline (10 μg) + BAA (300 μg/kg). The second subcutaneous injection
was 4 hours post the first intracerebroventricular injection. Withdrawal
signs were precipitated by intraperitoneal injection of naloxone (5
mg/kg) 40 minutes after subcutaneous injection. As shown in Fig. 6A-6E,
subcutaneous injection of BAA in morphine physical dependence mice
significantly attenuated naloxone-induced withdrawal signs including
shakes, jumps, genital licks, fecal excretions and body weight loss;
whereas intracerebroventricular minocycline failed to influence
morphine-induced physical dependence. However, pretreatment with
intracerebroventricular minocycline nearly completely restored systemic
BAA-suppressed withdrawal signs (P<0.05, by one-way ANOVA
followed by the post-hoc Student-Newman-Keuls test).
Additional four groups of morphine physical dependence mice (n=10 per
group) received the first intracerebroventricular injection followed by
the second subcutaneous injection of 1) saline (6 μL) + saline (10
mL/kg), 2) dynorphin A antiserum (1:30 dilution, 6 μL) + saline (10
mL/kg), 3) saline (6 μL) + BAA (300 μg/kg) and 4) dynorphin A antiserum
(1:30 dilution, 6 μL) + BAA (300 μg/kg). The second subcutaneous
injection was 30 minutes post the first intracerebroventricular
injection. Withdrawal signs were precipitated by intraperitoneal
injection of naloxone (5 mg/kg,) 40 minutes after subcutaneous
injection. Subcutaneous injection of BAA in morphine physical dependence
mice attenuated naloxone-induced withdrawal signs.
Intracerebroventricular injection of the dynorphin A antiserum did not
significantly affect baseline morphine physical dependence, but
reemerged naloxone-induced withdrawal syndrome from BAA inhibition
(P<0.05, by one-way ANOVA followed by the post-hoc
Student-Newman-Keuls test; Fig. 6F-6J).
Further four groups of morphine physical dependence mice (n=10 per
group) received
the
same pattern as above except that intracerebroventricular injection of
the dynorphin A antiserum was replaced with GNTI (5 μg). As displayed in
Fig. 6K-6O, intracerebroventricular injection of GNTI predominantly
restored BAA-suppressed morphine physical dependence (P<0.05,
by one-way ANOVA followed by the post-hoc Student-Newman-Keuls test),
although it did not significantly alter naloxone-induced withdrawal
signs in baseline physical dependence.
3.5. Brain microglial expression of dynorphin A mediated
BAA-inhibited CPP acquisition. Same as in the physical dependence
model, minocycline, dynorphin A antiserum and GNTI were
intracerebroventricularly injected separately into the morphine CPP
acquisition mice in order to determine whether microglial expression of
dynorphin A in brain contributed to BAA-inhibited morphine CPP
acquisition. Four groups of morphine-induced CPP mice (n=10 per group)
were first intracerebroventricularly injected followed 4 hours later by
subcutaneous injected with 1) saline (6 μL) + saline (10 mL/kg), 2)
minocycline (10 μg) + saline (10 mL/kg), 3) saline (6 μL) + BAA (300
μg/kg), and 4) minocycline (10 μg) + BAA (300 μg/kg). The place
preference test was assessed 50 minutes subsequent to subcutaneous
injection. As shown in Fig. 7A, subcutaneous injection of BAA but not
intracerebroventricular minocycline completely attenuated morphine CPP
acquisition. However, pretreatment with intracerebroventricular
minocycline entirely restored BAA-suppressed CPP acquisition
(P<0.05, by one-way ANOVA followed by the post-hoc
Student-Newman-Keuls test).
In addition, four groups of morphine CPP mice (n=10 per group) were
first intracerebroventricularly injected 0.5 hours later followed by
subcutaneous injected with 1) saline (6 μL) + saline (10 mL/kg), 2) the
dynorphin A (1:30 dilution, 10 μL) + saline (10 mL/kg), 3) saline (6 μL)
+ BAA (300 μg/kg), and 4) the dynorphin A (1:30 dilution, 10 μL) + BAA
(300 μg/kg). The place preference test was assessed 50 minutes
subsequent to subcutaneous injection. Subcutaneous injection of BAA but
not intracerebroventricular the dynorphin A antiserum totally inhibited
morphine CPP acquisition. However, pretreatment with
intracerebroventricular injection of the dynorphin A antiserum
completely attenuated BAA-suppressed morphine CPP acquisition
(P<0.05, by one-way ANOVA followed by the post-hoc
Student-Newman-Keuls test; Fig. 7B).
Last four groups of morphine CPP mice (n=10 per group) received the same
regimen as above except that intracerebroventricular injection of the
dynorphin A antiserum was replaced with GNTI (5 μg). As exhibited in
Fig. 7C, intracerebroventricular injection of GNTI did not have a
significant inhibitory effect on morphine CPP acquisition, but almost
totally restored systemic BAA-suppressed morphine-induced CPP
acquisition (P=0.09, by one-way ANOVA followed by the post-hoc
Student-Newman-Keuls test).