4. Discussion
Long-term application of morphine and other opioid narcotics induces
addiction composing physical and psychological dependence. In the
present study, physical dependence developed following bi-daily
subcutaneous injections of morphine in mice for 7 consecutive days, and
application of naloxone induced withdrawal signs including shakes,
jumps, genital licks, fecal excretion and body weight loss. In contrast,
bi-daily subcutaneous injections of BAA up to 300 μg/kg/day for 7 days
did not induce any physical dependence, consistent with the previous
finding in which daily subcutaneous BAA did not induce jumping responses
following nalorphine challenge (Tang et al., 1986). In addition, single
subcutaneous injection of BAA alleviated naloxone-induced withdrawal
signs in morphine physical dependence mice. In doses ranging from 30 to
300 μg/kg in morphine dependence mice, BAA caused a dose-related
inhibition of abrupt withdrawal signs, typically shakes and body weight
loss with ED50 values of 74.4 and 105.8 μg/kg
respectively. Consistently, the BAA analog lappaconitine was reported to
alleviate morphine and cocaine physical dependence (Qu and Qu, 1994). On
the other respect, daily subcutaneous injections of morphine but not BAA
(300 μg/kg/day) for 5 consecutive days induced remarkable CPP
acquisition with high conditioning scores. Single subcutaneous injection
of BAA (300 μg/kg) entirely abolished morphine-induced CPP acquisition.
All these results indicate that BAA does not induce withdrawal signs and
CPP acquisition, moreover, it markedly alleviates morphine-induced both
physical and psychological dependence.
Opiates such as morphine and heroin, act at the
mesolimbic
dopamine pathway projecting from ventral tegmental (VTA) to NAc (Patyal
et al., 2012). Opiates drugs effectively stimulate dopamine release in
NAc within 1 hour after administering intracerebroventricularly or
locally into VTA (Murphy et al., 1996; Sebastian et al., 2016).
Hippocampal input to the shell of NAc (NAcSh) is important for driving
NAc activity which may play a role in reward behaviors (LeGates et al.,
2018). Thus, it can be seen that NAc
and
hippocampus, located around the cerebroventricular area, are important
sites for drugs to produce addiction. Dynorphin A regulates the activity
of dopamine neurons by acting on κ-opioid receptors in mesolimbic, NAcSh
, prefrontal cortex and VTA that have been implicated in drug abuse
liability (Meshul and McGinty, 2000; Volkow et al., 2009). Extracellular
dopamine levels in NAc are increased in mice lacking κ-opioid receptors,
revealing that the dynorphin/κ-opioid receptor system inhibits dopamine
neurotransmission and counteracts the reward of opioid drugs of abuse
(Chefer et al., 2005; Shippenberg et al., 2007b). Hippocampal CA1 has
been demonstrated in reward-association of learning and memory
especially in the CPP model (Fanselow and Dong, 2010; Riahi et al.,
2013). The intra-CA1 administration of D1/D2 receptor antagonists
SCH23390 and sulpiride, and microinjection of the κ-opioid receptor
agonist U50,488H into NAcSh, resulted in a decrease in morphine
conditioning scores (Assar et al., 2016). In this study, we explored
whether the dynorphin A/κ-opioid receptor system in NAc and hippocampus
was closely associated with BAA-attenuated morphine physical dependence
and psychological dependence. Subcutaneous injection of BAA in
morphine-treated mice stimulated the expression of dynorphin A in NAc
and hippocampus at 1 hour after injection, which was in agreement with
the time-course of its anti-addictive effects. The results are parallel
to the previous findings in which intrathecal and subcutaneous injection
of BAA, bullatine A and lappaconitine stimulated the expression of
dynorphin but not β-endorphin in the spinal cord (Li et al., 2016a; Li
et al., 2016b).
The notion is further supported by the following intervention injections
through the cerebroventricular route which is located around NAc and
hippocampus. It was previously reported that single intravenous
injection of dynorphin A attenuated withdrawal symptoms of morphine
physical dependence (Takemori et al., 1993). The present study further
demonstrated that intracerebroventricular injection of the dynorphin A
antiserum totally eliminated systemic BBA-inhibited morphine withdrawal
signs and CPP acquisition. In addition, the highly specific κ-opioid
receptor antagonist GNTI, given intracerebroventricularly, also entirely
eliminated systemic BBA-inhibited morphine physical and psychological
dependence. The results are in agreement with the previous publications
that the κ-opioid receptor agonist salvinorin A punished
self-administration of cocaine and remifentanil in monkeys (Freeman et
al., 2014), and that addition of the
κ-opioid receptor agonist U69,593 to
fentanyl produced a proportion-dependent decrease of fentanyl
self-administration in rats (Negus et al., 2008). In agreement,
GNTI and other specific κ-opioid
receptor antagonist nor-BNI (but not the μ- or δ-opioid receptor
antagonist) blocked the antinociceptive effects of BAA (Li et al.,
2016b; Huang et al., 2020b), bullatine A (Huang et al., 2016) and
lappaconitine (Sun et al., 2018) in the rodent models of neuropathic
pain, bone cancer pain, inflammatory pain and visceral pain.
Cumulative evidence indicates that microglia in brain particularly in
NAc, VTA and hippocampus are influenced and activated by chronic
exposure to abuse drugs such as opiates and alcohol (Miguel-Hidalgo et
al., 2002; Hutchinson et al., 2009; Coller and Hutchinson, 2012). Drug
abuse activates microglia and produces a large number of inflammatory
factors, which affect synapse reconstruction, chemical changes in neural
signal transduction and phagocytosis of apoptotic neurons, and
ultimately regulate the dopamine reward signaling pathway and enhance
drug dependence and addiction (Kovacs, 2012; Garaschuk and Verkhratsky,
2019). On the other hand, recent studies have shown that microglia have
an alternative activation state or protective state, which activates
anti-inflammatory cascades and exhibits neuroprotection and
antinociception (Hu et al., 2012; Fan et al., 2015; Wu et al., 2017; Wu
et al., 2018). Our present study provides additional evidence that BAA
stimulates microglia to express dynorphin A to attenuate drug dependence
and addiction. Subcutaneous injection of BAA stimulated dynorphin A
expression only in microglia and not in astrocytes and neurons in NAcSh
and hippocampal CA1, identified by immunofluorescence staining of
dynorphin A with microglial cellular marker Iba-1, astrocytic marker
GFAP and neuronal marker NeuN. The results are in agreement with the
previous finding in which injection of the BAA analog bullatine A
specifically stimulated microglial (but not astrocytic or neural)
expression of dynorphin A in the spinal cords of neuropathic rats (Huang
et al., 2016). We further demonstrated that intracerebroventricular
injection of the microglial activation inhibitor minocycline entirely
blocked systemic BAA-inhibited morphine-induced physical and
psychological dependence. Consistently, the antinociceptive effects of
BAA and its analogs bullatine A and lappaconitine were also blocked by
intrathecal injection of minocycline in the rat models of pain
hypersensitivity (Huang et al., 2016; Sun et al., 2018a; Huang et al.,
2020b). These results highlight that stimulation of microglia expresses
and releases dynorphin A, in contrast to proinflammatory cytokines and
neurotrophins, and inhibits morphine physical and psychological
dependence and pain hypersensitivity.
Drug addiction is a chronically and relapsing disorder characterized by
compulsive seeking and taking drugs regardless of the adverse effects it
could be cause (Shippenberg et al., 2007b). The main treatment approach
for drug addiction is detoxification and relieves withdrawal symptoms,
and methadone substitution is most commonly used. This treatment,
however, is just provided to inpatients in well-equipped management
institutions due to its own physical dependence (Kampman and Jarvis,
2015; Tran et al., 2017). Clonidine is a non-opioid detoxifying agent
and diminishes withdrawal symptoms via activation of adrenergic
α2-receptors, and is superior to other treatments since
it is not addictive and does not produce euphoria, although the
side-effect of postural hypotension limits its clinical use (Wilson and
DiGeorge, 1993). On the other respect, psychotherapy for addiction
should be emphasized conceptually. However, only a few patients are
unfortunately willing to undergo it, and many patients are still carving
for addictive substances after improving withdrawal symptoms, which
eventually leads to relapse (Woody et al., 1983; Arevalo et al., 2008).
Thus, the approaches at present to solve opioid addiction especially
psychological dependence are limited. Our current study provides a solid
pharmacological base for BAA to alleviate both morphine physical and
psychological dependence at the animal level. In addition, oral
administration of the BAA analog lappaconitine over 6 days reduced or
eliminated withdrawal symptoms such as yawing, shedding tears,
chilliness, mydriasis and restlessness in drug addict patients whose
duration of heroin or opium addiction ranged from 1 to 4 years (Qu and
Qu, 1994). Taken together, all these results indicate that BAA is a
unique and promising clinical development candidate to treat opioid
addiction, especially for psychological dependence. The data also
suggest that targeting microglial expression and secretion of dynorphin
A is a potential approach for the opioid drug addiction treatment.