Naloxone-induced Withdrawal Symptoms Research Articles

Nucleus accumbens D1/D2 circuits control opioid withdrawal symptoms in mice.

The nucleus accumbens (NAc) is the most promising target for drug use disorder treatment. Deep brain stimulation (DBS) of NAc is effective for drug use disorder treatment. However, the mechanisms by which DBS produces its therapeutic effects remain enigmatic. Here, we define a behavioral cutoff criterion to distinguish depressive-like behaviors and non-depressive-like behaviors in mice after morphine withdrawal. We identified a basolateral amygdala (BLA) to NAc D1 medium spiny neuron (MSN) pathway that controls depressive-like behaviors after morphine withdrawal. Furthermore, the paraventricular nucleus of thalamus (PVT) to NAc D2 MSN pathway controls naloxone-induced acute withdrawal symptoms. Optogenetically induced long-term potentiation with κ-opioid receptor (KOR) antagonism enhanced BLA to NAc D1 MSN signaling and also altered the excitation/inhibition balance of NAc D2 MSN signaling. We also verified that a new 50 Hz DBS protocol reversed morphine withdrawal-evoked abnormal plasticity in NAc. Importantly, this refined DBS treatment effectively alleviated naloxone-induced withdrawal symptoms and depressive-like behaviors and prevented stress-induced reinstatement. Taken together, the results demonstrated that input- and cell type-specific synaptic plasticity underlies morphine withdrawal, which may lead to novel targets for the treatment of opioid use disorder.

Take-Home Naloxone and risk management from the perspective of people who survived an opioid overdose in Stockholm - An analysis informed by drug, set and setting.

Take-Home Naloxone (THN) programs were introduced in Sweden in 2018 - a country with one of the highest rates of overdose mortality in the EU and a severe stigmatisation of people who inject drugs. This qualitative study builds on the international research that has expanded a previously narrow and medical focus on overdose deaths. It uses Zinberg's framework to look beyond the role of the "drug" to include the attitudes and personality of the person ("set") and contextual factors ("setting"). This study explores the impacts of THN from the perspective of overdose survivors. Between November 2021 and May 2022 semi-structured interviews were conducted with 22 opioid overdose survivors, recruited among clients of the Stockholm needle and syringe program. All the participants had been treated with naloxone in an overdose situation. The interviews were processed through thematic analysis using deductive and inductive coding in accordance with the theoretical framework. Interviewees included men and women who used different types of drugs. THN has impacted on "drug" in terms of naloxone-induced withdrawal symptoms and peers having to deal with survivors' emotions. Exploring "set" revealed feelings of shame following naloxone revival for the person who overdosed. Despite such reactions, participants retained an overwhelmingly positive attitude towards THN. Participants integrated THN into their risk management practices ("setting") and some acknowledged that THN provided a new way to treat overdoses without necessarily needing to interact with authorities, especially the police. The THN program has influenced "drug, set and setting" for participants, providing increased safety at drug-intake and transferring overdose management and the burden of care to the community. The lived experience of participants also exposes the limitations of THN indicating that there are additional unmet needs beyond THN programs, particularly in terms of "setting".

Blockade of 5-Hydroxytryptamine 2A Receptor Attenuates Precipitation of Naloxone-Induced Withdrawal Symptoms in Opioid-Exposed Mice.

Heroin dependency has become a global problem and has caused significant clinical and socioeconomic burdens along with devastating medical consequences. Chronic drug exposure alters the expression and functional activity of 5-hydroxytryptamine (serotonin) 2A receptors (5-HT2ARs) in the brain. Furthermore, pharmacological blockade of 5-HT2ARs reduces cue-induced cocaine craving behaviors. In this study, we explored the influence of 5-HT2ARs on heroin-withdrawal behaviors in mice. Black C57BL/6J mice were given gradually increasing (10–50 mg/kg over 4.5 days) doses of heroin to induce heroin dependency, after which naloxone was given to precipitate withdrawal symptoms. MDL100907, a selective and potent 5-HT2AR antagonist, attenuated naloxone-precipitated withdrawal symptoms in these mice. In addition, 5-HT2AR protein levels increased significantly in the medial prefrontal cortex (mPFC), while phosphorylation of extracellular signal-regulated kinase (p-ERK) decreased in the mPFC after heroin exposure. In conclusion, these results suggest that 5-HT2ARs might be involved in the development of opioid dependency and that pharmacological blocking of 5-HT2ARs might be a new therapeutic strategy for heroin dependency.

Design, synthesis and preliminary bioactivity evaluation of bitopic benzopyranomorpholine analogues as selective dopamine D3 receptor ligands as anti-drug addiction therapeutic agents

Three series of bitopic benzopyranomorpholine analogues were designed, synthesized, and evaluated as a novel class of selective ligands for the dopamine D3 receptor. Binding affinities of target compounds were determined using the method of radioligand binding assay. Most compounds demonstrated considerable binding affinities and selectivity for D3 receptor. Besides, the compounds were screened for their ability to alleviate withdrawal symptoms of opioid addiction in animal behavioral models. The results showed that compound 20h displayed nanomolar affinity for the D3R, and exhibited anti-drug addiction efficacy in the animal model of of naloxone-induced withdrawal symptoms in morphine-dependent mice.

Bulleyaconitine A Inhibits Morphine-Induced Withdrawal Symptoms, Conditioned Place Preference, and Locomotor Sensitization Via Microglial Dynorphin A Expression.

Bulleyaconitine A (BAA), a C19-diterpenoid alkaloid, has been prescribed as a nonnarcotic analgesic to treat chronic pain over four decades in China. The present study investigated its inhibition in morphine-induced withdrawal symptoms, conditioned place preference (CPP) and locomotor sensitization, and then explored the underlying mechanisms of actions. Multiple daily injections of morphine but not BAA up to 300 μg/kg/day into mice evoked naloxone-induced withdrawal symptoms (i.e., shakes, jumps, genital licks, fecal excretion and body weight loss), CPP expression, and locomotor sensitization. Single subcutaneous BAA injection (30–300 μg/kg) dose-dependently and completely attenuated morphine-induced withdrawal symptoms, with ED50 values of 74.4 and 105.8 μg/kg in shakes and body weight loss, respectively. Subcutaneous BAA (300 μg/kg) also totally alleviated morphine-induced CPP acquisition and expression and locomotor sensitization. Furthermore, subcutaneous BAA injection also specifically stimulated dynorphin A expression in microglia but not astrocytes or neurons in nucleus accumbens (NAc) and hippocampal, measured for gene and protein expression and double immunofluorescence staining. In addition, subcutaneous BAA-inhibited morphine-induced withdrawal symptoms and CPP expression were totally blocked by the microglial metabolic inhibitor minocycline, dynorphin A antiserum, or specific KOR antagonist GNTI, given intracerebroventricularly. These results, for the first time, illustrate that BAA attenuates morphine-induced withdrawal symptoms, CPP expression, and locomotor sensitization by stimulation of microglial dynorphin A expression in the brain, suggesting that BAA may be a potential candidate for treatment of opioids-induced physical dependence and addiction.

Treatments to Combat Opiate Dependence

The current opioid epidemic is a pressing public health concern that has been difficult to address because there is no generally accepted hypothesis to explain the underlying neurophysiological mechanism(s) that lead to tolerance and withdrawal, which in turn could serve as the basis for developing therapeutic interventions. As a step to developing such a unifying mechanistic hypothesis we studied both the electrophysiological firing patterns of individual neurons and behaviors indicative of tolerance and withdrawal in rats following chronic administration of morphine. Neuronal activity recordings from the ventral tegmental area (VTA), nucleus accumbens (NAc), prefrontal cortex (PFC), thalamus, hypothalamus, hippocampus (HIPP), Amygdala (AMYG) and caudate nucleus (CN) following exposure of repetitive (chronic) morphine produced a new baseline pattern of neuronal firing rates, which we refer to as an “opioid-induced pattern” in the mesocorticolimbic neuronal activity circuit, which is thought to be involved in mediating the “reward” effects of opioid and other drugs of abuse. These changes in neuronal firing rate was paralleled by behavioral changes indicative of the development of dependence such as tolerance and withdrawal suggesting a possible cause-effect relationship between the opioid induced pattern change of baseline neuronal firing and the development of opioid tolerance and withdrawal. Briefly, (i) morphine produces a new baseline pattern of neuronal firing (i.e., an “opioid-induced pattern”), (ii) there is an intrinsic neurophysiological mechanism that seeks to maintain newly established patterns of baseline neuronal firing once established, (iii) continued morphine administration maintains the new pattern of baseline neuronal activity so that withdrawal behaviors do not occur, but (iv) eventual discontinuation of the drug leads to opioid withdrawal symptoms. Consistent with this proposed hypothesis, co-administration of the immunomodulator such as interferon, cyclosporin and cortisol attenuated both the development of an altered baseline pattern of neuronal firing and the parallel behavioral changes. This observation suggests that immunomodifiers treatment to morphine dependence subject restore the neuronal firing rate to its pre-morphine baseline and thus alleviate the withdrawal symptoms that make is so difficult for addicts to discontinue opioid use. The studies in this review describe in more detail the findings that led to our proposed hypothesis for the underlying neurophysiological basis of the development of opioid tolerance and withdrawal and the possible use of immunomodulators to decrease the development of dependence and thereby attenuate withdrawal symptoms that make it so difficult for addicts to discontinue drug use. Repetitive use of opioids results in dependence on the drug, a complex condition that is considered to be an opiate use disorder (OUD). The reduction or cessation of opioid consumption leads to severe withdrawal behaviors. The degree of opiate dependence can be assessed by the intensity of the withdrawal behavior. To prevent this devastating opiate withdrawal syndrome, the subject will continue to take the drug. Success in modifying the withdrawal behavior may shed light on the dynamics of OUD and help to curb the opiate epidemic. Classical therapeutic addiction research has focused on cellular and molecular alterations within neurons and their neuronal circuits. As such, most of the pharmacotherapies for opioid addiction are designed to target the neuronal processes known to be affected by drug intake. In addition to the pivotal role of neurons in the initiation, transition, and maintenance of opioid dependence, the glial cells within the central nervous system are also of particular importance. According to some studies, 60 to 80% of the cellular brain is composed of glial cells. Recent studies have shown that glial cells participate in synaptogenesis, neuronal excitability, and neurotransmission. Following opioid exposure, glial cells demonstrate robust changes in their morphology and physiology in key central nervous system regions known to contribute to drug dependence. They play a pivotal role in opioid-addiction like behaviors. Glial cells are also part of the immune system. This review summarizes studies demonstrating that the immune system participates in the expression of opiate withdrawal and that a single dose of immunological substances such as α-interferon, cyclosporine, or cortisol significantly attenuates the severity of the naloxone-induced withdrawal symptoms in opioid-dependent animals. These preclinical studies provide a new approach to treat opiate dependence using immunomodulators that do not belong to the opiate family. We hope that this review will encourage translational studies to use immunomodulators in combating the opioid epidemic and save lives.

“A Blessing and a Curse:” Opioid Users' Perspectives on Naloxone and the Epidemic of Opioid Overdose

Background: To address the alarming rise in opioid overdose deaths, states have increased public access to the overdose reversal medication, naloxone. While some studies suggest that increased naloxone accessibility reduces opioid overdose deaths, others raise concerns about unintended consequences, such as increases in risky drug use and opioid re-use post-overdose to counter naloxone-induced withdrawal symptoms. Few studies have examined the impact of expanded naloxone access on the attitudes and behaviors of opioid users. Methods: In this qualitative study, we conducted in-depth, semi-structured interviews with 36 English-speaking opioid users 18+ years of age. Informants were recruited from an urban methadone clinic, a needle exchange program and a residential treatment program. The approximately hour-long interviews focused on users’ attitudes and behaviors surrounding naloxone, opioid use and overdose. Transcribed audio-recordings of interviews were analyzed using NVivo. Results: Informants were ambivalent about naloxone, widely acknowledging its life-saving benefits while reporting such negative effects as severe withdrawal symptoms and the promotion of riskier drug use. Naloxone-induced withdrawal, coupled with misperceptions about naloxone’s pharmacological effects, prompted overdose survivors to rapidly re-use opioids and refuse hospitalization following an overdose reversal. About half the sample believed naloxone led to greater risk-taking by others, such as fentanyl use or use in higher quantities, but did not endorse riskier drug use themselves. Conclusions: The results suggest the need for targeted education about the pharmacological effects of naloxone and better strategies for managing naloxone-induced withdrawal. Future research should focus on the extent to which naloxone is associated with greater opioid risk-taking.

Cupping: a reasonable choice for attenuating morphine withdrawal symptoms in Wistar rats

Background: Nowadays, the sedative drugs are the main treatments to attenuate the opioid withdrawal symptoms. However, physical therapies are found a safe alternative treatment without any serious adverse effects. This study aimed to evaluate the effects of cupping treatment on the morphine withdrawal symptoms in rats.Methods: Eighty rats were divided into two main groups; treated with morphine or saline, and each group was also divided to cupping and sham cupping subgroups (with single or daily treatment). Injection of morphine and saline were done in the morphine and saline groups twice a day for nine days. On the ninth day, the naloxone was administered and the withdrawal symptoms including jumping, rearing, body grooming, abdominal writing, and wet-dog shaking were recorded for 60 minutes. Cupping or sham cupping was applied on the GV14 once before or daily after withdrawal induction in each group.Results: The results revealed that a single cupping before the withdrawal induction significantly attenuated the withdrawal symptoms in the morphine group (p<0.01).But, the daily cupping failed to decrease the withdrawal symptoms in this group. The results also indicated that the daily cupping in the saline group increased the naloxone-induced withdrawal symptoms significantly (p<0.01).Conclusions: Single session cupping before the withdrawal induction could decreased the withdrawal symptoms. This finding might be related to the modulation of GABAergic system.

Blockade and reversal of spinal morphine tolerance by P2X3 receptor antagonist.

In recent years, studies have substantiated the view that P2X3 receptors play a part in the generation and transmission of purinergic signals in inflammatory and chronic neuropathic pain. Data have also been presented to suggest that the process of P2X3 receptor antagonism inhibits inflammatory hyperalgesia, involving the spinal opioid system. The aim of this study was to investigate the effect of the selective P2X3 receptor antagonist A-317491 on the development of antinociceptive tolerance to chronic morphine administration in mice. Daily systemic injection of A-317491 attenuated the morphine-induced antinociceptive tolerance to von Frey and thermal stimuli. Repeated morphine injections alone led to a significant rightward shift in the morphine dose-response curve compared with that with A-317491. A single dose of A-317491 also showed a reversal effect in morphine-tolerant mice. In a withdrawal test, co-administration of A-317491 and morphine also reduced the naloxone-induced withdrawal symptoms compared with the morphine-alone group. Thus, we propose that the P2X3 receptor is involved in the process of morphine antinociceptive tolerance and may be a new therapeutic target in the prevention of tolerance to morphine-induced antinociception.

Yulangsan polysaccharide attenuates withdrawal symptoms and regulates the NO pathway in morphine-dependent rats

Yulangsan polysaccharide (YLSP) has been utilized as a phytomedicine to managing nervous dysfunction in China. Thus, this study aimed to evaluate the potential YLSP-mediated detoxification role against morphine dependence in rats. The results indicated that the morphine dependence model significantly increased withdrawal symptoms, levels of NO and NOS (P<0.05). Furthermore, monoaminergic neurotransmitters, including DA and NE, were detected at elevated levels in the ventral tegmental area (VTA), hippocampus (HIP) and prefrontal cortex (PFC), respectively, while the level of DA was decreased and NE was increased in the nucleus accumbens (NAc). Conversely, YLSP administration significantly reversed naloxone-induced withdrawal symptoms, expression of brain NO and NOS, and monoaminergic neurotransmitters (P<0.05). Interestingly, YLSP shows an even more effective trend in attenuating withdrawal symptoms than does clonidine, although without a significant difference. These findings indicate that YLSP attenuation of the naloxone-induced withdrawal symptoms of morphine dependence may be mediated by regulation of the NO pathway and modulation of monoaminergic neurotransmitters.

Role of 5-HT7 receptors in the anxiety-like behavior in naloxone-induced withdrawal symptoms in mice

Pharmacological studies have implicated anxiety behavior in the withdrawal of opiates. There is evidence demonstrating the role of serotonergic system and recently the role of 5-HT7 receptor regulation has been shown in anxiety behavior. The aim of the present study was to evaluate the effects of 5-HT7 receptor agonists and antagonists on anxiety behavior related to morphine withdrawal in male mice. Morphine dependence was induced by repeated treatment with morphine within 5 consecutive days. The anxiety responses were measured after the last morphine administration. Morphine-dependent mice were pretreated with AS19 (3 and 10 mg/kg, i.p.), SB269970 (1 and 10 mg/kg, i.p.) or saline, and subsequently with naloxone (3 mg/kg, s.c), prior to testing in elevated plus-maze. The number of entries into the open arms and the time spent in the open arms were measured. The results showed that there were no significant differences in the number of entries into the open arms and the time spent in open areas of plus-maze between the experimental groups. It was concluded that neither SB269970 nor AS19 had any effect on anxiety behavior related to morphine. The 5-HT7 receptor had no significant role in the anxiety behavior related to morphine withdrawal.

A drug-paired taste cue elicits withdrawal and predicts cocaine self-administration

Addiction is a chronic disease where periods of abstinence are riddled with instances of craving, withdrawal, and eventual relapse to escalated drug use. Cues previously associated with drug use can have a deleterious effect on this cycle by precipitating withdrawal symptoms. Here we focus specifically on the relationship between avoidance of a drug-paired taste cue and the ability of the drug-paired cue to elicit withdrawal and, ultimately, drug seeking and taking. We used a rat model of drug addiction and naloxone-induced loss of body weight to test whether a taste cue elicits withdrawal in anticipation of drug availability. Experiment 1 investigated the ability of a taste cue to elicit signs of withdrawal when it predicted experimenter-administered morphine (15mg/kg, i.p.). In Experiment 2, a saccharin taste cue was paired with the opportunity to actively self-administer cocaine (0.167mg/infusion, i.v.). The results show that presentation of a morphine- or cocaine-paired taste cue is sufficient to elicit naloxone-induced withdrawal symptoms, and greater withdrawal predicts greater cocaine self-administration in rats.

The effects of exogenous CCK-8 on the acquisition and expression of morphine-induced CPP

Cholecystokinin octapeptide (CCK-8) is the most potent endogenous anti-opioid peptide and regulates a variety of physiological processes. In our previous study, we found that exogenous CCK-8 attenuated naloxone-induced withdrawal symptoms, but the possible regulative effects of CCK-8 on the rewarding effects of morphine were not examined. In the present study, we aimed to determine the exact effects of exogenous CCK-8 at various doses on the rewarding action of morphine by utilizing the unbiased conditioned place preference (CPP) paradigm. We therefore examined the effects of CCK-8 on the acquisition, expression and extinction of morphine-induced CPP and on locomotor activity. The results showed that CCK-8 (0.01–1μg, i.c.v.), administered alone, induced neither CPP nor place aversion, but blocked the acquisition of CPP when administered with 10mg/kg morphine. The highest dose of CCK-8 (1μg) administered before CPP testing increased CPP and, along with lower doses (0.1μg), reduced its extinction. In addition, the highest dose (1μg) of CCK-8 suppressed locomotor activity. Our study provides the first behavioral evidence for the inhibitory effects of exogenous CCK-8 on rewarding activity and reveals significant effects of exogenous CCK-8 on various stages of place preference and the development of opioid dependence.

Antinociceptive potentiation and attenuation of tolerance by intrathecal β-arrestin 2 small interfering RNA in rats

Tolerance to the analgesic effect of opioids complicates the management of persistent pain states. We tested whether the intrathecal infusion of small interfering RNA (siRNA) against β-arrestin 2 would reduce tolerance to chronic morphine use and the severity of precipitated morphine withdrawal. Intrathecal β-arrestin 2 (2 μg siRNA per 10 μl per rat) was injected once daily for 3 days. Rats then received a continuous intrathecal infusion of morphine (2 nmol h⁻¹) or saline for 7 days. Daily tail-flick (TF) and intrathecal morphine challenge tests were performed to assess the effect of intrathecal β-arrestin 2 siRNA on antinociception and tolerance to morphine. Naloxone withdrawal (2 mg kg⁻¹) was performed to assess morphine dependence. In the daily TF test, the antinociception of intrathecal morphine was increased and maintained in rats receiving β-arrestin 2 siRNA compared with the control group (morphine alone). In the probe response test, rats receiving morphine infusion with β-arrestin 2 siRNA treatment showed a significant left shift in their dose-response curve, as measured by per cent maximal possible effect (MPE), such that the AD₅₀ was significantly decreased by a factor of 5.6 when compared with that of morphine-infused rats. In the naloxone-induced withdrawal tests, rats receiving β-arrestin 2 siRNA injection with morphine infusion showed a significant reduction in four of the six signs of withdrawal. We show here that intrathecal β-arrestin 2 siRNA in rats enhances analgesia and attenuates naloxone-induced withdrawal symptoms. This may warrant further investigation in the context of long-term use of intrathecal opioids for controlling chronic pain.

Exercise Dependence and Morphine Addiction: Evidence From Animal Models

This study used animal models to examine potential similarities between dependence on physical activity (i.e., exercise) and dependence on morphine. Using C57BL/6 mice, the study also tested the hypothesis that physical exercise (e.g., long-term wheel running) may enhance vulnerability to the development of morphine dependence. The existence of an endorphin-related dependence induced by physical activity was also assessed. Naloxone was used to precipitate morphine withdrawal in mice accustomed to morphine. Specifically, the study sought to assess the intensity of addiction provoked by injection of morphine in mice that engaged in wheel-running activity as opposed to inactive mice. After 25 days of free access to activity wheel, mice that engaged in wheel-running demonstrated increased vulnerability to naloxone-induced withdrawal symptoms, which may be linked to activation of peripheral, as opposed to central, opioid receptors. These results indicate a behavioral interaction in which engaging in wheel running appears to potentiate the effects of morphine addiction. Implications of these findings for understanding human behavior and exercise addiction are also discussed.

Effect of agmatine on the development of morphine dependence in rats: potential role of cAMP system

Agmatine is an endogenous amine derived from arginine that potentiates morphine analgesia and blocks symptoms of naloxone-precipitated morphine withdrawal in rats. In this study, we sought to determine whether treatment with agmatine during the development of morphine dependence inhibits the withdrawal symptoms and that the effect is mediated by cAMP system. Exposure of rats to morphine for 7 days resulted in marked naloxone-induced withdrawal symptoms and agmatine treatment along with morphine significantly decreasing the withdrawal symptoms. The levels of cAMP were markedly increased in morphine-treated rat brain slices when incubated with naloxone and this increase was significantly reduced in rats treated with morphine and agmatine. The induction of tyrosine hydroxylase after morphine exposure was also reduced in locus coeruleus when agmatine was administered along with morphine. We conclude that agmatine reduces the development of dependence to morphine and that this effect is probably mediated by the inhibition of cAMP signaling pathway during chronic morphine exposure.

Antinociceptive potentiation and attenuation of tolerance by intrathecal electric stimulation in rats.

We tested whether intrathecal electric stimulation would reduce the tolerance to chronic morphine use and the severity of precipitated morphine withdrawal. Rats received intrathecal electrode catheter implantation and a continuous intrathecal infusion of morphine (2 nmol/h) or saline for 7 days. Intrathecal electric stimulations (0, 20, or 200 V) were performed once daily during the same period. Daily tail-flick and intrathecal morphine challenge tests were performed to assess the effect of intrathecal electric stimulation on antinociception and tolerance to morphine. Naloxone withdrawal (2 mg/kg) was performed to assess morphine dependence, and changes in spinal neurotransmitters were monitored by microdialysis. The antinociceptive effect of intrathecal morphine was increased by 200 V of electric stimulation. The magnitude of tolerance was decreased in the rats receiving the 2 nmol/h infusion with 200 V of intrathecal electric stimulation compared with the control group (morphine 2 nmol/h alone) (AD(50), 13.6 vs 124.7 nmol). The severity of naloxone-induced withdrawal was less in the rats receiving 200 V of stimulation. Intrathecal stimulation thus enhances analgesia and attenuates naloxone-induced withdrawal symptoms in rats receiving chronic intrathecal morphine infusion. Increases in spinal glycine release may be the underlying mechanism. This method may merit further investigation in the context of the long-term use of intrathecal opioids for controlling chronic pain. Control of chronic pain is a major health problem. We show here that direct electrical stimulation of the spinal cord in rats enhances analgesia and attenuates naloxone-induced withdrawal symptoms. This may warrant further investigation in the context of long-term use of intrathecal opioids for controlling chronic pain.

Inverse agonists and neutral antagonists at mu opioid receptor (MOR): possible role of basal receptor signaling in narcotic dependence.

The mu opioid receptor, MOR, displays spontaneous agonist-independent (basal) G protein coupling in vitro. To determine whether basal MOR signaling contributes to narcotic dependence, antagonists were tested for intrinsic effects on basal MOR signaling in vitro and in vivo, before and after morphine pretreatment. Intrinsic effects of MOR ligands were tested by measuring GTPgammaS binding to cell membranes and cAMP levels in intact cells. beta-CNA, C-CAM, BNTX, and nalmefene were identified as inverse agonists (suppressing basal MOR signaling). Naloxone and naltrexone were neutral antagonists (not affecting basal signaling) in untreated cells, whereas inverse agonistic effects became apparent only after morphine pretreatment. In contrast, 6alpha- and 6beta-naltrexol and -naloxol, and 6beta-naltrexamine were neutral antagonists regardless of morphine pretreatment. In an acute and chronic mouse model of morphine-induced dependence, 6beta-naltrexol caused significantly reduced withdrawal jumping compared to naloxone and naltrexone, at doses effective in blocking morphine antinociception. This supports the hypothesis that naloxone-induced withdrawal symptoms result at least in part from suppression of basal signaling activity of MOR in morphine-dependent animals. Neutral antagonists have promise in treatment of narcotic addiction.

The role of histaminergic-noradrenergic axis in naloxone-induced withdrawal symptoms in mice

The effects of histamine antagonists on naloxone-precipitated withdrawal symptoms were studied in morphine-dependent mice. Chlorpheniramine (0.5–10 mg/kg), a H 1-blocker, given IP 30 min before naloxone challenge produced a dose-dependent potentiation of withdrawal body weight loss, burrowing, and hypothermia, but did not influence either jumping or wet-dog shakes. On the other hand, cimetidine (10–100 mg/kg), a H 2-blocker, produced dose-dependent potentiation of withdrawal hypothermia and jumping. Cimetidine was without effect on wet-dog shakes, burrowing, and body weight loss. The effect of Chlorpheniramine was investigated in mice injected with 6-hydroxydopamine (6-OHDA) intracerebrally to examine whether histamine-mediated effects are some-how linked to noradrenergic pathways. Intracerebral injection of 6-OHDA in 5-day-old mice pups resulted in hyperlocomotion by the end of 30 days before initiation of morphine dependence. Mice pretreated with 6-OHDA developed a higher degree of naloxone-induced withdrawal jumping than nontreated mice. 6-OHDA (50 μg) lesions completely blocked the potentiating effect of Chlorpheniramine on burrowing, hypothermia, and even reversed the effect on body weight loss. These findings suggest that both histamine H 1- and H 2-receptors may be involved in the expression of precipitated withdrawal in morphine-dependent mice and histamine receptors function as modulators of noradrenergic neurotransmission.

The role of cholinergic systems in the expression of morphine withdrawal

Both oxotremorine and physostigmine both in doses ranging from 25 to 100 μg/kg produced dose-dependent attenuation of withdrawal jumping and potentiation of ‘wet dog’ shakes, burrowing, hypothermia and body weight loss precipitated by naloxone (1 mg/kg, i.p.) in morphine-dependent mice. On the other hand, atropine sulphate (2–20 mg/kg) dose-dependently attenuated all naloxone precipitated withdrawal symptoms except withdrawal hypothermia which was further potentiated. However, the peripherally acting derivative atropine methyl nitrate (2–10 mg/kg) also attenuated all naloxone-induced withdrawal symptoms except jumping, which was not significantly modified. Hyoscine (0.2–20 mg/kg) exhibited a biphasic effect on withdrawal jumping. Withdrawal jumping was potentiated by low and attenuated by high doses of hyoscine. Withdrawal body weight loss was dose-dependently attenuated but ‘wet dog’ shakes, burrowing and hypothermia were markedly potentiated by hyoscine. Our results suggest that a combination of central muscarinic activation and peripheral muscarinic blockade can partially ameliorate precipitated morphine withdrawal. Differences observed between atropine and hyoscine with regard to their modifying effects on withdrawal symptoms may be explained on the basis that the drugs may be acting on the different subpopulations of the muscarinic receptor or through non-cholinergic systems.