Methadone induced physical dependence in the rat

Although the morphine withdrawal syndrome has been well described in the rat, a syndrome having similar characteristics has not been demonstrated following chronic methadone treatment. In this study we describe the behavioral effects produced by naloxone (4 mg/kg sc) following 72 hours of continuous iv infusion of methadone, (12.2 ug/kg/min), morphine (12.2 to 97.9 ug/kg/min) or saline. The cessation of methadone or morphine but not saline treatment followed by naloxone resulted in graded signs including wet dog shakes, escape attempts, self-stimulation and body weight loss and quantal signs including diarrhea, ear blanching, exophthalmos, ptosis, tachypnea and teeth chattering. These results indicate that this mode of methadone administration produces physical dependence characterized by a morphine-like withdrawal syndrome in the rat.     

Role of Corticotropin-Releasing Factor (CRF) Receptor-1 on the Catecholaminergic Response to Morphine Withdrawal in the Nucleus Accumbens (NAc)

Stress induces the release of the peptide corticotropin-releasing factor (CRF) into the ventral tegmental area (VTA), and also increases dopamine (DA) levels in brain regions receiving dense VTA input. Since the role of stress in drug addiction is well established, the present study examined the possible involvement of CRF1 receptor in the interaction between morphine withdrawal and catecholaminergic pathways in the reward system. The effects of naloxone-precipitated morphine withdrawal on signs of withdrawal, hypothalamo-pituitary-adrenocortical (HPA) axis activity, dopamine (DA) and noradrenaline (NA) turnover in the nucleus accumbens (NAc) and activation of VTA dopaminergic neurons, were investigated in rats pretreated with vehicle or CP-154,526 (selective CRF1R antagonist). CP-154,526 attenuated the increases in body weight loss and suppressed some of withdrawal signs. Pretreatment with CRF1 receptor antagonist resulted in no significant modification of the increased NA turnover at NAc or plasma corticosterone levels that were seen during morphine withdrawal. However, blockade of CRF1 receptor significantly reduced morphine withdrawal-induced increases in plasma adrenocorticotropin (ACTH) levels, DA turnover and TH phosphorylation at Ser40 in the NAc. In addition, CP-154,526 reduced the number of TH containing neurons expressing c-Fos in the VTA after naloxone-precipitated morphine withdrawal. Altogether, these results support the idea that VTA dopaminergic neurons are activated in response to naloxone-precipitated morphine withdrawal and suggest that CRF1 receptors are involved in the activation of dopaminergic pathways which project to NAc.     

Genes and Pathways Co-associated with the Exposure to Multiple Drugs of Abuse,Including Alcohol,Amphetamine/Methamphetamine,Cocaine, Marijuana,Morphine, and/or Nicotine: a Review of Proteomics Analyses

Drug addiction is a chronic neuronal disease. In recent years, proteomics technology has been widely used to assess the protein expression in the brain tissues of both animals and humans exposed to addictive drugs. Through this approach, a large number of proteins potentially involved in the etiology of drug addictions have been identified, which provide a valuable resource to study protein function, biochemical pathways, and networks related to the molecular mechanisms underlying drug dependence. In this article, we summarize the recent application of proteomics to profiling protein expression patterns in animal or human brain tissues after the administration of alcohol, amphetamine/methamphetamine, cocaine, marijuana, morphine/heroin/butorphanol, or nicotine. From available reports, we compiled a list of 497 proteins associated with exposure to one or more addictive drugs, with 160 being related to exposure to at least two abused drugs. A number of biochemical pathways and biological processes appear to be enriched among these proteins, including synaptic transmission and signaling pathways related to neuronal functions. The data included in this work provide a summary and extension of the proteomics studies on drug addiction. Furthermore, the proteins and biological processes highlighted here may provide valuable insight into the cellular activities and biological processes in neurons in the development of drug addiction.     

多巴胺对吗啡成瘾大鼠海马区痛觉调制的研究进展

当今社会日益增长的吗啡等阿片类药物的非法滥用已经严重威胁到人类的健康。然而,迄今为止尚没有找到能够较为有效的防治阿片成瘾的方法。目前研究已知,阿片成瘾的形成所涉及的脑区及核团包括中脑腹侧被盖区(VTA)、伏隔核(NAc)、海马等,其成瘾涉及的神经递质系统包括多巴胺、5-羟色胺等。本文将就多巴胺及海马在痛觉调制及药物成瘾过程中的作用进行综述,为吗啡的成瘾与戒断的进一研究及治疗提供线索。     

Regulation by chronic clonidine of adenylate cyclase and cyclic AMP-dependent protein kinase in the rat locus coeruleus

Clonidine and morphine are known to produce tolerance and dependence in rat locus coeruleus (LC) neurons after chronic administration based on electrophysiological criteria. Previous studies have shown that morphine tolerance and dependence is associated with increases in levels of adenylate cyclase, pertussis toxin-mediated ADP-ribosylation of G-proteins, and cyclic AMP-dependent protein kinase in this brain region. The present study was aimed at investigating whether clonidine tolerance and dependence is also associated with alterations in these intracellular messengers. It was found that, similar to chronic morphine, chronic (2 weeks) clonidine administration, under conditions that produce electrophysiological evidence of tolerance and dependence in LC neurons, increased levels of adenylate cyclase activity and cyclic AMP-dependent protein kinase activity in this brain region, but not in several other regions studied, which included the frontal cortex, neostriatum, and dorsal raphe. However, the changes induced by chronic clonidine in the LC, at maximal doses and duration of treatment, were only approximately 50% in magnitude of those observed in response to morphine. Unlike chronic morphine, chronic clonidine produced no change in G-protein ADP-ribosylation levels in the LC. Chronic administration of a number of other drugs, namely diazepam, chloral hydrate, and dextromethorphan, which produce electrophysiological actions distinct from those of clonidine and morphine in the LC, failed to alter adenylate cyclase and cyclic AMP-dependent protein kinase in this brain region. The results indicate that increased levels of adenylate cyclase and cyclic AMP-dependent protein kinase represent common adaptations by LC neurons to chronic clonidine and morphine, and raise the possibility that such changes contribute to the development of clonidine and morphine tolerance and dependence in these neurons.     

Influence of clonidine on the acute dependence response elicited in naive rats by naloxone

Clonidine has been used successfully in the treatment of opiate dependence. The discomforting effects of withdrawal are attenuated by the drug. The question of whether the more central process of dependence is affected by clonidine was tested in the present study. Change in plasma corticosterone was used as the indication of the stress of acute withdrawal from morphine. Conscious, unrestrained male rats showed a dose-related, though somewhat delayed, increase in plasma corticosterone after clonidine (0.01-0.1 mg/kg). The suggested mechanism for this effect involves presynaptic inhibition of noradrenergic neurons inhibiting CRF (corticotropin-releasing factor) release. Similar animals showed an elevation of plasma corticosterone after naloxone (0.4 mg/kg) was administered 3 hrs following a single morphine-priming (10 mg/kg). The naloxone-precipitated response was unaffected by clonidine (0.04 mg/kg). This dose of clonidine did not substitute for morphine-priming to produce the naloxone-precipitated response. The data suggests that clonidine elevated plasma corticosterone by an indirect mechanism. Further, the stress associated with acute withdrawal is unaffected by clonidine suggesting that the drug does not alter dependence development.     

Chronic morphine alters the presynaptic protein profile: identification of novel molecular targets using proteomics and network analysis

Opiates produce significant and persistent changes in synaptic transmission; knowledge of the proteins involved in these changes may help to understand the molecular mechanisms underlying opiate dependence. Using an integrated quantitative proteomics and systems biology approach, we explored changes in the presynaptic protein profile following a paradigm of chronic morphine administration that leads to the development of dependence. For this, we isolated presynaptic fractions from the striata of rats treated with saline or escalating doses of morphine, and analyzed the proteins in these fractions using differential isotopic labeling. We identified 30 proteins that were significantly altered by morphine and integrated them into a protein-protein interaction (PPI) network representing potential morphine-regulated protein complexes. Graph theory-based analysis of this network revealed clusters of densely connected and functionally related morphine-regulated clusters of proteins. One of the clusters contained molecular chaperones thought to be involved in regulation of neurotransmission. Within this cluster, cysteine-string protein (CSP) and the heat shock protein Hsc70 were downregulated by morphine. Interestingly, Hsp90, a heat shock protein that normally interacts with CSP and Hsc70, was upregulated by morphine. Moreover, treatment with the selective Hsp90 inhibitor, geldanamycin, decreased the somatic signs of naloxone-precipitated morphine withdrawal, suggesting that Hsp90 upregulation at the presynapse plays a role in the expression of morphine dependence. Thus, integration of proteomics, network analysis, and behavioral studies has provided a greater understanding of morphine-induced alterations in synaptic composition, and identified a potential novel therapeutic target for opiate dependence.     

The Use of Propoxyphene Napsylate in the Treatment of Heroin and Methadone Addiction

The use of methadone in the treatment of heroin addiction continues to be controversial. Propoxyphene napsylate (Darvon N®) is a possible alternative and a pilot study was conducted to test its acceptability, safety and clinical efficacy in treating long term, “multi-relapse” heroin addicts.Findings indicate that propoxyphene napsylate suppresses many of the symptoms associated with opioid withdrawal phenomena. It should be viewed as a very promising therapeutic tool to be used in conjunction with psychological counseling and socio-vocational rehabilitation in detoxification and maintenance therapy for heroin or methadone addiction.     

Intermittent administration of morphine alters protein expression in rat nucleus accumbens

Repeated exposure to drugs of abuse causes time-dependent neuroadaptive changes in the mesocorticolimbic system of the brain that are considered to underlie the expression of major behavioral characteristics of drug addiction. We used a 2-D gel-based proteomics approach to examine morphine-induced temporal changes in protein expression and/or PTM in the nucleus accumbens (NAc) of morphine-sensitized rats. Rats were pretreated with saline [1 mL/kg subcutaneously (s.c.)] or morphine (10 mg/kg, s.c.) once daily for 14 days and the animals were decapitated 1 day later. The NAc was extracted and proteins resolved by 2-DE. Several protein functional groups were found to be regulated in the morphine-treated group, representing cytoskeletal proteins, proteins involved in neurotransmission, enzymes involved in energy metabolism and protein degradation, and a protein that regulates translation.     

Hypothalamic orexin--a neurons are involved in the response of the brain stress system to morphine withdrawal

Both the hypothalamus-pituitary-adrenal (HPA) axis and the extrahypothalamic brain stress system are key elements of the neural circuitry that regulates the negative states during abstinence from chronic drug exposure. Orexins have recently been hypothesized to modulate the extended amygdala and to contribute to the negative emotional state associated with dependence. This study examined the impact of chronic morphine and withdrawal on the lateral hypothalamic (LH) orexin A (OXA) gene expression and activity as well as OXA involvement in the brain stress response to morphine abstinence. Male Wistar rats received chronic morphine followed by naloxone to precipitate withdrawal. The selective OX1R antagonist SB334867 was used to examine whether orexins' activity is related to somatic symptoms of opiate withdrawal and alterations in HPA axis and extended amygdala in rats dependent on morphine. OXA mRNA was induced in the hypothalamus during morphine withdrawal, which was accompanied by activation of OXA neurons in the LH. Importantly, SB334867 attenuated the somatic symptoms of withdrawal, and reduced morphine withdrawal-induced c-Fos expression in the nucleus accumbens (NAc) shell, bed nucleus of stria terminalis, central amygdala and hypothalamic paraventricular nucleus, but did not modify the HPA axis activity. These results highlight a critical role of OXA signalling, via OX1R, in activation of brain stress system to morphine withdrawal and suggest that all orexinergic subpopulations in the lateral hypothalamic area contribute in this response.     

Signaling pathway adaptations and novel protein kinase A substrates related to behavioral sensitization to cocaine

Behavioral sensitization is an animal model for aspects of cocaine addiction. Cocaine-sensitized rats exhibit increased AMPA receptor (AMPAR) surface expression in the nucleus accumbens (NAc) which may in turn enhance drug seeking. To identify signaling pathways contributing to AMPAR up-regulation, we measured AMPAR surface expression and signaling pathway activation in the NAc of cocaine-sensitized rats, cocaine-exposed rats that failed to sensitize and saline controls on withdrawal days (WD) 1, 7, and 21. We focused on calcium/calmodulin-dependent protein kinase II (CaMKII), extracellular signal-regulated protein kinase (ERK), and protein kinase A (PKA). In sensitized rats, AMPAR surface expression was elevated on WD7 and WD21 but not WD1. ERK2 activation followed a parallel time-course, suggesting a role in AMPAR up-regulation. Both sensitized and non-sensitized rats exhibited CaMKII activation on WD7, suggesting that CaMKII activation is not sufficient for AMPAR up-regulation. PKA phosphorylation, measured using an antibody recognizing phosphorylated PKA substrates, increased gradually over withdrawal in sensitized rats, from below control levels on WD1 to significantly greater than controls on WD21. Using proteomics, novel sensitization-related PKA substrates were identified, including two structural proteins (CRMP-2 and α-tubulin) that we speculate may link PKA signaling to previously reported dendritic remodeling in NAc neurons of cocaine-sensitized rats.     

Beneficial effects of co-treatment with dextromethorphan on prenatally methadone-exposed offspring

Background

Heroin use among young women of reproductive age has drawn much attention around the world. Although methadone is widely used in maintenance therapy for heroin/morphine addiction, the long-term effects of prenatal exposure to methadone and preventative therapy remain unclear. For revealing this question, female pregnant Sprague–Dawley rats were sub-grouped to receive (1) vehicle, (2) methadone 5 mg/kg at embryonic day 3 (E3) and then 7 mg/kg from E4 to E20, (3) dextromethorphan (DM) 3 mg/kg, and (4) methadone + DM (the rats received methadone followed by DM treatment), subcutaneously, twice a day from E3 to E20. The body weight, natural withdrawal, pain sensitivity, ED50, conditioned place preference and water maze were conducted at different postnatal stages (P1 to P79) of offspring. The quantitative real-time RT-PCR and electrophysiology were also used to measure the gene expression of opioid receptors in the spinal cord and changes of LTP/LTD in the hippocampus, separately.

Results

Prenatal exposure to methadone or DM did not affect survival rate, body weight, water maze and LTP or LTD of offspring. However, prenatal methadone significantly increased the withdrawal symptoms, pain sensitivity, addiction liability and decreased the mRNA expression of pain related opioid receptors. Co-administration of DM with methadone in the maternal rats effectively prevented these abnormalities of offspring induced by methadone.

Conclusions

Our study clearly showed that co-administration of dextromethorphan with methadone in the maternal rats prevented the adverse effects induced by prenatal methadone exposure. It implies that dextromethorphan may have a potential to be used in combination with methadone for maintenance treatment in pregnant heroin-addicted women to prevent the adverse effects induced by methadone on offspring.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0126-2) contains supplementary material, which is available to authorized users.     

Reduced dosing and liability in methadone maintenance treatment by targeting oestrogen signal for morphine addiction

Methadone maintenance treatment (MMT) is the major tapering therapy for morphine addictive patients. There have gender differences reported in response to MMT. This study discovered that the estrogen‐response element single nucleotide polymorphism (ERE‐SNP; rs16974799, C/T) of cytochrome 2B6 gene (cyp2b6; methadone catabolic enzyme) responded differently to MMT dosing. Oestradiol was associated with high MMT dosing, high enantiomer (R‐ or S‐) of 2‐ethylidene‐1,5‐dimethyl‐3,3‐dipheny‐pyrrolidine (EDDP; methadone metabolite) to methadone ratio and increased drug‐seeking behaviour, implicating oestradiol‐CYP‐EDDP/methadone axis decreasing MMT efficacy. In mouse model, oestrogen mitigates methadone antinociceptive response, facilitates methadone catabolism and up‐regulates methadone‐associated metabolizing enzymes. Oestrogen also ablates chronic methadone administration‐induced rewarding response. Mechanism dissection revealed the CC genotype of CYP2B6‐ERE‐SNP exerts higher ERE sequence alignment score, higher estrogenic response as compared to TT genotype. At last, preclinical study via targeting estrogen signal that tamoxifen (TMX; selective estrogen receptor modulator, SERM) could facilitate the tolerance phase rewarding response of methadone. Strikingly, TMX also reduces tapering/abstinence phases methadone liability in mice. In conclusion, this study demonstrates altering methadone metabolism through targeting estrogen signals might be able to free morphine addictive patients from the addiction of opioid replacement therapy. Therefore, the add‐on therapy clinical trial introducing SERM in MMT regimen is suggested.     

慢性吗啡依赖树鼩模型的建立

吗啡是一种有效的镇痛药,但易使动物产生耐受性和成瘾性。在该实验中,中缅树鼩(Tupai a belangeri chinensis)连续7d,每天接受三次肌肉注射递增剂量(5、10、15、20mg/kg体重)吗啡后对吗啡产生耐受和依赖;吗啡注射完成后,腹腔注射纳洛酮(1.25mg/kg体重)催瘾,可诱导其条件性位置厌恶(conditioned place aversion,CPA)及相应吗啡戒断症状的出现。该结果提示树鼩慢性吗啡依赖模型的建立可用于研究吗啡依赖和耐受的生物学机制,以及减轻戒断症状药物的筛选。     

慢性吗啡给予、戒断及再给药过程中大鼠海马感觉门控的动态变化

药物成瘾被认为是药物长期作用于脑而产生的一种慢性复吸性脑疾病,长期反复的药物（如吗啡）滥用会导致一系列严重后果,如药物依赖、药物耐受、强迫性药物寻求等。本实验利用条件化位置偏好（conditioned place preference,CPP）模型来检测大鼠对吗啡依赖和心理渴求等过程;采用双声刺激听觉诱发电位来研究大鼠在慢性吗啡给予、戒断以及再给药过程中海马感觉门控（N40）的动态变化。吗啡组大鼠注射吗啡（10mg／kg,i．p．）12d,经历第一次戒断12d,再次注射吗啡（2．5mg／kg,i．P．）1d,之后经历第二次戒断2d;对照组大鼠注射同体积生理盐水,其余实验条件与吗啡组相同。CPP实验表明,这种药物给予方法促使大鼠对吗啡产生药物依赖和心理渴求。双声刺激诱发电位实验表明,吗啡组大鼠在吗啡给予期间海马感觉门控受到损伤;第一次戒断期的第1～2天海马感觉门控能力减弱,第3天增强,第4～12天逐渐恢复到正常水平;再次给予吗啡后海马感觉门控能力与对照组相比显著降低,并且随后2d的戒断期内海马感觉门控能力也一直保持较低水平,表明再次给药使大鼠海马感觉门控对吗啡更加敏感化。结果提示,长期反复的吗啡给予及再给药干扰了海马的感觉门控能力,吗啡成瘾对大脑可能产生长期影响。     

Chronic morphine exposure and its abstinence alters dendritic spine morphology and upregulates Shank1

Exposure to chronic drugs of abuse has been reported to produce significant changes in postsynaptic protein profile, dendritic spine morphology and synaptic transmission. In the present study we demonstrate alterations in dendritic spine morphology in the frontal cortex and nucleus accumbens of mice following chronic morphine treatment as well as during abstinence for two months. Such alterations were accompanied with significant upregulation of the postsynaptic protein Shank1 in synaptosomal enriched fractions. mRNA levels of Shank1 was also markedly increased during morphine treatment and during withdrawal. Studies of the different postsynaptic proteins at the protein and mRNA levels showed significant alterations in the morphine treated groups compared to that of saline treated controls. Taken together, these observations suggest that Shank1 may have an important role in the regulation of spine morphology induced by chronic morphine leading to addiction.     

Expression changes of hippocampal energy metabolism enzymes contribute to behavioural abnormalities during chronic morphine treatment

Dependence and impairment of learning and memory are two well-established features caused by abused drugs such as opioids. The hippocampus is an important region associated with both drug dependence and learning and memory. However, the molecular events in hippocampus following exposure to abused drugs such as opioids are not well understood. Here we examined the effect of chronic morphine treatment on hippocampal protein expression by proteomic analyses. We found that chronic exposure of mice to morphine for 10 days produced robust morphine withdrawal jumping and memory impairment, and also resulted in a significant downregulation of hippocampal protein levels of three metabolic enzymes, including Fe-S protein 1 of NADH dehydrogenase, dihydrolipoamide acetyltransferase or E2 component of the pyruvate dehydrogenase complex and lactate dehydrogenase 2. Further real-time quantitative PCR analyses confirmed that the levels of the corresponding mRNAs were also remarkably reduced. Consistent with these findings, lower ATP levels and an impaired ability to convert glucose into ATP were also observed in the hippocampus of chronically treated mice. Opioid antagonist naltrexone administrated concomitantly with morphine significantly suppressed morphine withdrawal jumping and reversed the downregulation of these proteins. Acute exposure to morphine also produced robust morphine withdrawal jumping and significant memory impairment, but failed to decrease the expression of these three proteins. Intrahippocampal injection of D-glucose before morphine administration significantly enhanced ATP levels and suppressed morphine withdrawal jumping and memory impairment in acute morphine-treated but not in chronic morphine-treated mice. Intraperitoneal injection of high dose of D-glucose shows a similar effect on morphine-induced withdrawal jumping as the central treatment. Taken together, our results suggest that reduced expression of the three metabolic enzymes in the hippocampus as a result of chronic morphine treatment contributes to the development of drug-induced symptoms such as morphine withdrawal jumping and memory impairment.     

Regulation of G Protein-Coupled Receptor Kinase 2 in Brains of Opiate-Treated Rats and Human Opiate Addicts

Abstract: The effects of opiate drugs (heroin, morphine, and methadone) on the levels of G protein-coupled receptor kinase 2 (GRK2) were studied in rat and human brain frontal cortices. The density of brain GRK2 was measured by immunoblot assays in acute and chronic opiate-treated rats as well as in opiate-dependent rats after spontaneous or naloxone-precipitated withdrawal and in human opiate addicts who had died of an opiate overdose. In postmortem brains from human addicts, total GRK2 immunoreactivity was not changed significantly, but the level of the membrane-associated kinase was modestly but significantly increased (12%) compared with matched controls. In rats treated chronically with morphine or methadone modest increases of the enzyme levels (only significant after methadone) were observed. Acute treatments with morphine and methadone induced dose- and time-dependent increases (8–22%) in total GRK2 concentrations [higher increases were observed for the membrane-associated enzyme (46%)]. Spontaneous and naloxone-precipitated withdrawal after chronic morphine or methadone induced a marked up-regulation in the levels of total GRK2 in the rat frontal cortex (18–25%). These results suggest that GRK2 is involved in the short-term regulation of μ-opioid receptors in vivo and that the activity of this regulatory kinase in brain could have a relevant role in opiate tolerance, dependence, and withdrawal.