Effects of harman and harmine on naloxone-precipitated withdrawal syndrome in morphine-dependent rats

The effects of the beta-carbolines, harman and harmine, on naloxone-precipitated withdrawal syndrome in morphine-dependent rats were investigated. Two morphine pellets containing 75 mg morphine base were implanted subcutaneously in the scapular area of adult male Wistar rats (200-250 g) under light ether anesthesia. Rats were then assigned to several groups (n = 12 for each group). Seventy-two hours after morphine implantation, harman (5 and 10 mg/kg), harmine (5 and 10 mg/kg) or saline was injected to rats intraperitoneally (ip). After 45 min, a morphine withdrawal syndrome was precipitated by naloxone (2 mg/kg, ip), and morphine withdrawal signs were observed and evaluated for 15 min. Harmine (5 and 10 mg/kg) attenuated significantly the intensity of all signs of morphine withdrawal except for jumping. While jumping behaviour appearing in morphine withdrawal was intensified by harman (5 and 10 mg/kg) treatment, harmine administration did not produce any significant change in the intensity of this sign. Harman attenuated significantly the intensity of wet dog shakes, writhing, defecation, tremor and ptosis. However, it produced no significant changes in the intensity of teeth chattering and diarrhea. Our results suggest that harman and harmine, beta-carbolines, have some beneficial effects on naloxone-precipitated morphine withdrawal syndrome in rats. Findings from the present study also indicated that harmine was more effective than harman on morphine abstinence syndrome.     

脊髓水平iNOS在吗啡依赖大鼠戒断反应中的作用

目的:探讨脊髓水平诱导型一氧化氮合酶在吗啡依赖大鼠戒断反应中的作用。方法:健康雄性SD大鼠72只,体重200~250 g,吗啡剂量每次10 mg/kg,每日2次,隔日每次增加10 mg/kg,至第6天末次注射50 mg/kg,大鼠腹腔注射纳洛酮4 mg/kg建立吗啡依赖及戒断模型,在纳洛酮激发戒断前30 min鞘内注射iNOS特异性抑制剂氨基胍(AG)150μg。分为正常对照组、吗啡依赖组、吗啡戒断组、AG组。采用行为学(n=8)、免疫组织化学(n=6)和Western blot(n=4)方法观察鞘内应用iNOS特异性抑制剂氨基胍对吗啡依赖大鼠纳洛酮催促戒断反应和脊髓神经元iNOS表达的影响。结果:AG组戒断症状评分和戒断组促诱发痛评分均低于戒断组(P<0.05)。免疫组织化学和Western blot显示戒断组大鼠脊髓iNOS阳性神经元的数目和蛋白的表达增高,而AG组大鼠脊髓iNOS阳性神经元的数目和iNOS蛋白的表达低于戒断组(P<0.05)。结论:脊髓水平iNOS表达上调可能参与介导吗啡戒断反应。     

Circadian Rhythm of the Liver of Male Rats Dosed with Phenobarbital—I. Organ Weight,Cellular Structures,Glycogen Contents and Mitotic Activity

The circadian rhythm of the liver, namely organ weight, cellular structures (by light-microscopy), glycogen content (by periodic acid-Schiff (PAS) reaction) and mitotic activity, was studied in 166 male Sprague-Dawley rats orally treated daily at 0800-0900 with 70 (study 1) or 50 (study 2) mg/kg phenobarbital (PB) for 7days. Thereafter, eight (study 1) or five (study 2) rats each were studied at 4-hr intervals at 1000, 1400, 1800, 2200, 0200, 0600 and 1000 through till the following day. The lighting schedule in the colony was 12:12, light:dark (light from 0600 to 1800). The liver weight was raised in PB-treated rats at all times of the day compared to controls and showed a distinct circadian rhythm with a peak at 1000 and a minimum at 2200 in PB-treated rats and the controls. The circadian rhythm of cellular structures was closely related to the hepatic glycogen content which was in good agreement with the controls, but at 1400 and 1800 the glycogen particles were more distinctly diminished in the enlarged centrilobular hepatocytes of PB-treated rats. The mitotic activity of hepatocytes was markedly increased in rats treated with PB but showed the same circadian rhythm as controls with a peak at 1000.     

Circadian Rhythm of the Liver of Male Rats Dosed with Phenobarbital—I. Organ Weight, Cellular Structures, Glycogen Contents and Mitotic Activity

The circadian rhythm of the liver, namely organ weight, cellular structures (by light-microscopy), glycogen content (by periodic acid-Schiff (PAS) reaction) and mitotic activity, was studied in 166 male Sprague-Dawley rats orally treated daily at 0800-0900 with 70 (study 1) or 50 (study 2) mg/kg phenobarbital (PB) for 7days. Thereafter, eight (study 1) or five (study 2) rats each were studied at 4-hr intervals at 1000, 1400, 1800, 2200, 0200, 0600 and 1000 through till the following day. The lighting schedule in the colony was 12:12, light:dark (light from 0600 to 1800). The liver weight was raised in PB-treated rats at all times of the day compared to controls and showed a distinct circadian rhythm with a peak at 1000 and a minimum at 2200 in PB-treated rats and the controls. The circadian rhythm of cellular structures was closely related to the hepatic glycogen content which was in good agreement with the controls, but at 1400 and 1800 the glycogen particles were more distinctly diminished in the enlarged centrilobular hepatocytes of PB-treated rats. The mitotic activity of hepatocytes was markedly increased in rats treated with PB but showed the same circadian rhythm as controls with a peak at 1000.     

Circadian Rhythm of the Liver of Male Rats Pre-Treated with Phenobarbital—ii. Hexobarbital Sleeping Time and Lipids Content in Liver and Serum

The circadian rhythm of hexobarbital sleeping time and lipids content in liver and serum were studied in 226 male Sprague-Dawley rats pretreated daily at 0800-0900 with 70 mg/kg (study 1 or 3) or 50 mg/kg (study 2) phenobarbital (PB) orally for 7 days. Thereafter, eight (study 1) or five (study 2 and 3) rats each were studied at 4-hr intervals at 1000, 1400, 1800, 2200, 0200, 0600 and 1000 through the following day. The lighting schedule in the colony was 12:12 ± light:dark (light from 0600 to 1800). The hexobarbital sleeping times of PB-pretreated rats were generally shortened compared to the controls and no circadian rhythm was observed. PB-treatment increased slightly the liver content of cholesterol, and significantly that of triglycerides and phospholipids. Liver cholesterol and phospholipids showed circadian rhythms with peaks during the dark phase. No circadian rhythm of liver triglycerides existed. In serum, levels of triglycerides and phospholipids were slightly lowered by PB-treatment, while levels of cholesterol and beta-lipoprotein were not influenced. Serum values did not exhibit circadian rhythms.     

Effect of sex and time of sampling on selenium and glutathione peroxidase activity in tissues of mature rats

Sprague-Dawley rats were used to investigate variations in measures of glutathione peroxidase (GSH-Px) and selenium (Se) concentration resulting from diurnal cycles and sex. Mature rats (equal numbers of males and females) were killed at 4 h intervals over a 48 h period (0200, 0600, 1000, 1800 and 2200 h each day). Selenium and GSH-Px were measured in plasma, erythrocytes, and liver and kidney cytosols. Selenium concentrations did not vary diurnally, but plasma GSH-Px activities were higher during the light than dark periods. Males had greater plasma GSH-Px activities and Se concentrations (42 EU and .45 mg/kg, respectively) than females (35 EU and .41 mg/kg respectively). GSH-Px activities were also higher in male kidney cytosols than females (117 and 76 EU, respectively). Selenium and GSH-Px activities, however, were lower in male liver cytosols (.48 mg/kg and 272 EU) than females (1.19 mg/kg and 795 EU, respectively). These data suggest that Se is distributed differently in male and female rats and the difference in Se distribution is accomplished by differences in GSH-Px activities.     

Monoamine Metabolism in the Locus Coeruleus Measured Concurrently with Behavior During Opiate Withdrawal: An In Vivo Microdialysis Study in Freely Moving Rats

Abstract: Using microdialysis, changes in monoamine metabolism were monitored in the locus coeruleus of freely moving rats during opiate withdrawal concomitantly with behavioral symptoms. Rats were infused with morphine (2 mg/kg/h, s.c.) or saline for 5 days and challenged with naltrexone (100 mg/kg, s.c.) on day 6. Following naltrexone challenge, the classic behavioral symptoms of morphine withdrawal were observed in rats treated with morphine but not in saline-infused rats. In morphine-dependent rats, naltrexone induced a marked increase (280%) in dialysate concentrations of 3,4-dihydroxyphenylacetic acid, an index of the functional activity of the noradrenergic neurons in the locus coeruleus. The local concentrations of the serotonin metabolite 5-hydroxyindoleacetic acid were also increased (70%) during morphine withdrawal. Taken together, these results (a) confirm in unanesthetized rats the hypothesis of an activation by opiate withdrawal of noradrenergic neurons in the locus coeruleus and (b) suggest an increase in serotonergic transmission in the same nucleus during morphine withdrawal.     

Circadian Rhythm of Liver Parameters (Cellular Structures,Mitotic Activity,Glycogen and Lipids in Liver and Serum) During Three Consecutive Cycles in Phenobarbital-Treated Rats

The circadian rhythm of gastric content, serum alkaline phosphatase (alk.P.), serum lipids, body weight (wt), relative (rel.) liver wt, cellular structures (by light- and electron-microscopy), mitotic activity of hepatocytes, glycogen content, protein and lipids in liver was studied in 180 male Sprague-Dawley rats orally treated at 0830-1030 with 50 mg/kg phenobarbital (PB) for 7 days. Thereafter, five PB-treated males and five controls each were studied at 4-hr intervals at 0600, 1000, 1400, 1800, 2200 and 0200 on 3 consecutive days. The lighting schedule in the colony was 12:12 = light/dark (light from 0600 to 1800). Following the rhythm of gastric emptying, the rel. liver wt showed a clear circadian rhythm with a peak at 0800. The rel. liver wt was raised in PB-treated rats at all times of the day. The circadian rhythm of cellular structures was closely related to the hepatic glycogen content which exhibited a clear rhythm with the peak also at 0800, but lowered values were found in PB-treated rats. The mitotic activity of hepatocytes was significantly increased in PB-treated rats but displayed the same circadian rhythm as controls with peaks at noon and troughs at midnight. The well-known hypertrophy of the smooth endoplasmic reticulum in PB-treated rats was not found at 0600, but was fully developed at 1400 and 2200. PB-treatment increased significantly the liver content of cholesterol, triglycerides and phospholipids. Liver cholesterol showed a clear circadian rhythm with peaks at 1800. No rhythm of liver protein, triglycerides and phospholipids was observed. In serum, levels of cholesterol were significantly elevated, those of triglycerides and alk.P. significantly lowered, while those of phospholipids were not affected by the treatment. The three serum lipids, alk.P. and beta-lipoprotein exhibited a clear circadian rhythm, while serum glucose and non-esterified fatty acids did not.     

Circadian Rhythm of Liver Parameters (Cellular Structures, Mitotic Activity, Glycogen and Lipids in Liver and Serum) During Three Consecutive Cycles in Phenobarbital-Treated Rats

The circadian rhythm of gastric content, serum alkaline phosphatase (alk.P.), serum lipids, body weight (wt), relative (rel.) liver wt, cellular structures (by light- and electron-microscopy), mitotic activity of hepatocytes, glycogen content, protein and lipids in liver was studied in 180 male Sprague-Dawley rats orally treated at 0830-1030 with 50 mg/kg phenobarbital (PB) for 7 days. Thereafter, five PB-treated males and five controls each were studied at 4-hr intervals at 0600, 1000, 1400, 1800, 2200 and 0200 on 3 consecutive days. The lighting schedule in the colony was 12:12 = light/dark (light from 0600 to 1800). Following the rhythm of gastric emptying, the rel. liver wt showed a clear circadian rhythm with a peak at 0800. The rel. liver wt was raised in PB-treated rats at all times of the day. The circadian rhythm of cellular structures was closely related to the hepatic glycogen content which exhibited a clear rhythm with the peak also at 0800, but lowered values were found in PB-treated rats. The mitotic activity of hepatocytes was significantly increased in PB-treated rats but displayed the same circadian rhythm as controls with peaks at noon and troughs at midnight. The well-known hypertrophy of the smooth endoplasmic reticulum in PB-treated rats was not found at 0600, but was fully developed at 1400 and 2200. PB-treatment increased significantly the liver content of cholesterol, triglycerides and phospholipids. Liver cholesterol showed a clear circadian rhythm with peaks at 1800. No rhythm of liver protein, triglycerides and phospholipids was observed. In serum, levels of cholesterol were significantly elevated, those of triglycerides and alk.P. significantly lowered, while those of phospholipids were not affected by the treatment. The three serum lipids, alk.P. and beta-lipoprotein exhibited a clear circadian rhythm, while serum glucose and non-esterified fatty acids did not.     

脊髓水平细胞外信号调节激酶激活参与吗啡依赖的形成及戒断反应的表达

在大鼠吗啡依赖和戒断模型上,采用行为学、免疫组织化学和Western blot方法观察吗啡依赖及戒断大鼠脊髓神经元磷酸化细胞外信号调节激酶（phospho-extracellular signal-regulated kinase,pERK）表达的变化,及鞘内注射促分裂原活化蛋白激酶激酶（mitogen-activated protein kinase kinase,MEK）抑制剂U0126或ERK反义寡核苷酸对吗啡依赖大鼠纳洛酮催促戒断反应、触诱发痛及脊髓神经元pERK表达的影响,探讨脊髓水平pERK在介导吗啡依赖和戒断过程中的作用。结果显示：（1）在吗啡依赖形成过程中,大鼠脊髓胞浆与胞核非磷酸化ERK表达没有改变,但pERK表达逐渐增加,纳洛酮催促戒断后,仍有进一步增加的趋势,戒断1h后,其表达量明显下降,但仍高于对照组。（2）鞘内预先注射MEK抑制剂U0126或ERK反义寡核苷酸能明显抑制吗啡戒断反应和戒断引起的痛觉异常;与行为学结果一致,脊髓背角pERK阳性神经元表达与脊髓胞浆和胞核pERK表达也明显降低。上述结果提示,脊髓水平ERK激活和核转位参与吗啡依赖的形成及戒断反应的表达。     

Dorsal noradrenergic bundle lesions fail to alter opiate withdrawal or suppression of opiate withdrawal by clonidine

Previous work has shown that clonidine effectively supresses many of the signs of opiate withdrawal. The present study was designed to test the hypothesis that the supression of opiate withdrawal by clonidine is mediated by forebrain noradrenergic projections of the locus coeruleus. Two groups of 24 rats each were subjected to either a 6-hydroxydopamine lesion of the dorsal noradrenergic bundle (Lesion group) or a sham, vehicle injection (Sham group). All rats were made dependent on morphine by subcutaneous implantation of one 75 mg silastic morphine pellet for three days followed by 3 more days with two additional 75 mg pellets. Following removal of the morphine pellet, withdrawal was precipitated in all rats by subcutaneous injection of 4 mg/kg of naloxone. Pretreatment 10 min. before withdrawal with clonidine (0.1 or 0.2 mg/kg) produced a significant attenuation of withdrawal signs as compared to saline injected rats; this effect was equally significant in both sham and lesion groups. Lesions of the locus coeruleus had no effect on withdrawal, nor did they affect the ameliorating action of clonidine. These results substantiate the observation that clonidine can effectively attenuate signs of opiate withdrawal in the rat, but fail to support the hypothesis that these effects are mediated by the forebrain projections of the locus coeruleus.     

慢性吗啡处理及戒断后大鼠杏仁核中Parvalbumin的表达变化

目的:观察慢性吗啡处理及戒断后大鼠杏仁核中Parvalbumin(PV)的表达变化,为其功能的研究提供形态学依据。方法:将30只健康雄性SD大鼠随机分为吗啡依赖组和生理盐水对照组。吗啡依赖组大鼠腹膜腔注射吗啡,2次/d,起始剂量为5 mg/kg,逐日递增5mg,至第10d为50mg/kg;对照组注射同体积的生理盐水。于末次注射后动物分别存活3h、3 d和14d。用免疫组化方法和相对平均灰度值检测杏仁核内PV的表达。结果:在生理盐水处理组各存活时间点,杏仁核内PV的表达相同。和生理盐水对照组相比,3h时杏仁核内PV的表达明显增加(P<0.05)。第3d时,杏仁核内PV的表达减少,明显低于第3 h组(P<0.05)。至第14d时,PV的表达又开始增加,明显高于第3 d组(P<0.05)。结论:本结果提示慢性吗啡处理及戒断后杏仁核PV的表达具有时相特异性;这种变化在戒断早期可能主要与躯体依赖相关,而戒断晚期主要与精神依赖相关。     

Effects of morphine dependence,withdrawal and tolerance on prolactin and growth hormone secretion in the rat

The effects of morphine dependence and withdrawal on prolactin (Prl) and growth hormone (GH) secretion were examined in the rat. Morphine dependence, induced by morphine pellet implantation, had no effect on nonstress concentrations of plasma Prl or GH, but it potentiated the response of Prl secretion to the stress associated with blood collection + injection of saline. Naloxone-induced withdrawal had no demonstrable effect on the changes in Prl and GH secretion produced by stress. In addition, signs of tolerance to both the Prl- and GH-stimulating effects of morphine injection were observed in morphine-dependent rats.     

Orphanin FQ/nociceptin inhibits morphine withdrawal

The influence of orphanin FQ/nociceptin (OFQ/N) on the morphine-withdrawal symptom was investigated. Withdrawal syndrome was induced in the morphine-dependent rats by an intraperitoneal (i.p.) injection of 2 mg/kg naloxone hydrochloride--an opioid receptors antagonist. Wet-dog shakes were used as a measure of the abstinence syndrome. Intraventricular injections of OFQ/N (5-20 microg/animal) caused significant inhibition of the withdrawal signs at doses between 15-20 microg, in the morphine-dependent rats. OFQ/N alone did not change behavior of the morphine-dependent animals. The obtained results indicate that OFQ/N can inhibit the morphine withdrawal symptoms induced by naloxone.     

Behavioral effects of MK-801 in morphine-dependent and non-dependent mice

The behavioral effects of MK-801 were compared in morphine-dependent and non-dependent mice. The dose of MK-801 selected for these studies was previously demonstrated to attenuate some of the morphine withdrawal signs. Subjects were repeatedly exposed to morphine (8 days, b.i.d., 10–100 mg/kg, s.c.). Twenty-four hours after last morphine injection mice received naloxone (0.1 mg/kg, s.c.) and the observation was commenced. Animals were pretreated with either MK-801 (0.1 mg/kg, i.p.) or saline 30 min prior to testing. It was found that the behavioral effects of MK-801 (decreased sociability and increased rate of transitions between behavioral elements, locomotion, grooming) were less pronounced in morphine-dependent compared to non-dependent subjects. However, the intensified almost stereotypic eating possibly reflected increased psychotomimetic potency of MK-801 in morphine-withdrawn animals.     

鞘内注射M受体和GDNF反义寡脱氧核苷酸抑制吗啡戒断大鼠蓝斑c—Fos表达

The antisense approach and immunohistochemistry were used to study the effects of different muscarinic receptor (M) subtypes and glial cell derived neurotrophic factor (GDNF) on the scores of morphine-withdrawal syndrome and the expression of c-Fos in locus coeruleus (LC). Intrathecal injection of M2 receptor antisense oligonucleotides (M2AS-oligo) or GDNF antisense oligonucleotides (GDNFAS-oligo) decreased the scores of morphine withdrawal syndrome. The expression of c-Fos positive neurons in the LC increased in morphine-dependent rats and increased to a greater extent after the injection of naloxone (4mg/kg, ip) in morphine dependent rats. Intrathecal injection of M2AS-oligo or GDNFAS-oligo inhibited the increase of c-Fos expression in LC during morphine withdrawal, but there was no effect in case of M1AS-oligo. The results suggest that M2 receptor of spinal cord mediates the neural activation of LC during morphine withdrawal. And the interaction between neurons and glial cells may be involved in the ascending activation process.     

鞘内注射M受体和GDNF反义寡脱氧核苷酸抑制吗啡戒断大鼠蓝斑c-Fos表达

应用免疫组化方法观察鞘内注射毒蕈碱型乙酰胆碱(muscarinic acetylcholine receptor,M) 受体和胶质细胞源性神经营养因子(glial cell derived neurotrophic factor,GDNF)反义寡脱氧核苷酸对吗啡戒断大鼠蓝斑(locus coeruleus,LC)区内Fos表达的影响。结果显示,鞘内注射M_2受体和GDNF反义寡脱氧核苷酸明显减少大鼠吗啡戒断症状评分值(n=6,P<0.05)。正常大鼠LC区神经元Fos基础表达较低,吗啡依赖大鼠LC区神经元Fos表达增加,吗啡依赖大鼠纳酪酮(4mg/Kg,ip)催促戒断后,Fos表达进一步增加;鞘内注射M_2受体和GDNF反义寡脱氧核苷酸处理后均减少吗啡戒断大鼠LC区神经元Fos表达(n=5,P<0.05)。而鞘内注射M_1受体反义寡脱氧核苷酸处理组LC 区神经元Fos表达较吗啡戒断组没有显著差异(n=5,P>O.05)。结果提示:脊髓M_2受体调节吗啡戒断时LC区的神经元激活,而这种神经上行性激活涉及神经元与胶质细胞之间的适应性调节。     

脊髓水平nNOS和iNOS在吗啡戒断反应中的作用差异

目的:观察鞘内注射选择性一氧化氮合酶(nNOS)和诱导型一氧化氮合酶(iNOS)抑制剂对吗啡依赖大鼠纳洛酮催促戒断反应、脊髓Fos蛋白表达和脊髓神经元nNOS和iNOS表达的影响,以探讨nNOS和iNOS在吗啡依赖和戒断反应中的作用。方法:在大鼠吗啡依赖和戒断模型上,采用行为学、免疫组织化学和Western blot方法观察鞘内应用nNOS抑制剂7-硝基吲哚(7-Ni)和iNOS抑制剂氨基胍(AG)对吗啡依赖大鼠纳洛酮催促戒断反应、脊髓Fos蛋白表达和脊髓神经元nNOS和iNOS表达的影响。结果:①鞘内注射7-Ni、AG可明显减轻吗啡依赖大鼠戒断症状,戒断组戒断症状评分为28.6±4.89,7-Ni组为16.2±3.99(P<0.01),AG组为22.94±4.0(P<0.05);戒断组TEA评分为13.5±2.55,7-Ni、AG组分别为7.5±2.56、10.5±2.71(P<0.05);②鞘内注射7-Ni、AG可减少脊髓背角Fos阳性神经元的数目,7-Ni、AG组为228.2±49.5、296.8±50.6,低于戒断组(380±71,P<0.05);③7-Ni、AG组nNOS和iNOS阳性神经元的数目分别为169±32、10.2±2.85,均低于戒断组(239±45,16.8±5.1,P<0.05),两给药组脊髓NOS蛋白的表达也显著减少。结论:nNOS和iNOS抑制剂能减轻吗啡依赖及戒断大鼠的戒断症状和在脊髓水平抑制nNOS和iNOS的表达,nNOS起主要作用而iNOS可能起辅助作用。     

Effect of withdrawal of diazepam or morphine treatment on gastric motility (charcoal meal test) in mice: possible role of different central and peripheral receptors

Increased gastrointestinal motility in mice as one of the withdrawal symptoms of commonly abused drugs like diazepam or morphine and its possible mechanism of action was studied. Male Laka mice (20-25 g) were made addict to either diazepam (20 mg/kg, ip for 7 days) or morphine (10 mg/kg, sc for 9 days). Withdrawal symptoms were noted 24 hr after the last injection of diazepam or morphine. The animals were injected with Ro 15-1788 (flumazenil) (1 mg/kg, ip) or naloxone (2 mg/kg, ip) in the respective group to precipitate the withdrawal symptoms. Gastrointestinal motility was assessed by charcoal-meal test. Animals developed tolerance to acute sedative effect of diazepam, and similarly to the acute nociceptive action of morphine. On abrupt cessation of these drugs after chronic treatment the animals showed hyperlocomotion and hyperreactivity in diazepam withdrawal group and hyperalgesia on hot plate in morphine withdrawal groups, respectively. Increase in gastrointestinal motility was observed in all the drug withdrawal groups. Treatment with respective antagonists, Ro 15-1788 (flumazenil) and naloxone precipitated the withdrawal symptoms. The results suggest the involvement of both central and peripheral receptors of benzodiazepines and opioid (mu) receptors in the withdrawal symptoms of the benzodiazepines and morphine, respectively.