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.     

The extended amygdala system and self-stimulation of the lateral hypothalamus in rats: modulation with opiates and opioids

Wistar male rats were implanted with bipolar electrodes in the lateral hypothalamus to study self-stimulation reaction in the Skinner box. Simultaneously, the microcanules were implanted into the central nucleus of the amygdala to inject the drugs studied (1 microl in volume for each injection). The blockade of CRF receptors (astressin 1 microg) or sodium influx ionic currents (xycaine, or lidocain 1 microg) by means of intrastructural administration of drugs into the amygdala descreased self-stimulation reaction of the lateral hypothalamus in rats by 29-55%. The inhibition of D2 and D2 dopamine receptors in the amygdala with SCH23390 (1 microg) or sulpiride (1 microg), respectively. reduced self-stimulation too, but in less degree. On the background of blockade of CRF (astressin) and dopamine (sulpiride) receptors, as well as sodium influx ionic currents (lidocain) in the amygdala neurons, psychomotor stimulant amphetamine (1 mg/kg) and barbiturate sodium ethaminal (5 mg/kg) supported their psychoactivating effect on self-stimulation (+30-37%), but fentanyl (0.1 mg/kg) had got no effect. Fentanyl activated self-stimulation moderately only after blockade D1 dopamine receptors with SCH23390. After blockade of CRF receptors, leu-enkephaline strengthened its depressant effect on self-stimulation reaction (-89%). Therefore, if the modulating influence of the amygdala on the hypothalamus is diminished, the reinforcing effects of opiated (fentanyl) and opioids (leuencephaline) will block, but there will be no effect for psychomotor stimulant amphetamine and barbiturate sodium ethaminal.     

The effect of pentobarbital on the antinociceptive action of morphine in morphine-tolerant and non-tolerant rats

The interaction of sodium pentobarbital with morphine sulfate in both morphine-tolerant and non-tolerant rats was investigated using the tail-compression test for analgesia. Male Sprague-Dawley rats (300–350 g) were given pentobarbital (4, 8, or 16 mg/kg) 5 min before morphine (2, 4, 6, or 8 mg/kg). Control animals received two saline injections, or pentobarbital plus saline, or saline plus morphine. All injections were subcutaneous. Prior to the first injection, a baseline nociceptive threshold was determined for each rat by applying a modified micrometer to its tail and increasing the pressure until a squeak was elicited. Test readings were taken every half-hour for 2 hr beginning 30 min after the second injection. For the chronic studies, animals were first made tolerant to morphine by the administration of the narcotic twice a day for 3 days, increasing the dose from 10 to 50 mg/kg/injection. Identical testing procedures were then followed with these rats except that the test dose of morphine given on day 4 was in the range 8–128 mg/kg. It was found that Na pentobarbital, in the subanesthetic doses used, had neither antinociceptive nor hyperalgesic properties. Furthermore, the barbiturate had no effect on the antinociceptive action of morphine in either morphine-tolerant or non-tolerant rats.     

Attenuation of morphine dependence and withdrawal in rats by venlafaxine, a serotonin and noradrenaline reuptake inhibitor.

The effects of venlafaxine, a novel serotonin and adrenaline reuptake inhibitor, on the morphine withdrawal and activation of morphine conditioned place preference (CPP), were investigated in rats. Our results showed that the most morphine withdrawal signs, including jumping, writhing, shakes, exploring, lacrimation, piloerection, irritability, and diarrhea, were attenuated by pretreatment with 10 or 20 mg/kg venlafaxine. To investigate the effects of venlafaxine on relapse to opiate dependence, the morphine CPP was used and a dopamine D2 antagonist sulpiride was selected as a control drug. The morphine CPP disappeared following a 28-day drug-free period and appeared again after given a single injection of 1 mg/kg morphine. Acute treatment with sulpiride (25 or 50 mg/kg, i.p.) 30 min prior to 1 mg/kg morphine injection significantly blocked the reacquisition of CPP, while venlafaxine (10 or 20 mg/kg, i.p.) did not show significant effect. However, chronic treatment with venlafaxine (5 or 10 mg/kg, i.p. twice, daily, for seven consecutive days) significantly attenuated the reacquisition of morphine CPP, whereas chronic treatment with sulpiride (10 or 20 mg/kg, i.p.) have no significant effect. Our results demonstrated for the first time that venlafaxine strongly attenuates morphine withdrawal and morphine-induced reaquisition of     

Effect of methamphetamine on the development of acute morphine tolerance and dependence in mice

The effect of methamphetamine on morphine analgesia (tail-flick assay) was studied in non-tolerant mice and in mice made acutely tolerant to morphine following a single injection of 100 mg/kg morphine. The analgesic potency of morphine was increased in non-tolerant and tolerant mice to the same extent by 3.2 mg/kg methamphetamine (3.3 and 4.4 fold increases, respectively). In contrast, the ED50's for morphine analgesia and naloxone-precipitated jumping in mice pretreated with either 100 mg/kg morphine or both morphine and 3.2 mg/kg methamphetamine were not significantly different, indicating that methamphetamine had no effect on the development of acute morphine tolerance and dependence. Although methamphetamine had no effect on the development of acute tolerance to morphine, 4-day pretreatment with methamphetamine produced cross-tolerance to morphine analgesia. However, cross-tolerance to morphine was not accompanied by enchanced sensitivity to naloxone.     

Conditioned-reflex activation of electrical self-stimulation of the brain: a model of a situational craving for narcotics]

Rats with electrodes implanted into lateral hypothalamus were trained to press a lever to obtain electrical stimulation of the brain. After elaboration of self-stimulation (SS) conditioning of morphine-induced activation of SS-response was carried out. Five pairings were performed of morphine (3 mg/kg, i.p.) administration and SS in the box with distinct environmental cues (brightness, color, floor texture, background noise). After morphine withdrawal activation of SS was reproduced after simple placing of the animal in "conditioned" chamber. This effect was naloxone-sensitive. The observed effect is considered to be the adequate model of addictive substances craving.     

Stimulation of either cholecystokinin receptor subtype reduces while antagonists potentiate or sensitize a morphine-induced excitatory response.

Cholecystokinin peptides (CCK) have been shown to antagonize many opioid-mediated effects. The present study was undertaken to determine whether peripheral injections of cholecystokinin sulphated octapeptide (CCK8), cholecystokinin tetrapeptide (CCK4), the CCK(1) (lorglumide) and the CCK(2) (PD-135,158 and LY-225910) receptor antagonists can influence a classic morphine excitatory effect, i.e. the display of Straub tail reaction in mice (STR). A total of 570 female Balb/C mice were tested. Experiment 1 was undertaken to determine whether i.p. injections of CCK8 or CCK4 can influence STR. Each animal was treated with i.p. injections of saline or CCK8 (10 and 20 nmol/kg) or CCK4 (20 and 40 nmol/kg). After 30 min all animals received an i.p. injection of morphine hydrochloride (10.0 mg/kg). The highest doses of both CCK8 (35% STR) and CCK4 (40% STR) significantly reduced STR as compared to saline (85% STR) treated mice (Fisher test; P < 0.01). In experiment 2 each animal was treated with ip injections of saline or 1.0 mg/kg lorglumide or PD-135,158 fifteen minutes before an injection of morphine at doses ranging from 1.0 to 50.0 mg/kg. In experiment 3 animals were treated with injections of saline, 0.1 or 10.0 mg/kg lorglumide or LY-225910 before an injection of a fixed MC dose (2.0 mg/kg). Both lorglumide and PD-135,158 induced a significant shift to the left in the morphine dose-response curves as well as a significant decrease in ED50 of the STR. ED50 for lorglumide was significantly lower than ED50 for PD-135,158. Both doses of lorglumide and the highest dose of LY-225910 significantly increased the percent of animals displaying STR. Experiment 4 was undertaken to determine whether repeated peripheral injections of morphine or the morphine-potentiating agents CCK(1) (lorglumide) and the CCK(2) (LY-225910) receptor antagonists can induce morphine sensitization. Each animal was treated with 5 daily i.p. injections of saline (control group), 1.5 mg/Kg morphine hydrochloride (group morphine), and 1.0 mg/Kg lorglumide (group LOR) or LY-225910 (group LY). One, two, three and four weeks after the last treatment day, all animals were challenged with one i.p. injection of morphine (1.5 mg/Kg). The morphine, LOR groups and group LY showed a significant increase in percentage of animals displaying STR. These data demonstrate that the blockade of endogenous CCK actions leads to morphine sensitization probably through both CCK receptors. The present data are consistent with the antagonistic effects of CCK and opioids in the control of morphine-induced STR. In addition, these results suggest that both CCK receptors are involved in the modulatory effects of CCK on this morphine effect.     

Role of the GABA-ergic systems of the brain in manifesting the activating effect of diazepam]

It was shown in experiments on rats that the selective blocker of GABA receptors bicuculline (2 mg/kg) does not decrease the activating effect of diazepam as to the reaction of self-stimulation. The GABA-mimetic muscimol (0.5 and 1 mg/kg) had no effect on self-stimulation rate, while in the dose of 2 mg/kg causing behavioral changes produced a powerful decrease in it (by 93.3%). During the combined administration of diazepam and muscimol (1 mg/kg and 0.5 mg/kg, respectively) no potentiation of diazepam effect was observed. It is suggested that diazepam-induced facilitation of the reaction of self-stimulation is not due to the alteration in the activity of GABA-ergic processes.     

A comparison of the self-stimulation reaction and of conditioned place preference with fenamine administration in rats]

The reinforcing properties of different doses of amphetamine (1 and 5 mg/kg) were examined using two variants of self-stimulation reaction (in the Skinner box and locomotor self-stimulation in a shuttle box) and place preference test. Amphetamine in dose of 1 mg/kg increased the frequency of self-stimulation in the Skinner box and prolonged the time of rat staying in active zone of a shuttle box to a greater degree than 5 mg/kg of the drug. On the contrary, the aversive phase of self-stimulation, determined by a coefficient of "disagreement", grew higher after 5 mg/kg amphetamine than following 1 mg/kg. The study of effects by place preference test revealed the other regularity: the most positive reinforcing properties possessed the drug in a dose of 5 mg/kg. Thus, there are dissociation between the two doses of amphetamine (1 and 5 mg/kg) in their action on different physiological conditioned responses. The mechanisms of this dissociation are discussed.     

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。     

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。     

Nω-硝基-L-精氨酸甲酯抑制吗啡镇痛耐受大鼠中脑和脊髓一氧化氮合酶/N-甲基-D-天冬氨酸受体表达上调

采用热甩尾测痛法观察全身应用非特异性一氧化氮合酶（nitric oxide synthase,NOS）抑制剂——N^ω-硝基-L-精氨酸甲酯（L-NAME）对吗啡镇痛耐受形成的影响,并通过观察脊髓和中脑神经元型NOS（nNOS）和N-甲基-D-天冬氨酸（NMDA）受体亚单位表达的变化来阐释NO／NMDA受体在吗啡镇痛耐受形成中的作用。将36只健康成年Sprague-Dawley大鼠平均分为6组（每组6只）：1组为对照组,皮下注射生理盐水1ml;2、3、4、5和6组为处理组,分别皮下注射L-NAME10mg／kg、L-NAME20mg／kg、吗啡10mg／kg、L-NAME10mg／kg＋吗啡10mg／kg、L-NAME20mg／kg＋吗啡10mg／kg,每天2次。在注射前测量大鼠的热甩尾潜伏期（tail-flick latency,TFL）基础值,随后每天第一次给药50min后测量其TFL。第8天最后一次给药80min后（除2组和5组之外）断头取脊髓和中脑,采用RT-PCR技术测量nNOS以及NMDA受体1A（NR1A）和2A（NR2A）亚单位的表达。结果显示,2组大鼠第1天至第7天的TFL与基础值相比无显著差异;3组第7天时的TFL仍显著高于基础值;4组的TFL在第1天时最高,第2至第6天期间逐渐降低,第6天时与基础值相比无显著差异：5组的TFL在实验过程中呈下降趋势,虽然第7天时较第1天有所降低,但是仍然显著高于基础值;6组的TFL变化趋势与5组相同。PT—PCR分析结果显示,与1组相比,3组脊髓和中脑的nNOS mRNA表达显著降低,但NR1A mRNA和NR2A mRNA表达无显著改变;4组的nNOS mRNA、NR1A mRNA和NR2A mRNA表达均显著高于1组。与4组相比,6组的nNOS mRNA、NR1A mRNA和NR2A mRNA表达均显著降低。结果提示,吗啡镇痛耐受大鼠脊髓和中脑的nNOS和NMDA受体表达增加,联合应用L—NAME可抑制长期应用吗啡所致的nNOS表达增加和NMDA受体上调,延缓吗啡镇痛耐受的形成。本研究结果提示,脊髓和中脑的NO／NMDA受体与吗啡镇痛耐受形成密切相关。     

Long-term enhancement of morphine hypalgesia as a result of periodic blockade of opiate receptors using naloxone

A significant enhancement of the analgetic effect of morphine (6 mg/kg, subcutaneously; tail withdrawal reflex at 60 degrees C) was observed in rats 3-4 hours after single naloxone (1 mg/kg) administration. Periodical naloxone injection (0.5 mg/kg, subcutaneously, 3 times per day at 3.5-hour intervals for 3 days) led to a prominent and long-term (testing on the 20th and 105th hour after the last naloxone administration) enhancement of morphine analgesia (2.6 mg/kg subcutaneously) and insignificant inhibition of stress analgesia during two-hour immobilization of animals. These modifications of morphine and stress analgetic effects are considered a result of adaptive changes of opiate receptors after their blockade.     

脊髓蛋白激酶Cα和γ亚型在吗啡依赖和戒断反应中的不同作用

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方法观察鞘内应用蛋白激酶C(protien kinase C,PKC)抑制剂chelerythrine chloride(CHE)对吗啡依赖大鼠纳洛酮催促成断反应、脊髓Fos蛋白表达和脊髓神经元胞膜和胞浆PKCα、γ表达的影响,以探讨不同亚型PKC在吗啡依赖和戒断反应中的作用。结果表明,鞘内注射CHE能明显减轻吗啡成断症状的评分和吗啡戒断引起的痛觉异常,抑制吗啡成断期间脊髓Fos蛋白表达的增加;吗啡依赖可引起脊髓神经元PKCα和γ表达的上调和转位：吗啡戒断期间存在明显的且可被鞘内注射CHE抑制的PKCα转位,但未观察到明显的PKCγ转位。上述结果表明,脊髓PKC表达上调和转何可能参与吗啡依赖的形成和戒断反应的表达,且PKCα和γ亚型在吗啡依赖和戒断反应中的作用存在差异。     

The effect of alpha- and beta-adrenergic antagonists of the self-stimulation phenomenon

In an attempt to evaluate the possible existence of alpha- and/or beta- adrenergic components of the self-stimulation reward system, rats were injected (i.p.) with chlorpromazine hydrochloride (2.5 mg/kg), phentolamine hydrochloride (5 mg/kg), and propranolol hydrochloride (10 mg/kg). The alpha- adrenergic antagonists (chlorpromazine and pehntolamine) inhibited self-stimulation but the beta-adrenergic blocker (propranolol) was without significant effect. Self-stimulation is apparently mediated by the alpha-adrenergic system.     

Effects of acute and chronic morphine and narcotic antagonists on brain energy metabolism

Morphine sulphate (4 mg/kg to 32 mg/kg) produced a dose-dependent decrease in brain malate as antinociception increased. Decreased brain malate persisted 72 hours after implantation of morphine pellets by which time mice had become tolerant to antinociception. This finding suggests that malate decrease, unlike changes of other metabolites in other studies, might not be simply a result of general metabolic changes. Malate change as well as antinociception was prevented by prior injection of naloxone (3.0 mg/kg) or naltrexone (0.6 mg/kg) in acute experiments. Malate decrease in pelleted mice was no longer present if withdrawal was produced by naloxone or naltrexone in mice implanted with morphine pellets for 72 hours. Brain P-creatine was elevated in all mice implanted with morphine pellets even after withdrawal, thus, apparently, representing a more generalized effect than malate change.     

Naloxone reversal of morphine elicited hyperactivity

When naloxone is administered during morphine elicited hyperactivity, hyperactivity is reversed and hypoactivity occurs in its place. The present experiment tested the hypothesis that this effect is the result of morphine induced supersensitivity to naloxone. Two groups of hamsters received equivalent pretreatment with 15 mg/kg morphine (Groups M/M and M/S) for three days while a third group received saline (Group S/S). During subsequent testing one group received a morphine injection (Group M/M) while the others received saline (Groups M/S and S/S) before being placed in running wheels for a three hour session. Two hours later half the animals in each group received an injection of 0.4 mg/kg naloxone and half received saline. Naloxone produced hypoactivity in animals running under the influence of morphine (Group M/M), but neither in those with an equivalent history of morphine pre-treatment (Group M/S), nor in saline controls (Group S/S). These results are inconsistent with the hypothesis under test, but congruent with a modified dual-action hypothesis.     

Pupillary effects of leucine and methionine enkephalin in rats after intraperitoneal administration

Changes in pupil size after peripheral administration of met-enkephalin, leu-enkephalin, or morphine were studied in the rat. With a simple pupillographic technique, the pupil diameter of male, S.D. rats (250–300 g) was measured by a series of photographs taken every 60 sec for at least 45 min after the last drug injection. Morphine (8 mg/kg, SC) caused mydriasis characterized by rapid and marked fluctuations of pupil size. Mydriasis also occurred after leu-enkephalin (5 and 10 mg/kg, IP) and met-enkephalin (20 mg/kg, IP). Both peptides induced morphine-like fluctuations. When given 15 min after morphine, leu-enkephalin (5 and 10 mg/kg) increased the mydriatic effect of morphine from 172 percent of control to 224 and 272 percent, respectively. Met-enkephalin (20 mg/kg, but not 10 mg/kg) also enhanced the mydriatic response of morphine, to 244 percent of control. These interactions appear to represent simple addition rather than potentiation. The effects of both peptides were reversed by naloxone (1 mg/kg, SC), suggesting an opiate receptor interaction for the pupillary effects of the enkephalins. The rat pupil thus provides one of the few in vivo models permitting quantification of enkephalin action after parenteral administration.     

巴氯芬对慢性吗啡依赖后条件化位置偏好和戒断症状的抑制

小鼠连续7天腹腔注射吗啡(40mg/kg)建立条件化位置偏好模型,连续皮下递增注射吗啡(25、50、75、100、125、150mg/kg),成瘾后腹腔注射纳络酮(6mg/kg)诱导戒断症状(跳跃行为)建立戒断模型。腹腔注射GABAB受体激动剂巴氯芬(2mg/kg)可以有效地抑制吗啡诱导的条件化位置偏好和减轻纳络酮诱导的戒断症状,结果表明GABA系统参与动物成瘾后渴求和戒断过程,激动GABAB受体可以在一定程度上抑制成瘾的心理和生理戒断症状。