Neuropeptide trefoil factor 3 attenuates naloxone-precipitated withdrawal in morphine-dependent mice

Rationale

The persistence of physical dependence and craving in addicts is considered to contribute to relapse. Increasing evidence indicates that neuropeptide systems are associated with several phases of drug addiction, but little is known about whether the neuropeptide trefoil factor affects withdrawal symptoms.

Objectives

This study aims to investigate the potential effects of the neuropeptide trefoil factor 3 (TFF3) on naloxone-precipitated withdrawal symptoms in morphine-dependent mice.

Results

Mice received increasing doses of morphine over 3 days. On day 4, the mice were injected with TFF3 (1.0 mg/kg, i.p.) 30 min after the last dose of morphine. Thirty minutes after TFF3 treatment, naloxone (1 mg/kg, i.p.) was injected, and body weight, jumping behavior, wet-dog shakes, and locomotor activity were assessed 30 min later. Naloxone caused significant weight loss and increased jumping behavior and wet-dog shakes in morphine-dependent mice. TFF3 (1.0 mg/kg) reversed these behavioral symptoms caused by morphine withdrawal, suggesting that TFF3 might ameliorate physical dependence associated with opiate addiction. Furthermore, TFF3 pretreatment significantly reduced morphine withdrawal-induced increases in plasma corticosterone and adrenocorticotropic hormone levels. The glucocorticoid receptor agonist RU486 blocked the behavioral effects of TFF3 on morphine withdrawal symptoms. Finally, Fos expression in the medial prefrontal cortex which was decreased during morphine withdrawal was increased by TFF3 pretreatment.

Conclusion

These findings indicate that TFF3 might be a potential therapeutic candidate for opiate addiction by regulating glucocorticoid secretion and neuronal activation in the prefrontal cortex.     

Effects of dapiprazole, clonidine and yohimbine on the development of dependence and withdrawal behaviour in mice

There is evidence that central noradrenergic hyperactivity is involved in the manifestation of the major signs and symptoms of the opiate withdrawal syndrome. In order to assess whether or not the noradrenergic system is also implicated in the development of opiate dependence, we studied dapiprazole, an alpha-1 selective adrenoceptor antagonist, clonidine and yohimbine using the acute dependence model in mice. When administered just before naloxone, after dependence development, all three drugs reduce abstinence signs. When injected 15 min before morphine to observe the drugs' effects on the development of dependence, dapiprazole depresses all the symptoms registered while clonidine decreases only jumping, but increases paw and head shakes. None of these drugs affects the naloxone precipitated withdrawal syndrome when injected 1 h before morphine. It is suggested that the noradrenergic system is involved in both the manifestation of the withdrawal syndrome and in the development of opiate dependence. Diapiprazole may be a useful tool in patients and in pharmacological studies of dependence and abstinence.     

A comparison of the cardiovascular and the behavioral changes following naloxone as measures of the degree of physical dependence on morphine in rats

Behavioral and cardiovascular changes evoked by naloxone (0.5 mg/kg) were evaluated in unrestrained rats made physically dependent by a constant infusion of morphine at increasing doses over 7 days. The relationship between the duration of morphine administration and the naloxone-induced cardiovascular changes was investigated to determine the reliability and sensitivity of blood-pressure measurements in assessing the degree of physical dependence produced in these animals. No significant behavioral or cardiovascular changes from preaddiction levels were observed during morphine infusion. Arterial pressure increased significantly in a dose- and time-dependent manner in response to naloxone injected on days 1, 3, 5, or 7 of the morphine infusion schedule. Heart rate changes which accompanied the increase in blood pressure, however, were similar in magnitude over the course of increasing physical dependence. The frequency of counted behavioral signs precipitated by naloxone over the same infusion schedule indicated a significant, progressive increase only for escape attempts, whereas withdrawal body shakes and writhing peaked early in the schedule and actually declined to very low frequencies by day 7. A high dose of morphine (50 mg/kg) was injected following naloxone-precipitated withdrawal in an attempts to reverse the abstinence symptoms. The only symptoms significantly inhibited by morphine were the increase in arterial pressure and the occurrence of diarrhea. These results indicate that the absolute increase in arterial blood pressure produced by naloxone in opiate-dependent animals may serve as a simple, objective, and sensitive measurement, along with traditional behavioral signs, in the assessment of narcotic dependence liability.     

Cardiovascular changes during morphine withdrawal in the rat: Effects of clonidine

Arterial blood pressure and heart rate were measured in unrestrained rats as an index of the autonomic component of the morphine withdrawal syndrome. Physical dependence was produced by a constant infusion of morphine at increasing doses over 7 days. Signs of physical dependence observed during abrupt withdrawal included classical behavioral symptoms such as withdrawal body shakes (WBS) and increased autonomic responsiveness which was indicated by a sustained increase in mean arterial pressure (MAP) up to 23 mmHg. Injection of naloxone in morphine dependent rats also evoked a dose-related increase in MAP to about 40 mmHg. The antiwithdrawal effects of clonidine were tested in this model pretreating dependent rats with this agent (6–60 μg/kg). Clonidine inhibited the pressor response produced by naloxone by 23–60%. These findings indicate that the increase in MAP during opiate withdrawal provides an objective and quantitative index of the intensity of the narcotic withdrawal syndrome in dependent rats.     

维生素K3中枢镇痛效应的探讨

经由大鼠、小鼠甩尾及兔甩头法测痛,证实k₃具有剂量依赖的镇痛作用。兔侧脑室微量注射k₃亦有显著镇痛效应。k₃的镇痛作用可被阿片拮抗剂纳络酮所拮抗。实验观察到k₃与吗啡的镇痛效应间存在交叉耐受现象。一定浓度的k₃可抑制电场刺激所致豚鼠回肠纵肌标本的收缩,这一效应亦可被纳络酮部分逆转。小鼠经k₃预处理后对k₃的镇痛产生耐受;连续k₃大剂量预处理后纳络酮激发不产生跳跃。     

Antidiuretic effect of morphine in the rat: tolerance and physical dependence.

1 Injection of rats with morphine or methadone, before they received a water load equivalent to 5% of their body weight, produced a dose-dependent antidiuretic effect. Following the antidiuresis, urine was eliminated with kinetics similar to control untreated rats. 2 The antidiuretic effect of morphine or methadone was blocked by naloxone administered before the opiate, or reversed when given after the opiate. 3 Rats implanted with morphine pellets developed a marked degree of tolerance to the antidiuretic effect of morphine. Tolerance was also obtained on injection of three daily doses of morphine or methadone over two days. 4 Withdrawal symptoms were precipitated by naloxone in rats implanted with pellets of morphine; under these conditions the animals showed a marked reduction in urine production as compared to naive rats.     

Increase of extracellular dopamine in the medial prefrontal cortex during spontaneous and naloxone-precipitated opiate abstinence

Abstinence from chronic morphine has been shown to reduce extracellular dopamine in the nucleus accumbens as measured by brain microdialysis (Acquas et al. 1991). In the present study, we investigated if similar changes take place in the prefrontal cortex. Withdrawal from a schedule of increasing doses of morphine administered intraperitoneally twice a day for 9 days up to a daily dose of 60 mg/kg resulted in doubling of basal extracellular concentrations of dopamine in the prefrontal cortex and in a mild withdrawal syndrome (ptosis, piloerection, hunched-back posture). Administration of a low dose of naloxone (0.5 mg/kg SC) to rats withdrawn from chronic morphine resulted in a full withdrawal syndrome with wet dog shakes and diarrhoea and an increase of extracellular dopamine that peaked at 40 min and returned to the pre-naloxone values by 80 min. The results show that dopamine neurotransmission in the medial prefrontal cortex responds to opiate withdrawal in a manner opposite to dopamine transmission in the nucleus accumbens and indicate that the dopamine system is affected by abstinence in a topographically specific manner, consistent with a different functional role of mesocortical as compared to mesolimbic dopamine systems.     

Antidiuretic effect of beta-endorphin and morphine in Brattleboro rats: development of tolerance and physical dependence after chronic morphine treatment.

1 beta-Endorphin (2 micrograms injected into the lateral ventricles) produced a significant decrease in the urine outflow and in the excretion of Na+ and K+ in Brattleboro rats, animals suffering from severe diabetes insipidus. Morphine intracerebrally also produced antidiuresis, as compared to saline-treated controls. 2 Morphine injected intraperitoneally caused a dose-dependent decrease in the urine outflow, and in the excretion of Na+ and K+. 3. Rats chronically treated with morphine (72 h of morphine pellet implantation) were less sensitive to the antidiuretic effect of a challenge dose of morphine than control Brattleboro rats implanted with placebo pellets, but otherwise treated similarly. 4 After chronic morphine administration, Brattleboro rats became dependent on morphine. Challenge with 1 mg/kg naloxone (s.c.) precipitated an abrupt opiate withdrawal syndrome characterized, among other symptoms, by increased urination in contrast to the antidiuresis observed before naloxone.     

Effects of tizanidine administration on precipitated opioid withdrawal signs in rats

An opioid withdrawal syndrome was precipitated by naloxone administration in rats treated with morphine. The withdrawal caused alteration of several physiological signs. The aim of the study was to investigate whether the altered physiological profiles were modified by utilising tizanidine, an α₂ adrenergic receptor agonist which is capable of affecting faecal and urinary excretion, rectal temperature, pain threshold levels and salivation. To induce an opioid withdrawal syndrome, morphine was administered in three daily intraperitoneal injections for four days at doses of 9, 16 and 25 mg/kg (1st day), 25, 25 and 50 mg/kg (2nd day), 50, 50 and 50 mg/kg (3rd day) and 50, 50 and 100 mg/kg (4th day): naloxone was injected (30 mg/kg) i.p. 180 min after the last morphine injection. Tizanidine was administered orally at 0.17, 0.35 and 0.7 mg/kg, 60 min after the last morphine injection. Signs such as faecal and urine excretion, rectal temperature and latency times to thermal stimulus, salivation, jumping and wet dog shakes were affected in different ways by morphine, naloxone, tizanidine and by the combination of these agents. Notably, the administration of tizanidine in rats receiving morphine and naloxone decreased the intensity of certain withdrawal symptoms, including altered excretion of faeces and urine, salivation and wet dog shake behaviour. Body temperature levels and nociceptive threshold values were also modified. The effects caused by tizanidine administration may be due to its α₂ receptor agonist activity interfering with a mechanism involved in the regulation of these previously mentioned withdrawal symptoms. Thus, the use of this drug may be indicated as a possible control of the acute phase of opioid withdrawal in heroin addicts.     

Chronic morphine administration: Plasma levels and withdrawal syndrome in rats

Morphine, administered to Sprague-Dawley rats over a period of 65 hr either by the simultaneous implantation of two 75 mg pellets, or by a series of twice daily 20 or 30 mg/kg injections, produced dependence as indicated by the precipitation of the abstinence syndrome with the antagonist, naloxone. Plasma morphine levels, analyzed fluorometrically at various times during the treatment procedures, revealed peak concentrations that were 3 or 4 fold higher for injected animals than the maximum steady-state level established in the pellet-implanted animals. The calculated plasma concentration of the drug over time was not statistically different for the groups. It is noted that although the 2 methods of morphine administration produce a qualitatively identical dependent state, the pellet implantation technique causes greater weight loss and a higher incidence of jumping and wet-dog shakes during withdrawal.     

Clonidine reverses the behavioral and respiratory effects of continuous naloxone infusion

Opiate naive rats received 24 hours of continuous subcutaneous infusion of 0.67 mg/kg/hr naloxone via osmotic minipump. As in previous studies, this induced an opiate-abstinence-like syndrome of significantly increased oxygen consumption and behavioral signs (wet-dog shakes, abdominal writhes, etc.). Clonidine, which selectively reduces central noradrenergic activity, has been shown to reverse opiate abstinence syndrome. Subcutaneous injection of 0.033 and 0.01 mg/kg clonidine totally reversed the abstinence-like behaviors and respiratory activity induced by naloxone infusion. This constitutes an additional point of similarity between opiate abstinence syndrome and the "endorphin blockade syndrome" or withdrawal from endogenous opioids resulting from chronic naloxone treatment. It is consistent with the hypothesis that hyperactivity of central noradrenergic mechanisms may contribute to both phenomena.     

Continuous infusion of naloxone: effects on behavior and oxygen consumption

Twenty-eight hours of endorphin receptor blockade by subcutaneous naloxone infusion produced behavioral and respiratory symptoms resembling opiate abstinence syndrome. Rats were implanted subcutaneously with two Alzet osmotic minipumps delivering 0.7 mg/kg per hour naloxone or with two control minipumps containing distilled water only. They were observed for 10 minutes under blind conditions at 16 and 28 hours post-implantation. The naloxone-infused rats showed significantly more wet dog shakes, abdominal writhes and overall abstinence-like symptoms than did the control rats. These symptoms decreased after 28 hours despite continued naloxone infusion. Acute administration of naloxone failed to produce abstinence-like symptoms, even when combined with the trauma of carrying two implanted water-filled minipumps for 28 hours. In another experiment, naloxone-infused rats showed a highly significant 53.4% elevation of O2 consumption over water-infused control rats in a pure O2 atmosphere at 28 hours after implantation. This difference disappeared at 48 hours post-implantation. In contrast to the effect of naloxone infusion, acute administration of three different doses of naloxone failed to significantly increase O2 consumption.     

Chronic variable stress or chronic morphine facilitates immobility in a forced swim test: reversal by naloxone

The behaviors displayed in a forced swim test were investigated in rats previously exposed to a chronic variable stress treatment or chronic administration of morphine. In addition, to further explore the participation of an endogenous opiate mechanism in these behavioral effects, naloxone was either administered during the chronic treatment (prior to each stress or morphine exposure) or immediately prior to the forced swim test. Animals were submitted daily to a different stressor for 1 week or injected with morphine (10 mg/kg, IP) for 6 days, whereas controls were unmanipulated except for the injection process. On the day following the last stressor, control and stressed animals were administered saline or naloxone (2 mg/kg, IP) 15 min prior to the forced swim test. Morphine treated animals were similarly tested on the third day following the last morphine injection. In a separate group of rats, naloxone (2 mg/kg, IP) was administered daily 10 min prior to each stressor of the chronic stress regime or each daily morphine injection. A significant increase in the time spent in immobility was observed in stressed animals as well as in rats chronically treated with morphine. In both groups, this potentiated immobility was attenuated by naloxone pretreatment prior to the forced swim test or when given before each daily stressor or morphine injection. In addition, the concurrent exposure to stress or morphine along with naloxone administration enhanced struggling in the first 5 min of the forced swim test. Taken together, the results of these experiments support the conclusion that the increase in immobility seen following chronic variable stress or repeated morphine exposure is modulated by the activation of an endogenous opiate mechanism, given that this effect is attenuated by naloxone administration.     

Effect of morphine on 'wet-dog' shakes caused by cerebroventricular injection of serotonin

Intraventricular administration of serotonin to rats causes 'wet-dog' shakes, a sign of morphine withdrawal. The frequency of shakes is dose-dependent. Shaking is potentiated by pretreatment with an inhibitor of monoamine oxidase or with 5,7-dihydroxytryptamine, and is depressed by morphine or serotonin receptor blockers. Depression of serotonin-induced shaking by morphine is reversed rapidly by naloxone. However, naloxone did not reverse the inhibition of 'wet-dog' shakes caused by serotonin receptor blockers.     

Age-related differences in the effect of chronic administration of naloxone on opiate binding in rat brain

Infant and adult rats were injected chronically with either naloxone or saline for 21 consecutive days. At various intervals after cessation of the pretreatment with naloxone, animals were sacrificed and assessed for specific binding of [3H]naloxone in different regions of the CNS. Infants displayed an increase in opiate binding in the spinal cord, hypothalamus, striatum and cortex one day after cessation of the pretreatment with naloxone, but the increase in opiate binding was dissipated within one week after cessation of the pretreatment. The increase in opiate binding in infants was accompanied by an increase in the antinociceptive efficacy of morphine. In contrast to infants, adults failed to display any alteration in opiate binding following the chronic pretreatment with naloxone. Infants may be especially susceptible to naloxone-induced receptor supersensitivity because infants excrete naloxone more slowly than adults, and thus their opiate receptors may be blocked for a longer duration following an injection of naloxone.     

Micturition in naive and morphine-dependent rats.

Voiding responses were recorded in conscious water-loaded rats. Morphine sulphate (5 mg kg-1) elevated the volume threshold for micturition (MV); the group mean MV of 16 rats after morphine was 40% larger than control. Micturition was nevertheless complete since no urine remained in the bladder afterwards. The implantation of 2 or 4 morphine-base pellets (150 or 300 mg morphine) elevated for 12 days the MV in water-loaded rats. On the 3rd to the 10th day following implantation the group mean was approximately twice that of untreated controls. After micturition was over no residual urine was found in the bladder. Within 3 days the rats became tolerant to the antinociceptive action of the morphine-base pellets but little apparent tolerance developed to their action on micturition. On the 1st day after the pellets were removed, the mean MV was reduced. When withdrawal was precipitated by the administration of naloxone the MV was often too small to measure. This component of a withdrawal syndrome could be elicited in the rats throughout the 12 days of morphine pellet implantation. The administration of 20 mg kg-1 morphine sulphate to anaesthetized rats did not decrease the contractions of the urinary bladder to repetitive stimulation of its motor nerves at 1 and 20 Hz.     

Intermittent naloxone attenuates the development of physical dependence on methadone in rhesus monkeys

Rhesus monkeys that received 15 daily injections of methadone (2 mg/kg i.m.) exhibited a characteristic opiate withdrawal syndrome after injection of naloxone (0.5 mg/kg i.m) on the 16th day. In comparison, injection of naloxone (0.5 mg/kg i.m.) once every 2 days during a similar 15 day methadone treatment period in these same monkeys significantly attenuated the severity of the opiate withdrawal syndrome exhibited after naloxone injection on the 16th day. Each naloxone administration during the 15 day methadone treatment period elicited an opiate withdrawal syndrome that did not significantly differ on each of the 7 days it was given and was less severe than the syndrome precipitated by naloxone following 15 days of methadone without intermittent naloxone. The lack of increments in the withdrawal response to the seven naloxone injections during the 15 days of methadone treatment and the attenuation of the withdrawal response to naloxone on day 16 after intermittent naloxone administration during the 15 day methadone treatment period support the hypothesis that naloxone modifies opiate receptor mechanisms so that they revert to an agonist-naive state following antagonist exposure. These findings suggest that various agonist and antagonist drugs opiate combinations or mixed agonist-antagonist drug could be clinically useful in the management of situations where physical dependence on opiates is a problem.     

Ethanol attenuation of morphine dependence: comparison to dizocilpine

Recent studies indicate that morphine dependence, assessed as the severity of naloxone-precipitated opiate withdrawal in rats, is attenuated by dizocilpine, a non-competitive, excitatory amino acid antagonist. Because ethanol is a putative excitatory amino acid antagonist, the present study compared the effects of co-administration of ethanol to that of dizocilpine on morphine dependence. Rats were administered morphine (10 mg/kg) twice daily for 9 days. One group received ethanol (1 g/kg) co-administration, another received dizocilpine (0.05 mg/kg) co-administration, and a third served as vehicle controls. On day 10, all rats received naloxone (4 mg/kg) injections and ratings of several classic signs of opiate withdrawal were made. Both ethanol-and dizocilpine-treated rats showed significantly less severe precipitated opiate withdrawal overall, with the ethanol group showing reduced ratings of some specific signs. These results demonstrate that ethanol, like dizocilpine, attenuates the development of morphine dependence. The results are consistent with the action of ethanol at glutamate receptors. Received: 4 February 1997/Final version: 8 May 1997     

三氟拉嗪抑制吗啡依赖大、小鼠纳洛酮催促的戒断症状

目的　研究三氟拉嗪对吗啡依赖大、小鼠纳洛酮催促戒断症状的影响 ,并探讨其机制。方法　吗啡依赖大、小鼠纳洛酮催促实验。结果　三氟拉嗪 (2～ 2 0mg·kg-1)呈剂量依赖性抑制吗啡依赖小鼠纳洛酮催促所致的跳跃、湿狗样抖动、前爪震颤和体重下降。三氟拉嗪 5～ 2 0mg·kg-1ip ,对吗啡依赖大鼠大部分纳洛酮催促的阳性戒断症状均具有明显的抑制作用 ,其中包括跳跃、湿狗样抖动、排泄物、体重下降、咬牙、流涎、腹泻、上睑下垂、激惹。作为DA1/DA2 受体激动剂 ,阿朴吗啡 (2～ 8mg·kg-1)对三氟拉嗪抑制吗啡依赖小鼠纳洛酮催促戒断症状无明显影响 ,而钙通道阻滞剂硝苯吡啶 (5～ 2 0mg·kg-1)则呈剂量依赖性加强三氟拉嗪对吗啡依赖小鼠纳洛酮催促戒断症状的抑制作用。结论　三氟拉嗪对吗啡依赖大、小鼠纳洛酮催促的戒断症状具有明显的抑制作用。对受体后钙调素生物活性的抑制作用可能是三氟拉嗪抗吗啡依赖大、小鼠躯体戒断症状主要机制 ,而中枢神经系统DA2 受体可能不参与三氟拉嗪对吗啡躯体戒断症状的抑制作用     

Prevention by the 5-HT3 receptor antagonist, ondansetron, of morphine-dependence and tolerance in the rat.

1. The effect of ondansetron, a selective 5-hydroxytryptamine3 (5-HT3) receptor antagonist, was studied in morphine-addicted rats. Morphine-dependence and tolerance, induced by drinking increasing concentrations of morphine sulphate in 5% sucrose solution for 3 weeks, were demonstrated by the naloxone-precipitated withdrawal syndrome and tail flick response to a thermal noxious stimulus (water at 50 degrees C), respectively. 2. Morphine-dependence, assessed by naloxone precipitated withdrawal, was undetectable by the 6th day, when the animals drank only tap water for 7 days after the 3-week induction period. 3. When detoxified rats were offered sucrose and morphine solutions for 10 days, the recurrence of opiate solution preference with relapse to dependence and tolerance was observed. 4. Giving ondansetron (0.1 or 1 microgram kg-1; i.p.; twice daily) on the 14th day of, or 7 days prior to, the 3-week induction period reduced dependence and tolerance seen during the 3-week morphine induction and the 10-day drinking preference periods. 5. 5-Hydroxytryptamine2 (5-HT2) receptor antagonism by cyproheptadine (100 or 250 micrograms kg-1; i.p.; twice daily) did not influence morphine-dependence and tolerance. 6. These findings suggest that ondansetron may be useful for treating opiate addiction and lowering the recidivism rate.