Naloxone-precipitated Withdrawal Syndrome Research Articles

Effects of α4β2 and α7 nicotinic acetylcholine receptor antagonists on place aversion induced by naloxone in single-dose morphine-treated rats

Acute dependence can be observed when naloxone is administered 24 h after even a single dose of morphine, and nicotine attenuates this naloxone-precipitated withdrawal syndrome. This acute dependence has been hypothesized to be associated with a dopaminergic mechanism. In the present study, the role of nicotinic acetylcholine receptor subtypes in the place aversion induced by naloxone in single-dose morphine-treated rats was investigated. Methyllycaconitine (1, 2 and 5 mg/kg), an α7 nicotinic acetylcholine receptor subtype inhibitor, significantly and dose dependently inhibited the attenuating effect of nicotine on naloxone-induced place aversion. In contrast, dihydroxy-β-erithroidine (1, 2 and 5 mg/kg), an α4β2 nicotinic acetylcholine receptor subtype inhibitor, did not have any effect on the attenuating effect of nicotine on naloxone-induced place aversion. These findings suggested that the α7 nicotinic acetylcholine receptor subtype is associated with the place aversion induced by naloxone in single-dose morphine-treated rats. Nicotinic acetylcholine receptor subtype inhibitors warrant further study as possible treatment for acute dependence.

Serotonin reuptake inhibitors attenuate morphine withdrawal syndrome in neonatal rats passively exposed to morphine

Previous investigations had shown that inhibitor of serotonin reuptake transporter (SERT) could attenuate morphine withdrawal syndrome in adult animals. In the present study, we determined whether postnatal injection of serotonin reuptake inhibitors, fluoxetine, clomipramine, or citalopram, is able to attenuate the expression of the naloxone-precipitated morphine withdrawal syndrome in 5-day-old neonatal Sprauge–Dawley rats born to dams rat that received morphine injection since a week before mating till 5 days after delivery. Withdrawal syndrome of morphine, manifested as frequent abdominal stretching and yawning, was generated by injection of naloxone on postnatal day 5. Pre-injection with fluoxetine, clomipramine, or citalopram, significantly attenuated the naloxone-precipitated syndrome in a dose-dependent manner without apparent side effect. The rank order of inhibitory potency is citalopram=clomipramine>fluoxetine. This result suggests that inhibitor of SERT may be of potential in treating neonatal morphine withdrawal syndrome.

Suppression of morphine withdrawal syndrome by interleukin-2 and its gene

The naloxone-precipitated withdrawal syndrome in mice and rats after intrathecal injection of recombinant human interleukin-2 protein (rIL-2) or its gene was studied. The results showed that rIL-2 could significantly decrease the number of jumps in mice. In rats, rIL-2 significantly suppressed irritating, diarrhea, weight loss, abnormal posture and salivation. Tendencies towards reductions in teeth chewing and dog-shaking were also observed. Furthermore, pcDNA3-IL-2 (8 microg DNA) had a similar effect as 1x10 IU rIL-2 protein on inhibition of morphine withdrawal syndrome in mice, and the expression of rIL-2 protein in spinal cord could be detected for 6 days. These findings provided further evidence for the neuroregulatory function of an immunological molecule such as IL-2.

Molecular mechanisms in dizocilpine-induced attenuation of development of morphine dependence: an association with cortical Ca 2+/calmodulin-dependent signal cascade

We investigated how dizocilpine, a non-competitive N-methyl- d-aspartate (NMDA) receptor antagonist, affects the development of morphine dependence in mice. Co-administration of dizocilpine (0.25 mg/kg) and morphine (10 mg/kg) for 5 days attenuated the development of tolerance to the antinociceptive effects of morphine. The withdrawal manifestation induced by the naloxone-challenge (5 mg/kg) was significantly reduced in mice that were treated with a combination of dizocilpine and morphine, compared to the mice treated with morphine and saline. The present study revealed a significant increase in c-Fos protein expression in the cortex and thalamus of mice showing naloxone-precipitated withdrawal syndrome. The combination of dizocilpine and morphine prevented the increase of c-Fos protein expression in the cortex and thalamus. Interestingly, repeated co-administration of dizocilpine and morphine prevented the withdrawal-induced phosphorylation of Ca 2+/calmodulin kinase II (p-CaMK II) in the cortex, but not in the thalamus. Acute dizocilpine treatment prior to the naloxone-challenge and repeated treatment with dizocilpine alone had no effect on analgesia, withdrawal manifestations, p-CaMK II levels or c-Fos protein levels. These results showed that co-administration of dizocilpine and morphine prevented the development of morphine tolerance and dependence and suggested that the preventive effect of dizocilpine results from the regulation of c-Fos protein expression, which is possibly involved in the activation of the Ca 2+/calmodulin-dependent signal cascade in the cortex.

Inhibitory effect of harmane on morphine-dependent Guinea pig ileum.

Studies on the occurrence and properties of b-carbolines structurally related to harmala alkaloids have gained attention since it was hypothesized that some of these compounds play a role in processes of substance abuse and dependence. This study investigates the effects of harmane on naloxone-precipitated withdrawal syndrome in morphine-dependent guinea pig ileum. Segments of ilea from starved male guinea pigs were obtained and fixed at a resting tension of 1 g in an organ bath containing 10(-6) M morphine in Tyrode solution at 37 degrees C, which was bubbled with 95% O(2) and 5% CO(2). Tissues were incubated in 10(-6) M morphine containing Tyrode solution for 4 hours before harmane was added. Naloxone and harmane had no effect on naive ilea. Naloxone (10(-6) M) contracted morphine-dependent ilea. Harmane significantly inhibited the contractile response to naloxone in a dose-dependent manner (10(-7) M = 24%; 10(-6) M = 49.3%; 10(-5) = 70%). These results suggest that harmane may have beneficial effects on morphine withdrawal syndrome.

Agmatine inhibits naloxone-induced contractions in morphine-dependent Guinea pig ileum.

This study investigates the effects of agmatine on naloxone-precipitated withdrawal syndrome in morphine-dependent guinea pig ileum. Male guinea pigs that were starved for 24 hours were decapitated after cervical dislocation, and terminal portions of the ilea were removed. Segments were fixed at a resting tension of 1 g in an organ bath containing 1 x 10(-6) M morphine in Tyrode solution at 37 degrees C, which was bubbled with 95% O(2) and 5% CO(2). Tissues were incubated in morphine containing Tyrode solution for 4 hours before agmatine was added. Naloxone and agmatine had no effect on naive ilea. Naloxone (1 x 10(-6) M) contracted morphine-dependent ilea. Agmatine significantly inhibited the contractile response to naloxone in a dose-dependent manner (1 x 10(-7) M, 44%; 1 x 10(-6) M, 80%; 1 x 10(-5) M, 95%). This effect of agmatine was partly abolished by pretreatment with yohimbine and was almost completely abolished by idazoxan.

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, β-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.

Endomorphin-1 and -2 induce naloxone-precipitated withdrawal syndromes in rats

In 1997, endomorphin-1 (EM-1) and -2 (EM-2) were identified as the most specific endogenous μ-opioid ligands. These two peptides have shown analgesic effects and many other opioid functions. In the present study, we attempt to investigate the possible ability of endomorphins to induce naloxone-precipitated withdrawal in comparison with that induced by morphine. Using the previously established scoring system in rats, 12 withdrawal signs (chewing, sniffing, grooming, wet-dog shakes, stretching, yawning, rearing, jumping, teeth grinding, ptosis, diarrhea, and penile erection) were observed and scored following naloxone (4 mg/kg, i.p.) challenge. Compared with the sham control, EM-1 and EM-2 (20 μg, i.c.v., b.i.d. for 5 days) both produced significant naloxone-induced withdrawal syndromes with similar severity to that induced by the same dose of morphine. There was no significant difference between EM-1, EM-2, and morphine-treated group for naloxone-induced withdrawal signs, except for grooming. EM-1 and EM-2 induced more grooming than that caused by morphine. Although EM-1 and EM-2 both led to the withdrawal, they displayed different potency for certain signs and suggest their distinct regulations. The present results indicate EM-1 and EM-2 could initiate certain mechanism involved opiate dependence.

Increase of morphine withdrawal in mice lacking A2a receptors and no changes in CB1/A2a double knockout mice

CB1 cannabinoid and A2a adenosine receptors are highly expressed in the central nervous system where they modulate numerous physiological processes including emotional behaviour and the responses of several drugs of abuse. To investigate the contribution of these receptors in emotional-like responses and opioid dependence we have generated CB1/A2a double deficient mice (CB1-/-/A2a-/-). The spontaneous locomotor activity was reduced in double knockout as compared to wild-type animals. Emotional-like responses of CB1-/-/A2a-/- mice were investigated using the elevated plus-maze and the lit-dark box. Mutant mice exhibited an increased level of anxiety in both behavioural models. The specific involvement of CB1 and A2a receptors in morphine dependence was evaluated by using A2a knockout mice and CB1/A2a double mutant mice. The severity of naloxone-precipitated morphine withdrawal syndrome was significantly increased in the absence of A2a adenosine receptors whereas no modifications were observed in the double knockout mice. These results suggest that both receptors participate in the control of emotional behaviour and seem to play an opposite role in the expression of opioid physical dependence.

Attenuation of spontaneous opiate withdrawal in mice by the anandamide transport inhibitor AM404

The endogenous cannabinoid, anandamide, has been shown to attenuate naloxone-precipitated opiate withdrawal in rodents. Here we show that the spontaneous, but not the naloxone-precipitated withdrawal syndrome in morphine-dependent mice is attenuated by the inhibitor of carrier-mediated anandamide transport N-(4-hydroxyphenyl) arachidonylethanolamide (AM404) (2 and 10 mg/kg, i.p.). These results suggest that spontaneous but not opioid antagonist-precipitated withdrawal is associated with dynamic changes in endogenous cannabinoid signaling.

Dextromethorphan attenuates morphine withdrawal syndrome in neonatal rats passively exposed to morphine

We had previously found that co-injection of dextromethorphan, an antitussive drug and a non-competitive NMDA receptor antagonist, with morphine into dam rats throughout the pregnancy period could attenuate the naloxone-precipitated morphine withdrawal syndrome in their offspring. In the present study, we further tested whether postnatal injection of dextromethorphan into the neonatal rats or a 3-day co-injection of dextromethorphan with morphine into the dam rats before delivery is also effective. Female Sprague–Dawley rats were bi-daily injected with escalating doses of morphine from a week before mating till the first postnatal week. Withdrawal syndrome of morphine in the offspring, manifested mainly as abdominal stretching, was generated by injection of naloxone on postnatal day 5. Direct injection of dextromethorphan into the offspring effectively reduced the severity of naloxone-precipitated abdominal stretching in a dose-dependent manner. A 3-day co-treatment with dextromethorphan given to the dam rat before delivery also had a similar attenuating effect, but the efficacy was lower than that produced by postnatal injection. Thus, the results from the present study support that dextromethorphan is of potential in treating or preventing neonatal morphine withdrawal syndrome.

Dorsal and median raphe serotonergic system lesion does not alter the opiate withdrawal syndrome

Previous pharmacological studies have implicated serotonergic brain systems in opiate withdrawal. To test the hypothesis that serotonin (5-HT) has a critical role in the development of opiate withdrawal, we have employed a near-total brain 5-HT system lesion technique (90% depletion) using 5,7-dihydroxytryptamine combined with induction of opiate dependence by implantation of morphine pellets or by repeated injections of increasing doses of morphine. The effects of serotonergic neuron lesion were examined on spontaneous opiate withdrawal (changes in circadian locomotor activity) and naloxone-precipitated opiate withdrawal syndrome (the somatic aspect). The antiwithdrawal properties of clonidine, an α 2-adrenoceptor agonist currently used for clinical treatment for the somatic signs of opiate withdrawal, were tested also in the lesioned rats. Our findings show that serotonergic lesions in morphine-dependent rats did not alter either the spontaneous or the naloxone-induced withdrawal syndrome (with exception of jumping behavior). Moreover, clonidine alleviated the naloxone-induced withdrawal syndrome in lesioned as well as in sham-operated morphine-dependent rats. These results demonstrate that 5-HT systems are not directly responsible for the development of the somatic opiate withdrawal syndrome in morphine-dependent rats.

Gender-linked differences in the expression of physical dependence in the rat

In earlier studies, it was shown that there were gender differences in several aspects of the pharmacological profile of morphine, including its antinociceptive activity, discriminative stimulus properties and its reinforcing effects. The purpose of the present studies was to examine whether there might also be gender-related differences in the development of physical dependence, as reflected in the expression of an opiate withdrawal syndrome upon cessation of chronic morphine administration. We found that a more severe spontaneous withdrawal syndrome was produced by chronic morphine injections or morphine pellet implantation in male rats than in females. The duration of the withdrawal syndrome was also longer. In contrast to our observations with spontaneous withdrawal, we found no gender differences in the naloxone-precipitated withdrawal syndrome induced by chronic morphine administration. These observations suggest that there are gender differences only in the expression of the spontaneous withdrawal syndrome, but not in the neuro-adaptive changes associated with the generation of physical dependence as reflected by naloxone-precipitated withdrawal.

Dansyl-PQRamide, a possible neuropeptide FF receptor antagonist, induces conditioned place preference

Neuropeptide FF (NPFF) is an endogenous anti-opioid peptide. NPFF could potentiate the naloxone-precipitated morphine withdrawal syndromes in morphine-dependent rats, indicating the possible involvement of the endogenous NPFF system in opioid analgesia and dependence. The present study was performed to examine the effects of dansyl-PQRamide (dns-PQRa), a putative NPFF antagonist, on conditioned place preference (CPP), in addition, its interaction with the opioid system. Two CPP experiments were conducted. First, rats were treated with dns-PQRa (4–13 mg/kg, i.p.) and paired with the non-preferred compartment while the vehicle was paired with the preferred compartment. Second, similar to experiment 1 except naloxone (1 mg/kg, i.p.) was given 10 min prior to each dns-PQRa administration. The post-drug place preference was examined after 4 alternative pairings. Another group of animals after repetitive dns-PQRa treatments were analyzed for levels of neurotransmitters in discrete brain areas. Dns-PQRa (4–13 mg/kg, i.p.) induced a significant dose-dependent CPP. The dns-PQRa-induced CPP was completely blocked by pretreatment with 1 mg/kg i.p. naloxone, while naloxone alone did not induce any place aversion. The chronic dns-PQRa-treated (13 mg/kg, i.p., b.i.d.) rats caused a significant increase in 3,4-dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid in the olfactory tubercle compared to the vehicle-treated controls. There was also an increase in the turnover of serotonin in the olfactory tubercle, nucleus accumbens and medial prefrontal cortex. These results suggest that blockade of the NPFF system produces rewarding, possibly via an inhibition of the anti-opioid action of NPFF. These results also reveal a close relationship between NPFF, drug rewarding and the dopaminergic and serotoninergic neurons in the mesolimbic system.

Lithium inhibits the development of physical dependence to clonidine in mice.

Based on our previous finding that chronic lithium treatment reduced naloxone-precipitated withdrawal syndrome in morphine-treated mice, the effect of chronic lithium treatment was evaluated on the development of dependence to clonidine. Dependence was induced by injection of either morphine (50, 50 and 75 mg/kg, intraperitoneally with 3 hr interval for 3 consecutive days), or clonidine (2 mg/kg/day, intraperitoneally for 10 days). Naloxone (4 mg/kg, intraperitoneally) precipitated withdrawal signs in both morphine- and clonidine-treated mice. Yohimbine (5 mg/kg, intraperitoneally) precipitated withdrawal signs in the clonidine-treated mice, similar to morphine withdrawal signs; but failed to precipitate any significant sign in the morphine-treated mice. Coadministration of lithium was carried out by adding lithium chloride to drinking water (600 mg/l for 20 days; 10 days before the beginning of clonidine administration and 17 days before the administration of morphine to allow the lithium concentration to reach steady-state). The results indicated that chronic lithium administration significantly attenuated the withdrawal signs, precipitated either by yohimbine or naloxone, in clonidine-treated mice. As a conclusion, clonidine withdrawal signs are very similar to opioid withdrawal signs, and lithium is able to prevent the development of physical dependence to clonidine.

Clonidine attenuates naloxone-induced opioid-withdrawal syndrome in cholestatic mice.

Cholestasis is associated with elevated plasma level of endogenous opioid peptides. Naloxone-precipitated withdrawal syndrome has been described in a mouse model of acute cholestasis. Thus we aimed at determining whether central noradrenergic hyperactivity is involved in manifestation of naloxone-precipitated withdrawal syndrome in mice with obstructive cholestasis. Acute cholestasis was induced by bile duct resection in mice and physical dependence was observed by precipitating a withdrawal syndrome with naloxone (2 mg/kg, intraperitoneally) 5 days after induction of cholestasis. Administration of clonidine (0.1 mg/kg, intraperitoneally), an alpha2-adrenoceptor agonist, 15 min. before naloxone injection significantly alleviates withdrawal severity in cholestatic mice. However, pretreatment of animals with yohimbine (3 mg/kg, intraperitoneally), an alpha2-adrenoceptor antagonist, 15 min. before clonidine blocked this ameliorative effect of clonidine. The results of this study support the evidence for involvement of the alpha2-adrenoceptors in the withdrawal syndrome of cholestasis in a mouse model.

Clonidine Attenuates Naloxone‐Induced Opioid‐Withdrawal Syndrome in Cholestatic Mice*

Abstract: Cholestasis is associated with elevated plasma level of endogenous opioid peptides. Naloxone‐precipitated withdrawal syndrome has been described in a mouse model of acute cholestasis. Thus we aimed at determining whether central noradrenergic hyperactivity is involved in manifestation of naloxone‐precipitated withdrawal syndrome in mice with obstructive cholestasis. Acute cholestasis was induced by bile duct resection in mice and physical dependence was observed by precipitating a withdrawal syndrome with naloxone (2 mg/kg, intraperitoneally) 5 days after induction of cholestasis. Administration of clonidine (0.1 mg/kg, intraperitoneally), an α2‐adrenoceptor agonist, 15 min. before naloxone injection significantly alleviates withdrawal severity in cholestatic mice. However, pretreatment of animals with yohimbine (3 mg/kg, intraperitoneally), an α2‐adrenoceptor antagonist, 15 min. before clonidine blocked this ameliorative effect of clonidine. The results of this study support the evidence for involvement of the α2‐adrenoceptors in the withdrawal syndrome of cholestasis in a mouse model.

Involvement of cyclic AMP systems in morphine physical dependence in mice: prevention of development of morphine dependence by rolipram, a phosphodiesterase 4 inhibitor.

In this study, we examined whether morphine dependence was inhibited by rolipram, a cyclic AMP selective phosphodiesterase inhibitor in mice, since a role for the cyclic AMP systems in the development of morphine dependence has been reported. Mice, which received morphine (10 mg kg(-1) s.c.) twice a day for 5 days showed withdrawal syndromes such as jumping, rearing and forepaw tremor following naloxone challenge (5 mg kg(-1) i.p.) on the 6th day. Such mice exhibited a significant elevation of cyclic AMP levels in the thalamus compared to control mice. However, co-administration of rolipram (1 mg kg(-1) i.p.) with morphine for 5 days significantly attenuated the severity of the withdrawal syndrome and the increase in the cyclic AMP levels after the administration of naloxone. In naïve mice, acute morphine treatment (10 mg kg(-1) s.c.) decreased cyclic AMP levels in the thalamus and cerebral cortex 10 min later. The decrease of cyclic AMP levels induced by acute morphine treatment was blocked by co-administration of rolipram (1 mg kg(-1) i.p.). However, acute rolipram did not affect the naloxone-precipitated morphine withdrawal syndrome. These results suggest that the elevation of the cyclic AMP levels is involved in the development of morphine withdrawal syndrome and that blockade of the morphine-induced reduction of cyclic AMP levels by chronic rolipram inhibits the development of dependence and the behavioural and biochemical changes induced by naloxone. Furthermore, rolipram may be a useful drug for attenuating the development of morphine dependence.

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

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

A protein kinase inhibitor, H-7, blocks naloxone-precipitated changes in dopamine and its metabolites in the brains of opioid-dependent rats

The influence of an inhibitor of cAMP-dependent protein kinase and protein kinase C, H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine], on naloxone (an opioid receptor antagonist)-precipitated withdrawal signs and changes in levels of dopamine (DA) and its metabolites in morphine- or butorphanol-dependent rats was investigated. Animals were infused continuously with morphine (a μ-opioid receptor agonist) or butorphanol (a μ/δ/κ mixed opioid receptor agonist) for 3 days. Naloxone precipitated withdrawal syndrome and decreased the levels of DA in the cortex, striatum, and midbrain; 3,4-dihydroxyphenylacetic acid (DOPAC) in the cortex, striatum, limbic areas, and midbrain; and homovanilic acid (HVA) in the striatum, limbic areas, and midbrain regions. In animals rendered dependent on butorphanol, the results obtained were similar to those of morphine-dependent rats except for the changes in DOPAC levels. Concomitant infusion of H-7 and opioid blocked both the expression of withdrawal signs and the decreases in DA, DOPAC, and HVA levels in a dose-dependent manner. These results suggest that the enhancement of cAMP-dependent protein kinase and/or protein kinase C activity accompanying the increase of DA neuron activity during continuous infusion of opioids leads to an abrupt reduction in levels of DA and its metabolites precipitated by naloxone, which is intimately involved in the expression of physical dependence on opioids.