Reversal Effect Of Melatonin On Rewarding Properties Of Morphine And Its Mechanisms Within The Central Nervous System

Opioids are prescription drugs with a high risk of addiction. Opioids-induced addiction has been recognized increasingly as a leading cause of death, morbidity and lost productivity. The development of addiction caused by opioids involves of physical dependence and psychological dependence. To date, no effective drug occurs in the treatment of psychological dependence. To develop new effective drugs or therapy to treat psychological dependence is an urgent task.The rewarding properties of opioids are one of the main causes of psychological dependence to opioids. Conditioned place preference (CPP) test has been widely used to assess the rewarding properties of drugs with addiction liability (including opioids). The nucleus accumbens (NAc) is one of the most important central nervous system (CNS) regions involved in the rewarding effects of opioids.Melatonin has been known as a neuroendocrine hormone synthesized and secreted primarily by the pineal gland. Its chemical structure is N-acetyl-5-methoxytryptamine which can be synthesized chemically at present. Melatonin receptors are found in the body, especially in the brain, and melatonin is the endogenous ligand for these receptors. The MT₁ and MT₂ melatonin receptors on cell membrane have been cloned successfully. To date, quite a few of selective agonists and antagonists on melatonin receptor subtypes have been developed. For example, luzindole is a non-selective antagonist for melatonin MT₁ and MT₂ receptors, K185{N-butanoyl-2-(5,6,7-trihydro-11-methoxybenzo[3,4]cyclohept[2,1-α]indol-13-yl)ethanamine is the selective antagonist for melatonin MT₂ receptor. Finding some useful substance from melatonin and its homologue for treatment is one of the most interesting research fields at present. Melatonin has been involved in some neuropsychopharmacological actions. The study on the function of melatonin has been one of the most interesting research fields. Recently, a growing body of evidence suggests that melatonin may have some reversal effects on different drugs of abuse. For instance, melatonin is shown to reverse the development of tolerance and physical dependence to morphine and to inhibit the morphine withdrawal syndromes in mice without producing physical dependence by itself. Interestingly, several lines of evidence indicate the effect of melatonin on psychological dependence induced by opiods: Huang M et al showed that melatonin inhibits the development of morphine-induced CPP; Yahyavi-Firouz-Abadi et al showed that the combination of melatonin and sub-effective dose of morphine led to significant CPP, while intraperitoneal administration of melatonin alone did not induce CPP; Cong B et al showed melatonin has an reversal effect on the relapse behavior induced by priming injection of morphine after extinction of CPP. These results indicate melatonin may have some inhibition effects on rewarding properties of morphine. But the characteristic of the effects need to be further confirmed. Hence, the first step of the present study was designed to observe the effects of melatonin on morphine-induced CPP in mice and rats, in order to make clear further the effects of melatonin on the rewarding properties of morphine, to provide evidence for the likely clinical application of melatonin and its homologue in the treatment of psychological dependence opioids.Melatonin can penetrate the blood-brain barrier. CNS is one of the most important sites for melatonin to exert its neuropsychopharmacologic effects. Acting through receptors is an important mechanism of melatonin to exert its effects. Whether melatonin activates its receptors within CNS to produce its reversal effect on the rewarding properties of morphine remains to be clarified. Hence, the presents study was designed to investigate whether CNS is the primary site for melatonin to exert its reversal effect on the rewarding properties of morphine and the involvement of melatonin receptors within CNS in this effect in mice, to provide drug targets to design new drugs for the treatment of opioids-induced psychological dependence. The moleculer mechanism of the reversal effect of melatonin on the rewarding properties of morphine is still obsecure. It has been showed the transcriptional factor CREB andâ–³FosB are the most important moleculers during the development of drug addiction. The activation of CREB andâ–³FosB in the NAc mediate different aspects of the addicted state. Therefore, the present study was designed to investigate the changes of trascrptional factorâ–³FosB and CREB/p-CREB during melatonin exerts its reversal effect on the rewarding properties of morphine, to make clear the moleculer mechanisms of the effect, providing fundamental basis for the likely clinical application of melatonin and its homologue in the treatment of psychological dependence to opioids and contributing to the understanding of the neurobiology of opioids addiction.1. Establishment of morphine-induced CPP modelTo establish morphine-induced conditioned place preference model and observe the expression of this effect, the mice were divided into control group (NS-paired group) and model group (morphine-paired group), and received NS (10ml/kg, s.c.) and Mor (3mg/kg,s.c.) respectively for 5 consecutive days. Conditioned place preference was tested for 3 days following the last drug-paired conditioning trail, and evaluated as the difference in post-conditioning vs. pre-conditioning time spent in the drug-paired compartment, which is termed as the CPP score. The results showed that morphine was able to induce significant place preference in mice, indicated by a significant increase in the CPP score (P < 0.01 compaired with control group). The expression of this effect maintained at least 3 days. Conditioned place preference in rats was induced in the similar procedure as that in mice except that Mor (5mg/kg, s.c.) was administrated for 6 consecutive days and the place preference was tested for 2 days. The results showed that morphine induced significant place preference in rats (P < 0.01), and the expression of this effect maintained at least 2 days. As is described by most references, the place preference was tested in the first day following the last drug-paired conditioning trail in all subsequent studies, to observe the effect of melatonin on the development and expression of morphine-induced CPP and investigate the involvement of melatonin receptors in CNS in this effect.2. The effects of melatonin on rewarding properties of morphine2.1 Effect of i.p. administration of melatonin on the development of morphine-induced CPP in miceThe CPP in mice was induced as described above. Melatonin (50 and 25 mg/kg, i.p.) was co-administrated with morphine everyday. The results showed that co-administrated with morphine, melatonin didn't alter the CPP score, indicating that melatonin may have no effect on the development of morphine induced CPP.Another scheme of melatonin administration was adopted to further analysis the effect of melatonin on the development of morphine-induced CPP. The CPP in mice was induced under the same schedule as the above. Melatonin (50 mg/kg, i.p.) was co-administrated with morphine from the third day of the drug-paired conditioning trail. The results showed that melatonin didn't alter the CPP score either in this scheme, indicating further that melatonin may have no effect on the development of morphine-induced CPP.2.2 Effect of i.p. administration of melatonin on the expression of morphine-induced conditioned place preference in miceThe CPP in mice was induced under the same schedule as described in item 1. Melatonin (50, 25 and 12.5 mg/kg, i.p.) was administrated 20 min before the CPP testing to observe the effect of melatonin on the expression of morphine-induced CPP. The results showed that the CPP score in the group treated with melatonin either 25 mg/kg or 50 mg/kg was significantly less than that in the model group (P < 0.01), indicating that melatonin was likely to reverse the expression of morphine-induced CPP in mice in a dose dependent manner.2.3 Effect of i.p. administration of melatonin on the expression of morphine-induced conditioned place preference in ratsThe CPP in rats was induced as described in item 1. Melatonin (50 and 25 mg/kg, i.p.) was administrated 20 min before the CPP testing to observe the effect of melatonin on the expression of morphine-induced CPP in rats. The results showed that the CPP score in the group treated with melatonin 50 mg/kg was significantly less than that in the model group (P < 0.01), indicating that melatonin may reverse the expression of morphine-induced CPP in rats in a dose dependent manner.2.4 Effect of melatonin on conditioned place preference inductionThe CPP paradigm underwent as described in item 1. The mice were treated with melatonin (50mg/kg, i.p. once daily for 5 consecutive days) instead of morphine. The results showed that although melatonin seemed to increase the CPP score, statistical analysis revealed that there was no significant difference between the melatonin paired group and the control group, indicating that melatonin may not induce CPP.3. Melatonin receptor subtypes within the CNS mediating the reversal effect of melatonin on the expression of morphine-induced CPP3.1 Effects of i.c.v. administration of melatonin on the expression of morphine-induced CPPMice after recovery from surgery were conditioned with morphine as described in item 1. Melatonin (0.5, 0.25 and 0.125 mg/kg) was intracerebroventricular (i.c.v.) administrated 10 min before the CPP testing. The results showed that morphine was able to induce significant place preference (P < 0.01) after recovery from surgery for i.c.v. injection. The CPP score in the group treated with melatonin either 0.25 mg/kg or 0.5 mg/kg was significantly less than that in the model group (P < 0.01). Compared with the results of item 2.2, the effective dose for melatonin administrated i.c.v. was roughly about 1/100 of that for melatonin administrated i.p.. The data further indicated that melatonin may reverse the expression of morphine-induced CPP and implied that the CNS may be the primary site for melatonin to exert the effect.3.2 Effect of melatonin receptor antagonists on the reversal effect of melatonin on the expression of morphine-induced conditioned place preference Luzindole is the non-selective antagonists on the MT₁ and MT₂ subtype. K185 is the selective antagonist on the MT₂ subtype. The present study was designed to evaluate the effect of i.c.v. luzindole or K185 on the reversal effect of melatonin on the expression of morphine-induced CPP, so as to clarify which of the melatonin receptor subtype within the CNS was mediating the action. It was found that the i.c.v. administration of either K185 (5, 20μg ) or luzindole (6.25, 12.5μg) significantly antagonized the reversal effect of melatonin(50 mg/kg, i.p.) on the expression of morphine-induced CPP, while i.c.v. 20μg of K185 or 12.5μg of luzindole by itself did not alter the expression of morphine-induced CPP. These results suggest that the reversal effect of melatonin on the expression of morphine-induced CPP may be mediated by the activation of MT₁ and MT₂, especially MT₂ subtype within the CNS.4. The molecular mechanism of the reversal effect of melatonin on the expression of morphine-induced rewarding effect4.1 Changes of the intensity of CREB immunoreactivity in the nucleus accumbens and the hippocampus during reversal of melatonin on the expression of morphine-induced CPP in rats.The experimental rats were divided into three groups: the Control group, the Model group and the Mel group. The rats in the Model and Mel group received morphine (5 mg/kg, s.c., once each day for 6 consecutive days). From day 7, they received an i.p. administration of vehicle or Mel (50mg/kg) twice daily (in the morning and the afternoon) for 3 times. 6 hrs after the last i.p. administration, the rats were sacrificed and the brain sections of the nucleus accumbens (NAc) and the hippocampus (Hip) were prepared. The immunoreactivities of CREB were detected by immunohistochemistry analysis. The average gray scale was measured using the computer-assisted image processing technique. It was observed that CREB-like immunoreactivity of the neurons in the NAc and Hip was much increased in sections treated with morphine. The gray scale values were also decreased significantly (P < 0.01). Compared with Model group, the CREB-like immunoreactivity of the neurons in the NAc and Hip was not altered in sections of Mel group. There was no significant change of the gray scale (P > 0.05). The results suggest that there is an upregulation of CREB protein level in the NAc and Hip during the expression of morphine-induced rewarding effect in rats, while melatonin may have no influence on it.4.2 Changes of the intensity of p-CREB immunoreactivity in the nucleus accumbens and the hippocampus during reversal of melatonin on the expression of morphine-induced CPP in rats.The experimental protocol was the same as item 4.1. It was observed that the p-CREB-like immunoreactivity of the neurons in the NAc and the Hip was much increased in sections treated with morphine. The gray scale values were also decreased significantly (P < 0.01). The p-CREB-like immunoreactivity of the neurons in the NAc and the Hip of the Mel group was much decreased compared with Model group. The gray scale values were increased significantly (P < 0.01). The results suggest that melatonin may decrease the upregulation of p-CREB protein level in the NAc and the Hip during it reverses the expression of morphine-induced rewarding effect in rats.4.3 Changes of the FosB protein level in the nucleus accumbens and the hippocampus during reversal of melatonin on the expression of morphine-induced CPP in rats.4.3.1 Changes of the intensity of FosB immunoreactivity in the nucleus accumbens and the hippocampus during reversal of melatonin on the expression of morphine-induced CPP in rats.The experimental protocol was the same as item 4.1. No differences of the FosB-like immunoreactivity of the neurons in the NAc and the Hip were observed in the sections of the three groups. There was no significant change in the gray scale values among groups (P>0.05).4.3.2 Changes of theâ–³FosB protein level in the nucleus accumbens and the hippocampus during reversal of melatonin on the expression of morphine-induced CPP in rats.As the antibody we used in the immunohistochemistry test recognize both FosB and its truncated splice variantâ–³FosB, the changes ofâ–³FosB may not be able to be observed in the immunohistochemistry sections. Hence, we use the Western Blotting method to evaluate the effects of melatonin on theâ–³FosB protein levels in the NAc and the Hip during the expression of morphine-induced rewarding effect.The experimental protocol was the same as item 4.1. 6 hrs after the last Mel administration, the rats were sacrificed and the NAc and the Hip were separated. The tissue lysate was prepared and analyzed by Western blotting using the FosB antibody and the protein bands were determined by densitometric analysis. There was no difference among groups (P > 0.05).Summary1. Melatonin may have no influence on the development of morphine-induced rewarding effect; while it reverses the morphine-induced rewarding effect without inducing rewarding effect.2. The CNS may be the primary site for melatonin to exert its reversal effect on the rewarding properties of morphine; and this action may be mediated by the activation of MT₁ and MT₂, especially MT₂ melatonin receptor subtype within the CNS.3. The reversal effect of melatonin on rewarding properties of morphine may be relavent to its ability to decrease the upregulation of p-CREB protein level in the nucleus accumbens and hippocampus during the expression of morphine-induced rewarding effect.