Effects of melatonin in a place preference conditioning depend on the time of administration

Transcription

1 Pharmacological Reports 2010, 62, ISSN Copyright 2010 by Institute of Pharmacology Polish Academy of Sciences Effects of melatonin in a place preference conditioning depend on the time of administration Mariusz Papp, Ewa Litwa, Magdalena asoñ-tyburkiewicz, Piotr Gruca Institute of Pharmacology, Polish Academy of Sciences, Smêtna 12, PL Kraków, Poland Correspondence: Mariusz Papp, nfpapp@cyf-kr.edu.pl Abstract: A conditioned place preference paradigm was used to assess potential rewarding properties of melatonin. The conditioning with melatonin was carried out at two periods of the 12-h light/dark cycle: in the morning ( ) and in the evening ( ). Morning administration of melatonin (2.5, 5 and 10 mg/kg) did not support conditioned place preference. In contrast, evening conditioning with melatonin caused a clear shift towards the drug-paired side. This effect was dose-dependent; higher doses of 2.5, 5, 10 and 50 mg/kg induced conditioned preference while lower doses of 0.5 and 1 mg/kg were ineffective. The increase in the side preference induced by the two most effective doses of melatonin (10 and 50 mg/kg) were comparable to that induced by 1 mg/kg of amphetamine, and was significantly attenuated by the melatonin antagonist, S (20 mg/kg). In chronic experiment, melatonin (10 mg/kg) caused similar increase of the time spent on conditioned side both in animals administered vehicle for 7 days and in rats receiving 10 mg/kg of melatonin for the same period of time. Potent activity in the conditioned preference model suggests that melatonin may have rewarding properties, which moreover, is not tolerated following repeated pre-exposure to the drug. These findings may indicate potential abuse liability of melatonin, and therefore, its use by humans should require a careful monitoring for abuse and/or dependency. Key words: conditioned preference, reward, melatonin, S 22153, amphetamine, acute and repeated administration, circadian, rats Introduction Melatonin is a neurohormone that in humans as well as in animals is mainly synthesized by the pineal gland. The production and physiological activity of the melatonin system are regulated by diurnal and/or circadian rhythms, and are sensitive to light, which suppresses its activity in both diurnal and nocturnal organisms [34]. The best described physiological function of melatonin is the coordination of various biological rhythms, and therefore, the majority of animal and clinical studies with this drug concentrate on its chronobiotic or resynchronizing properties. In consequence of these studies, several therapeutic indications of melatonin have been suggested for the treatment of various circadian rhythms and sleep disorders and in some countries the use of the drug is currently quite extensive [1, 34]. In addition to chronobiotic and sleep promoting activities of melatonin, a number of other effects have been recently described in animals. For example, melatonin, as well as some of its synthetic agonists, has been reported to be active in models for anticonvulsant [8], analgesic [6, 33] and anxiolytic [9, 17, 18, 21] actions of drugs. Moreover, a clear antidepressant-like properties of melatonin has been reported in forced swim test [22] and chronic stress models [4, 20]. If all these animal activities of melatonin find confirmation in controlled clinical trials, the range of Pharmacological Reports, 2010, 62, !

2 therapeutic indications for both acute and chronic treatment with this drug may increase dramatically. This raises the problem of toxicology and adverse side-effects in patients undergoing short- and longterm melatonin therapy, and although this drug appears to be well tolerated and to produce relatively mild unwanted effects, the number of studies evaluating this problem is surprisingly small [1, 11, 14, 34]. Also in animal studies a very little attention has been paid to this issue, including the lack of any information on its putative psychomimetic properties. Therefore, the purpose of the present study was to evaluate the action of melatonin in a place preference conditioning (PPC) paradigm. This procedure is frequently used in studies on rewarding properties of psychoactive agents, and several drugs of abuse (e.g., psychostimulants, benzodiazepines, opiates, alcohol) have been shown to support the conditioned preference, while agents with little or no potential for abuse (e.g., antihistaminergic, neuroleptics) are generally inactive in this model [for review see: 5, 24, 28, 29]. The activity of various doses of melatonin in the PPC model was compared to the effect of amphetamine, a drug with known rewarding properties. According to the literature data, many, if not all, effects of melatonin in both humans and animals depends not only on the dose but also on the time of administration [see: 6 9, 34]. Therefore, in order to evaluate the role of diurnal rhythm in the action of melatonin in PPC, the drug was tested in the morning (i.e., when it is devoid of chronobiotic activity) and in the evening (see below for details). Finally, since in the clinical situation melatonin is used both acutely and chronically, the PPC study was conducted on naive animals and following 7 days of melatonin pretreatment. It was found in this study that while morning administration of melatonin was ineffective, the evening conditioning with melatonin caused a potent (i.e., comparable to that of amphetamine) conditioned place preference, and that this effect was not affected by 7 day pretreatment with the same dose of the drug. Materials and Methods Animals The experiments were carried out on male Wistar rats (Charles River, Germany) weighing g. The animals were housed in groups of 6 with food and water freely available, and were maintained on a 12-h light/dark cycle (lights on at 08.00) in a constant temperature (22 ± 2 C) and humidity (50 ± 5%) conditions. The study was conducted in compliance with the Polish Animal Protection Law of August 21, Apparatus The animals were trained and tested in ten wooden chambers containing white and black arms ( cm) with a different floor texture (plain wood or wire mash) and a grey central area ( cm). Both compartments were separated from the grey area by guillotine doors. Procedure On the first 3 days the animals were placed in the central area. After 5 s, the guillotine doors were raised and each rat was allowed freely to explore the whole chamber for 10 min. On day 4, the procedure was repeated and the time spent by animals in each part was manually recorded (through a mirror located 1.5 m above the chambers) in a 10 min pre-conditioning test. This measure was used to establish initial preference for either of the two arms of the chamber. Only those animals, which showed a clear preference for the black arm, were used for further testings. On days 5 12 the animals (n = 8 9 rats per group) were confined on alternate days for 60 min to either of the two arms (white or black), following injection of an active drug or vehicle. The drugs were injected prior to confinement to the white arm (initially non-preferred) and vehicle prior to confinement to the black arm (initially preferred). Changes in place preference were measured drug-free on day 13. The animals were again placed in the central area, after 5 s the guillotine doors were raised, and the time spend in each arm was measured manually in a 10 min post-conditioning test. As mentioned above, the conditioning with melatonin was carried out at two periods of the 12-h light/dark cycle: in the morning ( ) and in the evening ( ). In the evening experiment, an effect of melatonin antagonist, S (20 mg/kg), injected alone and in combination with melatonin, was also studied. The conditioning with amphetamine was carried out in the morning period. In a separate evening experiment, the place preference procedure was conducted as described above but the " Pharmacological Reports, 2010, 62,

3 Melatonin in place preference conditioning Mariusz Papp et al. animals were administered vehicle or 10 mg/kg of melatonin for 7 days before the conditioning. Drugs Melatonin (Sigma Aldrich) was suspended in 0.5% hydroxyethylcellulose (Merck), which was used as vehicle. D-amphetamine sulfate (Sigma Aldrich) was dissolved in distilled water. All injections were ip in a volume of 1 ml/kg body weight and were administered 30 min before confinement to the chamber. Statistics The results were analyzed by the two-way analysis of variance, with test (pre- and post-conditioning) and treatment (vehicle and drugs) as grouping variables and the time spent on conditioned side as dependent variable. The Fisher s Least Significant Difference (LSD) test was used for post-hoc comparisons of means; p values lower than 0.05 were considered statistically significant. Results The times spent on initially non-preferred side before and after evening conditioning with melatonin are shown in Figure 3. In contrast to the morning experiment, evening administration of melatonin caused a clear shift towards the drug-paired side. In consequence, when compared to vehicle injections, the overall effect of evening conditioning with melatonin led to significant effects of treatment [F(6,110) = 5.226; p < 0.001], test [F(1,110) = ; p < 0.001] and interaction [F(6,110) = 4.281; p < 0.001]. This effect was dose-dependent; high dose regimen of melatonin (2.5, 5, 10 and 50 mg/kg) was required to produce a significant increase in conditioned preference whilst the animals receiving lower doses of 0.5 and 1 mg/kg did not show any changes in place preference (Fig. 3). An increase in the side preference induced by the two most effective dose of melatonin (i.e., 10 and 50 mg/kg) were comparable to that induced by 1 mg/kg of amphetamine [F(2,23) = 0.065; NS]. The melatonin antagonist, S 22153, alone slightly and not significantly increased the post-conditioning time on the drug-paired side [test effect: F(3,28) = 1.579; NS]. However, when S was co-administered during evening acquisition of melatonin conditioned response, the shift in place preference was attenuated, resulting in a significant effects of treatment and test, In the pre-conditioning test, all animals showed a strong preference for the black side of the chamber and the times spent on the white (i.e., to-be-conditioned) side before conditioning did not differ significantly between any groups [morning exp.: F(5,43) = 0.035; NS, evening exp.: F(6,55) = 0.107; NS]. Amphetamine (1 mg/kg) administered in association with the initially non-preferred side caused a significant increase in the time spent on conditioning side in the post-conditioning test, resulting in a significant effects of treatment, test and interaction [F(1,30) = 8.317; p = 0.007, F(1,30) = 9.984; p = 0.004, F(1,30) = 8.428; p = 0.007, respectively, Fig. 1). As shown in Figure 2, morning administration of melatonin did not support conditioned place preference. This means that the times spent on initially non-preferred side following morning conditioning with any of the three doses of melatonin (2.5, 5 and 10 mg/kg) were not significantly different from those measured in the pre-conditioning test [treatment effect: F(3,56) = 0.010; NS, test effect: F(1,56) = 2.425; NS, interaction: F(3,56) = 0.013; NS]. Fig. 1. The effect of amphetamine (AMP, 1 mg/kg) on the conditioned place preference. Values are the means (± SEM) of time spent on the conditioning. *** p < 0.001; relative to pre-conditioning scores Pharmacological Reports, 2010, 62, #

4 Fig. 2. The effect of morning administration of melatonin (Mel, mg/kg) on the conditioned place preference. The drug was injected at am, i.e., two hours after the dark phase of the 12-h light/dark cycle. Values are the means (± SEM) of time spent on the conditioning Fig. 4. The effect of S22153 (S22, 20 mg/kg) on the conditioned place preference induced by melatonin (Mel, 10 mg/kg). Values are the means (± SEM) of time spent on the drug-associated side before (open bars) and after (hatched bars) conditioning. *** p < 0.001; relative to pre-conditioning scores, p < 0.001; relative to Mel alone Fig. 3. The effect of evening administration of melatonin (Mel, mg/kg) on the conditioned place preference. The drug was injected at pm, i.e., two hours before the dark phase of the 12-h light/dark cycle. Values are the means (± SEM) of time spent on the conditioning. *** p < 0.001; relative to pre-conditioning scores Fig. 5. The effect of chronic melatonin (Mel) pretreatment (10 mg/kg for 7 days) on the conditioned place preference induced by Mel (10 mg/kg). Values are the means (± SEM) of time spent on the conditioning. ** p < 0.01, *** p < 0.001; relative to pre-conditioning scores and interaction [F(1,28) = 7.457; p < 0.05, F(1,28) = ; p < 0.001, F(1,28) = 6.829; p < 0.05, Fig. 4]. In the chronic experiment (Fig. 5), melatonin (10 mg/kg) caused a similar increase of the time spent on conditioned side both in animals administered vehicle for 7 days (p = 0.003) and in rats receiving 10 mg/kg of melatonin for the same period of time (p < 0.001), leading to a significant test effect [F(1,64) = ; p < 0.001] and test treatment interaction [F(3,64) = 3.263; p < 0.05]. Discussion The PPC paradigm has been demonstrated to have high predictive validity for detecting compounds with an abuse potential in humans [5, 28, 29]. The present study shows that, in addition to a number of psychoactive drugs found active in this model, also melatonin used at doses effective in animal models of $ Pharmacological Reports, 2010, 62,

5 Melatonin in place preference conditioning Mariusz Papp et al. anxiety and depression, can increase subsequent preference for the environment in which it was administered. The magnitude of this effect is comparable to that of amphetamine, a drug with known rewarding properties. The ability of melatonin to support PPC clearly depends on the time of its administration. In other words, a shift in the conditioned preference was observed only in the evening experiment, while when injected in the morning, melatonin failed to elicit the conditioned response. This is consistent with its known chronobiotic properties in humans and with many other reports showing that most of behavioral effects of melatonin in animals show similar time-dependency. For example, in a series of elegant studies Golombek demonstrated that analgesic [6], anticonvulsant [8] and anxiolytic [9] actions of melatonin are substantially stronger if the drug was administered around the beginning of dark phase of the diurnal light/dark cycle [see also: 10]. Also in the chronic mild stress model of depression, melatonin was active only following evening administration; five weeks of its morning injections were ineffective against the stress-induced anhedonia [20]. Finally, a morning or early afternoon administrations of melatonin and other agonists of the MT1 and MT2 receptors were found inactive in the PPC paradigm as well as in other models for the preclinical abuse potential evaluation, as reported by Hirai [13] and Wiley [32]. Thus, our findings confirm that, like other behavioral activities of melatonin, its involvement in the brain reward processes is also subject to diurnal temporal regulation. The ability of melatonin to support PPC was sustained following 7 days of pretreatment, suggesting that no tolerance develops to its action in this paradigm. Also, as compared to acute experiment, the melatonin-induced PPC was not enhanced in pretreated animals, indicating that repeated administration does not cause sensitization, either. These data are in line with other reports showing that the behavioral activity of melatonin is comparable following both acute and chronic exposure to the drug [3, 15, 22, 25]. The acquisition of the melatonin-induced PPC was significantly attenuated by the MT1/MT2 receptors antagonist, S [31], confirming the involvement of these melatonin receptors in the development of this conditioned response. Similar effect was observed in the study, where S also potently antagonized the antidepressant-like activity of melatonin in the chronic mild stress model [20]. The melatonin MT1 and MT2 receptors have been cloned, identified as members of the G proteincoupled receptor family, and their localization in several brain regions was shown in a number of studies [19, 23]. Among others, melatonin receptors were identified in the mesolimbic areas (e.g., ventral tegmental area, ventral striatum), where they are colocalized with the dopaminergic neurons in both rodents and humans [30]. Although the effect of melatonin on the turnover of dopamine (DA) in nucleus accumbens and striatum is not clear [16, 36], the drug was shown to increase the affinity of D2 receptors in the rat striatum [12], providing further evidence for an intimate interaction between those two systems. Accordingly, in a number of behavioral and biochemical studies, convincing evidence were presented that melatonin can modify various behaviors controlled by the mesolimbic dopaminergic system, including locomotor and exploratory activities and emotional responses as well as that dopaminergic ligands can modify the effects of melatonin [2, 26, 35]. The above data suggest, however that melatonin inhibits, rather than potentiates, the activity of dopaminergic neurotransmission [27, 36]. Therefore, the interaction of melatonin with the mesolimbic DA system cannot explain the activity of melatonin in the PPC paradigm, suggesting an involvement of other systems. One of the candidates appears to be the benzodiazepine-gaba ) receptor complex; these receptors are also under a clear influence of melatonin and are believed to play an important role in mechanism of many of behavioral and biochemical effects of melatonin [6 8, 10, 22, 27]. Interestingly, the ligands acting at the benzodiazepine-gaba ) receptor complex have been found to share many behavioral properties of melatonin, and this includes their ability to support the place preference conditioning [28, 29]. Potent activity in the PPC model suggests that melatonin may have rewarding properties, which moreover, is not tolerated following repeated (7 days) pre-exposure to the drug. These findings may indicate abuse liability of melatonin, and therefore, its use by humans should require a careful monitoring for abuse and/or dependency. It should be, however, noted that in this study we used a biased procedure, in which animals showed strong, unconditioned preference for one side of the apparatus. Thus, in the pre-conditioning test, the rats spent substantially more time in the black Pharmacological Reports, 2010, 62, %

6 arm of the chamber (> 6 min) than in the initially non-preferred, white arm (< 2 min). Therefore, the changes in place preference induced by melatonin could alternatively be attributed to its antiaversive or antianxiety activity [see: 5, 27, 28]. This issue is particularly important for interpretation of the present results, since many reports show that melatonin is active in animal models of anxiety [9, 17, 18, 21]. In other words, the finding that melatonin supports the PPC does not necessarily mean that the drug is rewarding and as such has abuse potential; a shift of preference towards the initially non-preferred compartment could also occur through anxiolytic action of melatonin, resulting in more rapid habituation to the aversive properties of that environment. This possibility requires further studies using experimental procedures, which would allow differentiation of these two activities. References: 1. 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