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Effects of melatonin on behavioral dopaminergic supersensitivity
Life Sciences 72 (2003) 3003 – 3015 www.elsevier.com/locate/lifescie
Effects of melatonin on behavioral dopaminergic supersensitivity Vanessa C. Abı´lio, Joa˜o A.R. Vera Jr., Leonardo S.M. Ferreira, Carlo R.M. Duarte, Ce´sar R. Martins, Danila Torres-Leite, Rosana de A. Ribeiro, Roberto Frussa-Filho * Department of Pharmacology, Escola Paulista de Medicina/UNIFESP, Edifı´cio Jose´ Leal Prado-Rua Botucatu, 862 CEP 04023-062, Sa˜o Paulo, Brazil Received 11 July 2002; accepted 5 December 2002
Abstract This study examines the effects of melatonin on dopaminergic supersensitivity induced by long-term treatment with haloperidol in rats. Enhancements of spontaneous general activity in an open-field and of stereotyped behavior induced by apomorphine after abrupt withdrawal from long-term treatment with haloperidol were used as experimental parameters for dopaminergic supersensitivity. Experiment 1 was conducted to investigate the effects of melatonin on the development of dopaminergic supersensitivity, and experiment 2 was conducted to investigate the effects of melatonin on the development as well as on expression of dopaminergic supersensitivity. Rats of both experiments were long-term treated with saline or haloperidol concomitant to saline or melatonin. In experiment 1 behavioral observations were performed after abrupt withdrawal from long-term treatment. In experiment 2 behavioral observations were performed 1 hour after an acute injection of saline or melatonin, administered after the abrupt withdrawal from long-term treatment. Both behavioral parameters used showed the development of central dopaminergic supersensitivity in rats treated with haloperidol since 24 hours after abrupt withdrawal. Concomitant treatment with melatonin intensified haloperidol-induced dopaminergic supersensitivity, observed 72 hours after withdrawal. Melatonin treatment per se also induced behavioral supersensitivity evaluated by both open-field and stereotyped behaviors, although it was more fugacious than that presented by haloperidol. Acute treatment with melatonin reverted the enhancement of the haloperidol-induced dopaminergic supersensitivity produced by concomitant long-term treatment with melatonin, as well as melatonin-induced dopaminergic supersensitivity per se. Our results support previous evidence of antidopaminergic effects of melatonin and demonstrate that repeated administration of this hormone modifies the plasticity of behaviors mediated by central dopaminergic systems. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Melatonin; Dopamine; Dopaminergic supersensitivity; Rat; Behavior
* Corresponding author. Fax: +55-11-273-1766. E-mail address: [email protected] (R. Frussa-Filho). 0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(03)00231-5
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Introduction The central nervous system is not static and immutable. Neuronal circuitry is highly adaptable and can maintain homeostasis in response to internal and/or external perturbation. In rats, abrupt withdrawal from long-term administration of dopaminergic antagonists such as haloperidol [1,7,45,61], bromopride [20], metoclopramide [23] or sulpiride [24] enhanced not only the general activity observed in an open-field but also the responses to apomorphine-induced stereotyped behavior. These effects have been considered to be a consequence of the development of supersensitivity of central dopaminergic pathways [40]. Indeed, behavioral supersensitivity is thought to result from receptor site proliferation in mesolimbic and striatal brain tissues in response to chronic dopamine receptor blockade [11,22,39,62]. From a clinical point of view, whereas striatal dopaminergic supersensitivity has been proposed as a possible contributing factor to the development of tardive dyskinesia in schizophrenics receiving long-term neuroleptic treatment [12,18,28,31,41], mesolimbic dopaminergic supersensitivity has been suggested to be related to neuroleptic-induced supersensitivity psychosis [13,14] as well as to the increased responsiveness to drugs such as cocaine in human stimulant abusers chronically treated with neuroleptics [33]. Recently, we have described that rats abruptly withdrawn from a long-term haloperidol treatment concomitant to continuous exposure to light presented an enhancement in dopaminergic behaviors but an attenuation of the development of dopaminergic supersensitivity suggesting that continuous exposure to light leads to an increase in dopaminergic function that can attenuate the compensatory supersensitivity induced by long-term blockade of dopaminergic receptors [1]. In this respect, the suppression of melatonin secretion, a hormone produced by the pineal gland, is one of the most consistent biochemical effects of light exposure in mammals [2,9,34–37,42,48,50,52,53]. In addition, melatonin receptors have been widely identified in the central nervous system of mammals [5,17,44] including dopaminergic areas as the substantia nigra [29] and the striatum [32,64]. The aim of the present study was to verify the effects of a long-term treatment with exogenous melatonin on haloperidol-induced dopaminergic supersensitivity using both open-field general activity and apomorphine-induced stereotyped behavior in rats as experimental parameters.
Method Subjects Male Wistar EPM-1 rats, born and raised under our laboratory conditions, weighing 250-300 g and about 3 months of age at the beginning of the experiments were used. Groups of 5 or 6 animals were kept in Plexiglass cages with free access to food and water in a room with controlled temperature (22 F 1 jC) and under a 12 h light/dark cycle with lights on at 7:00 am. The animals were maintained and used in accordance to the guidelines of the Committee on Care and Use of Laboratory Animal Resources, National Research Council, USA. Drugs and treatment Haloperidol (Crista´lia, Sa˜o Paulo, Brazil) and melatonin (RBI, Natick, MA) were freshly diluted in distilled water. Melatonin was previously suspended in Tween 80 (3%). Apomorphine hydrochloride
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(Sandoz Basal, Switzerland) was diluted in 0.2% ascorbic acid. Saline and saline + Tween 80 were used as haloperidol and melatonin control solutions, respectively. Haloperidol and melatonin were administered intraperitoneally between 8:00 and 9:00 am, whereas apomorphine was administered subcutaneously in volumes not exceeding 1.0 ml/kg body weight. Open-field measures The open-field was constructed as described by Broadhurst [10] and rats were observed individually for 5 minutes. Hand-operated counters and stopwatches were employed to score ambulation frequency (number of floor units entered), rearing frequency (number of times the animal stood on hind legs) and duration of immobility (time that the animal stood still without showing any movements). The open-field was washed with a solution of alcohol and water (5.0%) before the placement of each animal to obviate possible bias due to odor clues left by previous subjects. To minimize possible effects of circadian changes on open-field behavior, experimental and control observations were alternated, each rat being tested at the same time in each session. Stereotypy measures The animals were observed for stereotyped behavior in wire mesh cages (16 30 19 cm) without food or water. Stereotypy was quantified every 10 minutes for 100 minutes after apomorphine administration (0.6 mg/kg) according to the scoring system proposed by Setler et al. [56]. Briefly, scores ranging from 0 to 6 were attributed to an animal’s behavior. The grading system was as follows: 0- asleep or stationary, 1- active, 2- predominantly active but with bursts of stereotyped sniffing and/or rearing, 3- constant stereotyped activity such as sniffing or rearing but with locomotion activity still present, 4- constant stereotyped activity maintained in one location, 5- constant stereotyped activity but with bursts of licking and/or gnawing and biting, 6- continuously licking and/or gnawing of cage grids. This criterion was not subjective, as shown by the excellent scoring agreement (Pearson’s correlation, v = 0.98) of two different and independent observers. In each group, the total sum of stereotypy scores for each animal was used for statistical analysis. Experimental design Experiment 1 This experiment was performed to verify the effects of melatonin on the development of dopaminergic supersensitivity. Rats were divided at random into 4 groups: SAL + SAL, MEL + SAL, SAL + HAL and MEL + HAL, injected once daily for 30 days with saline (SAL) or 5.0 mg/kg melatonin (MEL) and, 30 minutes after these injections, with saline or 2.0 mg/kg haloperidol (HAL). Thirty minutes, 24, 48, 72, 240 and 480 hours (sessions 1, 2, 3, 4 and 5) after withdrawal from chronic treatment rats were placed individually in the center of the open-field arena for observation of behavioral parameters. Four hours after the last three open-field sessions (76, 244 and 484 h after withdrawal) all animals were injected with apomorphine (0.6 mg/kg) and observed for stereotyped behavior quantification.
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Experiment 2 This experiment was performed to verify the effects of melatonin on the development and expression of dopaminergic supersensitivity. Rats were divided at random into 4 groups: SAL + SAL - SAL, MEL + SAL - MEL, SAL + HAL - SAL and MEL + HAL - MEL, injected once daily for 30 days with saline (SAL) or 5.0 mg/kg melatonin (MEL) and, 30 minutes after these injections, with saline or 2.0 mg/kg haloperidol. Thirty minutes, 24 and 48 hours (sessions 1, 2 and 3) after withdrawal from chronic treatment rats were placed individually in the center of the open-field arena for observation of behavioral parameters, and 76 h after withdrawal from chronic treatment these animals were injected with apomorphine (0.6 mg/kg) and observed for stereotyped behavior. All these behavioral observations were performed one hour after an injection of saline (-SAL) or 5.0 mg/kg of melatonin (-MEL). This protocol was decided based on the results of experiment 1 in order to avoid that two behavioral tests were performed at the same day, which would implicate two injections of melatonin before the second observation. Each rat was used in only one experiment and in both experiments the observer was blind to the identity of the animal. Statistical analysis Since homocedasticity is necessary for the analysis of variance, Bartlet’s test was performed. It was concluded that the open-field data were parametric. An analysis of variance (ANOVA) followed by Duncan’s test was used to study the open-field data. Stereotyped scores were treated by Kruskal–Wallis analysis of variance followed by the two-tailed Mann–Whitney U test.
Results Experiment 1 The open-field behavior of rats abruptly withdrawn from long-term treatment with haloperidol or saline concomitant to melatonin or saline is shown in Fig. 1. Thirty minutes after the last injection, locomotion (A) and rearing (B) frequencies were reduced and duration of immobility (C) was increased in the SAL + HAL and MEL + HAL groups when compared to the SAL + SAL group [F(3,44) = 69.34, 62.32, 127.65, p < 0.0001, respectively]. When compared to the SAL + SAL group, rats of the SAL + HAL group showed increased locomotion frequency (A) in sessions 2, 3 and 4 [F(3,44) = 6.54, 5.93, 7.53, p < 0.005, respectively], and decreased duration of immobility (C) in session 2 [F(3,44) = 4.80, p < 0.05]. Rearing frequency was not altered by haloperidol treatment in any of the sessions. The increase in locomotion frequency produced by abrupt withdrawal from haloperidol treatment was intensified by melatonin co-treatment in session 4. Furthermore, when compared to the SAL + SAL group, rats of the MEL + HAL group showed increased locomotion frequency (A) in sessions 2, 3 and 4, increased rearing frequency in sessions 3 and 4 [F(3,44) = 5.78, 5.84, p < 0.05], and decreased duration of immobility in sessions 2, 3 and 4 [F(3,44) = 4.80, 5.38, 5.49, p < 0.05]. In addition, the MEL + HAL group when compared to the SAL + HAL group presented increased rearing frequency (B) in sessions 3 and 4, and decreased duration of immobility (C) in session 4. Melatonin withdrawal per se produced an slight increase in general activity. Thus, the MEL + SAL group when compared to the SAL + SAL group
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Fig. 1. Open-field locomotion frequency (A), rearing frequency (B) and duration of immobility (C) of rats abruptly withdrawn from long-term treatment with saline (SAL) or 2.0 mg/kg haloperidol (HAL) concomitant to saline or 5.0 mg/kg melatonin (MEL). Data are reported as mean F SEM. Analysis of variance followed by Duncan’s test. * p < 0.05 compared to the SAL + SAL group. 1 p < 0.05 compared to the SAL + HAL group.
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presented decreased duration of immobility in all sessions after withdrawal, reaching statistical significance in session 4. Apomorphine-induced stereotyped behavior of rats abruptly withdrawn from long-term treatment with haloperidol or saline concomitant to melatonin or saline is shown in Fig. 2. When compared to the SAL + SAL group, the SAL + HAL and MEL + HAL groups presented stereotypy scores significantly higher 76 and 244 hours after abrupt withdrawal (H = 31.61, 8.81, p < 0.05, respectively). Furthermore, when compared to SAL + SAL group, the MEL + HAL group also presented significantly higher stereotypy scores 484 hours after withdrawal. In addition, the MEL + HAL group when compared to the SAL + HAL group presented higher stereotypy scores 76 h after withdrawal. Melatonin withdrawal per se induced a slight increase in stereotypy behavior. Thus, 76 (but not 244 and 484) hours after withdrawal, the stereotypy intensity shown by rats of the MEL + SAL group was significantly higher than that of the SAL + SAL group. Experiment 2 The open-field behavior of rats acutely injected with saline or melatonin after an abrupt withdrawal from a long-term treatment with saline or haloperidol concomitant to saline or melatonin is shown in Fig. 3. Thirty minutes after withdrawal, locomotion (A) and rearing (B) frequencies were reduced and duration of immobility (C) was increased in the SAL + HAL - SAL and MEL + HAL - MEL groups when compared to the SAL + SAL - SAL group [F(3,35) = 8.72, 19.17, 19.87, p < 0.0005, respectively]. The SAL + HAL - SAL group, when compared to the SAL + SAL - SAL group, showed an increase in the locomotion frequency (A) in sessions 2 and 3 [F(3,35) = 9.62, 7.38, p < 0.001]. This increase was attenuated by melatonin treatment since the MEL + HAL - MEL group when compared to the SAL + SAL - SAL group presented an increase in locomotion frequency (A) only in session 2.
Fig. 2. Apomorphine-induced stereotyped behavior of rats abruptly withdrawn from long-term treatment with saline (SAL) or 2.0 mg/kg haloperidol (HAL) concomitant to saline or 5.0 mg/kg melatonin (MEL). Data are reported as mean F SEM. Kruskal – Wallis analysis of variance followed by the Mann – Whitney U test. * p < 0.05 compared to the SAL + SAL group. 1 p < 0.05 compared to the SAL + HAL group.
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Fig. 3. Effects of an acute injection of saline (-SAL) or 5.0 mg/kg melatonin (-MEL) on open-field locomotion frequency (A), rearing frequency (B) and duration of immobility (C) of rats abruptly withdrawn from a long-term treatment with saline (SAL) or 2.0 mg/kg haloperidol (HAL) concomitant to saline or 5.0 mg/kg melatonin (MEL). Data are reported as mean F SEM. Analysis of variance followed by Duncan’s test. * p < 0.05 compared to the SAL + SAL - SAL group.
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*
score
30
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20
10
0 76
time after withdrawal (h) SAL+SAL-SAL
MEL+SAL-MEL
SAL+HAL-SAL
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Fig. 4. Effects of an acute injection of saline (-SAL) or 5.0 mg/kg melatonin (-MEL) on apomorphine-induced stereotyped behavior of rats abruptly withdrawn from a long-term treatment with saline (SAL) or 2.0 mg/kg haloperidol (HAL) concomitant to saline or 5.0 mg/kg melatonin (MEL). Data are reported as mean F SEM. Kruskal – Wallis analysis of variance followed by the Mann – Whitney U test. * p < 0.05 compared to the SAL + SAL - SAL group.
Apomorphine-induced stereotyped behavior of rats acutely injected with saline or melatonin after an abrupt withdrawal from a long-term treatment with saline or haloperidol concomitant to saline or melatonin is shown in Fig. 4. When compared to the SAL + SAL - SAL group, the SAL + HAL - SAL and MEL + HAL - MEL groups presented stereotypy scores significantly higher (H = 23.18). Acute administration of melatonin abolished the increase in stereotypy presented by rats of the MEL + HAL group when compared to the SAL + HAL group as well as the increase in this behavior presented by rats of the MEL + SAL group as compared to the SAL + SAL group, observed in experiment 1, 76 hours after withdrawal.
Discussion The major findings of the present investigation were that: 1-) withdrawal from long-term treatment with haloperidol was able to produce dopaminergic supersensitivity behaviorally manifested through an increase in open–field general activity and in apomorphine-induced stereotyped behavior; 2-) long-term treatment with melatonin concomitant to haloperidol enhanced the development of this dopaminergic supersensitivity; 3-) withdrawal from long-term melatonin per se induced a fugacious increase in both open-field and apomorphine-induced stereotypy models; 4-) acute administration of melatonin after withdrawal abolished the expression of the potentiating effect of long-term melatonin treatment on haloperidol-induced behavioral supersensitivity as well as the expression of the melatonin-induced dopaminergic supersensitivity per se. As mentioned above, we have recently described that rats continuously exposed to light presented an enhancement of dopaminergic behaviors. On the other hand, the development of haloperidol-induced
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dopaminergic supersensitivity was attenuated by continuous exposure to light [1]. These data led to the proposal that continuous exposure to light could increase dopaminergic function. This increment would, in turn, produce an increase in dopaminergic behaviors and counteract the blockade of dopaminergic receptors produced by haloperidol attenuating the development of dopaminergic supersensitivity. Since continuous exposure to light determines suppression of melatonin secretion [2,9,34–37,42,48,50,52,53], we hypothesized that melatonin could play an important role in this process. The present data seem to corroborate this hypothesis. Thus, opposite to the effect of continuous exposure to light, long-term treatment with melatonin intensified the development of haloperidol-induced dopaminergic supersensitivity. Therefore, melatonin could be leading to a decrease in dopaminergic function that would potentiate the dopaminergic blockade produced by haloperidol. In this way, withdrawal from long-term melatonin treatment alone was also able to induce behavioral dopaminergic supersensitivity, although its magnitude and duration were lower than that observed after haloperidol withdrawal. In this respect, it is interesting to note that, although haloperidol-induced supersensitivity as demonstrated by open-field locomotion frequency data was observed since 24 h after abrupt withdrawal, the potentiating effect of melatonin on this supersensitivity was observed only at 72 h after withdrawal, when the supersensitivity induced by melatonin per se was also present (as demonstrated by open-field immobility duration data). In addition, as expected for a drug presenting inhibitory dopaminergic properties, the enhancement of the haloperidol-induced dopaminergic supersensitivity produced by concomitant long-term treatment with melatonin was reversed when melatonin administration was prolonged after abrupt withdrawal. In line with the idea that melatonin would present an inhibitory action on dopaminergic transmission and consequently on dopaminergic behaviors, melatonin receptors have been detected in the human substantia nigra [29], and the intranigral injection of melatonin in rats inhibited motor activity [8]. Furthermore, Willis et al. [63] have described that melatonin treatment potentiates the development of experimental Parkinson’s disease whereas pinealectomy or continuous exposure to light attenuate it. In this respect, Parkinson’s disease is related to loss of nigrostriatal dopaminergic function and melatonin, or its suppression, should produce opposite effects in this pathology as compared to their effects on the development of dopaminergic supersensitivity. Besides a direct melatonin-dopamine interaction, a melatonin-GABA-dopamine interaction could also be considered since melatonin enhances [3H] GABA binding in the rat brain [15] and striatal dopamine release is decreased by injection of GABA into the substantia nigra [49]. In neuroleptic-treated rats, increased GABA release [51] and increased binding to the GABA site of the GABAA receptor complex in substantia nigra reticulata [57] have been reported. Importantly, while Sasaki et al. [55] demonstrated an increased binding of [35S]TBPS (which labels the picrotoxin binding site inside the CL- channel of the GABAA receptor complex) in the ventrolateral caudate putamen of haloperidol-treated rats showing high vacuous chewing movements levels, melatonin acute administration has been reported to attenuate reserpine-induced oral dyskinesia in rats [2,47]. Alternatively, a melatonin-serotonin-dopamine interaction could also be considered. Indeed, melatonin increases brain serotonin concentration [4] and there is convincing evidence that raphe serotonergic projections inhibit dopamine function at two levels: at the level of midbrain they inhibit the firing of the dopamine cells projecting from the substantia nigra, and in the striatum and cortex they inhibit the synaptic release of dopamine (see [Ref. 26] for review). In this regard, Korsgard et al. [30] reported that serotonergic antagonists increased amphetamine-induced locomotor activity, reactivity and repetitive movements in monkeys. In addition, treatment with L-tryptophan, a precursor of serotonin, has been
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reported to produce dramatic improvement in the severity of tardive dyskinesia [54]. In further support of this assumption repeated treatment with the partial 5-HT1A receptor agonist buspirone has been reported to attenuate both tardive dyskinesia in humans [38] and reserpine-induced oral dyskinesia in rats [46]. Finally, the possibility could be raised that long-term melatonin treatment could be able to phase shift the biological clock of the rats. This possibility, however, seems unlikely since the effects of melatonin treatment were observed only 48 hours or more after the last injection. In addition, these effects were reverted by acute melatonin treatment indicating a direct pharmacological action rather than an effect on the biological clock of the animal. Furthermore, a possible change in the circadian rhythm would have affected other parameters of general activity, such as grooming behavior, an effect that was not observed in the present work (data not shown). In our previous work [1] we observed that continuous exposure to light produced its effects on spontaneous open-field behavior and on amphetamine-induced stereotyped behavior, but not on apomorphine-induced stereotyped behavior. Considering that spontaneous open-field behavior and amphetamine-induced stereotypy depend both on the endogenous dopamine availability and on the dopaminergic postsynaptic transmission, whereas stereotyped behavior induced by apomorphine (a direct agonist) only depends on the dopaminergic postsynaptic transmission [19,60], we stated that the stimulatory effect of light exposure was related to presynaptic mechanisms. Surprisingly, in the present study exogenous melatonin was able to attenuate apomorphine-induced stereotyped behavior suggesting that a pharmacological dose of exogenous melatonin could also act the postsynaptic level. In this respect, Tenn and Niles [58,59] verified that acute administration of melatonin (at the same dose used in our study) or clonazepam were able to inhibit apomorphine-induced turning behavior in a 6hydroxydopamine model of dopaminergic supersensitivity, being these effects blocked by flumazenil, a central type benzodiazepine antagonist. These facts suggest that melatonin would act at the postsynaptic level through gabaergic mechanisms. Indeed, GABA is a key neurotransmitter in both the efferent pathways from the striatum to the globus pallidus and substantia nigra/entopenducular nucleus [21,43]. The aim of the present study was not to define the mechanisms by which melatonin interacts to exert its antidopaminergic effect. Nevertheless, collectively the considerations above suggest that melatonin could decrease dopaminergic function at the presynaptic level via GABAergic and/or serotonergic interactions, and at the postsynaptic level via GABAergic interactions. Despite the exact mechanisms underlying the inhibitory action of melatonin on dopaminergic function, this effect seems to occur on both mesolimbic and nigrostriatal pathways since continuous exposure to light and melatonin treatment were able to modify locomotion, rearing and stereotypy behaviors which are differentially related to those dopaminergic systems: whereas the nigrostriatal pathway is crucial for stereotypy and seems to play a greater role than the mesolimbic pathway in the control of rearing activity, the mesolimbic pathway is critically involved in both spontaneous and dopaminergic stimulant-induced locomotion [3,6,16,25,27]. In conclusion, the present study taken together with our previous investigation concerning the effects of continuous exposure to light [1] shows that melatonin can modulate the development and expression of dopaminergic supersensitivity related to both nigrostriatal and mesolimbic pathways. Considering that these dopaminergic systems are strongly involved in a wide variety of pathologies, our results indicate that melatonin and manipulations of the light/dark cycle could play an important role in the prevention, development and treatment of clinical dopamine-related processes.
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Acknowledgements This research was supported by fellowships from FAPESP (Procs. 1995/9462-7 and 98/13448-8), CNPq (Proc. 522975/95), CAPES, AFIP and FADA. The authors would like to thank Mrs. Teotila R.R. Amaral, Mr. Cleomar S. Ferreira and Mr. Allan C. de Oliveira for capable technical assistance.
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Effects of melatonin on behavioral dopaminergic supersensitivity Vanessa C. Abı´lio, Joa˜o A.R. Vera Jr., Leonardo S.M. Ferreira, Carlo R.M. Duarte, Ce´sar R. Martins, Danila Torres-Leite, Rosana de A. Ribeiro, Roberto Frussa-Filho * Department of Pharmacology, Escola Paulista de Medicina/UNIFESP, Edifı´cio Jose´ Leal Prado-Rua Botucatu, 862 CEP 04023-062, Sa˜o Paulo, Brazil Received 11 July 2002; accepted 5 December 2002
Abstract This study examines the effects of melatonin on dopaminergic supersensitivity induced by long-term treatment with haloperidol in rats. Enhancements of spontaneous general activity in an open-field and of stereotyped behavior induced by apomorphine after abrupt withdrawal from long-term treatment with haloperidol were used as experimental parameters for dopaminergic supersensitivity. Experiment 1 was conducted to investigate the effects of melatonin on the development of dopaminergic supersensitivity, and experiment 2 was conducted to investigate the effects of melatonin on the development as well as on expression of dopaminergic supersensitivity. Rats of both experiments were long-term treated with saline or haloperidol concomitant to saline or melatonin. In experiment 1 behavioral observations were performed after abrupt withdrawal from long-term treatment. In experiment 2 behavioral observations were performed 1 hour after an acute injection of saline or melatonin, administered after the abrupt withdrawal from long-term treatment. Both behavioral parameters used showed the development of central dopaminergic supersensitivity in rats treated with haloperidol since 24 hours after abrupt withdrawal. Concomitant treatment with melatonin intensified haloperidol-induced dopaminergic supersensitivity, observed 72 hours after withdrawal. Melatonin treatment per se also induced behavioral supersensitivity evaluated by both open-field and stereotyped behaviors, although it was more fugacious than that presented by haloperidol. Acute treatment with melatonin reverted the enhancement of the haloperidol-induced dopaminergic supersensitivity produced by concomitant long-term treatment with melatonin, as well as melatonin-induced dopaminergic supersensitivity per se. Our results support previous evidence of antidopaminergic effects of melatonin and demonstrate that repeated administration of this hormone modifies the plasticity of behaviors mediated by central dopaminergic systems. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Melatonin; Dopamine; Dopaminergic supersensitivity; Rat; Behavior
* Corresponding author. Fax: +55-11-273-1766. E-mail address: [email protected] (R. Frussa-Filho). 0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(03)00231-5
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Introduction The central nervous system is not static and immutable. Neuronal circuitry is highly adaptable and can maintain homeostasis in response to internal and/or external perturbation. In rats, abrupt withdrawal from long-term administration of dopaminergic antagonists such as haloperidol [1,7,45,61], bromopride [20], metoclopramide [23] or sulpiride [24] enhanced not only the general activity observed in an open-field but also the responses to apomorphine-induced stereotyped behavior. These effects have been considered to be a consequence of the development of supersensitivity of central dopaminergic pathways [40]. Indeed, behavioral supersensitivity is thought to result from receptor site proliferation in mesolimbic and striatal brain tissues in response to chronic dopamine receptor blockade [11,22,39,62]. From a clinical point of view, whereas striatal dopaminergic supersensitivity has been proposed as a possible contributing factor to the development of tardive dyskinesia in schizophrenics receiving long-term neuroleptic treatment [12,18,28,31,41], mesolimbic dopaminergic supersensitivity has been suggested to be related to neuroleptic-induced supersensitivity psychosis [13,14] as well as to the increased responsiveness to drugs such as cocaine in human stimulant abusers chronically treated with neuroleptics [33]. Recently, we have described that rats abruptly withdrawn from a long-term haloperidol treatment concomitant to continuous exposure to light presented an enhancement in dopaminergic behaviors but an attenuation of the development of dopaminergic supersensitivity suggesting that continuous exposure to light leads to an increase in dopaminergic function that can attenuate the compensatory supersensitivity induced by long-term blockade of dopaminergic receptors [1]. In this respect, the suppression of melatonin secretion, a hormone produced by the pineal gland, is one of the most consistent biochemical effects of light exposure in mammals [2,9,34–37,42,48,50,52,53]. In addition, melatonin receptors have been widely identified in the central nervous system of mammals [5,17,44] including dopaminergic areas as the substantia nigra [29] and the striatum [32,64]. The aim of the present study was to verify the effects of a long-term treatment with exogenous melatonin on haloperidol-induced dopaminergic supersensitivity using both open-field general activity and apomorphine-induced stereotyped behavior in rats as experimental parameters.
Method Subjects Male Wistar EPM-1 rats, born and raised under our laboratory conditions, weighing 250-300 g and about 3 months of age at the beginning of the experiments were used. Groups of 5 or 6 animals were kept in Plexiglass cages with free access to food and water in a room with controlled temperature (22 F 1 jC) and under a 12 h light/dark cycle with lights on at 7:00 am. The animals were maintained and used in accordance to the guidelines of the Committee on Care and Use of Laboratory Animal Resources, National Research Council, USA. Drugs and treatment Haloperidol (Crista´lia, Sa˜o Paulo, Brazil) and melatonin (RBI, Natick, MA) were freshly diluted in distilled water. Melatonin was previously suspended in Tween 80 (3%). Apomorphine hydrochloride
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(Sandoz Basal, Switzerland) was diluted in 0.2% ascorbic acid. Saline and saline + Tween 80 were used as haloperidol and melatonin control solutions, respectively. Haloperidol and melatonin were administered intraperitoneally between 8:00 and 9:00 am, whereas apomorphine was administered subcutaneously in volumes not exceeding 1.0 ml/kg body weight. Open-field measures The open-field was constructed as described by Broadhurst [10] and rats were observed individually for 5 minutes. Hand-operated counters and stopwatches were employed to score ambulation frequency (number of floor units entered), rearing frequency (number of times the animal stood on hind legs) and duration of immobility (time that the animal stood still without showing any movements). The open-field was washed with a solution of alcohol and water (5.0%) before the placement of each animal to obviate possible bias due to odor clues left by previous subjects. To minimize possible effects of circadian changes on open-field behavior, experimental and control observations were alternated, each rat being tested at the same time in each session. Stereotypy measures The animals were observed for stereotyped behavior in wire mesh cages (16 30 19 cm) without food or water. Stereotypy was quantified every 10 minutes for 100 minutes after apomorphine administration (0.6 mg/kg) according to the scoring system proposed by Setler et al. [56]. Briefly, scores ranging from 0 to 6 were attributed to an animal’s behavior. The grading system was as follows: 0- asleep or stationary, 1- active, 2- predominantly active but with bursts of stereotyped sniffing and/or rearing, 3- constant stereotyped activity such as sniffing or rearing but with locomotion activity still present, 4- constant stereotyped activity maintained in one location, 5- constant stereotyped activity but with bursts of licking and/or gnawing and biting, 6- continuously licking and/or gnawing of cage grids. This criterion was not subjective, as shown by the excellent scoring agreement (Pearson’s correlation, v = 0.98) of two different and independent observers. In each group, the total sum of stereotypy scores for each animal was used for statistical analysis. Experimental design Experiment 1 This experiment was performed to verify the effects of melatonin on the development of dopaminergic supersensitivity. Rats were divided at random into 4 groups: SAL + SAL, MEL + SAL, SAL + HAL and MEL + HAL, injected once daily for 30 days with saline (SAL) or 5.0 mg/kg melatonin (MEL) and, 30 minutes after these injections, with saline or 2.0 mg/kg haloperidol (HAL). Thirty minutes, 24, 48, 72, 240 and 480 hours (sessions 1, 2, 3, 4 and 5) after withdrawal from chronic treatment rats were placed individually in the center of the open-field arena for observation of behavioral parameters. Four hours after the last three open-field sessions (76, 244 and 484 h after withdrawal) all animals were injected with apomorphine (0.6 mg/kg) and observed for stereotyped behavior quantification.
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Experiment 2 This experiment was performed to verify the effects of melatonin on the development and expression of dopaminergic supersensitivity. Rats were divided at random into 4 groups: SAL + SAL - SAL, MEL + SAL - MEL, SAL + HAL - SAL and MEL + HAL - MEL, injected once daily for 30 days with saline (SAL) or 5.0 mg/kg melatonin (MEL) and, 30 minutes after these injections, with saline or 2.0 mg/kg haloperidol. Thirty minutes, 24 and 48 hours (sessions 1, 2 and 3) after withdrawal from chronic treatment rats were placed individually in the center of the open-field arena for observation of behavioral parameters, and 76 h after withdrawal from chronic treatment these animals were injected with apomorphine (0.6 mg/kg) and observed for stereotyped behavior. All these behavioral observations were performed one hour after an injection of saline (-SAL) or 5.0 mg/kg of melatonin (-MEL). This protocol was decided based on the results of experiment 1 in order to avoid that two behavioral tests were performed at the same day, which would implicate two injections of melatonin before the second observation. Each rat was used in only one experiment and in both experiments the observer was blind to the identity of the animal. Statistical analysis Since homocedasticity is necessary for the analysis of variance, Bartlet’s test was performed. It was concluded that the open-field data were parametric. An analysis of variance (ANOVA) followed by Duncan’s test was used to study the open-field data. Stereotyped scores were treated by Kruskal–Wallis analysis of variance followed by the two-tailed Mann–Whitney U test.
Results Experiment 1 The open-field behavior of rats abruptly withdrawn from long-term treatment with haloperidol or saline concomitant to melatonin or saline is shown in Fig. 1. Thirty minutes after the last injection, locomotion (A) and rearing (B) frequencies were reduced and duration of immobility (C) was increased in the SAL + HAL and MEL + HAL groups when compared to the SAL + SAL group [F(3,44) = 69.34, 62.32, 127.65, p < 0.0001, respectively]. When compared to the SAL + SAL group, rats of the SAL + HAL group showed increased locomotion frequency (A) in sessions 2, 3 and 4 [F(3,44) = 6.54, 5.93, 7.53, p < 0.005, respectively], and decreased duration of immobility (C) in session 2 [F(3,44) = 4.80, p < 0.05]. Rearing frequency was not altered by haloperidol treatment in any of the sessions. The increase in locomotion frequency produced by abrupt withdrawal from haloperidol treatment was intensified by melatonin co-treatment in session 4. Furthermore, when compared to the SAL + SAL group, rats of the MEL + HAL group showed increased locomotion frequency (A) in sessions 2, 3 and 4, increased rearing frequency in sessions 3 and 4 [F(3,44) = 5.78, 5.84, p < 0.05], and decreased duration of immobility in sessions 2, 3 and 4 [F(3,44) = 4.80, 5.38, 5.49, p < 0.05]. In addition, the MEL + HAL group when compared to the SAL + HAL group presented increased rearing frequency (B) in sessions 3 and 4, and decreased duration of immobility (C) in session 4. Melatonin withdrawal per se produced an slight increase in general activity. Thus, the MEL + SAL group when compared to the SAL + SAL group
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Fig. 1. Open-field locomotion frequency (A), rearing frequency (B) and duration of immobility (C) of rats abruptly withdrawn from long-term treatment with saline (SAL) or 2.0 mg/kg haloperidol (HAL) concomitant to saline or 5.0 mg/kg melatonin (MEL). Data are reported as mean F SEM. Analysis of variance followed by Duncan’s test. * p < 0.05 compared to the SAL + SAL group. 1 p < 0.05 compared to the SAL + HAL group.
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presented decreased duration of immobility in all sessions after withdrawal, reaching statistical significance in session 4. Apomorphine-induced stereotyped behavior of rats abruptly withdrawn from long-term treatment with haloperidol or saline concomitant to melatonin or saline is shown in Fig. 2. When compared to the SAL + SAL group, the SAL + HAL and MEL + HAL groups presented stereotypy scores significantly higher 76 and 244 hours after abrupt withdrawal (H = 31.61, 8.81, p < 0.05, respectively). Furthermore, when compared to SAL + SAL group, the MEL + HAL group also presented significantly higher stereotypy scores 484 hours after withdrawal. In addition, the MEL + HAL group when compared to the SAL + HAL group presented higher stereotypy scores 76 h after withdrawal. Melatonin withdrawal per se induced a slight increase in stereotypy behavior. Thus, 76 (but not 244 and 484) hours after withdrawal, the stereotypy intensity shown by rats of the MEL + SAL group was significantly higher than that of the SAL + SAL group. Experiment 2 The open-field behavior of rats acutely injected with saline or melatonin after an abrupt withdrawal from a long-term treatment with saline or haloperidol concomitant to saline or melatonin is shown in Fig. 3. Thirty minutes after withdrawal, locomotion (A) and rearing (B) frequencies were reduced and duration of immobility (C) was increased in the SAL + HAL - SAL and MEL + HAL - MEL groups when compared to the SAL + SAL - SAL group [F(3,35) = 8.72, 19.17, 19.87, p < 0.0005, respectively]. The SAL + HAL - SAL group, when compared to the SAL + SAL - SAL group, showed an increase in the locomotion frequency (A) in sessions 2 and 3 [F(3,35) = 9.62, 7.38, p < 0.001]. This increase was attenuated by melatonin treatment since the MEL + HAL - MEL group when compared to the SAL + SAL - SAL group presented an increase in locomotion frequency (A) only in session 2.
Fig. 2. Apomorphine-induced stereotyped behavior of rats abruptly withdrawn from long-term treatment with saline (SAL) or 2.0 mg/kg haloperidol (HAL) concomitant to saline or 5.0 mg/kg melatonin (MEL). Data are reported as mean F SEM. Kruskal – Wallis analysis of variance followed by the Mann – Whitney U test. * p < 0.05 compared to the SAL + SAL group. 1 p < 0.05 compared to the SAL + HAL group.
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Fig. 3. Effects of an acute injection of saline (-SAL) or 5.0 mg/kg melatonin (-MEL) on open-field locomotion frequency (A), rearing frequency (B) and duration of immobility (C) of rats abruptly withdrawn from a long-term treatment with saline (SAL) or 2.0 mg/kg haloperidol (HAL) concomitant to saline or 5.0 mg/kg melatonin (MEL). Data are reported as mean F SEM. Analysis of variance followed by Duncan’s test. * p < 0.05 compared to the SAL + SAL - SAL group.
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*
score
30
*
20
10
0 76
time after withdrawal (h) SAL+SAL-SAL
MEL+SAL-MEL
SAL+HAL-SAL
MEL+HAL-MEL
Fig. 4. Effects of an acute injection of saline (-SAL) or 5.0 mg/kg melatonin (-MEL) on apomorphine-induced stereotyped behavior of rats abruptly withdrawn from a long-term treatment with saline (SAL) or 2.0 mg/kg haloperidol (HAL) concomitant to saline or 5.0 mg/kg melatonin (MEL). Data are reported as mean F SEM. Kruskal – Wallis analysis of variance followed by the Mann – Whitney U test. * p < 0.05 compared to the SAL + SAL - SAL group.
Apomorphine-induced stereotyped behavior of rats acutely injected with saline or melatonin after an abrupt withdrawal from a long-term treatment with saline or haloperidol concomitant to saline or melatonin is shown in Fig. 4. When compared to the SAL + SAL - SAL group, the SAL + HAL - SAL and MEL + HAL - MEL groups presented stereotypy scores significantly higher (H = 23.18). Acute administration of melatonin abolished the increase in stereotypy presented by rats of the MEL + HAL group when compared to the SAL + HAL group as well as the increase in this behavior presented by rats of the MEL + SAL group as compared to the SAL + SAL group, observed in experiment 1, 76 hours after withdrawal.
Discussion The major findings of the present investigation were that: 1-) withdrawal from long-term treatment with haloperidol was able to produce dopaminergic supersensitivity behaviorally manifested through an increase in open–field general activity and in apomorphine-induced stereotyped behavior; 2-) long-term treatment with melatonin concomitant to haloperidol enhanced the development of this dopaminergic supersensitivity; 3-) withdrawal from long-term melatonin per se induced a fugacious increase in both open-field and apomorphine-induced stereotypy models; 4-) acute administration of melatonin after withdrawal abolished the expression of the potentiating effect of long-term melatonin treatment on haloperidol-induced behavioral supersensitivity as well as the expression of the melatonin-induced dopaminergic supersensitivity per se. As mentioned above, we have recently described that rats continuously exposed to light presented an enhancement of dopaminergic behaviors. On the other hand, the development of haloperidol-induced
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dopaminergic supersensitivity was attenuated by continuous exposure to light [1]. These data led to the proposal that continuous exposure to light could increase dopaminergic function. This increment would, in turn, produce an increase in dopaminergic behaviors and counteract the blockade of dopaminergic receptors produced by haloperidol attenuating the development of dopaminergic supersensitivity. Since continuous exposure to light determines suppression of melatonin secretion [2,9,34–37,42,48,50,52,53], we hypothesized that melatonin could play an important role in this process. The present data seem to corroborate this hypothesis. Thus, opposite to the effect of continuous exposure to light, long-term treatment with melatonin intensified the development of haloperidol-induced dopaminergic supersensitivity. Therefore, melatonin could be leading to a decrease in dopaminergic function that would potentiate the dopaminergic blockade produced by haloperidol. In this way, withdrawal from long-term melatonin treatment alone was also able to induce behavioral dopaminergic supersensitivity, although its magnitude and duration were lower than that observed after haloperidol withdrawal. In this respect, it is interesting to note that, although haloperidol-induced supersensitivity as demonstrated by open-field locomotion frequency data was observed since 24 h after abrupt withdrawal, the potentiating effect of melatonin on this supersensitivity was observed only at 72 h after withdrawal, when the supersensitivity induced by melatonin per se was also present (as demonstrated by open-field immobility duration data). In addition, as expected for a drug presenting inhibitory dopaminergic properties, the enhancement of the haloperidol-induced dopaminergic supersensitivity produced by concomitant long-term treatment with melatonin was reversed when melatonin administration was prolonged after abrupt withdrawal. In line with the idea that melatonin would present an inhibitory action on dopaminergic transmission and consequently on dopaminergic behaviors, melatonin receptors have been detected in the human substantia nigra [29], and the intranigral injection of melatonin in rats inhibited motor activity [8]. Furthermore, Willis et al. [63] have described that melatonin treatment potentiates the development of experimental Parkinson’s disease whereas pinealectomy or continuous exposure to light attenuate it. In this respect, Parkinson’s disease is related to loss of nigrostriatal dopaminergic function and melatonin, or its suppression, should produce opposite effects in this pathology as compared to their effects on the development of dopaminergic supersensitivity. Besides a direct melatonin-dopamine interaction, a melatonin-GABA-dopamine interaction could also be considered since melatonin enhances [3H] GABA binding in the rat brain [15] and striatal dopamine release is decreased by injection of GABA into the substantia nigra [49]. In neuroleptic-treated rats, increased GABA release [51] and increased binding to the GABA site of the GABAA receptor complex in substantia nigra reticulata [57] have been reported. Importantly, while Sasaki et al. [55] demonstrated an increased binding of [35S]TBPS (which labels the picrotoxin binding site inside the CL- channel of the GABAA receptor complex) in the ventrolateral caudate putamen of haloperidol-treated rats showing high vacuous chewing movements levels, melatonin acute administration has been reported to attenuate reserpine-induced oral dyskinesia in rats [2,47]. Alternatively, a melatonin-serotonin-dopamine interaction could also be considered. Indeed, melatonin increases brain serotonin concentration [4] and there is convincing evidence that raphe serotonergic projections inhibit dopamine function at two levels: at the level of midbrain they inhibit the firing of the dopamine cells projecting from the substantia nigra, and in the striatum and cortex they inhibit the synaptic release of dopamine (see [Ref. 26] for review). In this regard, Korsgard et al. [30] reported that serotonergic antagonists increased amphetamine-induced locomotor activity, reactivity and repetitive movements in monkeys. In addition, treatment with L-tryptophan, a precursor of serotonin, has been
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reported to produce dramatic improvement in the severity of tardive dyskinesia [54]. In further support of this assumption repeated treatment with the partial 5-HT1A receptor agonist buspirone has been reported to attenuate both tardive dyskinesia in humans [38] and reserpine-induced oral dyskinesia in rats [46]. Finally, the possibility could be raised that long-term melatonin treatment could be able to phase shift the biological clock of the rats. This possibility, however, seems unlikely since the effects of melatonin treatment were observed only 48 hours or more after the last injection. In addition, these effects were reverted by acute melatonin treatment indicating a direct pharmacological action rather than an effect on the biological clock of the animal. Furthermore, a possible change in the circadian rhythm would have affected other parameters of general activity, such as grooming behavior, an effect that was not observed in the present work (data not shown). In our previous work [1] we observed that continuous exposure to light produced its effects on spontaneous open-field behavior and on amphetamine-induced stereotyped behavior, but not on apomorphine-induced stereotyped behavior. Considering that spontaneous open-field behavior and amphetamine-induced stereotypy depend both on the endogenous dopamine availability and on the dopaminergic postsynaptic transmission, whereas stereotyped behavior induced by apomorphine (a direct agonist) only depends on the dopaminergic postsynaptic transmission [19,60], we stated that the stimulatory effect of light exposure was related to presynaptic mechanisms. Surprisingly, in the present study exogenous melatonin was able to attenuate apomorphine-induced stereotyped behavior suggesting that a pharmacological dose of exogenous melatonin could also act the postsynaptic level. In this respect, Tenn and Niles [58,59] verified that acute administration of melatonin (at the same dose used in our study) or clonazepam were able to inhibit apomorphine-induced turning behavior in a 6hydroxydopamine model of dopaminergic supersensitivity, being these effects blocked by flumazenil, a central type benzodiazepine antagonist. These facts suggest that melatonin would act at the postsynaptic level through gabaergic mechanisms. Indeed, GABA is a key neurotransmitter in both the efferent pathways from the striatum to the globus pallidus and substantia nigra/entopenducular nucleus [21,43]. The aim of the present study was not to define the mechanisms by which melatonin interacts to exert its antidopaminergic effect. Nevertheless, collectively the considerations above suggest that melatonin could decrease dopaminergic function at the presynaptic level via GABAergic and/or serotonergic interactions, and at the postsynaptic level via GABAergic interactions. Despite the exact mechanisms underlying the inhibitory action of melatonin on dopaminergic function, this effect seems to occur on both mesolimbic and nigrostriatal pathways since continuous exposure to light and melatonin treatment were able to modify locomotion, rearing and stereotypy behaviors which are differentially related to those dopaminergic systems: whereas the nigrostriatal pathway is crucial for stereotypy and seems to play a greater role than the mesolimbic pathway in the control of rearing activity, the mesolimbic pathway is critically involved in both spontaneous and dopaminergic stimulant-induced locomotion [3,6,16,25,27]. In conclusion, the present study taken together with our previous investigation concerning the effects of continuous exposure to light [1] shows that melatonin can modulate the development and expression of dopaminergic supersensitivity related to both nigrostriatal and mesolimbic pathways. Considering that these dopaminergic systems are strongly involved in a wide variety of pathologies, our results indicate that melatonin and manipulations of the light/dark cycle could play an important role in the prevention, development and treatment of clinical dopamine-related processes.
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Acknowledgements This research was supported by fellowships from FAPESP (Procs. 1995/9462-7 and 98/13448-8), CNPq (Proc. 522975/95), CAPES, AFIP and FADA. The authors would like to thank Mrs. Teotila R.R. Amaral, Mr. Cleomar S. Ferreira and Mr. Allan C. de Oliveira for capable technical assistance.
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