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Antidepressant-like effects of melatonin in the mouse chronic mild stress model
European Journal of Pharmacology 607 (2009) 121–125
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Behavioural Pharmacology
Antidepressant-like effects of melatonin in the mouse chronic mild stress model Bernardo C. Detanico a,⁎, Ângelo L. Piato a,b, Jennifer J. Freitas a, Francisco L. Lhullier c, Maria P. Hidalgo d, Wolney Caumo d, Elaine Elisabetsky a a
Laboratório de Etnofarmacologia, ICBS, Universidade Federal do Rio Grande do Sul, Avenida Sarmento Leite 500/202, Porto Alegre, RS, 90050-170, Brazil Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Avenida Ipiranga 2752, Porto Alegre, RS, 90610-000, Brazil Faculdade de Farmácia, Pontifícia Universidade Católica do Rio Grande do Sul, Avenida Ipiranga 6681, Porto Alegre, RS, 90619-000, Brazil d Hospital de Clínicas de Porto Alegre, Universidade Federal do Rio Grande do Sul, Ramiro Barcelos, 2350, Porto Alegre, RS, 90035-903, Brazil b c
a r t i c l e
i n f o
Article history: Received 14 October 2008 Received in revised form 29 January 2009 Accepted 9 February 2009 Available online 26 February 2009 Keywords: Melatonin Depression Antidepressant Unpredictable chronic mild stress HPA axis Corticosterone
a b s t r a c t Melatonin is a hormone primarily synthesized by the pineal gland and has been shown to govern seasonal and circadian rhythms, as well as the immune system, certain behaviours, and responses to stress. Chronic exposure to stress is involved in the etiology of human depression, and depressed patients present changes in circadian and seasonal rhythms. This study investigated the effects of daily exogenous melatonin (1 and 10 mg/kg, p.o.) and imipramine (20 mg/kg, i.p.) on the changes in the coat state, grooming behaviour and corticosterone levels induced by the unpredictable chronic mild stress model of depression in mice. As expected, the 5 weeks of unpredictable chronic mild stress schedule induced significant degradation of the coat state, decreased grooming and increased serum corticosterone levels. All of these unpredictable chronic mild stress-induced changes were counteracted by melatonin (P b 0.05) and imipramine (P b 0.01). Especially in view of the relevance of stress as a major contributing factor in depression, as well as the alleged importance of normalizing a hyperfunctioning HPA axis and resynchronizing circadian rhythms for a successful treatment of depression, this study reassesses the potential of melatonin as an antidepressant. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Depression affects 17–20% of the global population with significant consequences for society (Kessler et al., 1994). Depressive disorders are characterized by psychological, behavioural and physiological alterations, including anhedonia, feelings of hopelessness and guilt, suicidal thoughts, disturbances of sleep and appetite, as well as cognitive changes (Cryan et al., 2002). Stressful life events often precede the onset of an affective episode, suggesting that stress has an important role in the development of human depression (Nestler et al., 2002). Accordingly, there is evidence indicating that marked alterations in the hypothalamic–pituitary–adrenal (HPA) axis coexist with depressive disorders, and that normalization of the HPA axis takes place during successful antidepressant pharmacotherapy (Barden, 2004). The numerous studies linking depression with the HPA system lead to the proposition that HPA hyperactivation may in fact be a marker of major depression (Barden, 2004; Thomson and Craighead, 2008). There have been numerous reports of abnormality in phase and amplitude of circadian rhythms in depressed patients (Tsujimoto et al., 1990; Barden, 2004; Lader, 2007), with sleep disturbances
⁎ Corresponding author. Tel.: +55 51 3308 3137; fax: +55 51 3308 3121. E-mail address: [email protected] (B.C. Detanico). 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.02.037
reported by 50–90% of depressive patients. Additionally, patients show more severe symptoms in the morning and suicidal episodes were more common during daylight (Lader, 2007). The notion that depression is frequently associated with desynchronization of circadian rhythms suggests that drugs that reset normal circadian rhythms may have antidepressant potential. Melatonin is a neurohormone primarily synthesized by the pineal gland during darkness (Pandi-Perumal et al., 2006) with a well established role in regulating seasonal and circadian rhythms. Endogenous melatonin synthesis depends on the suprachiasmatic nucleus responsible for noradrenalin release by sympathetic nerves in the pineal (Nowak and Zawilska, 1998). Renewed attention has been given to the role of melatonin in modulating behaviour, immune system, and responses to stress, cancer and aging (Macchi and Bruce, 2004). Additionally, exogenous melatonin can affect circadian rhythm/ sleep disorders, insomnia, cancer, neurodegenerative diseases, immune function disorders and oxidative damage (Pandi-Perumal et al., 2006). Sedative (Golombek et al., 1996), anxiolytic (Papp et al., 2006), anticonvulsant (Golombek et al., 1996), antinociceptive (Mantovani et al., 2006) and antidepressant (Mantovani et al., 2003; Ergun et al., 2006) actions have been described. Further linking melatonin and depression, decreased plasma night peaks and phase delay of melatonin rhythms have been reported in depressive patients (Claustrat et al., 1984; Beck-Friis et al., 1985; Brown et al., 1985; Pacchierotti et al., 2001; Crasson et al., 2004). Moreover, successful
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treatment with various antidepressants normalizes plasma melatonin levels (Carvalho et al., 2008). The unpredictable chronic mild stress procedure induces depression-like behaviours in rats and mice, mimicking some symptoms present in human depression, such as a decrease in consumption of palatable sucrose solution (interpreted as anhedonia) (Willner, 1997), and degradation of the physical state of the coat (interpreted as decreased self-care) (Ducottet et al., 2003). Changes induced by the unpredictable chronic mild stress can be reversed by chronic (but not acute) treatment with antidepressants (Ducottet et al., 2003). Relevant to this study, it has been shown that the unpredictable chronic mild stress protocol in rodents also induces disorganization of circadian rhythms (Moreau et al., 1995; Cheeta et al., 1997; Papp et al., 2003). Additionally, this model increases plasma levels of tumor necrosis factor-α, interleukin-1-beta, corticosterone and corticotropin-releasing factor (CRF), all of which can be altered in human depression (Grippo et al., 2005). The present study was designed to further scrutinize the antidepressant-like effects of exogenous melatonin, by examining its effects on the unpredictable chronic mild stress-induced behavioural (coat state and splash test with sucrose solution), and biochemical (corticosterone) changes in mice.
semi-randomized manner, so that the coat state and body weights were equivalent in all the groups in the third week. After the last measurements of coat state and body weight, behavioural and biochemical parameters were assessed on different days (Fig. 1). Imipramine (20 mg/kg) and saline were injected intraperitoneally (i.p.), whereas 1% ethanol and melatonin (1 and 10 mg/kg) were given orally (p.o.); all drugs were injected daily at 13:30 h in a volume of 0.1 ml/10 g body weight. 2.4. Unpredictable chronic mild stress The unpredictable chronic mild stress protocol was based on Yalcin et al. (2005). During 5 weeks mice were submitted daily to 2–4 of the following stressors: damp sawdust (90–180 min), 3 changes of sawdust (30–60 min), sawdust-free cage (90–180 min), sawdust-free cage with 200 ml water (90–180 min), transfer to a new clean cage, 45° cage tilting (90–180 min), 15 min cat meowing, social stress (switching the cage), inversion of light/dark cycle (for 48 h in a different room) and various 30 min periods of light during the dark phase. None of the stressors involved water or food deprivation. In order to prevent habituation and maintain the aspect of unpredictability, each week stressors and stressor sequences took place at different times. 2.5. Behavioural and biochemical measurements
2. Materials and methods 2.1. Animals The experiments were carried out with BALB/c (30–35 g) male mice 8 weeks old at the beginning of unpredictable chronic mild stress, obtained from Fundação Estadual de Produção e Pesquisa em Saúde (FEPPS), and maintained before and during the unpredictable chronic mild stress in small individual cages (30 cm × 19 cm × 13 cm), with an inverted 12 hr-light:dark cycle (lights on at 20:00 h), in a controlled environment (22 ± 1 °C). Food and water were available ad libitum, except for the 6 h preceding drug administration. Mice were habituated to the maintenance room for two weeks before the beginning of the unpredictable chronic mild stress protocol. A non-stressed group (n = 9), housed at 4–5 mice per cage (65 cm × 25 cm × 15 cm), was kept in the same room under identical controlled conditions, but not submitted to the stress protocol. All procedures were carried out in accordance with institutional policies on the handling of experimental animals (ethics committee approval # 2006543), which follow NIH guidelines (NIH Guide for Care and Use of Laboratory Animals, NIH publication no. 85-23, 1985). 2.2. Drugs Imipramine HCl and melatonin were acquired from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), and dissolved in saline (0.9% NaCl) and 1% ethanol (EtOH) (in saline), respectively. 2.3. Experimental designs and drug administration After two weeks of drug-free exposure to unpredictable chronic mild stress mice were assigned to different treatment groups in a
2.5.1. Coat state and body weight Before and during unpredictable chronic mild stress, the coat state and body weights were recorded every Monday. The coat state is used as an indirect measure of grooming. Coat state assessment was carried out by observers unaware of treatments, by evaluating the coat in the head, neck, dorsal, ventral and genital regions, and on the forepaws, hindpaws and tail. A score of 0 for a coat in a good state, or 1 for a dirty coat was given for each of these areas (Ducottet and Belzung, 2004). The final score for the coat state was achieved by adding the scores for each body part and dividing by the total number of body parts. 2.5.2. Splash test The splash test is used as a direct measure of grooming, and can be considered as an indirect measure of palatable solution intake. At the beginning of the 6th week of unpredictable chronic mild stress, a 10% sucrose solution was squirted onto the dorsal coat of mice, in their home cages, and animals were videotaped for 5 min (Ducottet and Belzung, 2004). Videos were independently analyzed by three observers unaware of the treatment, using the Observer-Noldus software (Netherlands) to record the frequency of grooming. Grooming bouts included nose/face grooming (strokes along the snout), head washing (semicircular movements over the top of the head and behind the ears), and body grooming (body fur licking) (Kalueff and Tuohimaa, 2004). The splash test was carried out 20 h after the drug administration. 2.5.3. Corticosterone levels 24 h after the last injection and 24 h after the splash test, mice were sacrificed by decapitation, and blood samples collected. Blood samples were centrifuged at 4 °C, and plasma was then stored at −20 °C. Serum corticosterone was measured with the ImmuChen™ 125I Corticosterone Radioimmunoassay Kit (MP Biomedicals, LLC, Orangeburg, NY, USA) according to the manufacturer's instructions. The sensitivity of the measurement was 7.7 ng/ml. The intra- and inter- assay coefficients of variation were 7.1% and 9.5%, respectively. 2.6. Statistical analysis
Fig. 1. Experimental design.
Results are expressed as mean ± S.E.M. The coat state and grooming frequency were compared by the Kruskal–Wallis test, followed by the Mann–Whitney U test when appropriate. Corticosterone levels and body weight were compared by ANOVA followed by Duncan's test.
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Fig. 2. Effects of melatonin and imipramine on the coat state of mice during the unpredictable chronic mild stress. Mean ± S.E.M., n = 9– 11. Melatonin (Mel, 1 and 10 mg/kg) and imipramine (Imi, 20 mg/kg). ⁎P b 0.01 × Non-stressed group; Kruskal–Wallis/Mann–Whitney.
Fig. 3. Effects of melatonin and imipramine on the frequency of grooming behaviour in the splash test. Melatonin (Mel, 1 and 10 mg/kg) and imipramine (Imi, 20 mg/kg). Mean± S.E.M., n = 9–11. ⁎P b 0.001 × non-stressed group; Kruskal–Wallis/Mann–Whitney.
SPSS 11.0 for Windows was used for statistical analysis, and significance was set at P b 0.05. 3. Results Fig. 2 shows the degradation of the coat state during the 5 weeks of unpredictable chronic mild stress. Kruskal–Wallis revealed significant treatment differences for melatonin (H = 19.6, P b 0.001), and imipramine (H = 12.8, P b 0.01). Mann–Whitney confirmed that unpredictable chronic mild stress significantly (P b 0.01 at 6th week) degraded the coat state as compared to the non-stressed group and that imipramine (20 mg/kg/day) and melatonin (10 mg/kg/day) (P b 0.05) counteracted this effect. The effects of imipramine and melatonin in the splash test are shown in Fig. 3. Kruskal–Wallis revealed significant treatment differences for melatonin (H = 22.4, P b 0.001) and imipramine (H = 21.8, P b 0.001). Mann–Whitney confirmed that unpredictable chronic mild stress significantly (P b 0.001) decreased grooming as compared to the non-stressed group, and that imipramine and melatonin (10 mg/kg/day) prevented (P b 0.001) this decrease.
The effects of melatonin and imipramine on serum corticosterone are shown in Fig. 4. The inset shows that corticosterone levels were significantly higher (84.6%) in mice submitted to the unpredictable chronic mild stress (F2,28 = 4.8, P b 0.05), and that imipramine-treated mice maintained levels comparable to the non-stressed group. Likewise, animals treated with melatonin (1 and 10 mg/kg/day) showed corticosterone levels comparable to the non-stressed group, whereas the control (1% ethanol) clearly indicates that the unpredictable chronic mild stress significantly (F3,31 = 5.6, P b 0.05) increased (104.6%) corticosterone levels. No differences were found in body weight gain between the groups (F6,46 = 0.5, P = 0.801) (data not shown), on average 0.84 g/week during the 8 weeks. 4. Discussion The present study reinforces the assertion that melatonin has antidepressant-like properties by showing its effects in the unpredictable chronic mild stress model in BALB/c mice. As expected, chronic sequential exposure to a variety of mild stressor regimens
Fig. 4. Effects of melatonin (Mel,1 and 10 mg/kg) and imipramine (Imi, 20 mg/kg) on corticosterone levels of mice submited to unpredictable chronic mild stress. Mean± S.E.M., n = 9–11. ⁎P b 0.05× non-stressed group; ANOVA/Duncan.
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induced significant coat state degradation, decreased grooming behaviour in the splash test, and increased serum corticosterone levels as compared to the non-stressed mice. The data also show that chronic (3 weeks) treatment with melatonin prevents these unpredictable chronic mild stress-induced abnormalities in a manner comparable with imipramine. It is noteworthy that melatonin was effective at doses that would reach the brain in minute concentrations, a result compatible with responses of endogenous hormonal systems to exogenous supplementation. For instance, it has been shown that oral treatment with doses as low as 0.51 ± 0.02 mg/kg can reverse the CA1/CA3 hippocampal cell loss in rats previously submitted to pinealectomy (De Butte and Pappas, 2007). The degradation of the coat state is one of the most significant changes induced by the unpredictable chronic mild stress. A degraded coat state is thought to be a consequence of decreased grooming, with animals prioritizing behaviours more directly oriented to counteracting the stressful circumstances (Ducottet et al., 2003). The splash test is both a direct measure of grooming, and an indirect evaluation of sucrose solution consumption (Willner, 2005). Comparable to our data with melatonin and imipramine, recent studies show that desipramine (Yalcin et al., 2005), fluoxetine, CRF1 antagonist (Ducottet et al., 2003), and vasopressin V1b antagonist (Griebel et al., 2002) are able to prevent the effects of the unpredictable chronic mild stress on the state of the coat and/or the splash test. Melatonin has been previously shown to prevent the reduction in sucrose consumption in chronically stressed mice (Kopp et al., 1999), while the non-selective melatonin MT1/MT2 receptor antagonist luzindole reversed melatonin's antidepressant-like activity in the forced swim test (Micale et al., 2006). Moreover, it has been verified that mice lacking MT1 receptors display depression-like behaviours in the forced swimming test (Weil et al., 2006). Nonetheless, the antidepressant properties of melatonin may involve a contribution from other neurotransmitters. Several studies suggest that there may be a hypofunction of the GABAergic system in depression, and that GABA agonists should produce antidepressant effects in animals and humans (Nowak et al., 2006). In this line, melatonin has been shown to increase the numbers of GABAA receptors and this up-regulation may contribute to its antidepressant-like action (Sanacora and Saricicek, 2007). In fact, peripheral benzodiazepine receptors (Raghavendra et al., 2000), central serotoninergic neurotransmission (Micale et al., 2006), NMDA glutamate receptors, and the L-arginine-nitric oxide pathway (Mantovani et al., 2003) have all been implicated in the antidepressant-like effects of melatonin in rodent models. In parallel with the observation in human depression, the unpredictable chronic mild stress protocol induced higher basal corticosterone levels in the stressed group, an increase that was reversed by imipramine and melatonin. These data are in agreement with the reported attenuation obtained with melatonin of the adrenocortical secretory response in rats submitted to acute and chronic immobilization stress (Konakchieva et al., 1997). The mechanism by which antidepressants may normalize the corticosterone secretion or the activity of the HPA axis is unclear. However, a dual mechanism has been proposed (Barden, 2004), including a direct stimulation of gene expression for corticosteroid receptors, and an indirect activation through an increased serotoninergic/noradrenergic post-synaptic activation. Chronically elevated levels of corticosterone induced by adrenocorticotropic hormone (ACTH) appear to up-regulate 5-HT2A receptors (Kuroda et al., 1992), and a high density of 5-HT2A receptors may be implicated in the etiology of depression (Arango et al., 1990). Accordingly, it has been shown that melatonin reduces 5-HT2A receptor transmission (Eison et al., 1995), and it is suggested that the antidepressant-like effect of melatonin may result from 5-HT2A antagonism (Gorzalka et al., 1999; Raghavendra and Kulkarni, 2000). Additionally, an in vitro study on primate adrenal cortex has demonstrated not only the expression but also high-amplitude diurnal variation of functional MT1 receptors; furthermore, the stimulation of
these receptors with physiological concentrations of melatonin inhibited the ACTH-induced production of cortisol in a clock time-dependent manner (Richter et al., 2008). We suggest that melatonin may possess a more significant role in depression than so far realized, since melatonin receptor (MT1 and MT2) mRNAs are significantly modified by prolonged treatment with antidepressants (fluoxetine, desipramine and clomipramine) (Imbesi et al., 2006). It is proposed that such treatment alters the brain ratio of MT1/MT2 receptors to enable endogenous melatonin action, promoting an improvement in antidepressant effects (Hirsch-Rodriguez et al., 2007). Agomelatine, an MT1/MT2 melatonin agonist and 5-HT2B and 5-HT2C serotonin antagonist, has been shown to exhibit robust antidepressant-like activity in several animal models (Papp et al., 2003; Bourin et al., 2004; Dubocovich, 2006) and shown to be effective in treating patients with major depression (Pandi-Perumal et al., 2006). Considering that disorganization of internal rhythms is a prominent feature in depressive disorder (Lader, 2007) and that disturbances in circadian rhythms are observed in animal models of epression (Cheeta et al., 1997), it has been argued that exogenous melatonin may act as a resynchronizer of circadian rhythms (Pandi-Perumal et al., 2006). Combining direct measurements of circadian rhythm with the assessment of behavioural changes induced by the unpredictable chronic mild stress in mice treated with melatonin would be useful to further substantiate this proposition. To the best of our knowledge, this is the first investigation that extends available data on melatonin antidepressive effects by indicating that a restoration of corticosterone levels may be involved in its mechanism of action. Further research is needed to clarify if the combination of circadian rhythm and HPA normalization underlies the purported antidepressive effects of melatonin. While low levels of melatonin have been repeatedly observed in depressed patients, overall results from the clinical assessment of melatonin as an antidepressive were unsatisfactory (Pandi-Perumal et al., 2006). Considering the heterogeneous nature of depressive disorders (Nestler et al., 2002), this study suggests that an analysis of the antidepressive effects of melatonin in a subset of patients with stress-triggered depression accompanied by high levels of cortisol may be warranted. Acknowledgements The authors are grateful to CNPq for scholarships and “Rede Instituto Brasileiro de Neurociência (IBN-Net)” # 01.06.0842-00 for financial support. References Arango, V., Ernsberger, P., Marzuk, P.M., Chen, J.S., Tierney, H., Stanley, M., Reis, D.J., Mann, J.J., 1990. Autoradiographic demonstration of increased serotonin 5-ht2 and beta-adrenergic-receptor binding-sites in the brain of suicide victims. Arch. Gen. Psychiatry 47, 1038–1047. Barden, N., 2004. Implication of the hypothalamic–pituitary–adrenal axis in the physiopathology of depression. J. Psychiatry Neurosci. 29, 185–193. Beck-Friis, J., Kjellman, B.F., Aperia, B., Unden, F., von Rosen, D., Ljunggren, J.G., Wetterberg, L., 1985. Serum melatonin in relation to clinical variables in patients with major depressive disorder and a hypothesis of a low melatonin syndrome. Acta Psychiatr. Scand. 71, 319–330. Bourin, M., Mocaer, E., Porsolt, R., 2004. Antidepressant-like activity of S 20098 (agomelatine) in the forced swimming test in rodents: involvement of melatonin and serotonin receptors. J. Psychiatry Neurosci. 29, 126–133. Brown, R., Kocsis, J.H., Caroff, S., Amsterdam, J., Winokur, A., Stokes, P.E., Frazer, A., 1985. Differences in nocturnal melatonin secretion between melancholic depressed patients and control subjects. Am. J. Psychiatry 142, 811–816. Carvalho, L.A., Gorenstein, C., Moreno, R., Pariante, C., Markus, R.P., 2008. Effect of antidepressants on melatonin metabolite in depressed patients. J. Psychopharmacol. doi:10.1177/0269881108089871. Cheeta, S., Ruigt, G., van Proosdij, J., Willner, P., 1997. Changes in sleep architecture following chronic mild stress. Biol. Psychiatry 41, 419–427. Claustrat, B., Chazot, G., Brun, J., Jordan, D., Sassolas, G., 1984. A chronobiological study of melatonin and cortisol secretion in depressed subjects: plasma melatonin, a biochemical marker in major depression. Biol. Psychiatry 19, 1215–1228.
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Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Behavioural Pharmacology
Antidepressant-like effects of melatonin in the mouse chronic mild stress model Bernardo C. Detanico a,⁎, Ângelo L. Piato a,b, Jennifer J. Freitas a, Francisco L. Lhullier c, Maria P. Hidalgo d, Wolney Caumo d, Elaine Elisabetsky a a
Laboratório de Etnofarmacologia, ICBS, Universidade Federal do Rio Grande do Sul, Avenida Sarmento Leite 500/202, Porto Alegre, RS, 90050-170, Brazil Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Avenida Ipiranga 2752, Porto Alegre, RS, 90610-000, Brazil Faculdade de Farmácia, Pontifícia Universidade Católica do Rio Grande do Sul, Avenida Ipiranga 6681, Porto Alegre, RS, 90619-000, Brazil d Hospital de Clínicas de Porto Alegre, Universidade Federal do Rio Grande do Sul, Ramiro Barcelos, 2350, Porto Alegre, RS, 90035-903, Brazil b c
a r t i c l e
i n f o
Article history: Received 14 October 2008 Received in revised form 29 January 2009 Accepted 9 February 2009 Available online 26 February 2009 Keywords: Melatonin Depression Antidepressant Unpredictable chronic mild stress HPA axis Corticosterone
a b s t r a c t Melatonin is a hormone primarily synthesized by the pineal gland and has been shown to govern seasonal and circadian rhythms, as well as the immune system, certain behaviours, and responses to stress. Chronic exposure to stress is involved in the etiology of human depression, and depressed patients present changes in circadian and seasonal rhythms. This study investigated the effects of daily exogenous melatonin (1 and 10 mg/kg, p.o.) and imipramine (20 mg/kg, i.p.) on the changes in the coat state, grooming behaviour and corticosterone levels induced by the unpredictable chronic mild stress model of depression in mice. As expected, the 5 weeks of unpredictable chronic mild stress schedule induced significant degradation of the coat state, decreased grooming and increased serum corticosterone levels. All of these unpredictable chronic mild stress-induced changes were counteracted by melatonin (P b 0.05) and imipramine (P b 0.01). Especially in view of the relevance of stress as a major contributing factor in depression, as well as the alleged importance of normalizing a hyperfunctioning HPA axis and resynchronizing circadian rhythms for a successful treatment of depression, this study reassesses the potential of melatonin as an antidepressant. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Depression affects 17–20% of the global population with significant consequences for society (Kessler et al., 1994). Depressive disorders are characterized by psychological, behavioural and physiological alterations, including anhedonia, feelings of hopelessness and guilt, suicidal thoughts, disturbances of sleep and appetite, as well as cognitive changes (Cryan et al., 2002). Stressful life events often precede the onset of an affective episode, suggesting that stress has an important role in the development of human depression (Nestler et al., 2002). Accordingly, there is evidence indicating that marked alterations in the hypothalamic–pituitary–adrenal (HPA) axis coexist with depressive disorders, and that normalization of the HPA axis takes place during successful antidepressant pharmacotherapy (Barden, 2004). The numerous studies linking depression with the HPA system lead to the proposition that HPA hyperactivation may in fact be a marker of major depression (Barden, 2004; Thomson and Craighead, 2008). There have been numerous reports of abnormality in phase and amplitude of circadian rhythms in depressed patients (Tsujimoto et al., 1990; Barden, 2004; Lader, 2007), with sleep disturbances
⁎ Corresponding author. Tel.: +55 51 3308 3137; fax: +55 51 3308 3121. E-mail address: [email protected] (B.C. Detanico). 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.02.037
reported by 50–90% of depressive patients. Additionally, patients show more severe symptoms in the morning and suicidal episodes were more common during daylight (Lader, 2007). The notion that depression is frequently associated with desynchronization of circadian rhythms suggests that drugs that reset normal circadian rhythms may have antidepressant potential. Melatonin is a neurohormone primarily synthesized by the pineal gland during darkness (Pandi-Perumal et al., 2006) with a well established role in regulating seasonal and circadian rhythms. Endogenous melatonin synthesis depends on the suprachiasmatic nucleus responsible for noradrenalin release by sympathetic nerves in the pineal (Nowak and Zawilska, 1998). Renewed attention has been given to the role of melatonin in modulating behaviour, immune system, and responses to stress, cancer and aging (Macchi and Bruce, 2004). Additionally, exogenous melatonin can affect circadian rhythm/ sleep disorders, insomnia, cancer, neurodegenerative diseases, immune function disorders and oxidative damage (Pandi-Perumal et al., 2006). Sedative (Golombek et al., 1996), anxiolytic (Papp et al., 2006), anticonvulsant (Golombek et al., 1996), antinociceptive (Mantovani et al., 2006) and antidepressant (Mantovani et al., 2003; Ergun et al., 2006) actions have been described. Further linking melatonin and depression, decreased plasma night peaks and phase delay of melatonin rhythms have been reported in depressive patients (Claustrat et al., 1984; Beck-Friis et al., 1985; Brown et al., 1985; Pacchierotti et al., 2001; Crasson et al., 2004). Moreover, successful
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treatment with various antidepressants normalizes plasma melatonin levels (Carvalho et al., 2008). The unpredictable chronic mild stress procedure induces depression-like behaviours in rats and mice, mimicking some symptoms present in human depression, such as a decrease in consumption of palatable sucrose solution (interpreted as anhedonia) (Willner, 1997), and degradation of the physical state of the coat (interpreted as decreased self-care) (Ducottet et al., 2003). Changes induced by the unpredictable chronic mild stress can be reversed by chronic (but not acute) treatment with antidepressants (Ducottet et al., 2003). Relevant to this study, it has been shown that the unpredictable chronic mild stress protocol in rodents also induces disorganization of circadian rhythms (Moreau et al., 1995; Cheeta et al., 1997; Papp et al., 2003). Additionally, this model increases plasma levels of tumor necrosis factor-α, interleukin-1-beta, corticosterone and corticotropin-releasing factor (CRF), all of which can be altered in human depression (Grippo et al., 2005). The present study was designed to further scrutinize the antidepressant-like effects of exogenous melatonin, by examining its effects on the unpredictable chronic mild stress-induced behavioural (coat state and splash test with sucrose solution), and biochemical (corticosterone) changes in mice.
semi-randomized manner, so that the coat state and body weights were equivalent in all the groups in the third week. After the last measurements of coat state and body weight, behavioural and biochemical parameters were assessed on different days (Fig. 1). Imipramine (20 mg/kg) and saline were injected intraperitoneally (i.p.), whereas 1% ethanol and melatonin (1 and 10 mg/kg) were given orally (p.o.); all drugs were injected daily at 13:30 h in a volume of 0.1 ml/10 g body weight. 2.4. Unpredictable chronic mild stress The unpredictable chronic mild stress protocol was based on Yalcin et al. (2005). During 5 weeks mice were submitted daily to 2–4 of the following stressors: damp sawdust (90–180 min), 3 changes of sawdust (30–60 min), sawdust-free cage (90–180 min), sawdust-free cage with 200 ml water (90–180 min), transfer to a new clean cage, 45° cage tilting (90–180 min), 15 min cat meowing, social stress (switching the cage), inversion of light/dark cycle (for 48 h in a different room) and various 30 min periods of light during the dark phase. None of the stressors involved water or food deprivation. In order to prevent habituation and maintain the aspect of unpredictability, each week stressors and stressor sequences took place at different times. 2.5. Behavioural and biochemical measurements
2. Materials and methods 2.1. Animals The experiments were carried out with BALB/c (30–35 g) male mice 8 weeks old at the beginning of unpredictable chronic mild stress, obtained from Fundação Estadual de Produção e Pesquisa em Saúde (FEPPS), and maintained before and during the unpredictable chronic mild stress in small individual cages (30 cm × 19 cm × 13 cm), with an inverted 12 hr-light:dark cycle (lights on at 20:00 h), in a controlled environment (22 ± 1 °C). Food and water were available ad libitum, except for the 6 h preceding drug administration. Mice were habituated to the maintenance room for two weeks before the beginning of the unpredictable chronic mild stress protocol. A non-stressed group (n = 9), housed at 4–5 mice per cage (65 cm × 25 cm × 15 cm), was kept in the same room under identical controlled conditions, but not submitted to the stress protocol. All procedures were carried out in accordance with institutional policies on the handling of experimental animals (ethics committee approval # 2006543), which follow NIH guidelines (NIH Guide for Care and Use of Laboratory Animals, NIH publication no. 85-23, 1985). 2.2. Drugs Imipramine HCl and melatonin were acquired from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), and dissolved in saline (0.9% NaCl) and 1% ethanol (EtOH) (in saline), respectively. 2.3. Experimental designs and drug administration After two weeks of drug-free exposure to unpredictable chronic mild stress mice were assigned to different treatment groups in a
2.5.1. Coat state and body weight Before and during unpredictable chronic mild stress, the coat state and body weights were recorded every Monday. The coat state is used as an indirect measure of grooming. Coat state assessment was carried out by observers unaware of treatments, by evaluating the coat in the head, neck, dorsal, ventral and genital regions, and on the forepaws, hindpaws and tail. A score of 0 for a coat in a good state, or 1 for a dirty coat was given for each of these areas (Ducottet and Belzung, 2004). The final score for the coat state was achieved by adding the scores for each body part and dividing by the total number of body parts. 2.5.2. Splash test The splash test is used as a direct measure of grooming, and can be considered as an indirect measure of palatable solution intake. At the beginning of the 6th week of unpredictable chronic mild stress, a 10% sucrose solution was squirted onto the dorsal coat of mice, in their home cages, and animals were videotaped for 5 min (Ducottet and Belzung, 2004). Videos were independently analyzed by three observers unaware of the treatment, using the Observer-Noldus software (Netherlands) to record the frequency of grooming. Grooming bouts included nose/face grooming (strokes along the snout), head washing (semicircular movements over the top of the head and behind the ears), and body grooming (body fur licking) (Kalueff and Tuohimaa, 2004). The splash test was carried out 20 h after the drug administration. 2.5.3. Corticosterone levels 24 h after the last injection and 24 h after the splash test, mice were sacrificed by decapitation, and blood samples collected. Blood samples were centrifuged at 4 °C, and plasma was then stored at −20 °C. Serum corticosterone was measured with the ImmuChen™ 125I Corticosterone Radioimmunoassay Kit (MP Biomedicals, LLC, Orangeburg, NY, USA) according to the manufacturer's instructions. The sensitivity of the measurement was 7.7 ng/ml. The intra- and inter- assay coefficients of variation were 7.1% and 9.5%, respectively. 2.6. Statistical analysis
Fig. 1. Experimental design.
Results are expressed as mean ± S.E.M. The coat state and grooming frequency were compared by the Kruskal–Wallis test, followed by the Mann–Whitney U test when appropriate. Corticosterone levels and body weight were compared by ANOVA followed by Duncan's test.
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Fig. 2. Effects of melatonin and imipramine on the coat state of mice during the unpredictable chronic mild stress. Mean ± S.E.M., n = 9– 11. Melatonin (Mel, 1 and 10 mg/kg) and imipramine (Imi, 20 mg/kg). ⁎P b 0.01 × Non-stressed group; Kruskal–Wallis/Mann–Whitney.
Fig. 3. Effects of melatonin and imipramine on the frequency of grooming behaviour in the splash test. Melatonin (Mel, 1 and 10 mg/kg) and imipramine (Imi, 20 mg/kg). Mean± S.E.M., n = 9–11. ⁎P b 0.001 × non-stressed group; Kruskal–Wallis/Mann–Whitney.
SPSS 11.0 for Windows was used for statistical analysis, and significance was set at P b 0.05. 3. Results Fig. 2 shows the degradation of the coat state during the 5 weeks of unpredictable chronic mild stress. Kruskal–Wallis revealed significant treatment differences for melatonin (H = 19.6, P b 0.001), and imipramine (H = 12.8, P b 0.01). Mann–Whitney confirmed that unpredictable chronic mild stress significantly (P b 0.01 at 6th week) degraded the coat state as compared to the non-stressed group and that imipramine (20 mg/kg/day) and melatonin (10 mg/kg/day) (P b 0.05) counteracted this effect. The effects of imipramine and melatonin in the splash test are shown in Fig. 3. Kruskal–Wallis revealed significant treatment differences for melatonin (H = 22.4, P b 0.001) and imipramine (H = 21.8, P b 0.001). Mann–Whitney confirmed that unpredictable chronic mild stress significantly (P b 0.001) decreased grooming as compared to the non-stressed group, and that imipramine and melatonin (10 mg/kg/day) prevented (P b 0.001) this decrease.
The effects of melatonin and imipramine on serum corticosterone are shown in Fig. 4. The inset shows that corticosterone levels were significantly higher (84.6%) in mice submitted to the unpredictable chronic mild stress (F2,28 = 4.8, P b 0.05), and that imipramine-treated mice maintained levels comparable to the non-stressed group. Likewise, animals treated with melatonin (1 and 10 mg/kg/day) showed corticosterone levels comparable to the non-stressed group, whereas the control (1% ethanol) clearly indicates that the unpredictable chronic mild stress significantly (F3,31 = 5.6, P b 0.05) increased (104.6%) corticosterone levels. No differences were found in body weight gain between the groups (F6,46 = 0.5, P = 0.801) (data not shown), on average 0.84 g/week during the 8 weeks. 4. Discussion The present study reinforces the assertion that melatonin has antidepressant-like properties by showing its effects in the unpredictable chronic mild stress model in BALB/c mice. As expected, chronic sequential exposure to a variety of mild stressor regimens
Fig. 4. Effects of melatonin (Mel,1 and 10 mg/kg) and imipramine (Imi, 20 mg/kg) on corticosterone levels of mice submited to unpredictable chronic mild stress. Mean± S.E.M., n = 9–11. ⁎P b 0.05× non-stressed group; ANOVA/Duncan.
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induced significant coat state degradation, decreased grooming behaviour in the splash test, and increased serum corticosterone levels as compared to the non-stressed mice. The data also show that chronic (3 weeks) treatment with melatonin prevents these unpredictable chronic mild stress-induced abnormalities in a manner comparable with imipramine. It is noteworthy that melatonin was effective at doses that would reach the brain in minute concentrations, a result compatible with responses of endogenous hormonal systems to exogenous supplementation. For instance, it has been shown that oral treatment with doses as low as 0.51 ± 0.02 mg/kg can reverse the CA1/CA3 hippocampal cell loss in rats previously submitted to pinealectomy (De Butte and Pappas, 2007). The degradation of the coat state is one of the most significant changes induced by the unpredictable chronic mild stress. A degraded coat state is thought to be a consequence of decreased grooming, with animals prioritizing behaviours more directly oriented to counteracting the stressful circumstances (Ducottet et al., 2003). The splash test is both a direct measure of grooming, and an indirect evaluation of sucrose solution consumption (Willner, 2005). Comparable to our data with melatonin and imipramine, recent studies show that desipramine (Yalcin et al., 2005), fluoxetine, CRF1 antagonist (Ducottet et al., 2003), and vasopressin V1b antagonist (Griebel et al., 2002) are able to prevent the effects of the unpredictable chronic mild stress on the state of the coat and/or the splash test. Melatonin has been previously shown to prevent the reduction in sucrose consumption in chronically stressed mice (Kopp et al., 1999), while the non-selective melatonin MT1/MT2 receptor antagonist luzindole reversed melatonin's antidepressant-like activity in the forced swim test (Micale et al., 2006). Moreover, it has been verified that mice lacking MT1 receptors display depression-like behaviours in the forced swimming test (Weil et al., 2006). Nonetheless, the antidepressant properties of melatonin may involve a contribution from other neurotransmitters. Several studies suggest that there may be a hypofunction of the GABAergic system in depression, and that GABA agonists should produce antidepressant effects in animals and humans (Nowak et al., 2006). In this line, melatonin has been shown to increase the numbers of GABAA receptors and this up-regulation may contribute to its antidepressant-like action (Sanacora and Saricicek, 2007). In fact, peripheral benzodiazepine receptors (Raghavendra et al., 2000), central serotoninergic neurotransmission (Micale et al., 2006), NMDA glutamate receptors, and the L-arginine-nitric oxide pathway (Mantovani et al., 2003) have all been implicated in the antidepressant-like effects of melatonin in rodent models. In parallel with the observation in human depression, the unpredictable chronic mild stress protocol induced higher basal corticosterone levels in the stressed group, an increase that was reversed by imipramine and melatonin. These data are in agreement with the reported attenuation obtained with melatonin of the adrenocortical secretory response in rats submitted to acute and chronic immobilization stress (Konakchieva et al., 1997). The mechanism by which antidepressants may normalize the corticosterone secretion or the activity of the HPA axis is unclear. However, a dual mechanism has been proposed (Barden, 2004), including a direct stimulation of gene expression for corticosteroid receptors, and an indirect activation through an increased serotoninergic/noradrenergic post-synaptic activation. Chronically elevated levels of corticosterone induced by adrenocorticotropic hormone (ACTH) appear to up-regulate 5-HT2A receptors (Kuroda et al., 1992), and a high density of 5-HT2A receptors may be implicated in the etiology of depression (Arango et al., 1990). Accordingly, it has been shown that melatonin reduces 5-HT2A receptor transmission (Eison et al., 1995), and it is suggested that the antidepressant-like effect of melatonin may result from 5-HT2A antagonism (Gorzalka et al., 1999; Raghavendra and Kulkarni, 2000). Additionally, an in vitro study on primate adrenal cortex has demonstrated not only the expression but also high-amplitude diurnal variation of functional MT1 receptors; furthermore, the stimulation of
these receptors with physiological concentrations of melatonin inhibited the ACTH-induced production of cortisol in a clock time-dependent manner (Richter et al., 2008). We suggest that melatonin may possess a more significant role in depression than so far realized, since melatonin receptor (MT1 and MT2) mRNAs are significantly modified by prolonged treatment with antidepressants (fluoxetine, desipramine and clomipramine) (Imbesi et al., 2006). It is proposed that such treatment alters the brain ratio of MT1/MT2 receptors to enable endogenous melatonin action, promoting an improvement in antidepressant effects (Hirsch-Rodriguez et al., 2007). Agomelatine, an MT1/MT2 melatonin agonist and 5-HT2B and 5-HT2C serotonin antagonist, has been shown to exhibit robust antidepressant-like activity in several animal models (Papp et al., 2003; Bourin et al., 2004; Dubocovich, 2006) and shown to be effective in treating patients with major depression (Pandi-Perumal et al., 2006). Considering that disorganization of internal rhythms is a prominent feature in depressive disorder (Lader, 2007) and that disturbances in circadian rhythms are observed in animal models of epression (Cheeta et al., 1997), it has been argued that exogenous melatonin may act as a resynchronizer of circadian rhythms (Pandi-Perumal et al., 2006). Combining direct measurements of circadian rhythm with the assessment of behavioural changes induced by the unpredictable chronic mild stress in mice treated with melatonin would be useful to further substantiate this proposition. To the best of our knowledge, this is the first investigation that extends available data on melatonin antidepressive effects by indicating that a restoration of corticosterone levels may be involved in its mechanism of action. Further research is needed to clarify if the combination of circadian rhythm and HPA normalization underlies the purported antidepressive effects of melatonin. While low levels of melatonin have been repeatedly observed in depressed patients, overall results from the clinical assessment of melatonin as an antidepressive were unsatisfactory (Pandi-Perumal et al., 2006). Considering the heterogeneous nature of depressive disorders (Nestler et al., 2002), this study suggests that an analysis of the antidepressive effects of melatonin in a subset of patients with stress-triggered depression accompanied by high levels of cortisol may be warranted. Acknowledgements The authors are grateful to CNPq for scholarships and “Rede Instituto Brasileiro de Neurociência (IBN-Net)” # 01.06.0842-00 for financial support. References Arango, V., Ernsberger, P., Marzuk, P.M., Chen, J.S., Tierney, H., Stanley, M., Reis, D.J., Mann, J.J., 1990. Autoradiographic demonstration of increased serotonin 5-ht2 and beta-adrenergic-receptor binding-sites in the brain of suicide victims. Arch. Gen. Psychiatry 47, 1038–1047. Barden, N., 2004. Implication of the hypothalamic–pituitary–adrenal axis in the physiopathology of depression. J. Psychiatry Neurosci. 29, 185–193. Beck-Friis, J., Kjellman, B.F., Aperia, B., Unden, F., von Rosen, D., Ljunggren, J.G., Wetterberg, L., 1985. Serum melatonin in relation to clinical variables in patients with major depressive disorder and a hypothesis of a low melatonin syndrome. Acta Psychiatr. Scand. 71, 319–330. Bourin, M., Mocaer, E., Porsolt, R., 2004. Antidepressant-like activity of S 20098 (agomelatine) in the forced swimming test in rodents: involvement of melatonin and serotonin receptors. J. Psychiatry Neurosci. 29, 126–133. Brown, R., Kocsis, J.H., Caroff, S., Amsterdam, J., Winokur, A., Stokes, P.E., Frazer, A., 1985. Differences in nocturnal melatonin secretion between melancholic depressed patients and control subjects. Am. J. Psychiatry 142, 811–816. Carvalho, L.A., Gorenstein, C., Moreno, R., Pariante, C., Markus, R.P., 2008. Effect of antidepressants on melatonin metabolite in depressed patients. J. Psychopharmacol. doi:10.1177/0269881108089871. Cheeta, S., Ruigt, G., van Proosdij, J., Willner, P., 1997. Changes in sleep architecture following chronic mild stress. Biol. Psychiatry 41, 419–427. Claustrat, B., Chazot, G., Brun, J., Jordan, D., Sassolas, G., 1984. A chronobiological study of melatonin and cortisol secretion in depressed subjects: plasma melatonin, a biochemical marker in major depression. Biol. Psychiatry 19, 1215–1228.
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