Effects of melatonin and agomelatine in anxiety-related procedures in rats: Interaction with diazepam

Abstract

The anxiolytic potential of melatonin and agomelatine, a potent MT_1 / 2 receptor agonist, and their combined effects with diazepam, were investigated in rats using the punished drinking test, the safety signal withdrawal operant paradigm, the elevated-plus-maze and hypophagia-induced novelty. In the punished drinking test, evening injections of melatonin (80 mg/kg, IP, but not 20 and 40 mg/kg) and agomelatine (40 mg/kg, IP) increased the number of foot shocks received. However, neither melatonin (40–80 mg/kg) nor agomelatine (20–40 mg/kg) released response suppression during the period associated with the safety signal withdrawal and affected rats' behaviour in the elevated-plus-maze. Furthermore, agomelatine (40 mg/kg) did not enhance food consumption in unfamiliar environment. However, the co-administration of melatonin (80 mg/kg) or agomelatine (20–40 mg/kg) with diazepam, at a dose (0.25 mg/kg) inactive on its own, induced an anxiolytic-like effect in the punished drinking test and the elevated plus-maze. These results indicate that, although mostly devoid of anxiolytic-like action per se, melatonin and agomelatine can potentiate the anxiolytic effects of diazepam.

Introduction

Melatonin (N-acetyl-5-methoxy-tryptamine), a neurohormone synthesized in and released from the pineal gland during the dark period, participates in several physiological functions such as regulation of circadian rhythms, sleep and reproduction (Reiter, 1991, Simonneaux and Ribelayga, 2003). Its effects are mediated through the activation of two melatonin G-protein-coupled receptors, MT₁ and MT₂ (Reppert et al., 1994, Reppert et al., 1995). Another melatonin binding site with distinct pharmacological properties, MT₃, has been recently characterized as a melatonin sensitive-form of the quinone reductase 2 enzyme (Nosjean et al., 2000).

In addition to its neuroendocrine functions, melatonin seems to exert psychotropic effects in rodents, such as sedative, analgesic, anticonvulsant, hypnotic, and anxiolytic effects (Sugden, 1983). In particular, a large body of studies suggested an involvement of melatonin in stress-and anxiety-related behaviour. It has been notably reported that melatonin secretion and/or synthesis are increased by physical stressful events such as cold, immobilization or hypoglycaemia (Yocca and Friedman, 1984, Champney et al., 1985, Lynch and Deng, 1986, Tannenbaum et al., 1988). In rats, melatonin prevented gastric ulcers caused by water immersion restraint stress (Kato et al., 1997), reversed lipopolysaccharide-induced anxiogenic-like responses in the elevated plus-maze (Nava and Carta, 2001), and protected from the deleterious effects of a chronic stressor on sexual behaviour (Brotto et al., 2001). Anxiolytic-like effects of melatonin have been described mainly in models based on exploratory behaviour. In mice, like benzodiazepines (BZDs), melatonin induced (i) reduction of neophobic responses in a free-exploratory test (Kopp et al., 1999a, Kopp et al., 1999b, Kopp et al., 2000), (ii) increase in time spent in the lit compartment in a light/dark box choice procedure, and (iii) decrease of the number of head-dips in the holeboard test (Pierrefiche et al., 1993). In rats, melatonin increased open arm entries in the elevated plus-maze (Golombek et al., 1993), though the overall relevance of this study can be questioned due to the very small number of total arm entries made by control rats (circa 2-4 per 5-min test session). By contrast, two studies investigating the effects of melatonin in the punished drinking test yielded inconsistent results (Naranjo-Rodriguez et al., 2000, Millan et al., 2005).

Interestingly, evidence exists for an interaction of melatonin with central γ-aminobutyric acid (GABA) neurotransmission. Indeed, melatonin enhanced in vitro [³H]GABA and [³H]muscimol (a GABA_A receptor agonist) binding in rat brain tissues (Niles et al., 1987, Coloma and Niles, 1988). In vivo, melatonin administration increased GABA levels in several brain regions in rats (Rosenstein and Cardinali, 1986, Xu et al., 1995). In mice, the BZD receptor antagonist, flumazenil, counteracted the anxiolytic-like activity of melatonin in the elevated plus-maze (Golombek et al., 1993), the free-exploratory test (Kopp et al., 1999a), the holeboard test and the light/dark box choice procedure (Pierrefiche et al., 1993). Finally, a combined treatment with ineffective doses of melatonin and diazepam caused an anxiolytic-like effect in the four-plate test (Guardiola-Lemaître et al., 1992).

Agomelatine is an agonist which presents a high affinity for both MT₁ (pKi = 10.0) and MT₂ (pKi = 9.9) receptors and exhibits moderate affinity for serotonin 5-HT_2B (pKi = 6.6) and 5-HT_2C (pKi = 6.2) receptors where it acts as an antagonist (Millan et al., 2003). In keeping with melatonin studies, anxiolytic-like effects of agomelatine have been recently described in rats subjected to the social interaction paradigm and the punished drinking test (Millan et al., 2005), though, on chronic administration in mice, a trend for anxiogenic-like effect has been reported in the elevated plus-maze (Barden et al., 2005). Clinical studies also concluded that agomelatine is endowed with a good efficacy in anxiety disorders, as evaluated by the Hamilton Anxiety Scale, at least in patients with major depressive disorders (Lôo et al., 2002a, Lôo et al., 2002b).

The aim of the present study was to investigate further the effects of melatonin and agomelatine in well-validated procedures of anxiety in rats. For this purpose, both compounds were tested in two conflict procedures, the punished drinking test (Vogel et al., 1971) and the safety signal withdrawal paradigm (Thiébot et al., 1991), and two ethological procedures, the elevated plus-maze (Pellow et al., 1985) and the novelty-induced hypophagia (Soubrié et al., 1975). As significant diurnal variations in the amplitude of MT_1 / 2 receptor agonist action may occur (Golombek et al., 1993), the effects of melatonin and agomelatine were assessed mostly at the same period of the day, i.e., in the evening, for all these anxiety-related paradigms. Finally, since it has been suggested that the psychopharmacological effects of melatonin may be due, at least in part, to its ability to interact with central GABAergic processes, experiments were also conducted to examine the effects of melatonin and agomelatine co-administered with diazepam.