Evidence for the involvement of the monoaminergic system in the antidepressant-like effect of magnesium

Progress in Neuro-Psychopharmacology & Biological Psychiatry 33 (2009) 235–242

Contents lists available at ScienceDirect

Progress in Neuro-Psychopharmacology & Biological Psychiatry j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / p n p b p

Evidence for the involvement of the monoaminergic system in the antidepressant-like effect of magnesium Chandra C. Cardoso a, Kelly R. Lobato a, Ricardo W. Binfaré a, Priscilla K. Ferreira a, Angelo O. Rosa a, Adair Roberto S. Santos b, Ana Lúcia S. Rodrigues a,⁎ a b

Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Campus Universitário, Trindade, 88040-900, Florianópolis-SC, Brazil Departamento de Ciências Fisiológicas, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Campus Universitário, Trindade, 88040-900, Florianópolis-SC, Brazil

a r t i c l e

i n f o

Article history: Received 5 September 2008 Received in revised form 25 October 2008 Accepted 14 November 2008 Available online 27 November 2008 Keywords: Antidepressant Depression Forced swimming test Magnesium Monoaminergic system

a b s t r a c t Literature data has shown that acute administration of magnesium reduces immobility time in the mouse forced swimming test (FST), which suggests potential antidepressant activity in humans. However, its mechanism of action is not completely understood. Thus, this study is aimed at investigating the antidepressant-like action of magnesium and the possible involvement of the monoaminergic system in its effect in the FST. The immobility time in the FST was significantly reduced by magnesium chloride administration (30–100 mg/kg, i.p.) without accompanying changes in ambulation when assessed in an open-field test. The pre-treatment of mice with NAN190 (0.5 mg/kg, i.p. a 5-HT1A receptor antagonist), WAY100635 (0.1 mg/kg, s.c., a selective 5-HT1A receptor antagonist), ritanserin (4 mg/kg, i.p., a 5-HT2A/2C receptor antagonist), ketanserin (5 mg/kg, a preferential 5-HT2A receptor antagonist), prazosin (1 mg/kg, i.p., an α1-adrenoceptor antagonist), yohimbine (1 mg/kg, i.p., an α2adrenoceptor antagonist), haloperidol (0.2 mg/kg, i.p., a non selective dopaminergic receptor antagonist), SCH23390 (0.05 mg/kg, s.c., a dopamine D1 receptor antagonist) or sulpiride (50 mg/kg, i.p., a dopamine D2 receptor antagonist) 30 min before the administration of magnesium chloride (30 mg/kg, i.p.) significantly prevented its anti-immobility effect in the FST. Moreover, the administration of sub-effective doses of fluoxetine (10 mg/kg, i.p., serotonin reuptake inhibitor), imipramine (5 mg/kg, i.p., a mixed serotonergic noradrenergic reuptake inhibitor), bupropion (1 mg/kg, i.p., dopamine reuptake inhibitor) was able to potentiate the action of sub-effective doses of magnesium chloride. In conclusion, the present study provides evidence indicating that the antidepressant-like effect of magnesium in the FST is dependent on its interaction with the serotonergic (5-HT1A and 5-HT2A/2C receptors), noradrenergic (α1- and α2- receptors) and dopaminergic (dopamine D1 and D2 receptors) systems. © 2008 Elsevier Inc. All rights reserved.

1. Introduction Magnesium (Mg+2) is an essential intracellular bioelement which plays an important role in a wide variety of metabolic reactions, in particular energy-requiring processes (Ryan, 1991). In the central nervous system (CNS) it is involved in signal transmission, blocking the N-methyl-D-aspartate (NMDA) receptor ion channel in a voltagedependent manner (Sobolevskii and Khodorov, 2002). Preclinical studies have shown that NMDA receptor antagonists display a variety

Abbreviations: ANOVA, analysis of variance; 5-HT, serotonin; FST, forced swimming test; i.p., intraperitoneal; NMDA, N-methyl-D-aspartate; MAOi, monoamine oxidase inhibitor; NAN-190, 1-(2-methoxyphenyl)-4[-(2-phthalimido)butyl] piperazine); PCPA, p-chlorophenylalanine methyl ester; SCH23390, (R)-(+)-7 chloro-8-hydroxy-3-methyl1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride; SSRI, selective serotonin reuptake inhibitor; WAY100635, N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}N-(2-pyridynyl) cyclohexanecarboxamide. ⁎ Corresponding author. Tel.: +55 48 37215043; fax: +55 48 37219672. E-mail addresses: [email protected], [email protected] (A.L.S. Rodrigues). 0278-5846/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2008.11.007

of pharmacological and behavioral effects, including anticonvulsant (Wong et al., 1986), anxiolytic (Dunn et al., 1990) and antidepressantlike activities (Maj et al., 1992; Skolnick, 1999, 2002). Furthermore, Mg+2 deficiency is associated with behavioral and physiological alterations in patients with affective disorders (Hall and Joffe, 1973; Kirov et al., 1994), and in experimental animal models (Bac et al., 1995; Malpuech-Brugère et al., 2000). Several studies have demonstrated that acute and chronic administration of Mg+2 reduces immobility time in the forced swimming test (FST) in mice and rats, and enhances the antiimmobility activity of imipramine in this model (Decollogne et al., 1997; Poleszak et al., 2004, 2005a,b, 2006). Recently, an indication that the serotoninergic system is involved in the antidepressant-like effect of Mg+2 was given by the fact that the pre-treatment of mice with an inhibitor of serotonin synthesis, p-chlorophenylalanine was able to reduce the anti-immobility effect of magnesium in the FST (Poleszak, 2007). Moreover, Mg+2 depletion in mice produces an increase in anxiety and depression-like behavior (Singewald et al., 2004). This data is consistent with clinical studies that demonstrated

236

C.C. Cardoso et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 33 (2009) 235–242

low serum Mg+2 levels in depressed patients (Hashizume and Mori, 1990; Rasmussen et al., 1989; Zieba et al., 2000), which suggests an important role of this ion in the pathophysiology of depression. However, its mechanism of action is not completely understood. The monoaminergic system is one of the most important targets in the pathophysiology and treatment of depression (Elhwuegi, 2004; Millan, 2004). The monoaminergic hypothesis indicates that the pathology of depression involves dysfunction of monoamine neurotransmitter circuits in the CNS. It is supported by great number of neurochemical findings (Booij et al., 2003; Ruhé et al., 2007) and by the successful treatment of major depression with classical antidepressants, compounds that enhance monoaminergic neurotransmission (Nemeroff and Owens, 2002). Considering that substances which reduced NMDA transmission have antidepressant-like effect probably due a modulation of the monoaminergic pathways in the CNS (Loscher et al., 1991; Wedzony et al., 1997) and that treatment with antidepressants alters NMDA function (Skolnick, 1999), Mg+2, which is a endogenous antagonist at NMDA receptors, may play a significant role in the modulation of depression. Thus, to further contribute to the understanding of the mechanisms underlying the antidepressant-like effects of Mg +2 , this study was aimed at investigating the possible involvement of the monoaminergic system in its effect in the FST.

To assess the possible involvement of the noradrenergic and the dopaminergic systems on the antidepressant-like effect of Mg+2 in the FST, independent group of animals were pretreated with prazosin (1 mg/ kg, i.p., an α1-adrenoreceptor antagonist), yohimbine (1 mg/kg, i.p., an α2-adrenoreceptor antagonist), haloperidol (0.2 mg/kg, i.p., a non selective dopamine receptor antagonist), SCH23390 (0.05 mg/kg, s.c., a dopamine D1 receptor antagonist), sulpiride (50 mg/kg, i.p., a dopamine D2 receptor antagonist) or vehicle and after 30 min they received MgCl2 (30 mg/kg, i.p.) or vehicle and were tested in the FST 30 min later. In a separate set of experiments, independent group of animals were pretreated with sub-effective doses of the antidepressants fluoxetine (10 mg/kg, i.p., serotonin reuptake inhibitor), imipramine (5 mg/kg, i.p., a mixed serotonergic noradrenergic reuptake inhibitor), bupropion (1 mg/kg, i.p., dopamine reuptake inhibitor) or vehicle and after 30 min they received a sub-effective dose of MgCl2 (10 mg/kg, i.p.) or vehicle. After 30 min the open-field test or the FST were carried out. Doses and administration schedule were chosen on the basis of experiments previously performed in our laboratory and the literature data confirm the selectivity and efficacy of the above-mentioned treatments at the concentrations used (Brocardo et al., 2008; Kaster et al., 2005; Machado et al., 2007; O'Neill and Conway, 2001; Redrobe et al., 1996; Redrobe and Bourin, 1997; Rodrigues et al., 2002). 2.3. Forced swimming test (FST)

2. Methods 2.1. Animals Male Swiss mice (30–40 g) were maintained at 22–24 °C with free access to water and food, under a 12:12 h light:dark cycle (lights on at 7:00 h). Twenty mice were housed per cage (40 × 34 × 17 cm), which were placed in the experimental room 24 h before the test for acclimatization. All experiments were carried out between 12:00 and 16:00 h. Each experimental group consisted of 6–8 animals, with each animal used only once. All procedures in this study were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. The experiments were performed after approval by the Ethics Committee of the Institution and all efforts were made to minimize animal suffering and to reduce the number of animals used in the experiments.

The test was conducted using the method of Porsolt et al. (1977) with some modifications. Mice were individually forced to swim in an open cylindrical container (diameter 10 cm, height 25 cm), containing 19 cm of water at 25 ± 1 °C; the total duration of immobility during a 6 min test was scored live. This test procedure was carried out according to the previously standardized and validated animal in our laboratory (Brocardo et al., 2008; Eckeli et al., 2000; Kaster et al., 2005, 2007a,b; Rosa et al., 2008; Zomkowski et al., 2002, 2004). Classical antidepressants are reported to decrease immobility time in this paradigm (Brocardo et al., 2008; Dhir and Kulkarni, 2007; Kaster et al., 2007a; Rosa et al., 2008; Yamada et al., 2004). Each mouse was judged to be immobile when it ceased struggling and remained floating motionless in the water, making only those movements necessary to keep its head above water. 2.4. Open-field test

2.2. Drugs and treatment The following drugs were used: magnesium chloride (MgCl2, Merck, Darmstadt, Germany), 1-(2-methoxyphenyl)-4[-(2-phthalimido)butyl] piperazine) (NAN-190), N-{2-[4-(2-methoxyphenyl)-1-piperazinyl] ethyl}-N-(2-pyridynyl) cyclohexanecarboxamide (WAY100635), ritanserin, ketanserin, prazosin, yohimbine, haloperidol, (R)-(+)-7-chloro-8hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH23390), sulpiride, and the antidepressants fluoxetine, imipramine and bupropion (all from Sigma Chemical Company, St. Louis, MO, U.S.A.). All drugs were administered by intraperitoneal (i.p.) route in a constant volume of 10 ml/kg body weight except SCH23390 and WAY100635, that were administered by subcutaneous (s.c.) route (10 ml/kg body weight). Appropriate vehicle-treated groups were also assessed simultaneously. MgCl2 was administered by i.p. route 30 min before the FST or the open-field test. These behavioral tests were performed by an observer blind to the drug treatment. In order to investigate the possible involvement of the serotonergic system in the antidepressant-like effect of Mg+2, independent group of mice were pretreated with NAN-190 (0.5 mg/kg, i.p. a 5-HT1A receptor antagonist), WAY100635 (0.1 mg/kg, s.c., a selective 5-HT1A receptor antagonist), ketanserin (5 mg/kg, i.p., a preferential 5-HT2A receptor antagonist), ritanserin (4 mg/kg, i.p., a 5-HT2A/2C receptor antagonist) or vehicle and 30 later they received MgCl2 (30 mg/kg, i.p.) or vehicle before being tested in the FST after 30 min.

To assess the possible effects of magnesium on locomotor activity, mice were evaluated in the open-field paradigm as previously described (Rodrigues et al., 2002). Mice were individually placed in the left corner of a wooden box (40 × 60 × 50 cm) with the floor divided into 12 rectangles. The number of rectangles crossed with the four paws was registered during a period of 6 min. The arena floor was cleaned between the trials with a solution of ethanol 10% and the test was carried out in a temperature and light controlled room. 2.5. Statistical analysis Comparisons between experimental and control groups were performed by one or two-way ANOVA followed by Tukey's HSD test when appropriate. A value of P b 0.05 was considered to be significant. 3. Results 3.1. Effect of magnesium on the immobility time in the FST The immobility time in the FST of animals treated with MgCl2 is shown in Fig. 1A. The one-way ANOVA revealed a significant effect of MgCl2 on immobility [F(3.28) = 22.18, P b 0.01]. MgCl2, at the doses of 30 and 100 mg/kg, i.p., significantly decreased the immobility time when mice were tested in the FST. As shown in Fig. 1B MgCl2 (10–

C.C. Cardoso et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 33 (2009) 235–242

237

Fig. 1. Effect of acute administration of MgCl2 (10–100 mg/kg, i.p.) in the forced swimming test (A) and open-field test (B). MgCl2 was administered 30 min before the tests. Each column represents the mean + SEM (n = 6–8) ⁎⁎P b 0.01 as compared with the vehicle-treated control.

100 mg/kg, i.p.) did not significantly altered the number of crossings in the open-field test as compared to control group, as revealed by oneway ANOVA [F(3.26) = 0.47, P = 0.70] indicating that its antidepressantlike effect in the FST is not due to a locomotor effect. 3.2. Involvement of the serotonergic system Fig. 2A shows that the pre-treatment of mice with NAN-190 (0.5 mg/ kg, i.p.) significantly prevented the decrease in the immobility time elicited by MgCl2 (30 mg/kg, i.p.). The two-way ANOVA revealed a main effect of MgCl2 treatment [F(1.20)=9.85, Pb 0.01], NAN-190 pre-treatment [F(1,20)=5.19, P b 0.05] and MgCl2 ×NAN-190 interaction [F(1,20)=17.7,

P b 0.01]. Moreover, as depicted in Fig. 2B, the pre-treatment of mice with WAY100635 (0.1 mg/kg, s.c.) also prevented the antidepressant-like effect elicited by MgCl2. A two-way ANOVA showed significant differences for MgCl2 treatment [F(1,20)=27.36, P b 0.01], WAY100635 pre-treatment [F (1,20) =21.52, Pb 0.01] and MgCl2 ×WAY100635 interaction [F(1,20)= 16.58, P b 0.01]. Fig. 2C shows that the pre-treatment of mice with ritanserin (4 mg/kg, i.p.) also prevented the action of MgCl2 in the FST. The two-way ANOVA revealed a main effect of the MgCl2 treatment [F(1,20)= 3.27, P =0.08], ritanserin pre-treatment [F(1,20) =14.53, P b 0.01] and MgCl2 ×ritanserin interaction [F(1,20)=11.19, Pb 0.01]. In addition, the results depicted in Fig. 2D show that the pre-treatment of mice with ketanserin (5 mg/kg, i.p.) prevented the effect of MgCl2 in the FST. A two-

Fig. 2. Effect of the pre-treatment of mice with NAN-190 (0.5 mg/kg, i.p. a 5-HT1A receptor antagonist, panel A), WAY100635 (0.1 mg/kg, s.c., a selective 5-HT1A receptor antagonist, panel B), ritanserin (4 mg/kg, i.p., a 5-HT2A/2C receptor antagonist, panel C), ketanserin (5 mg/kg, a preferential 5-HT2A receptor antagonist, panel D) on MgCl2 (30 mg/kg, i.p.)-induced reduction in immobility time in the FST. Each column represents the mean + SEM (n = 6) ⁎⁎P b 0.01 as compared with the vehicle-treated control. #P b 0.01 as compared with the group pretreated with vehicle and magnesium chloride.

238

C.C. Cardoso et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 33 (2009) 235–242

Fig. 3. Effect of pre-treatment of mice with prazosin (1 mg/kg, i.p., panel A) or yohimbine (1 mg/kg, i.p., panel B) on MgCl2 (30 mg/kg, i.p.)-induced reduction in immobility time in the FST. Values are expressed as mean + S.E.M. (n = 6–8). ⁎⁎P b 0.01 as compared with the control group (vehicle); #P b 0.01 as compared with the group pretreated with vehicle and MgCl2.

way ANOVA showed significant differences for MgCl2 treatment [F(1,20)= 14.44, P b 0.01], ketanserin pre-treatment [F(1,20)=24.88, P b 0.01] and MgCl2 ×ketanserin interaction [F(1,20)=12.53, Pb 0.01].

(1,22) = 23.75, P b 0.01], yohimbine pre-treatment [F(1,22) = 7.57, P b 0.05] and MgCl2 × yohimbine interaction [F(1,22) = 6.13, P b 0.05]. 3.4. Involvement of the dopaminergic system

3.3. Involvement of the noradrenergic system The results depicted in Fig. 3A shows that pre-treatment of mice with prazosin (1 mg/kg, i.p) was able to reverse the antidepressantlike effect of MgCl2 (30 mg/kg, i.p.) in the FST. The two-way ANOVA revealed a main effect of MgCl2 treatment [F(1,24) = 13.77, P b 0.01], MgCl2 × prazosin interaction [F(1,24) = 19.37, P b 0.01], but not of the prazosin pre-treatment [F(1,24) = 13.77, P = 0.51]. Fig. 3B shows that the pre-treatment of mice with yohimbine (1 mg/kg, i.p.) was also able to prevent the anti-immobility effect of magnesium chloride in the FST. The two-way ANOVA revealed a main effect of MgCl2 treatment [F

The anti-immobility effect of MgCl2 (30 mg/kg, i.p.) was significantly prevented by pre-treatment of mice with haloperidol (0.2 mg/ kg, i.p., Fig. 4A). The results obtained in this experiment were analyzed by a two-way ANOVA. There was a significant effect of MgCl2 treatment [F(1,22) = 23.75, P b 0.01], haloperidol pre-treatment [F(1,22) = 15.02, P b 0.01] and MgCl2 × haloperidol interaction [F(1,22) = 17.79, P b 0.01]. Fig. 4B shows that SCH23390 (0.05 mg/kg, s.c.) was also able to prevent the anti-immobility effect of magnesium chloride in the FST. The twoway ANOVA revealed a main effect of MgCl2 treatment [F(1,26) = 19.43, P b 0.01], SCH23390 pre-treatment [F(1,26) = 56.11, P b 0.01] and

Fig. 4. Effect of pre-treatment of mice with haloperidol (0.2 mg/kg, i.p., a non selective dopaminergic receptor antagonist, panel A), SCH23390 (0.05 mg/kg, s.c., a dopamine D1 receptor antagonist, panel B) or sulpiride (50 mg/kg, i.p., a dopamine D2 receptor antagonist, panel C) on the MgCl2 (30 mg/kg, i.p.)-induced reduction in immobility time in the FST. Values are expressed as mean + S.E.M. (n = 6–8). ⁎⁎P b 0.01 as compared with the control group (vehicle); #P b 0.01 as compared with the group pretreated with vehicle and MgCl2.

C.C. Cardoso et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 33 (2009) 235–242

239

Fig. 5. Effect of the administration of fluoxetine (F; 10 mg/kg, i.p.), imipramine (I; 5 mg/kg, i.p.) or bupropion (B; 1 mg/kg, i.p.) with a sub-effective dose of MgCl2 (30 mg/kg, i.p.) in the FST (panel A) or in the open-field test (panel B). Each column represents the mean + SEM (n = 6–8). ⁎⁎P b 0.01 as compared with the vehicle-treated group.

MgCl2 × SCH23390 interaction [F(1,26) = 24.81, P b 0.01]. Sulpiride administration (50 mg/kg, i.p., Fig. 4C) was also able to reverse the antidepressant-like effect of MgCl2 in the FST. The two-way ANOVA revealed a main effect of MgCl2 treatment [F(1,26) = 21.34, P b 0.01], sulpiride pre-treatment [F(1,26) = 15.01, P b 0.01] and of MgCl2 × sulpiride interaction [F(1,26) = 9.79, P b 0.01]. 3.5. Interaction of magnesium chloride with antidepressants in the FST Fig. 5A shows the influence of a sub-effective dose of MgCl2 (10 mg/ kg, i.p.) given in combination with a sub-effective dose of fluoxetine (10 mg/kg, i.p.), imipramine (5 mg/kg, i.p.) or bupropion (1 mg/kg, i.p.) on the immobility time in the FST. A two-way ANOVA showed significant differences for antidepressant treatment [F(1,40) = 96.82, P b 0.01], M g C l 2 p r e - t r e a t m e n t [ F ( 3 , 4 0 ) = 2 2 . 0 7, P b 0 . 0 1 ] a n d antidepressants × MgCl2 interaction [F(3,40)= 18.00, P b 0.01]. As shown in Fig. 5B, these antidepressants did not significantly alter the exploratory activity in the open-field test. A two-way ANOVA showed significant differences for MgCl2 pre-treatment [F(3,44) = 5.70, P b 0.01], but not for antidepressants treatment [F(1,44) = 0.80, P = 0.38] and antidepressants × MgCl2 interaction [F(3,44) = 0.26, P = 0.85]. 4. Discussion The present study confirms literature data (Poleszak et al., 2004, 2005a,b, 2007) that show that the administration of magnesium salts produces an antidepressant-like effect in the FST, a widely-accepted behavioral model predictive of antidepressant activity that is sensitive to all major classes of antidepressant drugs including tricyclics, serotonin-specific reuptake inhibitors, monoamine oxidase inhibitors and atypicals (Cryan et al., 2002; Porsolt et al., 1977). Of most importance, our study significantly extends literature data, clearly demonstrating the involvement of the monoaminergic system in the antidepressant-like effect of MgCl2 in the FST and also, the synergistic antidepressant-like effect of MgCl2 administration with antidepressants from different classes: fluoxetine, imipramine or bupropion. Indeed, previous interaction of imipramine, citalopram, reboxetine and tianeptine with Mg2+ was examined (Poleszak et al., 2005b, 2006; Poleszak, 2007) and a synergistic antidepressant-like effect of Mg2+ was shown only with imipramine, citalopram and tianeptine. The FST has some drawbacks represented by the possibility to obtain some false positive results when drugs that enhance locomotor activity are used (Borsini and Meli, 1988). However, our results show that the synergistic effect of MgCl2 with subeffective doses of antidepressants in this test cannot be attributed to a psychostimulant action of this compound, since no effects on locomotor activity in the open-field test were observed.

Mg+2 is an inorganic NMDA receptor antagonist, and its antidepressant-like effect is attributed to this property (Poleszak et al., 2007). In fact, several preclinical studies have demonstrated a link between NMDA antagonism and antidepressant action (Skolnick, 1999). Considering that NMDA antagonists can modulate monoamines turnover in the CNS and elevate synaptic biogenic amine levels (Loscher et al., 1991; Mathe et al., 1999; Wedzony et al., 1997), the involvement of the serotonergic, noradrenergic and dopaminergic systems in the antidepressant-like effect of magnesium was investigated. Several reports have suggested an involvement of the 5-HT1A receptors in the mechanism of action of several classes of antidepressant drugs, including tricyclics, SSRIs (selective serotonin reuptake inhibitors) and MAOi (monoamine oxidase inhibitors) and in the pathophysiology of depression (Hensler, 2002; Hirvonen et al., 2008). In the present study, the involvement of 5-HT1A receptors in the antidepressant-like effect of Mg+2 was indicated by the results showing that pre-treatment of mice with NAN-190 and WAY100635 significantly prevented the anti-immobility effect of MgCl2 in the FST. It should be noted that NAN-190, in addition to its 5-HT1A receptor antagonist properties, has a high affinity for α1-adrenoceptors (Claustre et al., 1991; Schneider and Simson, 2007). However, in this study, the selective 5-HT1A receptor antagonist WAY100635 was able to prevent the antidepressant-like effect of MgCl2 in the FST. Therefore, these experiments clearly indicate that the 5-HT1A receptor could be relevant for the antidepressant action of this ion in the FST. A role for 5-HT2 receptors in the action of some antidepressants has been shown. Preclinical data has shown that 5-HT2A/2C antagonism has a significant role in the mechanism underlying the antidepressant-like effect of several antidepressants (Cryan and Lucki, 2000; Elhwuegi, 2004; Redrobe and Bourin, 1997). Moreover, the preferential 5-HT2A receptor partial agonist DOI was reported to enhance the antidepressant-like effect of some compounds (Khisti and Chopde, 2000; Zomkowski et al., 2004). In this study, the antidepressant-like effect of MgCl2 was prevented by the pre-treatment with ketanserin, a preferential 5-HT2A receptor antagonist, and with ritanserin, a 5-HT2A/2C receptors antagonist. These results suggest that the antidepressant-like effect of MgCl2 in the FST depends, at least in part, on an interaction with 5-HT2A or 5HT/2C receptors. Another finding of this study was that the classical antidepressant fluoxetine, a selective 5-HT reuptake inhibitor (SSRI), was able to potentiate the action of subeffective dose of MgCl2 in the FST. This result suggests that the effect of MgCl2 in the FST in mice seems to be similar to the effect of fluoxetine, regarding the interaction with 5-HT receptor subtypes. This result is somewhat in accordance with the synergistic antidepressant-like effect observed when Mg2+ was given jointly with citalopram, a SSRI (Poleszak, 2007). These reports suggest that Mg+2 might be beneficial for treatment of depression and is in agreement with previous clinical data (Eby and Eby, 2006). Regarding

240

C.C. Cardoso et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 33 (2009) 235–242

the serotonergic system, present data extend literature data that has shown that the depletion of serotonin stores with PCPA was able prevent the antidepressant-like effect of MgCl2 (Poleszak, 2007), by indicating that 5-HT1A and 5-HT2 receptors are implicated in its mechanism of action in the FST. The role of noradrenaline in the pathophysiology of depression has also been extensively studied (Brunello et al., 2003; Delgado and Moreno, 2000; Elhwuegi, 2004). Depression seems to be associated with a hypofunction of the noradrenergic system (Brunello et al., 2003; Wang et al., 1999) and some antidepressants as reboxetine and mirtazapine act by increasing the synaptic availability of noradrenaline (Brunello et al., 2003). Moreover, Dziedzicka-Wasylewska et al. (2006) showed that knockout mice, lacking noradrenaline transporter, behaved like wild-type mice acutely treated with reboxetine, desipramine, and imipramine, in the FST and in the tail suspension test. In our study both prazosin (an α1-adrenoreceptor antagonist) and yohimbine (an α2-adrenoreceptor antagonist) were able to reverse the antidepressant-like effect of magnesium in the FST. This result indicates that the magnesium may exert its effect in the FST by interacting with both α1 and α2-adrenoceptors. The α1- and α2adrenoceptors have been shown to underlie some of the antidepressant-like responses of drugs in behavioral models of depression (Danysz et al., 1986; Kaster et al., 2007b; Masuda et al., 2001). Moreover, the blockade of α1-adrenoceptors mimics depressive states, which, like chronic stress, are associated with α1-adrenoceptor desensitization (Stone et al., 2003). In contrast, chronic antidepressants and electroconvulsive therapy enhance the density and functional activity of α1-adrenoceptors in structures such as frontal cortex and hippocampus (Stone et al., 2003). In addition, chronic antidepressant treatment gradually downregulates α2-adrenoceptor autoreceptors and its levels are elevated both in depressed patients and by long-term stress (Flügge et al., 2003; Ordway et al., 2003). In spite of the fact that in the present study the anti-immobility effect of Mg2+ was reversed by prazosin and yohimbine, a recent study has shown that reboxetine, a selective noradrenergic reuptake inhibitor, was not effective in causing a synergistic effect when given jointly with Mg2+ (Poleszak, 2007). Therefore, the involvement of noradrenergic system in the antidepressant-like effect of Mg2+ needs further studies. Tricyclic antidepressants have been available since the early 1960s and provided one of the first clues into the types of chemical changes in the brain that regulate depressive symptoms (Dwoskin et al., 2006; Nestler et al., 2002). Imipramine acts by inhibiting the plasma membrane transporters for serotonin and noradrenaline, increasing the activity of the brain's serotonergic or noradrenergic system (Nestler et al., 2002; Richelson and Pfenning, 1984). In the present study, a sub-effective dose of MgCl2 was able to potentiate a subeffective dose of imipramine in the FST; since no effects on locomotor activity in the open-field test were observed, the results indicate a specific enhancement of antidepressant-like activity by such combined treatment. This finding is in line with the study from Poleszak et al. (2005b) which has shown that Mg+2 enhances the antiimmobility activity of imipramine in the FST. The dopaminergic system is suggested to be implicated in the regulation of mood (Dailly et al., 2004; Millan, 2004). Clinical studies showed that the plasma levels of dopamine metabolites were significantly lower in the depressed patients, indicating a diminished dopamine turnover (Mitani et al., 2006; Sher et al., 2006). Moreover, a common trait of antidepressants is an enhancement in extracellular levels of dopamine in the frontal cortex (Millan, 2004). As shown in the results, the selective dopamine D1 receptor antagonist SCH 23390 and the dopamine D2 receptor antagonist sulpiride, prevented the antiimmobility effects of MgCl2 in the FST. Serra et al. (1990) showed that the administration of SKF 38393, a dopamine D1 receptor agonist, is able to reverse learned helplessness behavior and to reduce immobility time in the FST. Moreover, the blockade of inhibitory dopamine D2 autoreceptors, which enhances dopamine levels in the nucleus accumbens and

frontal cortex, may be involved in antidepressant properties of the antipsychotic, amisulpride (Cassano and Jori, 2002; Yamada et al., 2004). There is also a considerable amount of pharmacological evidence regarding the efficacy of antidepressants with dopaminergic effects in the treatment of depression (Papakostas, 2006). Indeed, Yamada et al. (2004) suggested that dopamine D1 and D2 receptors play a role in the effects of dopamine reuptake inhibitors in the FST. Besides the ability of MgCl2 to produce an antidepressant-like effect with fluoxetine and imipramine, our study also show that it was able to cause a synergistic antidepressant-like effect with bupropion. This antidepressant, a dopamine reuptake inhibitor with subtle activity on noradrenergic and serotonergic reuptake, is often proposed as an adjunct drug with SSRIs and SNRIs to treat resistant depression (Cooper et al., 1980; Prica et al., 2008; Richelson, 2003). Pre-clinical studies suggested that an increase of 5-HT availability in the synapse, leading to 5-HT receptors stimulation on different neuronal systems might be necessary to the development of antidepressant-like effects of classical antidepressants co-administered with bupropion (Clenet et al., 2001; Prica et al., 2008; Redrobe and Bourin, 1997). In our study, the co-administration of sub-effective doses of bupropion and MgCl2 produced a synergistic effect in the FST, without modifying the ambulatory behavior. This effect suggests that Mg+2 could be helpful for the improvement of the conventional antidepressant therapy of depression, which remains inadequate for many individuals and presents a delay of onset of action, lack of efficacy and several side effects (Nemeroff and Owens, 2002; Nestler et al., 2002). However, in order to ascertain the antidepressant effect of Mg+2, further investigations in other animal models of depression and in clinical models are needed. 5. Conclusion Our study extends literature data regarding the mechanisms underlying the antidepressant-like effect of MgCl2 in the FST. We have shown that its antidepressant-like effect is dependent on its interaction with the serotonergic (5-HT1A, 5-HT2A/2C receptors), noradrenergic (α1 and α2-receptors) and dopaminergic (D1 and D2 receptors) systems. Moreover, the synergistic antidepressant-like effect obtained when mice were treated with MgCl2 in combination with fluoxetine, imipramine and bupropion has reinforced the hypothesis of the involvement of the monoaminergic system in the mechanism of the antidepressant-like action of magnesium. Moreover, our results suggest that MgCl2 shares with established antidepressants some pharmacological effects, at least at a preclinical level and that Mg+2 might improve the effectiveness of these antidepressant compounds in the therapy of human depression. Acknowledgements This study was supported by the FINEP research grant “Rede Instituto Brasileiro de Neurociência (IBN-Net)” # 01.06.0842-00, CNPq and CAPES (Brazil). References Bac P, Pages N, Herrenknecht C, Teste JF. Inhibition of mouse-killing behaviour in magnesium-deficient rats: effect of pharmacological doses of magnesium pidolate, magnesium aspartate, magnesium lactate, magnesium gluconate and magnesium chloride. Magnes Res 1995;8:37–45. Booij L, Van der Does AJ, Riedel WJ. Monoamine depletion in psychiatric and healthy populations: review. Mol Psychiatry 2003;8:951–73. Borsini F, Meli A. Is the forced swimming test a suitable model for revealing antidepressant activity? Psychopharmacology 1988;94:147–60. Brocardo PS, Budni J, Kaster MP, Santos ARS, Rodrigues ALS. Folic acid administration produces an antidepressant-like effect in mice: evidence for the involvement of the serotonergic and noradrenergic systems. Neuropharmacology 2008;54:464–73. Brunello N, Blier P, Judd LL, Mendlewicz J, Nelson CJ, Souery D, Zohar J, Racagni G. Noradrenaline in mood and anxiety disorders: basic and clinical studies. Int Clin Psychopharmacol 2003;18:191–202.

C.C. Cardoso et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 33 (2009) 235–242 Cassano GB, Jori MC. Efficacy and safety of amisulpride 50 mg versus paroxetine 20 mg in major depression: a randomized, double-blind, parallel group study. Int Clin Psychopharmacol 2002;17:27–32. Claustre Y, Rouquier L, Serrano A, Bénavidès J, Scatton B. Effect of the putative 5-HT1A receptor antagonist NAN-190 on rat brain serotonergic transmission. Eur J Pharmacol 1991;204:71–7. Clenet F, De Vos A, Bourin M. Involvement of 5-HT2C receptors in the antiimmobility effects of antidepressants in the forced swimming test in mice. Eur Neuropsychopharmacol 2001;11:145–52. Cooper BR, Hester TJ, Maxwell RA. Behavioral and biochemical effects of the antidepressant bupropion (Wellbutrin): evidence for selective blockade of dopamine uptake in vivo. J Pharmacol Exp Ther 1980;215:127–34. Cryan JF, Lucki I. Antidepressant-like behavioral effects mediated by 5-Hydroxytryptamine(2C) receptors. J Pharmacol Exp Ther 2000;295:1120–6. Cryan JF, Markou A, Lucki I. Assessing antidepressant activity in rodents: recent developments and future needs. Trends Pharmacol Sci 2002;23:238–45. Dailly E, Chenu F, Renard CE, Bourin M. Dopamine, depression and antidepressants. Fundam Clin Pharmacol 2004;18:601–7. Danysz W, Kostowski W, Kozak W, Hauptmann M. On the role of noradrenergic neurotransmission in the action of desipramine and amitriptyline in animal models of depression. Pol J Pharmacol Pharm 1986;38:285–98. Decollogne S, Tomas A, Lecerf C, Adamowicz E, Seman M. NMDA receptor complex blockade by oral administration of magnesium: comparison with MK-801. Pharmacol Biochem Behav 1997;58:261–8. Delgado PL, Moreno FA. Role of norepinephrine in depression. J Clin Psychiatry 2000;1:5-12. Dhir A, Kulkarni SK. Effect of addition of yohimbine (α2-receptor antagonist) to the antidepressant activity of fluoxetine or venlafaxine in the mouse forced swim test. Pharmacology 2007;80:239–43. Dunn RW, Corbett R, Martin LL, Payack JF, Laws-Ricker L, Wilmot CA, Rush DK, Cornfeldt ML, Fielding S. Preclinical anxiolytic profiles of 7189 and 8319, novel noncompetitive NMDA antagonists. Prog Clin Biol Res 1990;361:495–512. Dwoskin LP, Rauhut AS, King-Pospisil KA, Bardo MT. Review of the pharmacology and clinical profile of bupropion, an antidepressant and tobacco use cessation agent. CNS Drug Rev 2006;12:178–207. Dziedzicka-Wasylewska M, Faron-Górecka A, Kuśmider M, Drozdowska E, Rogóz Z, Siwanowicz J, Caron MG, Bönisch H. Effect of antidepressant drugs in mice lacking the norepinephrine transporter. Neuropsychopharmacology 2006;31:2424–32. Eby GA, Eby KL. Rapid recovery from major depression using magnesium treatment. Med Hypotheses 2006;67:362–70. Eckeli AL, Dach F, Rodrigues ALS. Acute treatments with GMP produce antidepressantlike effects in mice. NeuroReport 2000;11:1839–43. Elhwuegi AS. Central monoamines and their role in major depression. Prog Neuropsychopharmacol Biol Psychiatry 2004;28:435–51. Flügge G, van Kampen M, Meyer H, Fuchs E. α2A and α2C-adrenoceptor regulation in the brain: α2A changes persist after chronic stress. Eur J Neurosci 2003;17:917–28. Hall RC, Joffe JR. Hypomagnesemia. Physical and psychiatric symptoms. JAMA 1973;224:1749–51. Hashizume N, Mori M. An analysis of hypermagnesemia and hypomagnesemia. Jpn J Med 1990;29:368–72. Hensler JG. Differential regulation of 5-HT1A receptors-G protein interactions in brain following chronic antidepressant administration. Neuropsychopharmacology 2002;26:565–73. Hirvonen J, Karlsson H, Kajander J, Lepola A, Markkula J, Rasi-Hakala H, Någren K, Salminen JK, Hietala J. Decreased brain serotonin 5-HT1A receptor availability in medicationnaive patients with major depressive disorder: an in-vivo imaging study using PET and [carbonyl-11C]WAY-100635. Int J Neuropsychopharmacol 2008;11:465–76. Kaster MP, Santos ARS, Rodrigues ALS. Involvement of 5-HT1A receptors in the antidepressant-like effect of adenosine in the mouse forced swimming test. Brain Res Bull 2005;67:53–61. Kaster MP, Budni J, Binfaré RW, Santos ARS, Rodrigues ALS. The inhibition of different types of potassium channels underlies the antidepressant-like effect of adenosine in the mouse forced swimming test. Prog Neuropsychopharmacol Biol Psychiatry 2007a;31:690–6. Kaster MP, Raupp I, Binfaré RW, Andreatini R, Rodrigues ALS. Antidepressant-like effect of lamotrigine in the mouse forced swimming test: evidence for the involvement of the noradrenergic system. Eur J Pharmacol 2007b;565:119–24. Khisti RT, Chopde CT. Serotonergic agents modulate antidepressant-like effect of the neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one in mice. Brain Res 2000;865:291–300. Kirov GK, Birch NJ, Steadman P, Ramsey RG. Plasma magnesium levels in a population of psychiatric patients: correlations with symptoms. Neuropsychobiology 1994;30:73–8. Loscher W, Annies R, Honack D. The N-methyl-D-aspartate receptor antagonist MK-801 induces increases in dopamine and serotonin metabolism in several brain regions. Neurosci Lett 1991;128:191–4. Machado DG, Kaster MP, Binfaré RW, Dias M, Santos ARS, Pizzolatti MG, Brighente IM, Rodrigues ALS. Antidepressant-like effect of the extract from leaves of Schinus molle L. in mice: evidence for the involvement of the monoaminergic system. Prog Neuropsychopharmacol Biol Psychiatry 2007;31:421–8. Maj J, Rogóz Z, Skuza G, Sowińska H. Effects of MK-801 and antidepressant drugs in the forced swimming test in rats. Eur Neuropsychopharmacol 1992;2:37–41. Malpuech-Brugère C, Nowacki W, Daveau M, Gueux E, Linard C, Rock E, Lebreton J, Mazur A, Rayssiguier Y. Inflammatory response following acute magnesium deficiency in the rat. Biochim Biophys Acta 2000;1501:91–8. Masuda Y, Ohnuma S, Sugiyama T. Alpha 2-adrenoceptor activity induces the antidepressant-like glycolipid in mouse forced swimming. Methods Find Exp Clin Pharmacol 2001;23:19–21.

241

Mathe JM, Nomikos GG, Blakemen KH, Svensson TH. Differential actions of dizocilpine (MK-801) on the mesolimbic and mesocortical dopamine systems: role of neuronal activity. Neuropharmacology 1999;38:121–8. Millan MJ. The role of monoamines in the actions of established and “novel” antidepressant agents: a critical review. Eur J Pharmacol 2004;500:371–84. Mitani H, Shirayama Y, Yamada T, Kawahara R. Plasma levels of homovanillic acid, 5hydroxyindoleacetic acid and cortisol, and serotonin turnover in depressed patients. Prog Neuropsychopharmacol Biol Psychiatry 2006;30:531–4. Nemeroff CB, Owens MJ. Treatment of mood disorders. Nat Neurosci 2002;5:1068–70. Nestler EJ, Barrot M, DiLeone RJ, Eisch AJ, Gold SJ, Monteggia LM. Neurobiology of depression. Neuron 2002;34:13–25. O'Neill MF, Conway MW. Role of 5-HT1A and 5-HT1B receptors in the mediation of behavior in the forced swim test in mice. Neuropsychopharmacology 2001;24:391–8. Ordway GA, Schenk J, Stockmeier CA, May W, Klimek V. Elevated agonist binding to alpha2-adrenoceptors in the locus coeruleus in major depression. Biol Psychiatry 2003;53:315–23. Papakostas GI. Dopaminergic-based pharmacotherapies for depression. Eur Neuropsychopharmacol 2006;16:391–402. Poleszak E. Modulation of antidepressant-like activity of magnesium by serotonergic system. J Neural Transm 2007;114:1129–34. Poleszak E, Szewczyk B, Kedzierska E, Wlaź P, Pilc A, Nowak G. Antidepressant- and anxiolytic-like activity of magnesium in mice. Pharmacol Biochem Behav 2004;78:7-12. Poleszak E, Wlaź P, Kedzierska E, Radziwon-Zaleska M, Pilc A, Fidecka S, Nowak G. Effects of acute and chronic treatment with magnesium in the forced swim test in rats. Pharmacol Rep 2005a;57:654–8. Poleszak E, Wláz P, Szewczykc B, Kedzierskaa E, Wyskad E, Librowskie T, SzymuraOleksiakd J, Fideckaa S, Pilccf A, Nowakc G. Enhancement of antidepressant-like activity by joint administration of imipramine and magnesium in the forced swim test: behavioral and pharmacokinetic studies in mice. Pharmacol Biochem Behav 2005b;8:1524–9. Poleszak E, Wlaź P, Kedzierska E, Nieoczym D, Wyska E, Szymura-Oleksiak J, Fidecka S, Radziwoń-Zaleska M, Nowak G. Immobility stress induces depression-like behavior in the forced swim test in mice: effect of magnesium and imipramine. Pharmacol Rep 2006;58:746–52. Poleszak E, Wlaź P, Kedzierska E, Nieoczym D, Wróbel A, Fidecka S, Pilc A, Nowak G. NMDA/glutamate mechanism of antidepressant-like action of magnesium in forced swim test in mice. Pharmacol Biochem Behav 2007;88:158–64. Porsolt RD, Bertin A, Jalfre M. Behavioral despair in mice: a primary screening test for antidepressants. Arch Int Pharmacodyn Ther 1977;229:327–36. Prica C, Hascoet M, Bourin M. Is co-administration of bupropion with SSRIs and SNRIs in forced swimming test in mice, predictive of efficacy in resistant depression? Behav Brain Res 2008;194:92–9. Rasmussen HH, Mortensen PB, Jensen IW. Depression and magnesium deficiency. Int J Psychiatry Med 1989;19:57–63. Redrobe JP, Bourin M. Partial role of 5-HT2 and 5-HT3 receptors in the activity of antidepressants in the mouse forced swimming test. Eur J Pharmacol 1997;325:129–35. Redrobe JP, MacSweeney CP, Bourin M. The role of 5-HT1A and 5-HT1B receptors in antidepressant drug actions in the mouse forced swimming test. Eur J Pharmacol 1996;318:213–20. Richelson E. Interactions of antidepressants with neurotransmitter transporters and receptors and their clinical relevance. J Clin Psychiatry 2003;64:5-12. Richelson E, Pfenning M. Blockade by antidepressants and related compounds of biogenic amine uptake into rat brain synaptosomes: most antidepressants selectively block norepinephrine uptake. Eur J Pharmacol 1984;104:277–86. Rodrigues ALS, Silva GL, Matteussi AS, Fernandes E, Miguel O, Yunes RA, Calixto JB, Santos ARS. Involvement of monoaminergic system in the antidepressant-like effect of the hydroalcoholic extract of Siphocampylus verticillatus. Life Sci 2002;70:1347–58. Rosa AO, Kaster MP, Binfaré RW, Morales S, Martín-Aparicio E, Navarro-Rico ML, Martinez A, Medina M, García AG, López MG, Rodrigues ALS. Antidepressant-like effect of the novel thiadiazolidinone NP031115 in mice. Prog Neuropsychopharmacol Biol Psychiatry 2008;32:1549–56. Ruhé HG, Mason NS, Schene AH. Mood is indirectly related to serotonin, norepinephrine and dopamine levels in humans: a meta-analysis of monoamine depletion studies. Mol Psychiatry 2007;12:331–59. Ryan MF. The role of magnesium in clinical biochemistry: an overview. Ann Clin Biochem 1991;28:19–26. Schneider AM, Simson PE. NAN-190 potentiates the impairment of retention produced by swim stress. Pharmacol Biochem Behav 2007;87:73–80. Serra G, Collu M, D'Aquila PS, De Montis GM, Gessa GL. Possible role of dopamine D1 receptor in the behavioural supersensitivity to dopamine agonists induced by chronic treatment with antidepressants. Brain Res 1990;527:234–43. Sher L, Mann JJ, Traskman-Bendz L, Winchel R, Huang YY, Fertuck E, Stanley BH. Lower cerebrospinal fluid homovanillic acid levels in depressed suicide attempters. J Affect Disord 2006;90:83–9. Singewald N, Sinner C, Hetzenauer A, Sartori SB, Murck H. Magnesium-deficient diet alters depression- and anxiety-related behavior in mice-influence of desipramine and Hypericum perforatum extract. Neuropharmacology 2004;47:1189–97. Skolnick P. Antidepressants for the new millennium. Eur J Pharmacol 1999;375:31–40. Skolnick P. Beyond monoamine-based therapies: clues to new approaches. J Clin Psychiatry 2002;2:19–23. Sobolevskii AI, Khodorov BI. Blocker studies of the functional architecture of the NMDA receptor channel. Neurosci Behav Physiol 2002;32:157–71. Stone EA, Grunewald GL, Lin Y, Ahsan R, Rosengarten H, Kramer HK, Quartermain D. Role of epinephrine stimulation of CNS α1-adrenoceptors in motor activity in mice. Synapse 2003;49:67–76.

242

C.C. Cardoso et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 33 (2009) 235–242

Wang YM, Xu F, Gainetdinov RR, Caron MG. Genetic approaches to studying norepinephrine function: knockout of the mouse norepinephrine transporter gene. Biol Psychiatry 1999;46:1124–30. Wedzony K, Mackowiak M, Czyrak A, Fijal K, Michalska B. Single doses of MK-801, a noncompetitive antagonist of NMDA receptors, increase the number of 5-HT1A serotonin receptors in the rat brain. Brain Res 1997;756:84–91. Wong EH, Kemp JA, Priestley T, Knight AR, Woodruff GN, Iversen LL. The anticonvulsant MK-801 is a potent N-methyl-D-aspartate antagonist. Proc Natl Acad Sci USA 1986;83:7104–8. Yamada J, Sugimoto Y, Yamada S. Involvement of dopamine receptors in the antiimmobility effects of dopamine re-uptake inhibitors in the forced swimming test. Eur J Pharmacol 2004;504:207–11.

Zieba A, Kata R, Dudek D, Schlegel-Zawadzka M, Nowak G. Serum trace elements in animal models and human depression: Part III. Magnesium. Relationship with copper. Hum Psychopharmacol 2000;15:631–5. Zomkowski AD, Hammes L, Lin J, Calixto JB, Santos ARS, Rodrigues ALS. Agmatine produces antidepressant-like effects in two models of depression in mice. NeuroReport 2002;13:387–91. Zomkowski ADE, Rosa AO, Lin J, Santos ARS, Calixto JB, Rodrigues ALS. Evidence for serotonin receptor subtypes involvement in agmatine antidepressant-like effect in the mouse forced swimming test. Brain Res 2004;1023:253–63.