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Antidepressant-like effect of lamotrigine in the mouse forced swimming test: Evidence for the involvement of the noradrenergic system
European Journal of Pharmacology 565 (2007) 119 – 124 www.elsevier.com/locate/ejphar
Antidepressant-like effect of lamotrigine in the mouse forced swimming test: Evidence for the involvement of the noradrenergic system Manuella Pinto Kaster a , Inara Raupp b , Ricardo Wabner Binfaré a , Roberto Andreatini b , Ana Lúcia S. Rodrigues a,⁎ a
Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Campus Universitário, Trindade — 88040-900, Florianópolis-SC, Brazil b Departamento de Farmacologia, Setor de Ciências Biológicas, Universidade Federal do Paraná, Centro Politécnico — 81531-990, Curitiba-PR, Brazil Received 12 December 2006; received in revised form 27 February 2007; accepted 6 March 2007 Available online 19 March 2007
Abstract Lamotrigine is an anticonvulsant drug that is also effective in the treatment of mood disorders, especially bipolar disorder. However, few studies have been conducted in animal models of depression to evaluate its mechanism of action. The present study investigated the effect of lamotrigine in the forced swimming test in mice and the involvement of the noradrenergic system in this effect. Lamotrigine (20–30 mg/kg, i.p.) decreased the immobility time in the forced swimming test and the number of crossings in the open-field test. In addition, the pretreatment of mice with the inhibitor of the enzyme tyrosine hydroxylase, α-methyl-p-tyrosine (100 or 250 mg/kg), prevented the antidepressant-like effect of lamotrigine (30 mg/kg, i.p.) in the forced swimming test. Besides that, the pretreatment of mice with prazosin (1 mg/kg, i.p., an α1-adrenoceptor antagonist) or yohimbine (1 mg/kg, i.p., an α2-adrenoceptor antagonist) also prevented the anti-immobility effect of lamotrigine (30 mg/kg, i.p.). Moreover, the administration of subeffective doses of phenylephrine (5 mg/kg, i.p., an α1-adrenoceptor agonist) or clonidine (0.06 mg/kg, i.p., an α2-adrenoceptor agonist) was able to potentiate the action of a subeffective dose of lamotrigine (10 mg/kg, i.p.) in the forced swimming test. Thus, the present study suggests that the antidepressant-like effect of lamotrigine in the forced swimming test is related to the noradrenergic system, likely due to an activation of α1- and α2-postsynaptic adrenoceptors. © 2007 Elsevier B.V. All rights reserved. Keywords: Lamotrigine; Noradrenergic system; Forced swimming test; Antidepressant
1. Introduction Lamotrigine [3,5-diamino-6(2,3-dichlorophenyl)-1,2,4-triazine] is an anticonvulsant agent used primarily in the treatment of generalized and partial seizures (Kwan and Brodie, 2001; Bazil, 2002). The best characterized actions of lamotrigine are associated with the inhibition of voltage gated sodium channels, inhibition of the release of excitatory amino acids such as glutamate and aspartate, and calcium-channel blockade, mechanisms that are believed to mediate its seizure suppressing activity (Cheung et al., 1992; Xie et al., 1995; Cunningham and Jones, 2000). ⁎ Corresponding author. Tel.: +55 48 3331 9692; fax: +55 48 3331 9672. E-mail addresses: [email protected], [email protected] (A.L.S. Rodrigues). 0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2007.03.003
In common with other anticonvulsant drugs, including sodium valproate and carbamazepine, lamotrigine exhibits mood-stabilizing properties (Bowden, 1998; Post et al., 1998). A series of randomized trials have shown that lamotrigine was effective for the treatment of patients with bipolar I disorder who were currently experiencing an episode of major depression (Calabrese et al., 1999a). In addition, it has a low potential for inducing the switch to mania, a risk that is often associated with the use of conventional antidepressants (Calabrese et al., 1999b, 2001). Interestingly, several studies have suggested that lamotrigine might have antidepressant properties in unipolar depression (Frye et al., 2000; Barbee and Jamhour, 2002; Rocha and Hara, 2003) and may accelerate the onset of action when given in combination with typical antidepressants such as fluoxetine and paroxetine (Normann et al., 2002; Barbosa et al., 2003). Southam et al.
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(1998) reported that the synaptosomal uptake of serotonin, noradrenaline and dopamine in rat brain was inhibited by lamotrigine, suggesting that its efficacy against bipolar depression could be a consequence of the inhibition of monoamines reuptake. Recently, some preclinical work with mice (Bourin et al., 2005) and rats (Consoni et al., 2006) have reported that lamotrigine exhibits antidepressant-like effects in the forced swimming test, a widely used model for assessing pharmacological antidepressant activity (Cryan et al., 2002). These effects seem to be mediated through an interaction with the serotonergic (Bourin et al., 2005; Consoni et al., 2006) and noradrenergic systems (Consoni et al., 2006). The suggestion that the effect of lamotrigine is due to an interaction with the noradrenergic system was given by the finding that it produced a similar result in the modified forced swimming test in rats as compared to the one produced by nortriptyline, a noradrenaline reuptake inhibitor (Consoni et al., 2006). Several experimental and clinical studies indicate that the noradrenergic system is widely implicated in the pathophysiology of depression (Frazer, 2000; Nutt, 2006). Drugs affecting the noradrenergic neurotransmission, such as those that inhibit noradrenaline reuptake at nerve terminals, or its metabolism (monoamine oxidase inhibitors), are effective in depression. Also, early studies have shown that the depletion of monoamines with reserpine leads to depressogenic effects in individuals already vulnerable to affective illness (Goodwin and Bunney, 1971). Taking into account that only few studies have been conducted with lamotrigine in animal models to clarify its mechanism of antidepressant action, the present work aimed to extend literature data by further investigating the involvement of the noradrenergic system in the antidepressant-like effect of lamotrigine in the forced swimming test in mice. 2. Materials and methods 2.1. Animals Female Swiss mice (30–40 g) were maintained at 22–27 °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. The cages were placed in the experimental room 24 h before the test for acclimatization. All manipulations were carried out between 9:00 and 17:00 h, with each animal used only once. All procedures in this study were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize animal suffering and the number of animals used. 2.2. Drugs and treatment The following drugs were used: clonidine, lamotrigine, α-methyl-p-tyrosine, phenylephrine, prazosin, yohimbine (Sigma Chemical Co, USA). Lamotrigine and α-methyl-p-tyrosine were dissolved in saline with 10% Tween 80, whereas all the other drugs were dissolved in isotonic saline solution (NaCl 0.9%)
immediately before use. Appropriate vehicle-treated groups were also assessed simultaneously. All drugs were administered by intraperitoneal (i.p.) route, in a volume of 10 ml/kg body weight. To test the hypothesis that the antidepressant-like effect of lamotrigine is mediated through an interaction with the noradrenergic system, animals were pretreated with α-methyl-p-tyrosine (100 mg/kg or 250 mg/kg, an inhibitor of the enzyme tyrosine hydroxylase) 4 h before the administration of lamotrigine (30 mg/kg, i.p.). After 30 min the animals were tested in the forced swimming test or in the open-field test. In a separate series of experiments, the animals were pretreated with prazosin (1 mg/kg, i.p., an α1-adrenoceptor antagonist) or yohimbine (1 mg/kg, i.p., an α2-adrenoceptor antagonist) 30 min before the administration of lamotrigine (30 mg/kg, i.p.) and submitted to the forced swimming test 30 min later. Alternatively, animals were pretreated with subeffective doses of phenylephrine (5 mg/kg, i.p., an α1-adrenoceptor agonist) or clonidine (0.06 mg/kg, i.p., an α2-adrenoceptor agonist) 20 min before the administration of lamotrigine (30 mg/kg, i.p), and 30 min later the forced swimming test and the open-field test 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 abovementioned treatments at the concentrations used (Asakura et al., 1994; Bourin et al., 1996; Zomkowski et al., 2002; Mantovani et al., 2006; Machado et al., 2007). 2.3. Forced swimming test 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 was recorded during the last 4 min of the 6-min period. 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. A decrease in the duration of immobility is indicative of an antidepressant-like effect (Porsolt et al., 1977). 2.4. Open-field test The ambulatory behavior was assessed in an open-field test as described previously (Rodrigues et al., 1996). The apparatus consisted of a wooden box measuring 40 × 60 × 50 cm. The floor of the arena was divided into 12 equal squares. The number of squares crossed with all paws (crossing) was counted in a 6-min session. 2.5. Statistical analysis All experimental results are given as the mean ± S.E.M. Comparisons between experimental and control groups were performed by one-way or two-way ANOVA followed by
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Fig. 1. Effect of lamotrigine (10–30 mg/kg, i.p.) in the forced swimming test (panel A) and in the open-field test (panel B). Values are mean + S.E.M. (n = 5–6). ⁎P b 0.05, ⁎⁎P b 0.01 compared with the control group. A: F3,19 = 5.77; P b 0.01; B: F3,20 = 6.59; P b 0.01.
Newman–Keuls test for post hoc comparison when appropriate. A value of P b 0.05 was considered to be significant.
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Fig. 3. Effect of pretreatment of animals with prazosin (1 mg/kg, i.p., an α1-adrenoceptor antagonist, panel A) or yohimbine (1 mg/kg, i.p., an α2-adrenoceptor antagonist, panel B), on the lamotrigine (30 mg/kg, i.p.)-induced reduction in immobility time in the forced swimming test in mice. Values are expressed as mean + S.E.M. (n = 5–10). ⁎⁎P b 0.01 compared with the control group; #P b 0.01 when comparing with the same group pretreated with saline. A: treatment: F1,19 = 19.86; P b 0.01; pretreatment: F1,19 = 9.95; P b 0.01; interaction: F1,19 = 4.64; P b 0.05. B: treatment: F1,27 = 5.55; P b 0.05; pretreatment: F1,27 = 4.31; P b 0.05; interaction: F1,27 = 11.04; P b 0.01.
3. Results 3.1. Antidepressant-like effect of lamotrigine in the forced swimming test The results depicted in Fig. 1A show the effect of the administration of lamotrigine (10–30 mg/kg, i.p.) in the forced swimming test. Lamotrigine dose dependently decreased the immobility time in the forced swimming test in mice, with a statistically significant decrease at 20 and 30 mg/kg (P b 0.05 and P b 0.01, respectively) indicating an antidepressant-like effect. In addition, Fig. 1B shows the effect of lamotrigine (10–30 mg/kg, i.p.) in the ambulatory behavior. Lamotrigine at 20–30 mg/kg significantly decreased the number of crossings in the open-field test (P b 0.01). At a lower dose (10 mg/kg) lamotrigine was devoid of effect in the forced swimming test and in the open-field test.
kg, i.p.) in the forced swimming test. Fig. 2B shows that both the pretreatment with α-methyl-p-tyrosine (100 or 250 mg/kg) and the treatment with lamotrigine significantly decreased the number of crossings in the open-field test. The group treated with α-methyl-p-tyrosine (100 mg/kg, i.p.) plus lamotrigine did not produce a significant effect in the open-field test when compared with the groups treated with α-methyl-p-tyrosine (100 mg/kg, i.p.) or lamotrigine alone. However, the pretreatment of mice with α-methyl-p-tyrosine (250 mg/kg) plus lamotrigine significantly reduced de ambulatory behavior as compared with the group treated with lamotrigine alone. 3.3. Involvement of α1–α2-adrenoceptors in the antidepressant-like effect of lamotrigine in the forced swimming test
Fig. 2A shows that the pretreatment of mice with α-methylp-tyrosine (100 mg/kg or 250 mg/kg, i.p.) significantly prevented the antidepressant-like effect of lamotrigine (30 mg/
The results presented in Fig. 3A show that the pretreatment of mice with prazosin (1 mg/kg, i.p.) was able to prevent the antiimmobility effect of lamotrigine (30 mg/kg, i.p.) in the forced swimming test. Fig. 3B shows the reversal of the antidepressantlike effect of lamotrigine (30 mg/kg, i.p.) in the forced swimming test by the pretreatment with yohimbine. As presented in Fig. 4A, the administration of subeffective doses of phenylephrine (5 mg/kg, i.p.,) and clonidine (0.06 mg/
Fig. 2. Effect of pretreatment of mice with α-methyl-p-tyrosine (AMPT, 100 mg/ kg or 250 mg/kg), on the lamotrigine (30 mg/kg)-induced reduction in the immobility time in the forced swimming test (panel A), or in the number of crossings in the open-field test (panel B). Values are mean + S.E.M. (n = 5–8). ⁎P b 0.05 compared with the control group; ⁎⁎P b 0.01 compared with the control group; #P b 0.01 compared with the same group pretreated with saline; + P b 0.01 compared with the group treated with vehicle/lamotrigine. A: treatment: F1,32 = 11.86; P b 0.01; pretreatment: F2,32 = 4.52; P b 0.05; interaction: F2,32 = 6.79; P b 0.01. B: treatment: F1,30 = 5.78; P b 0.05; pretreatment: F2,30 = 18.55; P b 0.01; interaction: F2,30 = 1.23; P = 0.30.
Fig. 4. Effect of phenylephrine (5 mg/kg, i.p., an α1-adrenoceptor agonist) or clonidine (0.06 mg/kg, i.p., an α2-adrenoceptor agonist) combined with a subeffective dose of lamotrigine (10 mg/kg, i.p.) in the forced swimming test (panel A) and in the open-field test (panel B). Values are expressed as mean +S.E.M. (n =5–6). ⁎⁎P b 0.01 compared with the control group. A: treatment: F1,27 =28.78; P b 0.01; pretreatment: F2,27 = 10.67; P b 0.01; interaction: F2,27 =6.02; P b 0.01. B: treatment: F1,27 =28.78; P b 0.01; pretreatment: F2,27 =28.88; P b 0.01; interaction: F1,27 = 2.35; P = 0.13.
3.2. Effect of lamotrigine on α-methyl-p-tyrosine-treated mice in the forced swimming test
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kg, i.p.) produced a synergistic antidepressant-like effect when combined with a subeffective dose of lamotrigine (10 mg/kg, i.p.) in the forced swimming test. The results presented in Fig. 4B show the effect of phenylephrine (5 mg/kg, i.p.,) and clonidine (0.06 mg/kg, i.p.) combined with lamotrigine (10 mg/kg, i.p.) in the open-field test. 4. Discussion The present study was carried out to evaluate the antidepressant-like effect of lamotrigine in the mouse forced swimming test and the involvement of the noradrenergic system in its effect. Our results confirm literature data, by showing that lamotrigine reduces the immobility time in the forced swimming test. In addition, our study extends literature data by providing convincing evidence that its effect in the forced swimming test is probably mediated by an interaction with the noradrenergic system, probably with postsynaptic α1- and α2-adrenoceptors. The administration of lamotrigine produced a decrease in the immobility time in the forced swimming test, a response that is consistent with an antidepressant-like action. Among all animal models the forced swimming test remains one of the most used tools for assessing pharmacological antidepressant activity (Petit-Demouliere et al., 2005). Antidepressant treatments reduce the immobility time of mice in this test. Indeed, the sensitivity of the forced swimming test to an impressively broad range of antidepressant drugs is one of the most important features supporting its primary use as a screen in antidepressant discovery research (Porsolt et al., 1977). It should be noted that the doses of lamotrigine that decreased the immobility time also produced a reduction in the locomotor activity assessed in the open-field test. Thus, these results demonstrated that the antidepressant-like effect of lamotrigine occurs despite its sedative effect. This assumption is in accordance with the literature data showing that lamotrigine produced an antidepressant-like effect in the forced swimming test in mice and rats in spite of impairing spontaneous locomotor activity (Bourin et al., 2005; Consoni et al., 2006). Indeed, the tricyclic antidepressants imipramine and desipramine, produce sedative effects, but also exhibit an antidepressant-like activity in the forced swimming test in mice (Hascoet et al., 1991; David et al., 2003). In order to investigate the possible involvement of the noradrenergic system in the antidepressant-like effect of lamotrigine, αmethyl-p-tyrosine, a selective inhibitor of the enzyme tyrosine hydroxylase, was used. This is the rate-limiting enzyme in the synthesis of noradrenaline and dopamine. In the present work the pretreatment of mice with α-methyl-p-tyrosine (100 and 250 mg/ kg) was able to prevent the anti-immobility effect of lamotrigine in the forced swimming test. However, the higher dose of α-methyl-p-tyrosine combined with lamotrigine induced a marked decrease in locomotor activity in the open-field. The sedative effect produced by α-methyl-p-tyrosine at this higher dose might be responsible for the reversal of the antidepressant-like effect of lamotrigine. However, an indication of the involvement of the noradrenergic system in the effect of lamotrigine was given by the
finding that the reversal of the anti-immobility effect of lamotrigine by α-methyl-p-tyrosine was observed even at the lower dose of α-methyl-p-tyrosine, which produced a reduction of the locomotion similar to the one produced by lamotrigine or α-methyl-p-tyrosine per se. Mayorga et al. (2001) demonstrated that α-methyl-p-tyrosine reduces dopamine and noradrenaline levels (57% and 53%, respectively) in mice, without affecting the levels of serotonin. In addition, the acute administration of α-methyl-p-tyrosine temporarily reversed the antidepressant response to desipramine, mazindol and mirtazapine in patients (Delgado et al., 1993, 2002; Miller et al., 1996). Southam et al. (1998) reported that the synaptosomal uptake of serotonin, noradrenaline and dopamine in rat brain was inhibited by lamotrigine. Thus, one possibility that we should not rule out is that lamotrigine may indirectly affect the noradrenergic system by releasing noradrenaline that may, in turn, interact with their receptors. The α1- and α2-adrenoceptors have been shown to underlie some of the antidepressant-like responses of drugs in behavioral models of depression (Kitada et al., 1983; Danysz et al., 1986; Masuda et al., 2001). The decrease in immobility time elicited by lamotrigine was reversed by the pretreatment of mice with prazosin and yohimbine, suggesting that these receptor subtypes underlie its action in the forced swimming test. Indeed, in a study of O'Neill et al. (2001) the antidepressant-like effect of clonidine in the mouse forced swimming test was reversed by yohimbine. Besides that, the antidepressant-like action of desipramine was prevented by the pretreatment of mice with prazosin (Danysz et al., 1986). Further suggesting that α1- and α2-adrenoceptors are implicated in the mechanism of action of lamotrigine in the forced swimming test are the experiments in which the pretreatment of mice with either phenylephrine or clonidine was able to produce a synergistic antidepressant-like effect when combined with a subeffective dose of lamotrigine. These findings reinforce the assumption that the activation of α1- and α2-adrenoceptors is involved in the mechanism of action of lamotrigine. Phenylephrine is a postsynaptic α1-adrenoceptor agonist that was shown to reduce the duration of immobility time in the forced swimming test (Kitada et al., 1983). In addition, the repeated treatment with the antidepressants imipramine, amitriptyline, citalopram and mianserin produced an increase in the affinity of α1-adrenergic receptors for phenylephrine (Klimek et al., 1991; DziedzickaWasylewska et al., 2004). Clonidine is an α2-adrenoceptor agonist that also reduces the immobility time in the forced swimming test in mice and rats (Cervo and Samanin, 1991; O'Neill et al., 2001). Similarly to our result, a subeffective dose of clonidine was able to produce a synergistic effect in the forced swimming test when combined with subeffective doses of various antidepressants such as imipramine, amitriptyline, maprotiline, citalopram, fluvoxamine and mianserin (Malinge et al., 1988). In addition, the pretreatment of mice with α-methyl-p-tyrosine did not prevent the antidepressant-like effect of clonidine in the forced swimming test, suggesting that like phenylephrine, its effect is mediated by postsynaptic receptors (Malinge et al., 1988). In this work, the administration of phenylephrine and clonidine produced a strong decrease in the locomotor activity, an effect that
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Southam, E., Kirkby, D., Higgins, G.A., Hagan, R.M., 1998. Lamotrigine inhibits monoamine uptake in vitro and modulates 5-hydroxytryptamine uptake in rats. Eur. J. Pharmacol. 358, 19–24. Sukul, N.C., Cherian, L., Klemm, W.R., 1988. Alpha noradrenergic agonists promote catalepsy in the mouse. Pharmacol. Biochem. Behav. 31, 87–91. Xie, X., Lancaster, B., Peakman, T., Garthwaite, J., 1995. Interaction of the antiepileptic drug lamotrigine with recombinant rat brain type IIA Na+ channels and with native Na+ channels in rat hippocampal neurones. Pflugers Arch. 430, 437–446. Zhang, W.P., Ouyang, M., Thomas, S.A., 2004. Potency of catecholamines and other L-tyrosine derivatives at the cloned mouse adrenergic receptors. Neuropharmacology 47, 438–449. Zomkowski, A.D., Hammes, L., Lin, J., Calixto, J.B., Santos, A.R.S., Rodrigues, A.L.S., 2002. Agmatine produces antidepressant-like effects in two models of depression in mice. Neuroreport 13, 387–391.
Antidepressant-like effect of lamotrigine in the mouse forced swimming test: Evidence for the involvement of the noradrenergic system Manuella Pinto Kaster a , Inara Raupp b , Ricardo Wabner Binfaré a , Roberto Andreatini b , Ana Lúcia S. Rodrigues a,⁎ a
Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Campus Universitário, Trindade — 88040-900, Florianópolis-SC, Brazil b Departamento de Farmacologia, Setor de Ciências Biológicas, Universidade Federal do Paraná, Centro Politécnico — 81531-990, Curitiba-PR, Brazil Received 12 December 2006; received in revised form 27 February 2007; accepted 6 March 2007 Available online 19 March 2007
Abstract Lamotrigine is an anticonvulsant drug that is also effective in the treatment of mood disorders, especially bipolar disorder. However, few studies have been conducted in animal models of depression to evaluate its mechanism of action. The present study investigated the effect of lamotrigine in the forced swimming test in mice and the involvement of the noradrenergic system in this effect. Lamotrigine (20–30 mg/kg, i.p.) decreased the immobility time in the forced swimming test and the number of crossings in the open-field test. In addition, the pretreatment of mice with the inhibitor of the enzyme tyrosine hydroxylase, α-methyl-p-tyrosine (100 or 250 mg/kg), prevented the antidepressant-like effect of lamotrigine (30 mg/kg, i.p.) in the forced swimming test. Besides that, the pretreatment of mice with prazosin (1 mg/kg, i.p., an α1-adrenoceptor antagonist) or yohimbine (1 mg/kg, i.p., an α2-adrenoceptor antagonist) also prevented the anti-immobility effect of lamotrigine (30 mg/kg, i.p.). Moreover, the administration of subeffective doses of phenylephrine (5 mg/kg, i.p., an α1-adrenoceptor agonist) or clonidine (0.06 mg/kg, i.p., an α2-adrenoceptor agonist) was able to potentiate the action of a subeffective dose of lamotrigine (10 mg/kg, i.p.) in the forced swimming test. Thus, the present study suggests that the antidepressant-like effect of lamotrigine in the forced swimming test is related to the noradrenergic system, likely due to an activation of α1- and α2-postsynaptic adrenoceptors. © 2007 Elsevier B.V. All rights reserved. Keywords: Lamotrigine; Noradrenergic system; Forced swimming test; Antidepressant
1. Introduction Lamotrigine [3,5-diamino-6(2,3-dichlorophenyl)-1,2,4-triazine] is an anticonvulsant agent used primarily in the treatment of generalized and partial seizures (Kwan and Brodie, 2001; Bazil, 2002). The best characterized actions of lamotrigine are associated with the inhibition of voltage gated sodium channels, inhibition of the release of excitatory amino acids such as glutamate and aspartate, and calcium-channel blockade, mechanisms that are believed to mediate its seizure suppressing activity (Cheung et al., 1992; Xie et al., 1995; Cunningham and Jones, 2000). ⁎ Corresponding author. Tel.: +55 48 3331 9692; fax: +55 48 3331 9672. E-mail addresses: [email protected], [email protected] (A.L.S. Rodrigues). 0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2007.03.003
In common with other anticonvulsant drugs, including sodium valproate and carbamazepine, lamotrigine exhibits mood-stabilizing properties (Bowden, 1998; Post et al., 1998). A series of randomized trials have shown that lamotrigine was effective for the treatment of patients with bipolar I disorder who were currently experiencing an episode of major depression (Calabrese et al., 1999a). In addition, it has a low potential for inducing the switch to mania, a risk that is often associated with the use of conventional antidepressants (Calabrese et al., 1999b, 2001). Interestingly, several studies have suggested that lamotrigine might have antidepressant properties in unipolar depression (Frye et al., 2000; Barbee and Jamhour, 2002; Rocha and Hara, 2003) and may accelerate the onset of action when given in combination with typical antidepressants such as fluoxetine and paroxetine (Normann et al., 2002; Barbosa et al., 2003). Southam et al.
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(1998) reported that the synaptosomal uptake of serotonin, noradrenaline and dopamine in rat brain was inhibited by lamotrigine, suggesting that its efficacy against bipolar depression could be a consequence of the inhibition of monoamines reuptake. Recently, some preclinical work with mice (Bourin et al., 2005) and rats (Consoni et al., 2006) have reported that lamotrigine exhibits antidepressant-like effects in the forced swimming test, a widely used model for assessing pharmacological antidepressant activity (Cryan et al., 2002). These effects seem to be mediated through an interaction with the serotonergic (Bourin et al., 2005; Consoni et al., 2006) and noradrenergic systems (Consoni et al., 2006). The suggestion that the effect of lamotrigine is due to an interaction with the noradrenergic system was given by the finding that it produced a similar result in the modified forced swimming test in rats as compared to the one produced by nortriptyline, a noradrenaline reuptake inhibitor (Consoni et al., 2006). Several experimental and clinical studies indicate that the noradrenergic system is widely implicated in the pathophysiology of depression (Frazer, 2000; Nutt, 2006). Drugs affecting the noradrenergic neurotransmission, such as those that inhibit noradrenaline reuptake at nerve terminals, or its metabolism (monoamine oxidase inhibitors), are effective in depression. Also, early studies have shown that the depletion of monoamines with reserpine leads to depressogenic effects in individuals already vulnerable to affective illness (Goodwin and Bunney, 1971). Taking into account that only few studies have been conducted with lamotrigine in animal models to clarify its mechanism of antidepressant action, the present work aimed to extend literature data by further investigating the involvement of the noradrenergic system in the antidepressant-like effect of lamotrigine in the forced swimming test in mice. 2. Materials and methods 2.1. Animals Female Swiss mice (30–40 g) were maintained at 22–27 °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. The cages were placed in the experimental room 24 h before the test for acclimatization. All manipulations were carried out between 9:00 and 17:00 h, with each animal used only once. All procedures in this study were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize animal suffering and the number of animals used. 2.2. Drugs and treatment The following drugs were used: clonidine, lamotrigine, α-methyl-p-tyrosine, phenylephrine, prazosin, yohimbine (Sigma Chemical Co, USA). Lamotrigine and α-methyl-p-tyrosine were dissolved in saline with 10% Tween 80, whereas all the other drugs were dissolved in isotonic saline solution (NaCl 0.9%)
immediately before use. Appropriate vehicle-treated groups were also assessed simultaneously. All drugs were administered by intraperitoneal (i.p.) route, in a volume of 10 ml/kg body weight. To test the hypothesis that the antidepressant-like effect of lamotrigine is mediated through an interaction with the noradrenergic system, animals were pretreated with α-methyl-p-tyrosine (100 mg/kg or 250 mg/kg, an inhibitor of the enzyme tyrosine hydroxylase) 4 h before the administration of lamotrigine (30 mg/kg, i.p.). After 30 min the animals were tested in the forced swimming test or in the open-field test. In a separate series of experiments, the animals were pretreated with prazosin (1 mg/kg, i.p., an α1-adrenoceptor antagonist) or yohimbine (1 mg/kg, i.p., an α2-adrenoceptor antagonist) 30 min before the administration of lamotrigine (30 mg/kg, i.p.) and submitted to the forced swimming test 30 min later. Alternatively, animals were pretreated with subeffective doses of phenylephrine (5 mg/kg, i.p., an α1-adrenoceptor agonist) or clonidine (0.06 mg/kg, i.p., an α2-adrenoceptor agonist) 20 min before the administration of lamotrigine (30 mg/kg, i.p), and 30 min later the forced swimming test and the open-field test 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 abovementioned treatments at the concentrations used (Asakura et al., 1994; Bourin et al., 1996; Zomkowski et al., 2002; Mantovani et al., 2006; Machado et al., 2007). 2.3. Forced swimming test 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 was recorded during the last 4 min of the 6-min period. 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. A decrease in the duration of immobility is indicative of an antidepressant-like effect (Porsolt et al., 1977). 2.4. Open-field test The ambulatory behavior was assessed in an open-field test as described previously (Rodrigues et al., 1996). The apparatus consisted of a wooden box measuring 40 × 60 × 50 cm. The floor of the arena was divided into 12 equal squares. The number of squares crossed with all paws (crossing) was counted in a 6-min session. 2.5. Statistical analysis All experimental results are given as the mean ± S.E.M. Comparisons between experimental and control groups were performed by one-way or two-way ANOVA followed by
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Fig. 1. Effect of lamotrigine (10–30 mg/kg, i.p.) in the forced swimming test (panel A) and in the open-field test (panel B). Values are mean + S.E.M. (n = 5–6). ⁎P b 0.05, ⁎⁎P b 0.01 compared with the control group. A: F3,19 = 5.77; P b 0.01; B: F3,20 = 6.59; P b 0.01.
Newman–Keuls test for post hoc comparison when appropriate. A value of P b 0.05 was considered to be significant.
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Fig. 3. Effect of pretreatment of animals with prazosin (1 mg/kg, i.p., an α1-adrenoceptor antagonist, panel A) or yohimbine (1 mg/kg, i.p., an α2-adrenoceptor antagonist, panel B), on the lamotrigine (30 mg/kg, i.p.)-induced reduction in immobility time in the forced swimming test in mice. Values are expressed as mean + S.E.M. (n = 5–10). ⁎⁎P b 0.01 compared with the control group; #P b 0.01 when comparing with the same group pretreated with saline. A: treatment: F1,19 = 19.86; P b 0.01; pretreatment: F1,19 = 9.95; P b 0.01; interaction: F1,19 = 4.64; P b 0.05. B: treatment: F1,27 = 5.55; P b 0.05; pretreatment: F1,27 = 4.31; P b 0.05; interaction: F1,27 = 11.04; P b 0.01.
3. Results 3.1. Antidepressant-like effect of lamotrigine in the forced swimming test The results depicted in Fig. 1A show the effect of the administration of lamotrigine (10–30 mg/kg, i.p.) in the forced swimming test. Lamotrigine dose dependently decreased the immobility time in the forced swimming test in mice, with a statistically significant decrease at 20 and 30 mg/kg (P b 0.05 and P b 0.01, respectively) indicating an antidepressant-like effect. In addition, Fig. 1B shows the effect of lamotrigine (10–30 mg/kg, i.p.) in the ambulatory behavior. Lamotrigine at 20–30 mg/kg significantly decreased the number of crossings in the open-field test (P b 0.01). At a lower dose (10 mg/kg) lamotrigine was devoid of effect in the forced swimming test and in the open-field test.
kg, i.p.) in the forced swimming test. Fig. 2B shows that both the pretreatment with α-methyl-p-tyrosine (100 or 250 mg/kg) and the treatment with lamotrigine significantly decreased the number of crossings in the open-field test. The group treated with α-methyl-p-tyrosine (100 mg/kg, i.p.) plus lamotrigine did not produce a significant effect in the open-field test when compared with the groups treated with α-methyl-p-tyrosine (100 mg/kg, i.p.) or lamotrigine alone. However, the pretreatment of mice with α-methyl-p-tyrosine (250 mg/kg) plus lamotrigine significantly reduced de ambulatory behavior as compared with the group treated with lamotrigine alone. 3.3. Involvement of α1–α2-adrenoceptors in the antidepressant-like effect of lamotrigine in the forced swimming test
Fig. 2A shows that the pretreatment of mice with α-methylp-tyrosine (100 mg/kg or 250 mg/kg, i.p.) significantly prevented the antidepressant-like effect of lamotrigine (30 mg/
The results presented in Fig. 3A show that the pretreatment of mice with prazosin (1 mg/kg, i.p.) was able to prevent the antiimmobility effect of lamotrigine (30 mg/kg, i.p.) in the forced swimming test. Fig. 3B shows the reversal of the antidepressantlike effect of lamotrigine (30 mg/kg, i.p.) in the forced swimming test by the pretreatment with yohimbine. As presented in Fig. 4A, the administration of subeffective doses of phenylephrine (5 mg/kg, i.p.,) and clonidine (0.06 mg/
Fig. 2. Effect of pretreatment of mice with α-methyl-p-tyrosine (AMPT, 100 mg/ kg or 250 mg/kg), on the lamotrigine (30 mg/kg)-induced reduction in the immobility time in the forced swimming test (panel A), or in the number of crossings in the open-field test (panel B). Values are mean + S.E.M. (n = 5–8). ⁎P b 0.05 compared with the control group; ⁎⁎P b 0.01 compared with the control group; #P b 0.01 compared with the same group pretreated with saline; + P b 0.01 compared with the group treated with vehicle/lamotrigine. A: treatment: F1,32 = 11.86; P b 0.01; pretreatment: F2,32 = 4.52; P b 0.05; interaction: F2,32 = 6.79; P b 0.01. B: treatment: F1,30 = 5.78; P b 0.05; pretreatment: F2,30 = 18.55; P b 0.01; interaction: F2,30 = 1.23; P = 0.30.
Fig. 4. Effect of phenylephrine (5 mg/kg, i.p., an α1-adrenoceptor agonist) or clonidine (0.06 mg/kg, i.p., an α2-adrenoceptor agonist) combined with a subeffective dose of lamotrigine (10 mg/kg, i.p.) in the forced swimming test (panel A) and in the open-field test (panel B). Values are expressed as mean +S.E.M. (n =5–6). ⁎⁎P b 0.01 compared with the control group. A: treatment: F1,27 =28.78; P b 0.01; pretreatment: F2,27 = 10.67; P b 0.01; interaction: F2,27 =6.02; P b 0.01. B: treatment: F1,27 =28.78; P b 0.01; pretreatment: F2,27 =28.88; P b 0.01; interaction: F1,27 = 2.35; P = 0.13.
3.2. Effect of lamotrigine on α-methyl-p-tyrosine-treated mice in the forced swimming test
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kg, i.p.) produced a synergistic antidepressant-like effect when combined with a subeffective dose of lamotrigine (10 mg/kg, i.p.) in the forced swimming test. The results presented in Fig. 4B show the effect of phenylephrine (5 mg/kg, i.p.,) and clonidine (0.06 mg/kg, i.p.) combined with lamotrigine (10 mg/kg, i.p.) in the open-field test. 4. Discussion The present study was carried out to evaluate the antidepressant-like effect of lamotrigine in the mouse forced swimming test and the involvement of the noradrenergic system in its effect. Our results confirm literature data, by showing that lamotrigine reduces the immobility time in the forced swimming test. In addition, our study extends literature data by providing convincing evidence that its effect in the forced swimming test is probably mediated by an interaction with the noradrenergic system, probably with postsynaptic α1- and α2-adrenoceptors. The administration of lamotrigine produced a decrease in the immobility time in the forced swimming test, a response that is consistent with an antidepressant-like action. Among all animal models the forced swimming test remains one of the most used tools for assessing pharmacological antidepressant activity (Petit-Demouliere et al., 2005). Antidepressant treatments reduce the immobility time of mice in this test. Indeed, the sensitivity of the forced swimming test to an impressively broad range of antidepressant drugs is one of the most important features supporting its primary use as a screen in antidepressant discovery research (Porsolt et al., 1977). It should be noted that the doses of lamotrigine that decreased the immobility time also produced a reduction in the locomotor activity assessed in the open-field test. Thus, these results demonstrated that the antidepressant-like effect of lamotrigine occurs despite its sedative effect. This assumption is in accordance with the literature data showing that lamotrigine produced an antidepressant-like effect in the forced swimming test in mice and rats in spite of impairing spontaneous locomotor activity (Bourin et al., 2005; Consoni et al., 2006). Indeed, the tricyclic antidepressants imipramine and desipramine, produce sedative effects, but also exhibit an antidepressant-like activity in the forced swimming test in mice (Hascoet et al., 1991; David et al., 2003). In order to investigate the possible involvement of the noradrenergic system in the antidepressant-like effect of lamotrigine, αmethyl-p-tyrosine, a selective inhibitor of the enzyme tyrosine hydroxylase, was used. This is the rate-limiting enzyme in the synthesis of noradrenaline and dopamine. In the present work the pretreatment of mice with α-methyl-p-tyrosine (100 and 250 mg/ kg) was able to prevent the anti-immobility effect of lamotrigine in the forced swimming test. However, the higher dose of α-methyl-p-tyrosine combined with lamotrigine induced a marked decrease in locomotor activity in the open-field. The sedative effect produced by α-methyl-p-tyrosine at this higher dose might be responsible for the reversal of the antidepressant-like effect of lamotrigine. However, an indication of the involvement of the noradrenergic system in the effect of lamotrigine was given by the
finding that the reversal of the anti-immobility effect of lamotrigine by α-methyl-p-tyrosine was observed even at the lower dose of α-methyl-p-tyrosine, which produced a reduction of the locomotion similar to the one produced by lamotrigine or α-methyl-p-tyrosine per se. Mayorga et al. (2001) demonstrated that α-methyl-p-tyrosine reduces dopamine and noradrenaline levels (57% and 53%, respectively) in mice, without affecting the levels of serotonin. In addition, the acute administration of α-methyl-p-tyrosine temporarily reversed the antidepressant response to desipramine, mazindol and mirtazapine in patients (Delgado et al., 1993, 2002; Miller et al., 1996). Southam et al. (1998) reported that the synaptosomal uptake of serotonin, noradrenaline and dopamine in rat brain was inhibited by lamotrigine. Thus, one possibility that we should not rule out is that lamotrigine may indirectly affect the noradrenergic system by releasing noradrenaline that may, in turn, interact with their receptors. The α1- and α2-adrenoceptors have been shown to underlie some of the antidepressant-like responses of drugs in behavioral models of depression (Kitada et al., 1983; Danysz et al., 1986; Masuda et al., 2001). The decrease in immobility time elicited by lamotrigine was reversed by the pretreatment of mice with prazosin and yohimbine, suggesting that these receptor subtypes underlie its action in the forced swimming test. Indeed, in a study of O'Neill et al. (2001) the antidepressant-like effect of clonidine in the mouse forced swimming test was reversed by yohimbine. Besides that, the antidepressant-like action of desipramine was prevented by the pretreatment of mice with prazosin (Danysz et al., 1986). Further suggesting that α1- and α2-adrenoceptors are implicated in the mechanism of action of lamotrigine in the forced swimming test are the experiments in which the pretreatment of mice with either phenylephrine or clonidine was able to produce a synergistic antidepressant-like effect when combined with a subeffective dose of lamotrigine. These findings reinforce the assumption that the activation of α1- and α2-adrenoceptors is involved in the mechanism of action of lamotrigine. Phenylephrine is a postsynaptic α1-adrenoceptor agonist that was shown to reduce the duration of immobility time in the forced swimming test (Kitada et al., 1983). In addition, the repeated treatment with the antidepressants imipramine, amitriptyline, citalopram and mianserin produced an increase in the affinity of α1-adrenergic receptors for phenylephrine (Klimek et al., 1991; DziedzickaWasylewska et al., 2004). Clonidine is an α2-adrenoceptor agonist that also reduces the immobility time in the forced swimming test in mice and rats (Cervo and Samanin, 1991; O'Neill et al., 2001). Similarly to our result, a subeffective dose of clonidine was able to produce a synergistic effect in the forced swimming test when combined with subeffective doses of various antidepressants such as imipramine, amitriptyline, maprotiline, citalopram, fluvoxamine and mianserin (Malinge et al., 1988). In addition, the pretreatment of mice with α-methyl-p-tyrosine did not prevent the antidepressant-like effect of clonidine in the forced swimming test, suggesting that like phenylephrine, its effect is mediated by postsynaptic receptors (Malinge et al., 1988). In this work, the administration of phenylephrine and clonidine produced a strong decrease in the locomotor activity, an effect that
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