The Role of the NMDA Receptors and L-Arginine–Nitric Oxide–Cyclic

Pharmacology, Biochemistry and Behavior 103 (2012) 408–417

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Pharmacology, Biochemistry and Behavior

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The role of the NMDA receptors and L-arginine–nitric oxide–cyclic guanosine monophosphate pathway in the antidepressant-like effect of duloxetine in the forced swimming test

Andréa D.E. Zomkowski, Daiane Engel, Mauricio P. Cunha, Nelson H. Gabilan, Ana Lúcia S. Rodrigues ⁎

Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis 88040‐900, SC, Brazil article info abstract

Article history: Duloxetine is a selective serotonin and noradrenaline reuptake inhibitor used as antidepressant. However, its Received 22 December 2011 mechanisms of action are not fully understood. This study investigated the effect of duloxetine in the mouse forced Received in revised form 10 September 2012 swimming test (FST) and in the tail suspension test (TST) and the involvement of the NMDA receptors and the Accepted 16 September 2012 L-arginine–NO–cGMP pathway in its effect in the FST. Duloxetine reduced the immobility time both in the FST Available online 23 September 2012 and in the TST (dose range of 1–30 mg/kg, i.p.), without changing locomotion in an open-ﬁeld. Duloxetine administered orally (1–30 mg/kg) also reduced the immobility time in the FST. The effect of duloxetine (10 mg/kg, p.o.) Keywords: μ Depression in the FST was prevented by pre-treatment with NMDA (0.1 pmol/site, i.c.v.), D-serine (30 g/site, i.c.v.), (L-arginine Duloxetine (750 mg/kg, i.p.), S-nitroso-N-acetyl-penicillamine (SNAP, 25 μg/site, i.c.v) or sildenaﬁl(5mg/kg,i.p.).Theadmin- Forced swimming test istration of MK-801 (0.001 mg/kg, i.p.), 7-nitroindazole(50mg/kg,i.p.),methyleneblue(20mg/kg,i.p.)or Nitric oxide 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) (30 pmol/site i.c.v.) in combination with a sub-effective dose NMDA of duloxetine (0.3 mg/kg, p.o.) reduced the immobility time in the FST. Moreover, the administration of duloxetine Tail suspension test (10 mg/kg) produced a reduction in NOx levels in the hippocampus and cerebral cortex. Altogether the results suggest that the effect of duloxetine in the FST is dependent on either a blockade of NMDA receptors or an inhibition of NO. In addition, our results further reinforce the role of NMDA receptors and L-arginine–NO–cGMP pathway, besides the monoaminergic systems, in the mechanism of action of current prescribed antidepressant agents. © 2012 Elsevier Inc. Open access under the Elsevier OA license.

1. Introduction used in the treatment of depression (Detke et al., 2004), anxiety (Carter and McCormack, 2009) and pain (Bellingham and Peng, Depression is a disorder that has a high lifetime prevalence 2010). It has been proposed that 5-HT and NE reuptake inhibitors (around 16%) and is often manifested with symptoms at the psycho- may result in higher remission rates of major depressive disorder logical, behavioral and physiological levels. Suicide is usually a conse- than therapy with selective inhibitors of 5-HT reuptake (Shelton et quence of depression and other deleterious health-related effects al., 2005; Thase et al., 2001; Smith et al., 2002). It has been reported have been reported (Wong and Licinio, 2001). Despite the devastat- that duloxetine has some advantages over other antidepressants, ing impact of depression, its etiology or pathophysiology is not well since it has greater effectiveness than ﬂuoxetine and paroxetine in established. The monoaminergic systems (serotonergic, noradrener- patients with moderate to severe depression (Thase et al., 2007) gic and dopaminergic) have received a great attention in neurobio- and has a better tolerability than venlafaxine (Stahl et al., 2005). logical studies of depression (Wong and Licinio, 2001). Some Moreover, it is generally well tolerated and has fewer adverse effects clinical reports suggest that the combination of selective serotonin such as dry mouth, nausea, fatigue (Hunziker et al., 2005), sexual dys- (5-HT) reuptake inhibitors with selective noradrenaline (NE) reup- function (Stahl et al., 2005) and little or no effect on the cardiovascu- take inhibitors may present a therapeutic advantage, resulting in a lar system (Stahl et al., 2005; Hunziker et al., 2005). Of note, more effective or rapid antidepressant effect in comparison to each duloxetine-treated patients demonstrated clinically meaningful im- drug given alone (Shelton et al., 2005; Thase et al., 2007). provements within the ﬁrst weeks of treatment (Brannan et al., Duloxetine is a drug that inhibits 5-HT and NE reuptake, with 2005). Regarding preclinical studies, it was shown that duloxetine re- weak effects on dopamine reuptake (Hunziker et al., 2005). It is duced the immobility time in the forced swimming test (FST) in rats (Ciulla et al., 2007; Rénéric and Lucki, 1998). Several clinical studies have demonstrated abnormalities in gluta- ⁎ Corresponding author at: Departamento de Bioquímica, CCB, Universidade Federal mate function in mood disorders. Some studies have indicated that com- de Santa Catarina, Campus Universitário, Trindade, Florianópolis 88040‐900, SC, Brazil. Tel.: +55 48 37215043; fax: +55 48 37219672. pounds that reduce transmission at N-methyl-D-aspartate (NMDA) E-mail address: [email protected] (A.L.S. Rodrigues). receptors and NMDA receptor antagonists exhibit antidepressant-like

0091-3057 © 2012 Elsevier Inc. Open access under the Elsevier OA license. http://dx.doi.org/10.1016/j.pbb.2012.09.011 A.D.E. Zomkowski et al. / Pharmacology, Biochemistry and Behavior 103 (2012) 408–417 409 effects (Sanacora et al., 2008; Skolnick, 1999; Zarate et al., 2006). De- 1-one (ODQ), 7-nitroindazole and D-serine (Sigma Chemical Co, USA), pressed patients also showed a significant improvement after the S-nitroso-N-acetyl-penicillamine (SNAP), sildenafil(Pfizer) and MK- acute administration of ketamine, a non-competitive NMDA receptor 801 (Research Biochemicals International, USA) were used. All drugs antagonist (Berman et al., 2000; Zarate et al., 2006). Moreover, it is were dissolved in saline, except ODQ which was dissolved in saline also noteworthy that NMDA receptor antagonists exert synergistic with 1% dimethylsulfoxide (DMSO) and 7-nitroindazole that was antidepressant-like effects with classical antidepressants in the FST dissolved in 5% Tween 80. All drugs were administered by intraperitone- (Rogóz et al., 2002). Also, the use of MK801, a non-competitive NMDA al (i.p.) route in a constant volume of 10 ml/kg body weight, except antagonist, during imipramine withdrawal following chronic imipra- NMDA, D-serine, ODQ and SNAP which were administered by intra- mine treatment resulted in significant suppression of swim stress cerebroventricular (i.c.v.) route (5 μl/site). immobility (Harvey et al., 2002). I.c.v. administration was performed under ether anesthesia as The NMDA receptor stimulation induces the activation of nitric oxide previously described (Brocardo et al., 2008). Briefly, mice were (NO) synthase (NOS). The activated NOS then converts L-arginine to NO placed inside a glass jar containing ether. The animals remained in- and L-citrulline (Esplugues, 2002). NO is a signaling molecule in the brain side the glass jar for 30 s and immediately after, the i.c.v. administra- and has been implicated in depression (Da Silva et al., 2000; tion was performed. Briefly, a 0.4 mm external diameter hypodermic Harkin et al., 1999, 2004; Heiberg et al., 2002). It has been demon- needle attached to a cannula, which was linked to a 25 μl Hamilton strated that the reduction of NO levels within the hippocampus syringe, was inserted perpendicularly through the skull and no can induce antidepressant-like effects, thus implicating endoge- more than 2 mm into the brain of the mouse. A volume of 5 μlwas nous hippocampal NO in the neurobiology of depression (Joca then administered in the left lateral ventricle. The injection was and Guimarães, 2006). In addition, plasma nitrate concentrations given over 30 s, and the needle remained in place for another 30 s were significantly increased in depressed patients, suggesting in order to avoid the reflux of the substances injected. To ascertain that NO production is enhanced in depression (Suzuki et al., that the drugs were administered exactly into the cerebral ventricle, 2001). Several studies have demonstrated that NOS inhibitors, the brains were dissected and examined macroscopically after the depending on their concentration, exert antidepressant-like ef- test. fects (Da Silva et al., 2000; Harkin et al., 2003, 2004; Volke et al., Duloxetine was also administered by oral (p.o.) route by gavage in 2003). Some of the physiological actions of NO are mediated a volume of 10 ml/kg body weight. through the enzyme guanylate cyclase (sGC) with the consequent In order to investigate the antidepressant-like effect of the duloxetine, increase in cyclic guanosine monophosphate (cGMP) levels. It is it was administered (dose range: 0.3–30 mg/kg, i.p.) 30 min before the currently accepted that the NO–cGMP pathway is the main effec- FST, TST or open-field test. Alternatively, duloxetine or vehicle was ad- tor of NO actions in the brain (Esplugues, 2002). ministered by oral (p.o.) route (dose range: 0.3–30 mg/kg, p.o.) 60 min Although the behavioral studies using duloxetine in the FST sup- before the FST or open-field test. port its antidepressant properties (Ciulla et al., 2007; Rénéric and In a separate series of experiments, we investigated whether the Lucki, 1998), its mechanisms of action besides the monoaminergic antidepressant-like effect of duloxetine in the FST is mediated system are not fully understood. The present study examined wheth- through the blockade of NMDA receptors. To this end, mice were er duloxetine produces antidepressant-like effect in the FST and also pretreated with NMDA (0.1 pmol/site, i.c.v.) or with D-serine in another model predictive of antidepressant activity, the TST, in (30 μg/site, i.c.v., a coagonist at the NMDA receptor) and 15 min mice. These behavioral tests have good predictive value for antide- after, duloxetine (10 mg/kg, p.o.) or vehicle was administered. Sixty pressant potency in man (Cryan et al., 2002). Considering that the minutes later the FST was carried out. blockade of NMDA receptor is associated with reduced levels of NO In another set of experiments, we investigated the synergistic and cGMP (Snyder, 1992) and the inhibition of both NOS and sGC effect of a sub-effective dose of duloxetine (0.3 mg/kg, p.o.) with a may, depending on the dose, elicit antidepressant-like effects in the sub-effective dose of MK-801 (0.001 mg/kg, i.p., a non-competitive FST (Eroglu and Caglayan, 1997; Da Silva et al., 2000; Harkin et al., NMDA receptor antagonist). Duloxetine or vehicle was administered 2003; Heiberg et al., 2002; Volke et al., 2003; Yildiz et al., 2000), we 30 min before MK-801. A further 30 min was allowed to elapse investigated whether NMDA receptors and L-arginine–NO–cGMP before the animals were tested in the FST. pathway may be involved in the effect of acute duloxetine adminis- To investigate the possible involvement of the L-arginine–nitric tration in mouse FST. oxide pathway in the anti-immobility effects of duloxetine in the FST, in an experimental protocol aimed at increasing NO levels, mice 2. Materials and methods were pre-treated with L-arginine, a precursor of NO (750 mg/kg, i.p., a dose that produces no effect in the FST) or with the NO donor 2.1. Animals SNAP (25 μg/site, i.c.v., a dose that produces no effect in the FST) or vehicle. Thirty minutes after L-arginine or 15 min after SNAP admin- Swiss albino female mice (30–40 g, 55–70 days old) were maintained istration, duloxetine (10 mg/kg, p.o.) or vehicle was injected, and at 21–23 °C with free access to water and food, under a 12:12 h light/dark 60 min later the FST was carried out. cycle (lights on at 7:00 h). Animals were obtained from the Central In another set of experiments, we investigated the effect of Biothery of Universidade Federal de Santa Catarina (UFSC). All manipula- the combined administration of a sub-effective dose of duloxetine tions were carried out between 9:00 a.m. and 4:00 p.m., with each animal (0.3 mg/kg, p.o.) with sub-effective doses of 7-nitroindazole used only once. All procedures in this study were performed in accor- (50 mg/kg, i.p., a specific neuronal NOS inhibitor), ODQ (30 pmol/ dance with the National Institute of Health Guide for the Care and Use site i.c.v., a specific sGC inhibitor) or methylene blue (20 mg/kg, of Laboratory Animals (NIH Publication No. 80‐23). The experiments i.p., an inhibitor of both NOS and sGC). Duloxetine or vehicle was were performed after approval of the protocol by the Institutional Ethics administered 30 min before of 7-nitroindazole, methylene blue Committee and all efforts were made to minimize animals' suffering or 40 min before ODQ. A further 30 min (after i.p. administration of and to reduce the number of animals used in the experiments. 7-nitroindazole or methylene blue) or 20 min (after i.c.v. ODQ administration) was allowed to elapse before the animals were tested 2.2. Drugs and pharmacological treatments in the FST. In order to investigate the role of cGMP in the antidepressant Duloxetine HCl (Eli Lilly & Co.), L-arginine, methylene blue, action of duloxetine, in an experimental protocol aimed at increasing N-methyl-D-aspartate (NMDA), 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin- cGMP levels, mice received an injection of sildenafil (5 mg/kg, i.p., a 410 A.D.E. Zomkowski et al. / Pharmacology, Biochemistry and Behavior 103 (2012) 408–417 phosphodiesterase 5 inhibitor) or vehicle 30 min before duloxetine 3. Results (10 mg/kg, p.o.) and 60 min later the FST was carried out. The doses of the drugs used were selected on the basis of literature 3.1. Effect of duloxetine in the immobility time in the FST and TST and on data and on previous results from our laboratory (Almeida et al., the locomotor activity in the open-field test 2006; Brocardo et al., 2008; Da Silva et al., 2000; Dhir and Kulkarni, 2007; Kaster et al., 2005a,2005b; Rosa et al., 2003; Zomkowski et al., The treatment of mice with duloxetine at the dose range 1–30 mg/kg 2010). The doses of duloxetine administered were chosen on the given by intraperitoneal route significantly decreased the immobility basis of experiments previously performed in our laboratory and lit- time both in the FST [F(5,44)=83.01, pb0.01; Fig. 1A] and in the TST erature data (Bomholt et al., 2005; Rénéric and Lucki, 1998). [F(5,30)=70.47, pb0.01; Fig. 1B]. The doses of duloxetine that In order to evaluate a putative effect of duloxetine on NO levels, an produced an anti-immobility effect in the FST and TST (1–30 mg/kg) indirect measurement of NO levels via the production of nitrite formed did not cause any change in ambulation in an open-field in a separate from its metabolism, was performed. Animals received distilled water experiment as compared to the control group [F(5,32)=1.56, p= or duloxetine (10 mg/kg, p.o.). After 60 min, animals were killed by de- 0.20; Fig. 1C]. Fig. 1D shows that duloxetine also caused a reduction in capitation and hippocampi and cerebral cortices were rapidly removed. the immobility time in the FST when administered by p.o. route at the Briefly, one cerebral cortex or a pool of three hippocampi were mixed dose range 1–30 mg/kg [F(5,30)=51.91, pb0.01], but did not produce with 25% trichoroacetic and centrifuged at 1800 g for 10 min. The anychangeinambulationinmicetestedinanopen-field in a separate supernatant was immediately neutralized with 2 M potassium bicar- experiment [F(5,30)=2.15, p=0.09; Fig. 1E]. bonate. Nitrate (NO3−) was reduced to nitrite (NO2−)bynitratereduc- tase. The total NO2− in the incubation was measured by a colorimetric 3.2. Involvement of NMDA receptors on the antidepressant-like effect of assay read at 540 nm, based on the Griess reaction. A standard curve duloxetine in the FST was performed using sodium nitrate (0–80 μΜ). Results were expressed as % of control (100%) (Hevel and Marletta, 1994). Fig. 2A shows that the pre-treatment of mice with NMDA (0.1 pmol/site, i.c.v.) was able to reverse the antidepressant-like effect of duloxetine (10 mg/kg, p.o.) in the FST. A two-way ANOVA 2.3. Behavioral tests revealed significant differences for the NMDA pre-treatment [F(1,20)= 102.15, pb0.01], duloxetine treatment [F(1,20)=95.78, pb0.01] and 2.3.1. Forced swimming test NMDA×duloxetine interaction [F(1,20)=64.39, pb0.01]. The admin- The FST was conducted using the method of Porsolt et al. (1977) istration of NMDA alone or in combination with duloxetine did not affect with some modifications. Mice were individually forced to swim in the ambulation in the open-field (Fig. 2B). A two-way ANOVA did not re- an open cylindrical container (diameter 10 cm, height 25 cm), veal significant differences for the NMDA pre-treatment [F(1,20)=1.26, containing 19 cm of water at 25±1 °C; the total duration of immo- p=0.27], duloxetine treatment [F(1,20)=2.00, p=0.17] and NMDA× bility during the 6-min test was scored as described previously duloxetine interaction [F(1,20)=0.25, p=0.62]. Fig. 2Cshowsthat (Brocardo et al., 2008). Each mouse was judged to be immobile D-serine (30 μg/site, i.c.v.) was able to prevent the reduction in the im- when it ceased struggling and remained floating motionless in the mobility time elicited by duloxetine (10 mg/kg, p.o.) in the FST. A water, making only those movements necessary to keep its head two-way ANOVA revealed significant differences for D-serine pre- above water. treatment [F(1,20)=30.60, pb0.01], duloxetine treatment [F(1,20)= 32.29, pb0.01] and D-serine×duloxetine interaction [F(1,20)=37.96, pb0.01]. The administration of D-serine alone or in combination with 2.3.2. Tail suspension test duloxetine did not affect locomotor activity in the open-field test. A The total duration of immobility induced by tail suspension was two-way ANOVA did not reveal significant differences for D-serine pre- measured according to the method described by Steru et al. (1985). treatment [F(1,20)=0.67, p=0.42], duloxetine treatment [F(1,20)= Briefly, mice both acoustically and visually isolated were suspended 0.07, p=0.80] and D-serine×duloxetine interaction [F(1,20)=3.50, 50 cm above the floor by adhesive tape placed approximately 1 cm p=0.08](Fig. 2D). The results illustrated in Fig. 2E show that the admin- from the tip of the tail. Immobility time was recorded during a istration of MK-801 (0.001 mg/kg, i.p., non-competitive NMDA receptor 6 min period (Binfaré et al., 2009). antagonist) in combination with duloxetine (0.3 mg/kg, p.o.) produced an antidepressant-like effect as compared with the administration of either drug alone. A two-way ANOVA revealed significant differences 2.3.3. Open-field behavior for the duloxetine pre-treatment [F(1,20)=23.62, pb0.01], MK-801 The ambulatory behavior was assessed in an open-field test as de- treatment [F(1,20)=31.62, pb0.01] and duloxetine×MK-801 interac- scribed previously (Brocardo et al., 2008). The apparatus consisted of tion [F(1,20)=12.48, pb0.01]. Fig. 2F shows that the administration of a wooden box measuring 40 ×60×50 cm high. The floor of the arena MK-801 alone or in combination with duloxetine did not affect was divided into 12 equal squares. The number of squares crossed locomotion in the open-field test. A two-way ANOVA did not reveal with all paws (crossings) was counted in a 6-min session. The light significant differences for the duloxetine pre-treatment [F(1,20)=3.72, was maintained at minimum to avoid anxiety behavior. The appara- p=0.07], MK-801 treatment [F(1,20)=1.18, p=0.29] and duloxetine× tus was cleaned with a solution of 10% ethanol between tests in order MK-801 interaction [F(1,20)=0.35, p=0.56]. to hide animal cues. 3.3. Involvement of L-arginine–NO–cGMP pathway in the anti-immobility effect of duloxetine in the FST 2.4. Statistical analysis The results depicted in Fig. 3A show that pre-treatment with Comparisons between treatment groups and control were per- L-arginine (750 mg/kg i.p., an NO precursor) prevented the formed by one-way (dose–response experiments) or two-way (experi- antidepressant-like effect of duloxetine (10 mg/kg, p.o.) in the FST. The ments dealing with the role of NMDA receptors and L-arginine–NO– two-way ANOVA showed significant differences for L-arginine pre- cGMP pathway in the antidepressant effect of duloxetine) analysis of treatment [F(1,20)=22.13, pb0.01], duloxetine treatment [F(1,20)= variance (ANOVA) followed by Tukey's HSD test when appropriate. A 43.96, pb0.01] and L-arginine×duloxetine interaction [F(1,20)=24.75, value of pb0.05 was considered to be significant. pb0.01]. The administration of L-arginine alone or in combination A.D.E. Zomkowski et al. / Pharmacology, Biochemistry and Behavior 103 (2012) 408–417 411

Fig. 1. Effect of intraperitoneal administration of duloxetine (0.3–30 mg/kg) in the immobility time in the FST (A), TST (B) and on the number of crossings in the open-field test (C) and the effect of oral administration of duloxetine (0.3–30 mg/kg) in the immobility time in the FST (D) and on the number of crossings in the open-field test (E) in mice. Values are expressed as mean±SEM. Values are expressed as mean±SEM, n=6–11. **Pb0.01 compared with the vehicle-treated control group. with duloxetine did not affect the ambulation in the open-field locomotor activity in the open-field test. A two-way ANOVA did not re- (Fig. 3B). A two-way ANOVA did not reveal significant differences veal significant differences for the duloxetine pre-treatment [F(1,20)= for the L-arginine pre-treatment [F(1,20)=1.29, p=0.27], duloxetine 2.27, p=0.15], 7-nitroindazole treatment [F(1,20)=1.28, p=0.27] and treatment [F(1,20)=0.08, p=0.77] and L-arginine×duloxetine inter- 7-nitroindazole×duloxetine interaction [F(1,20)=0.32, p=0.58]. action [F(1,20)=3.25, p=0.86]. Fig. 3C shows that SNAP (25 μg/site, Fig. 4C shows that methylene blue (20 mg/kg i.p., direct inhibitor of i.c.v., a NO donor) was able to prevent the reduction in the immo- both NOS and sGC) in combination with duloxetine (0.3 mg/kg, p.o.) bility time elicited by duloxetine (10 mg/kg, p.o.) in the FST. The two- also produced an anti-immobility effect in the FST as compared with way ANOVA showed significant differences for SNAP pre-treatment the administration of either drug alone. A two-way ANOVA revealed sig- [F(1,20)=53.68, pb0.01], duloxetine treatment [F(1,20)=65.99, nificant differences for the duloxetine pre-treatment [F(1,20)=16.48, pb0.01] and SNAP×duloxetine interaction [F(1,20)=109.42, pb0.01]. pb0.01], methylene blue treatment [F(1,20)=28.53, pb0.01] and The administration of SNAP alone or in combination with duloxetine duloxetine×methylene blue interaction [F(1,20)=10.27, pb0.01]. The did not affect the ambulation in the open-field test (Fig. 3D). A two- administration of methylene blue (20 mg/kg i.p.) alone or in combina- way ANOVA did not reveal significant differences for SNAP pre- tion with duloxetine did not affect the ambulation in the open-field treatment [F(1,20)=0.79, p=0.38], duloxetine treatment [F(1,20)= (Fig. 4D). A two-way ANOVA did not reveal significant differences for 0.55, p=0.47] and SNAP ×duloxetine interaction [F(1,20)=0.61, the duloxetine pre-treatment [F(1,20)=4.02, p=0.06], methylene p=0.44]. blue treatment [F(1,20)=0.86, p=0.36] and duloxetine×methylene The results illustrated in Fig. 4A show the administration of blue interaction [F(1,20)=1.16, P=0.29]. 7-nitroindazole (50 mg/kg i.p., a specific neuronal neuronal NOS inhibi- Corroborating the results presented above which suggest that a pu- tor) in combination with duloxetine (0.3 mg/kg, p.o.) produced an tative reduction in NO levels is implicated in the antidepressant-like antidepressant-like effect as compared with the administration of either effect of duloxetine, Fig. 4E shows that an effective dose of duloxetine drug alone. A two-way ANOVA revealed significant differences for the in the FST (10 mg/kg, p.o.) significantly reduced the NOx levels in the duloxetine pre-treatment [F(1,20)=34.36, pb0.01], 7-nitroindazole hippocampus [F(1,11)=10.42, Pb0.01] and cerebral cortex: [F(1,8)= treatment [F(1,20)=60.45, pb0.01] and duloxetine×7-nitroindozole in- 7.33, Pb0.05], as compared with the control group (100%). teraction [F(1,20)=26.31, pb0.01]. Fig. 4B shows that the administration The results depicted in Fig. 5A show that ODQ (30 pmol/site i.c.v., of 7-nitroindazole alone or in combination with duloxetine did not affect a specific inhibitor of sGC) in combination with duloxetine (0.3 mg/ 412 A.D.E. Zomkowski et al. / Pharmacology, Biochemistry and Behavior 103 (2012) 408–417

Fig. 2. Effect of the pre-treatment of mice with NMDA (0.1 pmol/site, i.c.v.) or D-serine (30 μg/site, i.c.v.) on the anti-immobility action of duloxetine (10 mg/kg, p.o.) in the FST (panels A and C, respectively) and in the open-ﬁeld test (panels B and D, respectively). Effect of MK-801 (0.001 mg/kg, i.p.) in combination with a sub-effective dose of duloxetine (0.3 mg/kg, p.o.) in the FST (E) and in the open-ﬁeld test (F). Values are expressed as mean±SEM, n=6. **pb0.01 compared with the vehicle-treated control group; #pb0.01 compared with the same group pretreated with vehicle.

kg, p.o.) also produced an anti-immobility effect in the FST as com- treatment [F(1,20)=66.88, pb0.01] and sildenafil×duloxetine inter- pared with the administration of either drug alone. A two-way action [F(1,20)=57.44, pb0.01]. The administration of sildenafil ANOVA revealed significant differences for the duloxetine (5 mg/kg, i.p.) alone or in combination with duloxetine did not affect pre-treatment [F(1,20)=19.33, pb0.01], ODQ treatment [F(1,20)= the locomotor activity in the open-field (Fig. 5D). A two-way ANOVA 21.47, pb0.01] and duloxetine×ODQ interaction [F(1,20)=15.11, did not reveal significant differences for the sildenafilpre-treatment pb0.01]. The administration of ODQ (30 pmol/site i.c.v.) alone or in [F(1,20)=0.05, p=0.83], duloxetine treatment [F(1,20)=1.72, p= combination with duloxetine did not affect the ambulation in 0.20] and sildenafil×duloxetine interaction [F(1,20)=3.56, p=0.07]. the open-field (Fig. 5B). A two-way ANOVA did not reveal significant differences for the duloxetine pretreatment [F(1,20)=1.20, p=0.28], ODQ treatment [F(1,20)=0.01, p=0.90] and duloxetine×ODQ interac- 4. Discussion tion [F(1,20)=0.04, p=0.85]. Fig. 5C shows that the anti-immobility effect of duloxetine (10 mg/kg, p.o.) was completely prevented by pre- The FST and the TST are quite sensitive and relatively specific treatment of animals with sildenafil (5 mg/kg, i.p., a phosphodiesterase for all major classes of antidepressant drugs including tricyclics, 5 inhibitor inhibitor). A two-way ANOVA revealed significant differences serotonin-specific reuptake inhibitors, monoamine oxidase inhibi- for the sildenafil pre-treatment [F(1,20)=47.54, pb0.01], duloxetine tors, and atypicals (Porsolt et al., 1977; Steru et al., 1985). A.D.E. Zomkowski et al. / Pharmacology, Biochemistry and Behavior 103 (2012) 408–417 413

Fig. 3. Effect of the pre-treatment of mice with L-arginine (750 mg/kg, i.p.) on the anti-immobility action of duloxetine (10 mg/kg, p.o.) in the FST (A) and on the number of crossings in the open-ﬁeld test (B). Effect of the pre-treatment of mice with SNAP (25 μg/site, i.c.v) on the anti-immobility action of duloxetine (10 mg/kg, p.o.) in the FST (C) and on the number of crossings in the open-ﬁeld test (D). Values are expressed as mean±SEM, n=6. **pb0.01 compared with the vehicle-treated control group; #pb0.01 compared with the same group pre-treated with vehicle.

Antidepressant drugs are reported to reduce the immobility time of models; they are stressor-based and although there is evidence for a mice in both tests (Porsolt et al., 1977; Steru et al., 1985). The results link between depression and stress, this does not always follow. In presented here show that duloxetine, given systemically (i.p. or p.o. addition, they respond to the acute antidepressant treatment (Cryan routes), is effective in producing significant antidepressant-like ef- et al., 2002; Cryan and Slattery, 2007). In spite of these limitations, fects, when assessed in the FST, which is the most widely used tool these tests are useful for studying the mechanisms underlying the for assessing antidepressant activity preclinically (Cryan et al., antidepressant-like effects of several compounds. 2002). The antidepressant-like action of duloxetine administered i.p. We investigated the participation of NMDA receptors and L-arginine– was confirmed in the TST. This study was performed in female mice, NO–cGMP pathway in the antidepressant-like effect of duloxetine in the since several studies have shown that the prevalence of depression FST. This test was chosen considering that the effects of the modulation of is about two fold higher in women than in men (Wong and Licinio, NMDA receptors and NOS–cGMP pathway on the antidepressant-like 2001). behavior in this test are well known, as compared to the TST (Almeida The results indicate that anti-immobility effect of duloxetine is not et al., 2006; Brocardo et al., 2008; Dhir and Kulkarni, 2007; Ghasemi et associated with any motor effects, since mice treated with duloxetine al., 2009, 2010; Harkin et al., 1999, 2003, 2004; Kaster et al., did not exhibit increased ambulation when tested in an open-field. 2005a,2005b; Rosa et al., 2003). For this aim, oral administration of This indicates that a psychostimulant effect is not responsible for duloxetine was selected because it is the most common route of admin- the decrease in the immobility elicited in the FST. A number of studies istration for antidepressants in psychiatric patients. In this study, we have demonstrated that several antidepressants administered by i.p. showed that the reduction of the immobility time elicited by duloxetine route produced antidepressant-like effect in the FST in rodents in the FST was prevented by NMDA and D-serine, a coagonist at the (Ciulla et al., 2007; Dhir and Kulkarni, 2007; Ghasemi et al., 2009; NMDA receptor. Similar to our results, the antidepressant-like effect of Zomkowski et al., 2010). Duloxetine was shown to produce antide- lithium and escitalopram in the FST was prevented by the pre- pressant effect after its administration by the i.p. (40 mg/kg) and sub- treatment with NMDA (Ghasemi et al., 2010; Zomkowski et al., 2010). cutaneous routes (10–40 mg/kg) to rats in the FST (Ciulla et al., 2007; Moreover, it was shown that NMDA receptor NR2A subunit knockout Rénéric and Lucki, 1998, respectively). In the present study, we con- mice exhibit anxiolytic-like effects in elevated plus-maze, light–dark ex- firm that duloxetine administered by the i.p. route produces an ploration, novel and familiar open field tests and home cage locomotor antidepressant-like effect in the FST in mice, but at a lower dose– activity and antidepressant-like effects in the FST and TST in mice response range (1–30 mg/kg). In order to further reinforce this anti- (Boyce-Rustay and Holmes, 2006). In addition, antidepressants have depressant effect, we tested duloxetine in the TST. been reported to reduce binding, expression and function of NMDA re- Although the FST and TST are widely used for assessing antidepres- ceptors (Boyer et al., 1998; Szasz et al., 2007). An evidence of a direct ac- sant activity preclinically, we should take into account that they have tion of fluoxetine and desipramine inhibiting NMDA-evoked currents in some limitations and their validity as simulations of the psychiatric con- rat cortical cell cultures was reported, suggesting that direct inhibition dition is questionable. Some of the limitations are related to the fact that of NMDA receptors may contribute to the clinical effects of antidepres- many symptoms of depression are not measurable in preclinical sants (Szasz et al., 2007). Furthermore, Poleszak et al. (2007) have 414 A.D.E. Zomkowski et al. / Pharmacology, Biochemistry and Behavior 103 (2012) 408–417

Fig. 4. Effect of 7-nitroindazole (50 mg/kg, i.p.) or methylene blue (20 mg/kg, i.p.) in combination with a sub-effective dose of duloxetine (0.3 mg/kg, p.o.) in the FST (panels A and C, respectively) and in the open-ﬁeld test (panels B and D, respectively). The effect of duloxetine administration (10 mg/kg, p.o., 1 h before decapitation) in the NOx levels in the hippocampus and cerebral cortex is shown in panel E. Values are expressed as mean±SEM, n=6. *pb0.05, **pb0.01 compared with the vehicle-treated control group.

shown that activation of NMDA receptors by NMDA and D-serine reverses a synergistic effect of NMDA receptor antagonists with antidepres- the anti-immobility effect of NMDA receptor antagonists in the FST. The sants and lithium in the FST (Ghasemi et al., 2010; Rogóz et al., antidepressant effects of ﬂuoxetine, imipramine and reboxetine were 2002). However, there are little data on the mechanisms by which also reversed by D-serine (Poleszak et al., 2011). Therefore, our results NMDA antagonists enhance the effects of antidepressants in the FST. are in line with the literature data reinforcing the role of the NMDA recep- A microdialysis study has demonstrated that acute administration of tors in the mechanism of action of antidepressant agents and in the noneffective doses of the NMDA antagonist amantadine combined pathophysiology of depression, since they clearly indicate that the with noneffective doses of several antidepressants (paroxetine, antidepressant-like effect of duloxetine is mediated through a blockade reboxetine, budipine and clomipramine) increases the cortical release of NMDA receptor. However, our behavioral results do not allow us to of 5-HT in freely moving rats (Owen and Whitton, 2005). Hence, we conclude about the mechanism by which duloxetine interacts with the cannot rule out the possibility that the antidepressant-like effects NMDA receptor and further studies are necessary to better clarify this elicited by treatment of mice with sub-effective doses of duloxetine issue. and NMDA antagonists may be due to their effects on the central The synergistic antidepressant-like effect observed when mice 5-HT release. were treated with the non-competitive NMDA receptor antagonist The L-arginine–NO–cGMP pathway has been implicated in the MK-801 combined with duloxetine reinforces the assumption that pathogenesis of depression (Kaster et al., 2005a, 2005b; Mantovani duloxetine exerts its antidepressant-like effect by blocking NMDA et al., 2003). NO plays a signiﬁcant role in the central nervous system receptor activation. It is known that MK-801 preferentially binds and pharmacological manipulation of the NO pathway may constitute to the activated NMDA receptor complex (Javitt and Zukin, 1989) a novel therapeutic approach for the treatment of depression (Harkin interacting with a site located within the NMDA channel pore. In et al., 2003, 2004). In this study, we showed that the reduction of im- concordance with our results, a study recently demonstrated that mobility time elicited by duloxetine in the FST was reversed by the combined treatment of sub-effective doses of paroxetine and pre-treatment of mice with L-arginine (NOS substrate) or SNAP, an MK-801 exerted antidepressant-like effect in the FST in mice NO donor. Similarly to our results, the antidepressant-like effects of (Ghasemi et al., 2009). Moreover, some studies have demonstrated imipramine, escitalopram, paroxetine and venlafaxine were also A.D.E. Zomkowski et al. / Pharmacology, Biochemistry and Behavior 103 (2012) 408–417 415

Fig. 5. Effect of ODQ (30 pmol/site i.c.v.) in combination with a sub-effective dose of duloxetine (0.3 mg/kg, p.o.) in the FST (panel A) and in the open-field test (panel B). Effect of the pre-treatment of mice with sildenafil (5 mg/kg, i.p.) on the anti-immobility effect of duloxetine (10 mg/kg, p.o.) in the FST (A) and on the number of crossings in the open-field test (B). Values are expressed as mean±SEM, n=6. **pb0.01 compared with the vehicle-treated control group; #pb0.01 compared with the same group pre-treated with vehicle.

blocked by pre-treatment with L-arginine (Dhir and Kulkarni, 2007; mouse FST (Almeida et al., 2006). These results reinforce our hy- Ghasemi et al., 2009; Harkin et al., 2004; Zomkowski et al., 2010). In pothesis that the anti-immobility effect of duloxetine in the FST is previous studies, our group demonstrated that L-arginine and SNAP dependent on the decrease in NO synthesis and also that it may be reversed the antidepressant-like effects of putative antidepressant mediated through the reduction of cGMP, likely as a consequence agents such as zinc chloride (Rosa et al., 2003), adenosine (Kaster et of the reduction of NO synthesis. Noteworthy, a study reported al., 2005b), memantine (Almeida et al., 2006), folic acid (Brocardo that L-arginine was able to partially prevent the inhibition of genital et al., 2008) and ascorbic acid (Moretti et al., 2011). Therefore, a vascular responses induced by duloxetine in rabbits, reinforcing the great amount of literature data points to a significant role of nitrergic notion that this antidepressant may act on NOS system (Angulo et system in the mechanism of action of antidepressant agents. Corrob- al., 2004). Although the present study does not allow us to know orating this notion, our results indicate that the effect of duloxetine exactly the mechanism responsible for the observed behavioral may be dependent on the inhibition of NO synthesis. Further effects, one possibility is that the effects are indirectly dependent reinforcing this assumption, in this study, the pre-treatment of mice on an increase in the release of 5-HT. It is interesting to note that with a sub-effective dose of 7-nitroindazole (a specific neuronal theeffectsofNOSinhibitorsintheFSTarereportedtobedependent NOS inhibitor), methylene blue (direct inhibitor of both NOS and on endogenous 5-HT, since the depletion of 5-HT caused by the sGC) or ODQ (a specific inhibitor of sGC) produced a synergistic treatment of rats with PCPA prevented the effects of NOS inhibitors antidepressant-like effect with duloxetine. Corroborating these re- in the FST (Harkin et al., 2003). Furthermore, some studies have sults, duloxetine treatment was able to decrease NOx levels in the demonstrated that inhibition of NOS can modulate the release of hippocampus and cerebral cortex. Together this set of results suggests central 5-HT (Kiss, 2000; Smith and Whitton, 2000; Wegener et that the inhibition of NO synthesis is implicated in the anti- al., 2000). immobility effect of duloxetine in the FST and is in accordance with Of note, we also showed that the pre-treatment of mice with sil- the fact that NOS inhibitors exert antidepressant-like effect in animal denafil (selective type 5 phosphodiesterase inhibitor) prevented the models of depression (Eroglu and Caglayan, 1997; Volke et al., 2003; antidepressant-like effect of duloxetine in the FST. Sildenafil increases Yildiz et al., 2000). In addition, in line with our results, studies have the cGMP level in target tissues (Beavo, 1995) and was previously demonstrated that 7-nitroindazole potentiated the antidepressant- reported to reverse the anti-immobility effect of ODQ in the mouse like effect of venlafaxine (Dhir and Kulkarni, 2007), imipramine and FST (Kaster et al., 2005b). Our results are somewhat in accordance fluoxetine (Harkin et al., 2004) in the FST. Similarly to our results, a with the literature data that showed that the reduction in the immo- study showed that methylene blue potentiated the antidepressant- bility time elicited by the antidepressants venlafaxine (Dhir and like effect of venlafaxine in the FST (Dhir and Kulkarni, 2007). More- Kulkarni, 2007) and escitalopram (Zomkowski et al., 2010) was also over, it has been reported that the antidepressant-like effect prevented by the pre-treatment with sildenafil. This finding reported produced by ODQ in the FST in rats was reversed by pre-treatment here reinforces the notion that duloxetine exerts its effect in the FST with L-arginine (Heiberg et al., 2002). 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