Ascorbic acid administration produces an antidepressant-like effect: Evidence for the involvement of monoaminergic neurotransmission

Progress in Neuro-Psychopharmacology & Biological Psychiatry 33 (2009) 530–540

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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

Ascorbic acid administration produces an antidepressant-like effect: Evidence for the involvement of monoaminergic neurotransmission Ricardo W. Binfaré a, Angelo O. Rosa a, Kelly R. Lobato a, Adair R.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

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Article history: Received 19 December 2008 Received in revised form 19 January 2009 Accepted 1 February 2009 Available online 11 February 2009 Keywords: Antidepressants Ascorbic acid Forced swimming test Monoaminergic system Tail suspension test

a b s t r a c t Ascorbic acid is highly concentrated in the brain, being considered as a neuromodulator. This study investigated the effect of ascorbic acid in the tail suspension test (TST) and in the forced swimming test (FST) in mice and the contribution of the monoaminergic system to its antidepressant-like effect. Moreover, the effects of fluoxetine, imipramine and bupropion in combination with ascorbic acid in the TST were investigated. Ascorbic acid (0.1–10 mg/kg, i.p., 1–10 mg/kg p.o. or 0.1 nmol/mice i.c.v.) produced an antidepressant-like effect in the TST, but not in the FST, without altering the locomotor activity. The effect of ascorbic acid (0.1 mg/kg, i.p.) in the TST was prevented by i.p. pre-treatment with NAN-190 (0.5 mg/kg), ketanserin (5 mg/kg), MDL72222 (0.1 mg/kg), prazosin (62.5 µg/kg), yohimbine (1 mg/kg), propranolol (2 mg/kg), haloperidol (0.2 mg/kg), sulpiride (50 mg/kg), but not with SCH23390 (0.05 mg/kg, s.c.). Additionally, ascorbic acid (1 mg/kg, p.o.) potentiated the effect of subeffective doses (p.o. route) of fluoxetine (1 mg/kg), imipramine (0.1 mg/kg), or bupropion (1 mg/kg) in the TST. The combined treatment of ascorbic acid with antidepressants produced no alteration in the locomotion in the open-field test. In conclusion, our results show that administration of ascorbic acid produces an antidepressant-like effect in TST, which is dependent on its interaction with the monoaminergic system. Moreover, ascorbic acid caused a synergistic antidepressant-like effect with conventional antidepressants. Therefore, the present findings warrant further studies to evaluate the therapeutical relevance of ascorbic acid for the treatment of depression and as a co-adjuvant treatment with antidepressants. © 2009 Elsevier Inc. All rights reserved.

1. Introduction Depression is a highly disabling condition associated with significant morbidity and mortality (Berton and Nestler, 2006; Holtzheimer and Nemeroff, 2006) with a lifetime prevalence approaching 17% (Kessler et al., 2005). It is recognized as a major health problem (Nestler and Carlezon, 2006) and has a substantial social impact, since depressives have impairment in their activities and wellbeing, what leads to incapability and loss of productivity (Ebmeier et al., 2006). Furthermore, the disease produces a raise in Abbreviations: 5-HT, serotonin; 8-OH-DPAT, (+)-8-hydroxy-2-(di-n-propylamino)-tetralin; AA, ascorbic acid; ACTH, adrenocorticotropic hormone; ANOVA, analysis of variance; CNS, central nervous system; CSF, cerebrospinal fluid; DOI, (+/ −)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCI; DMSO, dimethylsulfoxide; ECF, extracellular fluid; ECT, electroconvulsive therapy; FST, forced swimming test; GMP, guanosine monophosphate; MDL72222, tropanyl 3,5-dichlorobenzoate; MK-801, (+)-5methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate; NMDA, N-methyl-D-aspartate; NAN-190, 1-(2-methoxyphenyl)-4[-(2-phthalimido)butyl] piperazine); SCH23390, (R)-(+)-7 chloro-8-hydroxy-3-methyl-1phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride; SSRI, selective serotonin reuptake inhibitor; TCA, tricyclic antidepressant; TST, tail suspension test. ⁎ Corresponding author. Tel.: +55 48 3721 5043; fax: +55 48 3721 9672. E-mail addresses: [email protected], [email protected] (A.L.S. Rodrigues). 0278-5846/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2009.02.003

health system utilization (Nemeroff, 2007). Conventional available antidepressants are inadequate for many individuals and have frequent and persistent side effects. Another drawback is that available treatments remain sub-optimal, with a delay of 3–6 weeks before their clinical effects can be achieved and a lack of efficacy is also observed in many cases (Nemeroff and Owens, 2002; Nestler et al., 2002). For these reasons, the discovery of new drugs or innovative compounds that could further improve current depression therapies are welcomed (Berton and Nestler, 2006). Although the molecular alterations underlying the pathogenesis of depression remain to be clearly established, preclinical and clinical studies have suggested the involvement of monoamines, particularly the serotonergic and noradrenergic systems (Schildkraut, 1965). There are several lines of evidence indicating that serotonergic and noradrenergic neurotransmissions are involved in the expression of an antidepressant-like effect in the behavioral despair models of depression (Elhwuegi, 2004). In addition, there is a considerable amount of pharmacological evidence regarding the efficacy of antidepressants with dopaminergic effects in the treatment of depression (Papakostas, 2006) and studies that suggest an involvement of this system with antidepressant-like responses in preclinical models of depression (D'Aquila et al., 2000).

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Ascorbic acid, commonly known as vitamin C, is a water-soluble antioxidant vitamin that participates in a wide range of physiological reactions (Padh, 1990; Rebec and Pierce, 1994). It also acts as a cofactor for important hydroxylation reactions needed for synthesis of collagen, carnitine and catecholamines (Padh, 1990). This vitamin possesses antioxidant properties due to its action either as an electron donor or as broad-spectrum radical scavenger (Rice, 2000). Moreover, it was shown that ascorbic acid produces antinociceptive effects (Rosa et al., 2005) and it was also recently shown that it diminished growth of several aggressive cancer types in mice without causing apparent adverse effects (Chen et al., 2008). These data indicate that this vitamin could exert several relevant biological functions. At physiological pH, ascorbic acid exists as a monovalent anion and its endogenous form is known as ascorbate (Rice, 2000). In humans, the brain accumulates ascorbate from the blood supply and maintains it at a relatively high concentration (milimolar) under widely varying conditions (Agus et al., 1997; Grünewald, 1993; Rebec and Pierce, 1994; Schreiber and Trojan, 1991), where it is proposed to exert neuroprotective effects as a result of its antioxidant action (Padayatty et al., 2003). Besides that, ascorbate is supposed to act as a neuromodulator in the brain, modulating both dopamine- and glutamate-mediated neurotransmission (Grünewald, 1993; Rebec and Pierce, 1994; Rice, 2000). Despite the high and regulated levels of brain ascorbate, its physiological roles in the central nervous system (CNS) are not well established (Rice, 2000). A role for ascorbic acid in mood disorders was suggested from clinical findings in which this vitamin relieved depression symptoms associated with ACTH administration in one infant patient being treated for chronic hepatitis. In addition, ascorbic acid treatment (intravenous; 50 mg/kg/day) for two weeks was shown to lead to complete remission from depressive symptoms in depressive patients (Coochi et al., 1980). Furthermore, it was reported that the administration of ascorbic acid for 14 days decreased scores in a Beck Depression Inventory in healthy young adults, an indicative of mood improvement (Brody, 2002). In another study, Khanzode et al. (2003) showed that plasma ascorbic acid levels were decreased in depressive patients. In a more recent study Chang et al. (2007) reported a case of a patient with depression who developed scurvy, suggesting that an inadequate vitamin C intake and subsequent reduced plasma levels of ascorbate could be linked to the pathophysiology of depression. Furthermore, other studies have shown that depressive symptoms are present in scurvy disease (DeSantis, 1993; Nguyen et al., 2003; Stöger et al., 1994). Further suggesting that ascorbic acid may be implicated in mood regulation, Brody et al. (2002) showed that a sustained-release of ascorbic acid (3000 mg/ day) for 14 days in humans elicited a reduction on blood pressure, subjective stress, and state anxiety response to an acute interpersonal psychological stressor, and also produced faster recovery of salivary cortisol after the stressor. These protective effects may be dependent on the neuromodulatory properties of this vitamin (Grünewald, 1993; Rebec and Pierce, 1994; Rice, 2000). It should be mentioned that depression is a stress-related disorder and hypercortisolaemia and an impaired hypothalamic-pituitary-adrenal axis activation are linked to the development of depressive symptoms (Berton and Nestler, 2006). Although some clinical studies suggest antidepressant properties for ascorbic acid, there is no preclinical evidence indicating that this vitamin exerts antidepressant effects. Considering this and the need of discovery of compounds that could improve conventional therapies and given that ascorbic acid exhibits low toxicity, is inexpensive and is currently available within the community, we sought to investigate the effect of ascorbic acid in the tail suspension test (TST) and in the forced swimming test (FST), two widely used behavioral tests that predict the efficacy of antidepressant treatments (Bourin et al., 2005). The primary aim of the present study was to investigate the possible antidepressant-like effect of an acute intraperitoneal (i.p.) injection of ascorbic acid in these two models. The positive results obtained in the

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TST have conducted us to investigate the involvement of the monoaminergic system in the antidepressant-like effect of ascorbic acid in this model. Given the fact that our results suggest that ascorbic acid produce antidepressant actions, the next step of our study was to investigate the possible antidepressant-like effect of ascorbic acid administered by oral route (p.o.) and in addition, its effect in combination with conventional antidepressants. 2. Methods 2.1. Animals Adult Swiss mice of either sex (homogeneously distributed among groups) obtained from Universidade Federal de Santa Catarina's animal facility, weighing 30–40 g were maintained at 22–25 °C with free access to water and food, under a 12/12 h light–dark cycle (lights on at 07:00 hours). All manipulations were carried out between 09:00 and 16:00 hours, 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. The experimental protocols were approved by the Institutional Ethics Committee and all efforts were made to minimize animal suffering. 2.2. Drugs and treatment The following drugs were used: ascorbic acid, 1-(2-methoxyphenyl)-4[-(2-phthalimido)butyl]piperazine) (NAN-190), ketanserin tartarate, tropanyl 3, 5-dichlorobenzoate (MDL72222), prazosin hydrochloride, yohimbine hydrochloride, propranolol, haloperidol, (R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro1H-3-benzazepine hydrochloride (SCH23390), sulpiride, fluoxetine, imipramine and bupropion (from Sigma Chemical Co., St. Louis, U.S.A.). Drugs were dissolved in saline, except that NAN-190 and MDL72222 were diluted in saline with 1% Tween 80, sulpiride and prazosin were diluted in saline with 5% DMSO and haloperidol was diluted in saline with 5% ethanol. Appropriate vehicle treated groups were also assessed simultaneously. To investigate the hypothesis that ascorbic acid produces antidepressant-like effects in predictive tests of antidepressant action and to study its interaction with the monoaminergic system, drugs were administered by i.p. route, except SCH23390 that was administered by subcutaneous (s.c.) route. All drugs were administered in a constant volume of 10 ml/kg body weight. In the experiments designed to verify the effect of ascorbic acid in the predictive models of depression, mice were treated with vehicle or ascorbic acid (0.01– 100 mg/kg, i.p.) 30 min before being tested in the TST, FST or the openfield test (Kulkarni and Dhir, 2007; Mantovani et al., 2003; Rosa et al., 2003). In the experiments performed to investigate de interaction of ascorbic acid with the monoaminergic system, mice were pre-treated with vehicle, NAN-190 (0.5 mg/kg, a 5-HT1A receptor antagonist), ketanserin (5 mg/kg, a 5-HT2A/2C receptor antagonist), MDL72222 (0.1 mg/kg, a 5-HT3 receptor antagonist), prazosin (62.5 µg/kg, an α1adrenoceptor antagonist), yohimbine (1 mg/kg, an α2-adrenoceptor antagonist), propranolol (2 mg/kg, a β-adrenoceptor antagonist), haloperidol (0.2 mg/kg, a non-selective dopamine receptor antagonist), SCH23390 (0.05 mg/kg, a dopamine D1 receptor antagonist) or sulpiride (50 mg/kg, a dopamine D2 receptor antagonist), and 30 min later they received vehicle or ascorbic acid (0.1 mg/kg, i.p.) before being tested in the TST after 30 min, as described previously (Kaster et al., 2005; Zomkowski et al., 2002, 2004). In another set of experiments, to test the ability of ascorbic acid to provoke an antidepressant-like effect when administered orally, animals were treated with vehicle or with this vitamin (0.1–100 mg/ kg) and 60 min later, TST, FST or the open-field test were carried out (Machado et al., 2007). In addition, we investigated the effect of an intracerebroventricular (i.c.v.) injection of ascorbic acid in the TST and

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in the open-field test. In this protocol, vehicle or ascorbic acid (0.01– 10 nmol/mice) were injected and 15 min later TST or the open-field test were carried out. The i.c.v. administration was performed under light ether anesthesia. Briefly, a 0.4 mm external diameter hypodermic needle attached to a cannula, which was linked to a 25 µl Hamilton syringe, was inserted perpendicularly through the skull and no more than 2 mm into the brain of the mouse. A volume of 5 µl was then administered in the left lateral ventricle. The injection was given over 30 s, and the needle remained in place for another 30 s in order to avoid the reflux of the substances injected. The injection site was 1 mm to the right or left from the mid-point on a line drawn through to the anterior base of the ears (Kaster et al., 2007; Zomkowski et al., 2006). To ascertain that the drugs were administered exactly into the cerebral ventricle, the brains were dissected and examined macroscopically after the test. We also investigated the ability of ascorbic acid to potentiate the antidepressant-like effect of conventional antidepressants. To this end, mice received by p.o. route vehicle, fluoxetine (1 mg/kg, a serotonin reuptake inhibitor), imipramine (0.1 mg/kg, a serotonin and noradrenaline reuptake inhibitor) or bupropion (1 mg/kg, a dopamine reuptake inhibitor) and immediately after, ascorbic acid (0.1 mg/kg, p. o.) or vehicle were administered. Sixty minutes later, the TST was carried out (Brocardo et al., 2008; Cunha et al., 2008). The locomotor activity in the open-field test was assessed in an independent group of mice that was subjected to the same protocol. The doses of the drugs used were selected on the basis of literature data (O'Neill and Conway, 2001; Redrobe and Bourin, 1997; Yamada et al., 2004), on previous results from our group (Brocardo et al., 2008; Cunha et al., 2008; Kaster et al., 2005; Machado et al., 2007; Rodrigues et al., 2002; Zomkowski et al., 2002, 2004) and on pilot experiments carried out in our laboratory. 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 during a 6-min test was scored as described previously (Rosa et al., 2008; Zomkowski et al., 2002, 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. A decrease in the duration of immobility is indicative of an antidepressant-like effect (Porsolt et al., 1977). 2.4. Tail suspension test The total duration of immobility induced by tail suspension was measured according to the method described by Steru et al. (1985). Mice both acoustically and visually isolated were suspended 50 cm above the floor by adhesive tape placed approximately 1 cm from the tip of the tail. Immobility time was recorded during a 6-min period (Machado et al., 2007; Rodrigues et al., 2002).

Fig. 1. Effect of intraperitoneal administration of ascorbic acid (dose range 0.01– 100 mg/kg) in the TST (panel A), in the FST (panel B) and open-field test (panel C). Each column represents the mean + S.E.M. (n = 6–11). ⁎⁎p b 0.01 as compared with the vehicle-treated group (V).

2.6. 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 Newman–Keuls test for post hoc comparison when appropriate. A value of p b 0.05 was considered to be significant.

2.5. Open-field behavior 3. Results The ambulatory behavior was assessed in an open-field test as described previously (Rodrigues et al., 2002; Rosa et al., 2008). The apparatus consisted of a wooden box measuring 40 × 60 × 50 cm high. The floor of the arena was divided into 12 equal rectangles. At the start of each trial, a mouse was placed in the left corner of the field and was allowed to freely explore the arena. The number of rectangles crossed with all paws (crossing) was counted in a 6-min session. The light was maintained at minimum to avoid anxiety behavior. The apparatus were cleaned with a solution of 10% ethanol between tests in order to hide animal clues.

3.1. Effect of ascorbic acid on the immobility time in the TST and FST The result depicted in Fig. 1A shows that the administration of ascorbic acid by i.p. route decreased the immobility time in the TST, indicating that the systemic administration of ascorbic is effective in producing an antidepressant-like effect in this behavioral model. Oneway ANOVA revealed a significant effect of ascorbic acid [F(5,40) = 9.48, p b 0.01]. Post hoc analysis indicated a significant decrease in the immobility time elicited by the administration of ascorbic acid at the

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p b 0.05], ascorbic acid treatment [F(1,20) = 9.11, p b 0.01] and ketanserin pre-treatment × ascorbic acid treatment interaction [F(1,20) = 10.54, p b 0.01]. Fig. 2C shows the effect of pre-treatment of mice with MDL 72222 (0.1 mg/kg, a 5-HT3 receptor antagonist) on the reduction in immobility-time elicited by ascorbic acid (0.1 mg/kg, i.p.). A twoway ANOVA revealed significant differences of MDL 72222 pretreatment [F(1,28) = 6.40, p b 0.05] and MDL 72222 pre-treatment× ascorbic acid treatment interaction [F(1,28) = 4.30, p b 0.05], but not of ascorbic acid treatment [F(1,28) = 2.81, p = 0.10]. Post hoc analyses indicated that the pre-treatment of mice with NAN-190, ketanserin or MDL 72222 prevented (p b 0.01) the decrease in the immobility time in the TST elicited by ascorbic acid. 3.3. Involvement of the noradrenergic system Fig. 3A shows that the pre-treatment of mice with prazosin (62.5 µg/kg, an α1- adrenoceptor antagonist) prevented the antiimmobility effect of ascorbic acid (0.1 mg/kg, i.p.) in the TST. The twoway ANOVA revealed significant differences of ascorbic acid treatment

Fig. 2. Effect of the pre-treatment of mice with NAN-190 (0.5 mg/kg, i.p., panel A), ketanserin (5 mg/kg, i.p., panel B), and MDL72222 (0.1 mg/kg, i.p., panel C) on ascorbic acid (AA; 0.1 mg/kg, i.p.)-induced reductions in immobility time in the TST. Each column represents the mean + S.E.M. (n = 6–9) ⁎⁎ p b 0.01 as compared with the vehicle-treated control. # p b 0.01 as compared with the same group pre-treated with vehicle.

doses of 0.1; 1 and 10 mg/kg. Fig. 1B shows that the administration of ascorbic acid by i.p. route did not produce any effect in the immobility time in the FST [F(4,35) = 9.93, p = 0.45]. Fig. 1C shows that when ascorbic acid was administered at doses that produced an antiimmobility effect in the TST, no effect was observed in the open-field test [F(3,31) = 1.20, p = 0.32]. 3.2. Involvement of the serotonergic system The result presented in Fig. 2A shows that the pre-treatment of mice with NAN-190 (0.5 mg/kg, a 5-HT1A receptor antagonist) was able to prevent the anti-immobility effect of ascorbic acid (0.1 mg/kg, i. p.) in the TST. The two-way ANOVA revealed significant differences of NAN-190 pre-treatment [F(1,22) = 10.56, p b 0.01], ascorbic acid treatment [F(1,22) = 30.71, p b 0.01] and NAN-190 pre-treatment × ascorbic acid treatment interaction [F(1,22) = 4.58, p b 0.05]. Fig. 2B shows the results of pre-treatment of mice with ketanserin (5 mg/kg, a 5-HT2A/2C receptor antagonist, i.p.) against the anti-immobility effect of ascorbic acid (0.1 mg/kg, i.p.) in the TST. The two-way ANOVA revealed significant differences of ketanserin pre-treatment [F(1,20) = 4.83,

Fig. 3. Effect of the pre-treatment of mice with prazosin (62.5 µg/kg, i.p., panel A), yohimbine (1 mg/kg, i.p., panel B), and propranolol (2 mg/kg, i.p., panel C) on the ascorbic acid (AA; 0.1 mg/kg, i.p.)-induced reductions in immobility time in the TST. Each column represents the mean + S.E.M. (n = 6–10) ⁎⁎ p b 0.01 as compared with the vehicle-treated control. # p b 0.01 as compared with the same group pre-treated with vehicle.

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[F(1,27) = 5.71, p b 0.05] and prazosin pre-treatment × ascorbic acid treatment interaction [F(1,27) = 8.39, p b 0.01], but not of prazosin pretreatment [F(1,27) = 0.53, p = 0.47]. Fig. 2B shows the effect of pretreatment of mice with yohimbine (1 mg/kg, an α2-adrenoceptor antagonist) on the reduction in immobility-time produced by the administration of ascorbic acid (0.1 mg/kg, i.p.). A two-way ANOVA revealed significant differences of yohimbine pre-treatment [F(1,31) = 10.31, p b 0.01], ascorbic acid treatment [F(1,31) = 5.36, p b 0.05] and yohimbine pre-treatment× ascorbic acid treatment interaction [F(1,31) =7.70, p b 0.01]. Fig. 3C shows the results of pre-treatment of mice with propranolol (2 mg/kg, a β-adrenoceptor antagonist) on the reduction in immobility-time elicited by ascorbic acid (0.1 mg/kg, i.p.) in the TST. The two-way ANOVA revealed significant differences of propranolol pretreatment [F(1,23) = 16.55, p b 0.01], ascorbic acid treatment [F(1,23) = 14.02, p b 0.01] and propranolol pre-treatment × ascorbic acid treatment interaction [F(1,23)= 6.64, p b 0.05]. Post hoc analyses indicated that the pre-treatment of mice with prazosin, yohimbine or propranolol prevented (p b 0.01) the decrease in immobility-time in the TST elicited by ascorbic acid.

Fig. 5. Effect of p.o. administration of ascorbic acid (dose range 0.1–100 mg/kg) or (F) fluoxetine (10 mg/kg) in the TST (panel A), in the FST (panel B); (F) fluoxetine (20 mg/ kg) and open-field test (panel C). Each column represents the mean + S.E.M. (n = 6–10). ⁎ p b 0.05 and ⁎⁎ p b 0.01 as compared with the vehicle-treated group (V).

3.4. Involvement of the dopaminergic system

Fig. 4. Effect of the pre-treatment of mice with haloperidol (0.2 mg/kg, i.p., panel A), SCH23390 (0.05 mg/kg, s.c., panel B), and sulpiride (50 mg/kg, i.p., panel C) on the ascorbic acid (AA; 0.1 mg/kg, i.p.)-induced reductions in the immobility time in the TST. Each column represents the mean + S.E.M. (n = 6–8) ⁎⁎ p b 0.01 as compared with the vehicle-treated control. # p b 0.01 as compared with the same group pre-treated with vehicle.

The results presented in Fig. 4A shows that the pre-treatment of mice with haloperidol (0.2 mg/kg, a non-selective dopamine receptor antagonist, i.p.) prevented the anti-immobility effect of ascorbic acid (0.1 mg/kg, i.p.) in the TST. The two-way ANOVA showed significant differences of haloperidol pre-treatment [F(1,26) = 8.91, p b 0.01], ascorbic acid treatment [F(1,26) = 14,33, p b 0.01] and haloperidol pre-treatment × ascorbic acid treatment interaction [F(1,26) = 5.06, p b 0.05]. Fig. 4B shows that the pre-treatment of mice with SCH 23390 (0.05 mg/kg, a D1 receptor antagonist, s.c.) was not able to prevent the antidepressant-like effect of ascorbic acid (0.1 mg/kg, i.p.) in the TST. A two-way ANOVA revealed significant differences of ascorbic acid treatment [F(1,21)= 31.68, p b 0.01], but not of SCH 23390 pre-treatment [F(1,21) = 0.64, p = 0.43] and SCH 23390 pre-treatment × ascorbic acid treatment interaction [F(1,21) = 0.35, p = 0.55]. Fig. 4C shows that the pre-treatment of mice with sulpiride (50 mg/kg, a D2 receptor antagonist, i.p.) was able to prevent the reduction in

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3.6. Effect of intracerebroventricular administration of ascorbic acid on the immobility time in the TST Fig. 6A shows that ascorbic acid administration by i.c.v. route was able to produce an antidepressant-like effect in the TST. A one-way ANOVA revealed a significant effect of ascorbic acid [F(4,26) = 6.85, p b 0.01]. Post hoc analysis indicated a significant decrease in the immobility time elicited by the administration of ascorbic acid at the doses of 0.1 nmol/mice. Fig. 6B shows that when ascorbic acid was administered at the same doses (0.01–10 nmol/mice, i.c.v.), no effect was observed in the open-field test [F(4,25) = 0.10, p = 0.97]. 3.7. Interaction of ascorbic acid with antidepressants in the TST We also investigated the effect of the p.o. co-administration of subeffective doses of conventional antidepressants and ascorbic acid to mice. The result depicted in Fig. 7A shows that an administration of a subeffective dose of ascorbic acid (0.1 mg/kg, p.o.) was able to potentiate the effect of a subeffective dose of fluoxetine (1 mg/kg, p.o.).

Fig. 6. Effect of i.c.v. administration of ascorbic acid (dose range 0.01–10 nmol/mice) in the TST (panel A) and in the open-field test (panel B). Each column represents the mean + S.E.M. (n = 6–7). ⁎⁎ p b 0.01 as compared with the vehicle-treated group (V).

immobility-time produced by ascorbic acid (0.1 mg/kg, i.p.). The twoway ANOVA showed significant differences of sulpiride pre-treatment [F(1,21)= 18.04, p b 0.01] and sulpiride pre-treatment× ascorbic acid treatment interaction [F(1,21) = 6.98, p b 0.05], but not of ascorbic acid treatment [F(1,21)= 3.19, p = 0.08]. Post hoc analyses indicated that the pre-treatment of mice with haloperidol and sulpiride (p b 0.01) prevented the decrease in immobility-time in the TST elicited by ascorbic acid. 3.5. Effect of oral administration of ascorbic acid on the immobility time in the TST and FST Fig. 5A shows that the p.o. administration of ascorbic acid or the conventional antidepressant fluoxetine decreased the immobility time in the TST. A one-way ANOVA revealed a significant effect of ascorbic acid or fluoxetine [F(5,40) = 7.64, p b 0.01]. Post hoc analysis indicated a significant decrease in the immobility time elicited by the administration of ascorbic acid at the doses of 1 and 10 mg/kg, p.o. and fluoxetine (10 mg/kg, p.o.). Fig. 5B shows that the administration of ascorbic acid by p.o. route did not produce any effect in the immobility time in the FST, similarly to the result obtained when ascorbic acid was administered by i.p. route. However, fluoxetine administered by p.o. route produced an antidepressant-like effect in the FST. A one-way ANOVA showed a significant effect of treatment [F(5,33) = 13.13, p b 0.01]. Post hoc analysis indicated a significant decrease in the immobility time elicited by the administration of fluoxetine (20 mg/ kg, p.o.). Fig. 5C shows that when administered by p.o. route at doses active in the TST, ascorbic acid produced no effect in the open-field test [F(4,25) = 0.19, p = 0.94], indicating that the effect observed in the TST was a specific antidepressant-like effect. In addition, fluoxetine administration at any of the doses employed (10 or 20 mg/kg, p.o.) did not alter the locomotor behavior of the animals subjected to the openfield test (data not shown).

Fig. 7. Effect of the administration of a subeffective dose of ascorbic acid (AA; 0.1 mg/kg, p.o.) in potentiating the effect of a subeffective dose of fluoxetine (1 mg/kg, p.o., panel A), imipramine (0.1 mg/kg, p.o., panel B) and bupropion (1 mg/kg, p.o., panel C) in the TST. Each column represents the mean + SEM (n = 6–8). ⁎ p b 0.05 and ⁎⁎ p b 0.01 as compared with the vehicle-treated group.

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The effects of p.o. administration of a subeffective dose of ascorbic acid (0.1 mg/kg) and fluoxetine (1 mg/kg), imipramine (0.1 mg/kg) or bupropion (1 mg/kg) in the open-field test were also investigated. The result depicted in Fig. 8A shows that the administration of ascorbic acid and fluoxetine did not affect the locomotor activity. The two-way ANOVA showed no differences for fluoxetine pre-treatment [F(1,21) = 0.00, p = 0.93], ascorbic acid treatment [F(1,21) = 0.17, p = 0.67] and fluoxetine pre-treatment × ascorbic acid treatment interaction [F(1,21) = 2.71, p = 0.11]. Fig. 8B shows that the administration of ascorbic acid and imipramine did not alter the locomotor activity of mice in the open-field test. A two-way ANOVA revealed no differences for imipramine pre-treatment [F(1,20) = 2.81, p = 0.10], ascorbic acid treatment [F(1,20) = 0.20, p = 0.65] and imipramine pre-treatment × ascorbic acid treatment interaction [F(1,20) = 1.92, p = 0.18]. Finally, Fig. 8C shows that the administration of ascorbic acid and bupropion did not alter the ambulation of mice. A two-way ANOVA showed no differences for bupropion pre-treatment [F(1,20) =3.64, p = 0.07], ascorbic acid treatment [F(1,20) = 0.29, p = 0.59] and bupropion pre-treatment × ascorbic acid treatment interaction [F (1,20) = 0.95, p = 0.34]. 4. Discussion

Fig. 8. Effect of the administration of a subeffective dose of ascorbic acid (AA; 0.1 mg/kg, p.o.) and subeffective doses of fluoxetine (1 mg/kg, p.o., panel A), imipramine (0.1 mg/ kg, p.o., panel B) and bupropion (1 mg/kg, p.o., panel C) in the open-field test. Each column represents the mean + SEM (n = 6).

The two-way ANOVA revealed significant differences of fluoxetine pretreatment [F(1,22) = 29.39, p b 0.01], ascorbic acid treatment [F(1,22) = 46.48, p b 0.01] and fluoxetine pre-treatment × ascorbic acid treatment interaction [F(1,22) = 15.69, p b 0.01]. Fig. 7B shows that the effect of imipramine (0.1 mg/kg, p.o.) was potentiated by an administration of a subeffective dose of ascorbic acid (0.1 mg/kg, p.o.). The two-way ANOVA showed significant differences of imipramine pre-treatment [F(1,22) =9.56, p b 0.01], ascorbic acid treatment [F(1,22) = 15.05, p b 0.01] and imipramine pre-treatment × ascorbic acid treatment interaction [F(1,22) = 9.80, p b 0.01]. Fig. 7C shows that the administration of a subeffective dose of bupropion (1 mg/kg, p.o.) and a subeffective dose of ascorbic acid (0.1 mg/kg, p.o.) produces an antidepressant-like effect in the TST. The two-way ANOVA revealed significant differences of bupropion pre-treatment [F(1,24) = 4.85, p b 0.05], ascorbic acid treatment [F(1,24)= 6.91, p b 0.05] and bupropion pre-treatment × ascorbic acid treatment interaction [F(1,24) =4.26, p b 0.05]. Post hoc analyses showed a synergistic effect elicited by fluoxetine (p b 0.01), imipramine (p b 0.01) and bupropion (p b 0.05) when administrated together with ascorbic acid in the TST.

Our results show, to our knowledge for the first time, that ascorbic acid given systemically (by i.p. or p.o. route) or centrally (i.c.v. route) produces an antidepressant-like effect in the TST, which is dependent on an interaction with the monoaminergic system. In addition, we showed that ascorbic acid was able to potentiate the action of conventional antidepressants in this predictive test of antidepressant action, reinforcing the idea that this vitamin has antidepressant properties. Despite the lack of studies investigating the effect of ascorbic acid in animal models of depression, the antidepressant-like effect of this vitamin in the TST is somewhat in accordance with clinical studies that suggest that ascorbic acid could be beneficial for treating depression and is involved in the modulation of mood (Brody, 2002; Coochi et al., 1980). A recent study has indicated that chronic exposure to FST, a model of chronic fatigue, caused a depletion of adrenal ascorbic acid levels, which was reversed by the treatment with the antidepressant venlafaxine (Dhir and Kulkarni, 2008a). TST and FST are two of the most commonly animal models used to detect and characterize the efficacy of antidepressant drugs and are sensitive to these drugs after acute administration (Borsini and Meli, 1988; Cryan et al., 2005). In both tests, animals are placed in an inescapable situation and the antidepressant-like activity is expressed by the decrease of immobility time (Bourin et al., 2005), an effect that is exhibited by conventional antidepressants (Porsolt et al., 1977; Steru et al., 1985). In spite of the large use of these models to assess the antidepressant activity of new drugs and compounds, they are also important tools to study neurobiological mechanisms involved in antidepressant responses (Bourin et al., 2005; Steru et al., 1985). Taking this features into account and considering that these models are simple and reliable across laboratories, the identification and elucidation of antidepressant properties of endogenous compounds like adenosine (Kaster et al., 2005, 2007), agmatine (Zomkowski et al., 2002, 2004), magnesium (Cardoso et al., 2009), melatonin (Mantovani et al., 2003), putrescine (Zomkowski et al., 2006), zinc (Cunha et al., 2008) synthetic compounds (Rosa et al., 2008) and plant extracts (Machado et al., 2007) have been studied by our group. Although both TST and the FST are similar in their constructs and objectives, they are different in terms of the biological substrates that underlie the observed behavior (Cryan et al., 2005). TST was proposed to have a greater pharmacological sensitivity than FST (Thierry et al., 1986) and intra- and inter-strain differences in the performance in the TST and FST have been demonstrated and may indicate that the neural circuitry mediating behaviors in these tests is not identical (Bai et al., 2001; Cryan et al., 2005; Renard et al., 2003). These models could

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present differential sensitivity to the immobility-reducing effects of drugs or compound with antidepressant properties. For example, Bai et al. (2001) observed that the tricyclic antidepressant imipramine produced a U-shaped dose–response curve in the FST, whereas a dose–response effect was observed in the TST over the same dose range. Moreover, it has been postulated that the mouse FST is not a sensitive model for detecting SSRI activity, whereas these antidepressants are generally reported as active in the TST (Cryan et al., 2005). In this study, ascorbic acid elicited antidepressant-like effect in mice when administered systemically (i.p or p.o. route) or centrally (i.c.v. route) in the TST, but not in the FST. It is difficult to explain the reason by which the FST was not sensitive to the effect of ascorbic acid, but the differences in the biological substrates that underlie the observed behavior in the FST as compared to the TST may account for this result. The decrease of the immobility time elicited by the conventional antidepressant fluoxetine in both behavioral tests validates these behavioral approaches. It should be noted that the anti-immobility effect of ascorbic acid in the TST cannot be attributed to a psychostimulant action of this vitamin. This conclusion derives from the fact that in our study ascorbic acid administered at doses that produced a significant decrease in the immobility-time in the TST did not alter the locomotor activity in the open-field test, as compared to control animals. An interesting finding of the present study is that the antidepressant-like effect of ascorbic acid in the TST exhibited a dose-dependent U-shaped curve. The mechanism underlying this effect remains unclear and further investigations will be necessary to elucidate this question. However, some key points may contribute to the U-shaped trend observed. A point that may account for the U-shaped curve observed in this study is related to the glutamate-ascorbate heteroexchange in neurons. Ascorbate is released from glutamatergic neurons, in which the glutamate transporter exchanges ascorbate for glutamate (Rebec and Pierce, 1994). In this sense, manipulations that elevate ascorbate may prolong glutamate transmission by interfering with this heteroexchange process and the impaired and enhanced glutamatergic neurotransmission is associated with depression (Skolnick, 1999) as well as with a depressive phenotype in the FST (Rada et al., 2003). On the other hand, it is also possible that the non observed effect of ascorbic acid at its higher dose in this study was due to its NMDA receptor antagonist properties. Ascorbate operates on a regulatory site of the NMDA receptor that alters the electric charge of the receptor, reducing its activity (Majewska et al., 1990; Rebec and Pierce, 1994). Preclinical and clinical studies have shown that NMDA receptor antagonists may exert antidepressant action (Skolnick, 1999). Dhir and Kulkarni (2008b) have recently showed that the well known NMDA receptor antagonist MK-801, similar to the effect elicited by ascorbic acid in the TST, produced U-shape dose–response curve in the mouse FST. In addition, the U-shape trend observed in this study was also observed previously with endogenous modulatory compound such as agmatine (Zomkowski et al., 2002), putrescine (Zomkowski et al., 2006) and GMP (Eckeli et al., 2000) which produce their antidepressant-like effects in the mouse FST, at least in part, by inhibiting NMDA receptors (Eckeli et al., 2000; Zomkowski et al., 2002). Furthermore, it should be considered that even the conventional antidepressant imipramine produced a U-shaped dose– response curve in the FST (Bai et al., 2001). A large number of evidence indicates that the serotonergic system is closely implicated in the pathogenesis of depression and in the mechanism of action of antidepressants (Duman et al., 1997; Wong and Licinio, 2001). Most of the prescribed antidepressants directly affect serotonin turnover in the brain (Kreiss and Lucki, 1995), inhibit serotonin reuptake and also interact with 5-HT1A and 5-HT2 receptors (Cryan et al., 2005). Several reports have suggested that 5-HT1A receptors are involved in the mechanism of action of antidepressant drugs (Blier and Ward, 2003), including tricyclics, selective serotonin reuptake inhibitors and monoamine oxidase inhibitors (Hensler,

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2002). It was previously shown that the blockade of 5-HT1A receptors by NAN-190 prevented the antidepressant-like responses of serotonergic agonists (8-OH-DPAT and buspirone) and the tricyclic antidepressant desipramine in the FST in rats (Detke et al., 1995). NAN-190 is proposed as an antagonist at post-synaptic 5-HT1A receptors (Glennon et al., 1988; Przegalinski et al., 1990) and a partial agonist at pre-synaptic receptors (Gruel and Glaser, 1992; Hjorth and Sharp, 1990). Here we showed the involvement of 5-HT1A receptors in the antidepressant-like effect of ascorbic acid, since NAN-190 was able to prevent its anti-immobility effect in the TST. The antidepressant-like effect of ascorbic acid in the TST was also prevented by the pretreatment with ketanserin, a 5-HT2A/2C receptor antagonist. A role for the regulation of 5-HT2 receptors, especially 5HT2A and 5-HT2C subtypes, emerged when some studies showed that the administration of antidepressants caused a down-regulation of these receptors (Deakin, 1988). There are studies showing that 5-HT2A/ 2C antagonism has a role in the mechanism underlying the antidepressant-like effect of certain antidepressants in the FST (Cryan and Lucki, 2000; Elhwuegi, 2004; Redrobe and Bourin, 1997). On the other hand, there are reports showing that the preferential 5-HT2A receptor agonist DOI enhances the antidepressant-like effect of some compounds (Khisti and Chopde, 2000; Zomkowski et al., 2004). The reversal of the antiimmobility effect of ascorbic acid with ketanserin suggests that its effect in the TST is mediated through an interaction with 5-HT2A or 5-HT2C receptors. Despite the well known involvement of serotonergic neurotransmission in depression, there are only a few pieces of evidence regarding the participation of 5-HT3 receptors in the pathophysiology of this disease. The involvement of 5-HT3 receptors in the mechanism of action of antidepressants was indicated by the fact that different classes of antidepressants act as functional antagonists at the 5-HT3 receptors, suggesting that the inhibition of these receptors activity may contribute to the action of antidepressants (Eisensamer et al., 2003). However, there is evidence showing that electroconvulsive therapy, clinically used to treat drug resistant depression, was able to potentiate the function of 5-HT3 receptors in the hippocampus, a brain region closely related to the etiology of depression (Krishnan et al., 1991; Ishihara and Sasa, 2001). In the present study, our result is in agreement with the hypothesis that an activation of the 5-HT3 receptors can afford antidepressant-like responses, since the antidepressant-like effect of ascorbic acid was prevented by the pretreatment of mice with the 5-HT3 antagonist MDL72222. Experimental and clinical studies indicate that the noradrenergic system is strongly implicated in the pathophysiology of depression (Frazer, 2000; Nutt, 2006). Preclinically, the α1-, and α2-adrenoceptors have been shown to underlie some of the antidepressant-like responses of drugs in behavioral models of depression (Danysz et al., 1986; Kitada et al., 1983; Masuda et al., 2001). It was previously shown that the antidepressant-like action of desipramine was prevented by the pretreatment of mice with the α1-adrenoceptor antagonist prazosin (Danysz et al., 1986). In addition, O'Neill and Conway (2001) showed that the antidepressant-like effect of clonidine (an α2-adrenoceptor agonist) in the FST was reversed by the α2adrenoceptor antagonist yohimbine. Moreover, it was also reported that the β-adrenoceptor number and function are consistently decreased in the rat cortex by the repetitive administration (14 days) of desipramine, electroconvulsive therapy (Heal et al., 1987, 1989) or reboxetine (a noradrenaline reuptake inhibitor) (Harkin et al., 2000). In this study, the antidepressant-like effect elicited by ascorbic acid was inhibited by the pre-treatment of mice with prazosin, yohimbine or propranolol, suggesting that α1-, α2- and β-adrenoceptors underlie the action of the vitamin in the TST. In parallel with the serotonergic and noradrenergic systems, dopaminergic system is strongly implicated in regulation of mood (D'Aquila et al., 2000; Willner et al., 2005) and there are several evidences regarding the efficacy of antidepressants with

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dopaminergic effects in the treatment of depression (Papakostas, 2006). Our results showed that the dopaminergic system is involved in the antidepressant-like action of ascorbic acid in the TST, through an interaction with dopamine D2 receptors, since the pre-treatment of the animals with haloperidol (a non-selective dopamine receptor antagonist), or sulpiride (a selective dopamine D2 receptor antagonist) prevented the antidepressant-like effect evoked by ascorbic acid in the TST. The implication of D2 receptors in the pathophysiology of depression has been suggested by clinical studies which show that dopamine D2 receptor agonists are effective for treating depressive patients (Waehrens and Gerlach, 1981). Preclinical data indicate that dopamine D2 receptors are related to the anti-immobility action of antidepressants in the mouse FST (Borsini et al., 1988; Yamada et al., 2004). Moreover, electroconvulsive therapy (ECT), used to treat depressions which are resistant to the usual antidepressant drugs, causes a reduction in the immobility time in the TST, that was antagonized by sulpiride, implicating the dopamine D2 receptor in the antidepressant action of ECT (Teste et al., 1990). In our study, the pretreatment of mice with the dopamine D1 receptor antagonist SCH23390 did not prevent the anti-immobility effect elicited by ascorbic acid, suggesting that this receptor subtype is not implicated in the antidepressant-like effect of ascorbic acid in the TST. The role of dopamine D1 receptors in the mechanism of action of antidepressants is controversial. Some studies have indicated that SCH23390 prevented the antidepressant-like effect of some antidepressant agents in the FST and TST (Hirano et al., 2007; Machado et al., 2007; Yamada et al., 2004), but others have suggested that the activation of dopamine D1 receptors is not involved in the mechanism of action of antidepressants (Borsini et al., 1988; Kitamura et al., 2008). The interaction of ascorbic acid with the dopaminergic system has been studied and seems to be complex. It appears that the action of ascorbic acid is dose-dependent such that the administration of ascorbic acid (50–200 mg/kg) in rats enhances dopamine-mediated behavioral effects (Wambebe and Sokomba, 1986). On the other hand, higher doses of ascorbic acid seem to antagonize dopamine-mediated behavioral effects (Rebec and Pierce, 1994). Following the previous discussion, this may explain the U-shaped dose–response curve of ascorbic acid in the TST. It is important to highlight that in our study, the doses of ascorbic acid that was effective in the TST are very low (0.1–10 mg/kg, i.p and 1–10 mg/kg, p.o.) and our results suggest that an activation of dopamine D2 receptors is involved in the antidepressant-like effect elicited by the vitamin. Finally, an interesting finding of this study is that ascorbic acid potentiated the effect of subeffective doses of conventional antidepressants such as fluoxetine, imipramine and bupropion in the TST, without modifying the ambulatory behavior. Fluoxetine (a selective serotonin reuptake inhibitor, SSRI), imipramine (a serotonin/noradrenaline reuptake inhibitor, TCA) and bupropion (an atypical antidepressant that is a potent inhibitor of dopamine reuptake with subtle activity on noradrenergic reuptake) are commonly used for treating major depression (Cooper et al., 1980; Holtzheimer and Nemeroff, 2006; Wilkes, 2006). In several cases these antidepressants are ineffective, poorly tolerable and present a long delay to achieve its therapeutical effects (Nemeroff and Owens, 2002; Berton and Nestler, 2006; Holtzheimer and Nemeroff, 2006). Considering the need for faster acting, safer and more effective treatments for depression (Berton and Nestler, 2006), the synergistic antidepressant-like effect between ascorbic acid and the mentioned antidepressants suggest that this vitamin could be helpful for the improvement of the conventional pharmacotherapy (decreasing the doses of antidepressants prescribed and consequently the side effects). However, it should be considered that the TST is not a model of depression per se, and that the results obtained with this model should be considered with caution. Thus, our results support the rationale for further animal studies and also clinical studies to confirm the antidepressant effect of ascorbic acid.

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