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European Journal of 687 (2012) 21–27

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European Journal of Pharmacology

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Behavioural Pharmacology Involvement of different types of potassium channels in the antidepressant-like effect of ascorbic acid in the mouse tail suspension test

Morgana Moretti, Josiane Budni, Camille Mertins Ribeiro, 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: Considering that the administration of ascorbic acid elicits an antidepressant-effect in mice by a mechanism Received 20 March 2012 which involves an interaction with N-methyl-D-aspartate receptors and the L-arginine-nitric oxide-cGMP Received in revised form 19 April 2012 pathway and taking into account that the stimulation of this pathway is associated with the activation of po- Accepted 21 April 2012 tassium (K+) channels, this study investigated the involvement of different types of K+ channels on the ef- Available online 2 May 2012 fect of ascorbic acid in the mouse tail suspension test (TST). Intracerebroventricular administration of (TEA, a non-specific blocker of K+ channels, 25 pg/site), (an ATP- Keywords: + Antidepressant sensitive K channel blocker, 0.5 pg/site), (a large- and intermediate conductance calcium- + + Ascorbic acid activated K channel blocker, 25 pg/site) or apamin (a small-conductance calcium-activated K channel K+ channel blocker, 10 pg/site) was able to produce a synergistic effect with a sub-effective dose of ascorbic acid Tail suspension test (0.1 mg/kg) given orally (p.o.). The antidepressant-like effect of ascorbic acid (1 mg/kg, p.o.) in the TST was prevented by the pre-treatment of mice with (a K+ , 10 μg/site, i.c.v.) and (10 μg/site, i.c.v.). Moreover, cromakalim abolished the synergistic effect elicited by the combined treatment with sub-effective doses of ascorbic acid and 7-nitroindazole. The administration of the K+ chan- nel modulators alone or in combination with ascorbic acid did not affect the locomotion of mice. Together, our results show that the antidepressant-like effect of ascorbic acid in the TST may involve, at least in part, the modulation of neuronal excitability, via inhibition of K+ channels. © 2012 Elsevier B.V. Open access under the Elsevier OA license.

1. Introduction Reinforcing the notion that this vitamin may be an alternative approach in the management of depression symptoms, our group Depression is a debilitating and highly prevalent disorder that af- recently demonstrated that repeated ascorbic acid treatment, fects individuals worldwide and causes a significant negative impact similar to fluoxetine, reversed depressive-like behavior and brain ox- on public health. Although a range of antidepressants with varied idative damage induced by chronic unpredictable stress in mice mechanisms of action are available, a considerable number of patients (Moretti et al., 2012). exhibit partial, refractory or intolerant responses to the pharmacolog- Several subtypes of potassium (K+) channels with different phar- ical treatment. Ascorbic acid, a water-soluble vitamin with neuro- macological sensitivities and electro-physiological characteristics protective and antioxidant properties (Rice, 2000), is emerging as a have been reported in neurons (Aronson, 1992; Halliwell, 1990). novel putative compound to assist in the treatment of depression. In These channels are important regulators of physiological processes clinical studies, it was reported that the administration of ascorbic which include the regulation of neuronal excitability and signal prop- acid relieved adrenocorticotropic hormone-induced depression in a agation (MacKinnon, 2003; Shieh et al., 2000). The association of K+ child (Cocchi et al., 1980) and decreased scores in a Beck Depression channels with the pathophysiology of depression has been suggested Inventory in healthy young adults (Brody, 2002), indicating mood im- by several preclinical studies. The administration of a variety of K+ provement. In mice, the administration of ascorbic acid produced an channel blockers, such as tetraethylammonium (TEA), apamin, antidepressant-like effect in the tail suspension test (TST) by a mech- charybdotoxin, and glibenclamide, were reported to pro- anism that is dependent on the interaction of this vitamin with the duce an antidepressant-like effect in the forced swimming test systems (Binfaré et al., 2009), N-Methyl-D-aspartate (Galeotti et al., 1999; Kaster et al., 2005), whereas the treatment + (NMDA) receptors and the L-arginine-nitric oxide (NO)-cyclic with K channel openers, such as minoxidil or cromakalim, increased guanosine monophosphate (cGMP) pathway (Moretti et al., 2011). the immobility time in this test, indicative of a depressive-like effect (Galeotti et al., 1999). Moreover, literature data suggest that different classes of antidepressants, including fluoxetine, venfalaxine, imipra- ⁎ Corresponding author. Tel.: +55 48 3721 5043; fax: +55 48 3721 9672. mine, desipramine, , , , E-mail addresses: [email protected], [email protected] (A.L.S. Rodrigues). maprotiline, and may modulate neuronal

0014-2999 © 2012 Elsevier B.V. Open access under the Elsevier OA license. doi:10.1016/j.ejphar.2012.04.041 22 M. Moretti et al. / European Journal of Pharmacology 687 (2012) 21–27 excitability via K+ channel inhibition and this may be the final 95% of trials. Results from mice presenting cannula misplacement or common pathway of the pharmacological action of these any signs of cerebral hemorrhage (b5%) were discarded from statisti- (Bortolatto et al., 2010; Kobayashi et al., 2004, 2006; Nicholson et cal analysis. al., 2002; Tytgat et al., 1997). Some studies have reported that both NO and cGMP, produced 2.3. Pharmacological treatment through the activation of nitric oxide synthase (NOS) and soluble guanylate cyclase (sGC), respectively, can activate different types of To test the hypothesis that the antidepressant-like effect of ascorbic K+ channels in several tissues (Bolotina et al., 1994; H.J. Jeong et al., acid is mediated through the inhibition of K+ channels, distinct groups 2001; S.Y. Jeong et al., 2001). In addition, NO donors are also capable of mice were pretreated with a sub-effective dose of ascorbic acid of activating these channels (Fujino et al., 1991; Williams et al., 1988). (0.1 mg/kg, p.o.) or vehicle 45 min before the i.c.v. administration of Indeed, the inhibition of NMDA receptors with consequent reduction TEA (a non-specificblockerofK+ channels, 25 pg/site), glibenclamide in the NO levels prevents the activation of K+ channels, avoiding hy- (an ATP-sensitive K+ channel blocker, 0.5 pg/site), charybdotoxin (a perpolarization of neurons and reducing the effectiveness of excitato- large- and intermediate-conductance calcium-activated K+ channel ry inputs (Christie, 1995). blocker, 25 pg/site), apamin (a small-conductance calcium-activated K+ Given that the antidepressant-like effect of ascorbic acid in the TST channel blocker, 10 pg/site) or vehicle. A further 15 min were allowed is dependent on the inhibition of either NMDA receptors or NO and to elapse before the animals were tested in the TST. In another set of ex- cGMP synthesis, we hypothesized that an inhibition of K+ channels periments, mice were given an i.c.v. administration of cromakalim (a is implicated in its effect in the TST. K+ channel opener, 10 μg/site) or minoxidil (a K+ channel opener, 10 μg/site) 45 min after ascorbic acid administration (1 mg/kg, p.o.). Fif- 2. Material and methods teen minutes later the TST was carried out. In another experiment, we evaluated the effect of the adminis- 2.1. Animals tration of a K+ channel opener on the antidepressant-like effect elicited by the combined administration of sub-effective doses of The behavioral experiments were conducted using adult Swiss ascorbic acid and 7-nitroindazole (25 mg/kg, i.p., a neuronal NOS mice (2 months, 30–40 g) of either sex (homogeneously distributed inhibitor). To this end, ascorbic acid or vehicle was administered among groups), maintained at 20–22 °C with free access to water 30 min before 7-nitroindazole or vehicle. Fifteen minutes after and food, under a 12:12 h light/dark cycle, with lights on at administration of 7-nitroindazole, mice were administered with 7:00 a.m. The animals were caged in groups of 15 in a 41× cromakalim (a K+ channel opener, 10 μg/site, i.c.v.) or vehicle and 34×16 cm cage and were placed in the experimental room 24 h be- 15 min later they were tested in the TST. fore the test for acclimatization. All behavioral tests were carried The doses of ascorbic acid were selected according to previous out between 09.00 a.m. and 04.00 p.m. The animals were used studies conducted in our laboratory (Binfaré et al., 2009; Moretti et according to the NIH Guide for the Care and Use of Laboratory Ani- al., 2011). Doses and administration schedule of K+ channel modula- mals and the experiments were performed after approval of the pro- tors were chosen based on literature data and on time-course and tocol by the Ethics Committee of the Institution. All efforts were made dose–response experiments (Bortolatto et al., 2010; Budni et al., to minimize animal suffering and to reduce the number of animals 2007, 2012; Galeotti et al., 1999; Jesse et al., 2009; Kaster et al., 2005). used in the experiments. 2.4. Behavioral tests 2.2. Drugs 2.4.1. Tail suspension test The following drugs were used: ascorbic acid, TEA, 7-nitroindazole The total duration of immobility induced by tail suspension was (Sigma Chemical Co, USA), apamin, charybdotoxin, cromakalim, mi- measured according to the method described by Steru et al. (1985). noxidil and glibenclamide (Tocris Cookson, Ballwin, MO, USA). Mice both acoustically and visually isolated were suspended 50 cm Cromakalim, minoxidil and 7-nitroindazole were dissolved in saline above the floor by adhesive tape placed approximately 1 cm from with 10% Tween 80, ascorbic acid was dissolved in distilled water the tip of the tail. Mice were considered immobile only when they and all the other drugs were dissolved in isotonic saline solution hung passively and completely motionless. Immobility time was (NaCl 0.9%) immediately before use. Appropriate vehicle-treated manually recorded during a 6-min period by an experienced observ- groups were also assessed simultaneously. Most of the drugs were ad- er. The observer was in the room where experiments were performed ministered by intracerebroventricular (i.c.v.) route, in a volume of 5 μl and was blind to the animal condition. per mouse, except ascorbic acid, which was administered orally (p.o.) by gavage and 7-nitroindazole, which was administered intraperito- 2.4.2. Open-field test neally (i.p.) in a constant volume of 10 ml/kg body weight. Ten minutes after the tail suspension test, the locomotor activity The i.c.v. injections were performed by employing a “free hand” was assessed in an open-field test as described previously (Moretti method under light ether anesthesia according to the procedure de- et al., 2011). The apparatus consisted of a wooden box measuring scribed previously (Brocardo et al., 2008; Kaster et al., 2012). Briefly, 40×60×50 cm high. The floor of the arena was divided into 12 a 0.4 mm external diameter hypodermic needle attached to a cannu- equal squares. The number of squares crossed with all paws (cross- la, which was linked to a 25 μl Hamilton syringe, was inserted per- ings) was manually counted in a 6-min session. The light was pendicularly through the skull and no more than 2 mm into the maintained at minimum to avoid anxiety behavior. The apparatus brain of each mouse. A volume of 5 μl was then administered into were cleaned with a solution of 10% ethanol between tests in order the left lateral ventricle. The injection was given over 30 s, and the to hide animal clues. 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 left 2.5. Statistical analysis from the mid-point on a line drawn through to the anterior base of the ears. I.c.v. injections were performed by an experienced investiga- All data are presented as mean±S.E.M. Differences among exper- tor, who frequently controlled the regularity and the success of injec- imental groups were determined by two- or three-way ANOVA tions using a methylene blue dye and who observed, after killing and followed by Newman–Keuls post hoc test. A value of Pb0.05 was con- frontal brain sectioning that the injection was successful in more than sidered to be significant. M. Moretti et al. / European Journal of Pharmacology 687 (2012) 21–27 23

3. Results way ANOVA revealed significant differences for ascorbic acid [F(1,24)= 6.46, P=0.01], charybdotoxin [F(1,24)=14.76, Pb0.01] and ascorbic 3.1. Effects of combined administration of sub-effective doses of the K+ acid×charybdotoxin interaction [F(1,24)=11.05, Pb0.01]. Post hoc anal- channel blockers and ascorbic acid in the TST and open-field test ysis indicated that the pre-treatment with a sub-effective dose of ascorbic acid produced a synergistic effect with charybdotoxin in the TST. Addi- Fig. 1 A shows the effect of the administration of sub-effective tionally, charybdotoxin administration alone or in combination with doses of ascorbic acid and TEA (a non specific blocker of K+ channels, ascorbic acid did not modify the locomotor activity of mice in the open- 25 pg/site) in the TST. The two-way ANOVA revealed significant dif- field (Fig. 2B), since two-way ANOVA revealed no significant differences ferences for ascorbic acid [F(1,24)=10.85, Pb0.01], TEA [F(1,24)= for ascorbic acid [F(1,23)=0.51, P=0.48], charybdotoxin [F(1,23)= 5.11, P=0.03] and ascorbic acid×TEA interaction [F(1,24)5.33, 0.83, P>0.26] and ascorbic acid×charybdotoxin interaction [F(1,23)= P=0.02]. Post hoc analysis indicated that the treatment with ascorbic 1.27, P=0.26]. acid produced a synergistic antidepressant-like effect with TEA. Re- The results presented in Fig. 2C show the effect of apamin (a small- garding locomotor activity, the two-way ANOVA revealed no differ- conductance calcium-activated K+ channel blocker, 10 pg/site, i.c.v.) in ences for ascorbic acid [F(1,25)=0.80, P=0.37], TEA [F(1,25)= producing a synergistic anti-immobility effect with a sub-effective dose 0.46, P=0.50], and ascorbic acid×TEA interaction [F(1,25)=2.39, of ascorbic acid in the TST. The two-way ANOVA revealed significant dif- P=0.13] (Fig. 1B). ferences for ascorbic acid [F(1,28)=6.94, P=0.01], apamin [F(1,28)= The results depicted in Fig. 1C show that glibenclamide (an ATP- 7.01, P=0.01] and ascorbic acid×apamin interaction [F(1,28)= sensitive K+ channel blocker, 0.5 pg/site) was also able to produce a 21.21, Pb0.01].As revealed by the post-hoc analysis, the administra- synergistic action with a sub-effective dose of ascorbic acid in the tion of apamin, administered at a dose that did not produce an TST. The two-way ANOVA revealed significant differences for antidepressant-like effect in the TST, produced a synergistic effect ascorbic acid [F(1,27) = 12.28, Pb0.01], glibenclamide [F(1,27) = when combined with a sub-effective dose of ascorbic acid in the 7.67, Pb0.01] and ascorbic acid×glibenclamide interaction [F(1,27)= TST. Additionally, apamin administration alone or in combination 11.71, Pb0.01]. Post hoc analysis indicated that the treatment with with ascorbic acid did not modify the locomotor activity in the ascorbic acid produced a synergistic antidepressant-like effect with open-field (Fig. 2D). The two-way ANOVA revealed no significant glibenclamide in the TST. In addition, the administration of glibenclamide differences for ascorbic acid [F(1,27)=2.90, P=0,09], apamin alone or in combination with ascorbic acid did not affect the locomotion [F(1,27)=2.77, P=0.10] and ascorbic acid×apamin interaction of mice (Fig. 1D). The two-way ANOVA revealed no differences for [F(1,27)=0.37, P=0.54]. ascorbic acid [F(1,27)=1.04, P=0.31], glibenclamide [F(1,27)=0.49, P=0.48] and ascorbic acid x glibenclamide interaction [F(1,27)=0.42, 3.2. Effects of K+ channel openers on ascorbic acid-induced anti- P=0.51] in the locomotor activity. immobility effects in the TST and in the number of crossings in the As presented in Fig. 2A, the administration of charybdotoxin (a large- open-field test and intermediate conductance calcium-activated K+ channel blocker, 25 pg/site, i.c.v.) produced a synergistic antidepressant-like effect when Fig. 3A shows that the treatment of mice with cromakalim (a K+ combined with a sub-effective dose of ascorbic acid in the TST. The two- channel opener, 10 μg/site, i.c.v.) was able to reverse the antidepressant-

Fig. 1. Effect of the treatment of mice with TEA (25 pg/site, i.c.v.) or glibenclamide (0.5 pg/site, i.c.v.) in combination with a sub-effective dose of ascorbic acid (0.1 mg/kg, p.o.) in the TST (panels A and C) and in the open-field test (panels B and D). Values are expressed as mean±S.E.M. (n=7–8). * Pb0.01 compared with the vehicle-treated control group. 24 M. Moretti et al. / European Journal of Pharmacology 687 (2012) 21–27

Fig. 2. Effect of the treatment of mice with charybdotoxin (25 pg/site, i.c.v.) or apamin (10 pg/site, i.c.v.) in combination with a sub-effective dose of ascorbic acid (0.1 mg/kg, p.o.) in the TST (panels A and C) and in the open-field test (panels B and D). Values are expressed as mean±S.E.M. (n=7–8). *Pb0.01 compared with the vehicle-treated control group.

Fig. 3. Effect of the treatment of mice with cromakalim (10 μg/site, i.c.v.) or minoxidil (10 μg/site, i.c.v.) on the ascorbic-induced (1 mg/kg, p.o.) antidepressant-like effect in the TST (panels A and C) and on locomotor activity in the open-field test (panels B and D). Values are expressed as mean±S.E.M. (n=7–8). *Pb0.01 compared with the vehicle-treated control group. #Pb0.05 compared with the same group pre-treated with vehicle. M. Moretti et al. / European Journal of Pharmacology 687 (2012) 21–27 25 like effect of ascorbic acid (1 mg/kg, p.o.) in the TST. The two-way ANOVA revealed significant differences for cromakalim [F(1,25)=6.50, P=0.01] and ascorbic acid×cromakalim interaction [F(1,25)=13.58, Pb0.01], but not a main effect of ascorbic acid [F(1,25)=1.31, P=0.26]. Post-hoc anal- ysis indicated that the treatment with cromakalim abolished the decrease in the immobility time produced by ascorbic acid in the TST. Regarding lo- comotor activity, as showed in the Fig. 3B, charybdotoxin administration alone or in combination with ascorbic acid did not produce any change in the ambulatory behavior of mice, since the two-way ANOVA did not show significant differences for ascorbic acid [F(1,26)=0.06, P=0,80], cromakalim [F(1,26)=0.11, P=0.73] and ascorbic acid×cromakalim in- teraction [F(1,26)=2,32, P=0.13]. Fig. 3C indicates that minoxidil (a K+ channel opener, 10 μg/site, i.c.v.) also reversed the antidepressant-like effect of ascorbic acid (1 mg/kg, p.o.) in the TST. The two-way ANOVA revealed significant differences for ascorbic acid [F(1,26)=5.61, P=0.02], minoxidil [F(1,26)=8.63, Pb0.01] and ascorbic acid×minoxidil interaction [F(1,26)=7.38, P=0.01]. Post-hoc analysis indicated that the treat- ment with minoxidil abolished the reduction in immobility time pro- duced by ascorbic acid in the TST. Fig. 3Dshowsthatminoxidil administration alone or in combination with ascorbic did not change the locomotor activity of mice, since the two-way ANOVA did not show significant differences for ascorbic acid [F(1,26)=0.42, P=0,52], cromakalim [F(1,26)=1.04, P=0.31] and ascorbic acid×cromakalim interaction [F(1,26)=3,42, P=0.07].

3.3. Effect of cromakalim on the synergistic antidepressant-like effect induced by the combined administration of ascorbic acid and 7- nitroindazole

As shown in Fig. 4A, the administration of 7-nitroindazole (25 mg/kg i.p., a specific neuronal NOS inhibitor) in combination with ascorbic acid (0.1 mg/kg, p.o.) produced an antidepressant-like effect Fig. 4. Effect of the treatment of mice with cromakalim (10 μg/site, i.c.v.) on the synergis- in the TST. Administration of cromakalim (a K+ channel opener, tic antidepressant-like effect induced by the combined administration of ascorbic acid 10 μg/site, i.c.v.) was able to abolish the synergistic antidepressant- (0.1 mg/kg, p.o.) and 7-nitroindazole (25 mg/kg, i.p.) in the TST (panel A) and on loco- motor activity in the open-field test (panel B). Values are expressed as mean±S.E.M. like effect elicited by the combined administration of ascorbic acid (n=7–8). *Pb0.01 compared with the vehicle-treated control group. #Pb0.05 com- and 7-nitroindazole. The three-way ANOVA showed significant differ- pared with the same group pre-treated with vehicle. ences for ascorbic acid×7-nitroindazole×cromakalim interaction [F(1,49)=22.07, Pb0.01]. Post-hoc analysis indicated that the results observed in the TST could be influenced by differences in loco- treatment with cromakalim blocked the decrease in immobility motor behavior. time in the TST produced by combined administration of ascorbic The precise molecular mechanisms responsible for the antidepres- acid and 7-nitroindazole. Moreover, ascorbic acid, 7-nitroindazole sant action of ascorbic acid are still not fully understood, however, our and cromakalim administration alone or in combination did not group has previously demonstrated that it has antidepressant proper- modify the locomotor activity in the open-field (Fig. 4B). The ties in mice, as evidenced by its ability to decrease the immobility three-way ANOVA revealed no significant differences for ascorbic time in the TST (Binfaré et al., 2009; Moretti et al., 2011) and to abol- acid×7-nitroindazole×cromakalim interaction [F(1,49)=0.87, ish the depressive-like behavior induced by chronic unpredictable P=0.35]. stress in mice (Moretti et al., 2012). Besides, it was demonstrated that the mechanism by which ascorbic acid exerts its anti- 4. Discussion immobility effect in the TST in mice involves an interaction with monoaminergic systems (Binfaré et al., 2009), and an inhibition of ei- In the present study, through the use of several pharmacological ther the L-arginine-NO pathway or NMDA receptors (Moretti et al., tools, we provide evidence that the modulation of K+ channels contrib- 2011). Noteworthy, these targets have been progressively reported utes to the antidepressant-like effect of ascorbic acid in the TST. Treat- to be implicated in the pathophysiology of depression (Elhwuegi, ment of mice with sub-effective doses of different blockers of K+ 2004; Harkin et al., 1999). In the present work, we extend our previ- channels was able to produce a synergistic antidepressant-like effect ous findings by indicating that the antidepressant-like effect observed with a sub-effective dose of ascorbic acid in the TST, whereas the treat- after an acute administration of ascorbic acid in the TST might be also ment of animals with cromakalim and minoxidil, pharmacological com- related to a regulation of neuronal excitability via modulation of dif- pounds able to open K+ channels, abolished the antidepressant-like ferent types of K+ channels. effect elicited by an effective dose of ascorbic acid. In addition, the ad- K+ channels can be classified by their mechanism of activation- ministration of cromakalim was able to abolish the synergistic inactivation. The K+ channel blockers glibenclamide, charybdo- antidepressant-like effect elicited by the combined administration of toxin and apamin are known to selectively block ATP-sensitive, ascorbic acid and 7-nitroindazole, an inhibitor of neuronal NOS activity. large- and intermediate-conductance calcium-activated and small- It is important to note that neither ascorbic acid nor K+ channel modu- conductance calcium-activated K+ channels, respectively (Gehlert lators and 7-nitroindazole, administered alone or in combination, affect- and Gackenheimer, 1993). Apamin, a peptide contained in the ed the spontaneous locomotion, ruling out the hypothesis that the venom of the honey bee Apis mellifera (Stocker, 2004), selectively 26 M. Moretti et al. / European Journal of Pharmacology 687 (2012) 21–27 blocks calcium-dependent K+ conductance (Hugues et al., 1982). ascorbic acid in the TST could be dependent on the activation of the se- TEA is classified as a non selective blocker of K+ channels, since rotonergic system. This hypothesis is in agreement with the data pres- it blocks different types of K+ channels in neurons including ented by Binfaré et al. (2009), which demonstrated that the + calcium-activated and voltage-dependent K channels (Cook and administration of NAN-190 (a 5-HT1A antagonist), ketanserin Quast, 1990; Halliwell, 1990). Previous studies have demonstrated (a 5-HT2A/2C ) and MDL72222 (a 5-HT3 receptor an- that the K+ channel functionally appears fundamental in the mod- tagonist) prevented the antidepressant-like effect of ascorbic acid in the ulation of the mouse immobility time in the forced swimming test, mouse TST. a widely used test to predict the antidepressant action of drugs in The involvement of the L-arginine-NO-cGMP pathway has been humans. In mice, an antidepressant-like effect was produced by extensively reported to be implicated in the pathophysiology of de- blocking the K+ channels, whereas a depressive-like effect was pression (Dhir and Kulkarni, 2001). Various drugs known to produce obtained by opening K+ channels (Galeotti et al., 1999; Kaster et an antidepressant-like action in the animal models of behavioral de- al., 2005). spair are recognized to modulate this signaling pathway (Brocardo Despite the fact that modulation of the K+ channels can have con- et al., 2008; Rosa et al., 2003; Zomkowski et al., 2010). Of note, several siderable implications on neural function, the actions of ascorbic acid studies have reported that NO and cGMP can activate different types on these channels have not been studied thoroughly. Ascorbic acid of K+ channels (Bolotina et al., 1994; S.Y. Jeong et al., 2001; Shin et al., has been previously shown to reverse the hydrogen peroxide- 1997), and the inhibition of these channels might represent an im- induced inhibition of inward K+ currents in Vicia guard cells (Zhang portant role in the mechanisms involved in depressive disorders et al., 2001). Moreover, ascorbic acid was reported to reduce the tran- (Fernandes et al., 2002; Inan et al., 2004; Kaster et al., 2005). An inter- sient and sustained K+ currents in Drosophila neurons, which could esting finding of the present study is that the administration of increase neuronal excitability (Alshuaib and Mathew, 2006). In the cromakalim (a K+ channel opener, 10 μg/site, i.c.v.) was able to abol- present study, an important role played by ATP-sensitive and ish the synergistic antidepressant-like effect elicited by the combined calcium-activated K+ channels in the antidepressant-like effect of administration of ascorbic acid and 7-nitroindazole, an inhibitor of ascorbic acid in the TST was shown. This conclusion is derived from neuronal NOS activity. This result suggests that an indirect blockade + the fact that the administration of sub-effective doses of TEA, apamin, of the K channels by ascorbic acid via the NMDA-L-arginine-NO charybdotoxin, and glibenclamide in combination with a sub-effective pathway could account for the behavioral results observed in the dose of ascorbic acid provoked a robust reduction in the immobility present study. In view of these and previous results found by our time, indicative of an antidepressant-like behavioral profile. Altogether, group indicating that the antidepressant-like effect of ascorbic acid these results suggest that the blockade of ATP-sensitive and calcium- is mediated by inhibition of NMDA receptors and NO synthesis activated K+ channels contributes to the antidepressant-like effect of (Moretti et al., 2011), we can suggest that the blockade of K+ chan- this vitamin in the TST. nels may be a consequence of the inhibition of NMDA receptors and Rosati et al. (1998) showed that glibenclamide, an ATP-sensitive NO production induced by ascorbic acid. However, the possibility K+ channel inhibitor, blocks neural delayed rectifier K+ currents. that ascorbic acid also causes a direct inhibition of these channels On the other hand, Ca+-dependent K+ conductance plays a major cannot be ruled out. role in regulating the frequency of repetitive neuronal discharges Further reinforcing the assumption that the antidepressant-like and accelerating spike repolarization (Szente et al., 1988). Charyb- effect of ascorbic acid involves the inhibition of K+ channels, treat- dotoxin blocks K+ movement by occluding the pore of the Ca2+ acti- ment of mice with the K+ channel openers, cromakalim and minoxi- vated K+ channel, significantly prolonging the duration of the action dil abolished the decrease in the immobility time (antidepressant-like potential. Further, while there is less efflux of K+ the membrane po- effect) induced by an effective dose of ascorbic acid in the TST. These tential rises, and hence the likelihood of initiating an results support the involvement of K+ channels in the antidepressant- increases, causing hyperexcitability of the nervous system. It was like effect of ascorbic acid in mice TST and are in accordance with stud- demonstrated that TEA increases the ventricular threshold potential, ies showing that channel openers antagonize the anti-immobility effect prolongs the action potential and at high concentration (3 mM) in- of antidepressants such as imipramine, amitriptyline, desipramine, creases its amplitude (Doggrell and Bishop, 1996), effects that could paroxetine and venfalaxine in the FST (Bortolatto et al., 2010; Redrobe be due to the blocking of inward-rectifying or the delayed outward- et al., 1996). rectifying K+ channels (Cook and Quast, 1990). In light of these con- siderations and taking into account that K+ channel blockers may in- 5. Conclusion crease the excitability of central circuits, it is plausible to propose that the potentiation of the antidepressant-like effect of ascorbic acid in- The present study demonstrates that the pretreatment of mice duced by K+ channel blockers (TEA, apamin, charybdotoxin, and with different K+ channel blockers produces an antidepressant-like glibenclamide) observed in the present study may be due to an inhi- action with a sub-effective dose of ascorbic acid, whereas the treat- bition of the hyperpolarization elicited by this vitamin, thus leading ment with K+ channel openers abolished the antidepressant-like ef- to an increased excitatory response. However, it remains to be fect produced by an effective dose of this vitamin. Given that the established if ascorbic acid shares with the inhib- pharmacological modulation of these channels may be a putative itors the same mechanisms mentioned above. strategy in controlling central nervous system disorders, including It was reported that the stimulation of 5-hydroxytryptamine (5-HT) depression, and considering our results indicating that the modulato- receptors can affect several types of K+ channels in central neurons ry effects of ascorbic acid on neuronal excitability, via inhibition of K+ (Bobker and Williams, 1990) and, consistent with this notion, there is channels, we can suggest that this vitamin can be a promising agent substantial evidence indicating an association between K+ channels to assist in the treatment of depression. However, additional studies and the mechanism of action of antidepressants, especially those relat- aiming at investigating other mechanisms by which ascorbic acid in- ed to the 5-HT system, including fluoxetine, and venfalaxine duces antidepressant-like effects are essential. (Bortolatto et al., 2010; Choi et al., 2004; Inan et al., 2004). Recent find- ings indicate that these antidepressant drugs are potent inhibitors of K+ Disclosure/Conflict of interest channels, affecting the excitability of neurons (Choi et al., 2004). Moreover, it has been suggested that the blockade of K+ channels en- The authors declare that no financial support or compensation has hances the basal release of 5-HT in rat hippocampal slices (Schechter, been received from any individual or corporate entity over the past 1997). Hence, the synergistic action of the K+ channel blockers and three years for research or professional service and there are no M. 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