Antidepressant-like responses in the forced swimming test elicited by glutathione and redox modulation

Behavioural Brain Research 253 (2013) 165–172

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Antidepressant-like responses in the forced swimming test elicited by glutathione and redox modulation Juliana M. Rosa 1 , Alcir Luiz Dafre ∗ , Ana Lúcia S. Rodrigues Department of Biochemistry, Biological Sciences Centre, Federal University of Santa Catarina, 88040-900 Florianópolis, SC, Brazil

h i g h l i g h t s • • • •

GSH induces antidepressant-like effect in mice. Acivicin, a ␥-GT inhibitor, abolishes antidepressant-like effect of GSH. Thiol/disulfide reagents modulate antidepressant-like effect of GSH. Extracellular redox milieu participates in the antidepressant-like effect of GSH.

a r t i c l e

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Article history: Received 20 April 2013 Received in revised form 4 July 2013 Accepted 7 July 2013 Available online 11 July 2013 Keywords: Antidepressant Glutathione Forced swimming test Redox regulation Gamma-glutamyl transpeptidase Depressive disorders

a b s t r a c t Glutathione (GSH) displays a broad range of functions, among them a role as a neuromodulator with some neuroprotective properties. Taking into account that oxidative stress has been associated with depressive disorders, this study investigated the possibility that GSH, a major cell antioxidant, elicits an antidepressant-like effect in mice. Thus, GSH was administered by i.c.v. route to mice that were tested in the forced swimming test and in the tail suspension test, two predictive tests for antidepressant drug activity. In addition, GSH metabolism and the redox environment were modulated in order to study the possible mechanisms underlying the effects of GSH in the forced swimming test. The administration of GSH decreased the immobility time in the forced swimming test (300–3000 nmol/site) and tail suspension test (100–1000 nmol/site), consistent with an antidepressant-like effect. GSH depletion elicited by l-buthionine sulfoximine (3.2 ␮mol/site, i.c.v.) did not alter the antidepressant-like effect of GSH, whereas the inhibition of extracellular GSH catabolism by acivicin (100 nmol/site, i.c.v.) prevented the antidepressant-like effect of GSH. Moreover, a sub-effective dose (0.01 nmol/site, i.c.v.) of the oxidizing agent DTNB (5,5 -dithiobis(2-nitrobenzoic acid)) potentiated the effect of GSH (100 nmol/site, i.c.v.), while the pretreatment (25–100 mg/kg, i.p.) with the reducing agent DTT (dl-dithiothreitol) prevented the antidepressant-like effect of GSH (300 nmol/site, i.c.v.). DTNB (0.1 nmol/site, i.c.v.), produced an antidepressant-like effect, per se, which was abolished by DTT (25 mg/kg, i.p.). The results show, for the first time, that centrally administered GSH produces an antidepressant-like effect in mice, which can be modulated by the GSH metabolism and the thiol/disulfide reagents. The redox environment may constitute a new venue for future antidepressant-drug development. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The tripeptide ␥-glutamyl-l-cysteinyl-glycine, or glutathione (GSH), is a ubiquitous antioxidant thiol, which plays a major role in maintaining intracellular reduction–oxidation (redox) balance.

Abbreviations: BSO, l-buthionine-[S,R]-sulfoximine; DTNB, 5,5 -dithiobis(2nitrobenzoic acid); DTT, dl-dithiothreitol; FST, forced swimming test; GCL, glutamate cysteine ligase; ␥-GT, ␥-glutamyl transpeptidase; GSH-t, total glutathione (GSH plus GSSG in GSH-equivalents); GPx, glutathione peroxidase; GR, glutathione reductase; GST, glutathione-S-transferase; TST, tail suspension test. ∗ Corresponding author. Tel.: +55 48 37212817; fax: +55 48 37212827. E-mail address: [email protected] (A.L. Dafre). 1 Present address: Neurobiology Division, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK. 0166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2013.07.009

It is synthesized in the brain by both neurons and glial cells, being more abundant within astrocytes [1,2]. Besides its role as an antioxidant, GSH is essential for cell proliferation, modulation of signal transduction, immune response and regulation of apoptosis [2–6]. GSH is synthesized in the cell by the consecutive action of glutamate cysteine ligase (GCL) and glutathione synthetase. GCL, the rate limiting enzyme in GSH synthesis, uses glutamate and cysteine as substrates to form the dipeptide ␥-glutamylcysteine, whereas glutathione synthetase catalyzes the synthesis of GSH by adding glycine to ␥-glutamylcysteine [7]. The intracellular synthesis of GSH can be inhibited in vivo by buthionine sulfoximine (BSO), a specific GCL inhibitor [8]. Extracellular GSH is a substrate for the ectoenzyme ␥-glutamyl transpeptidase (␥-GT), which catalyzes the transfer of the ␥glutamyl moiety from GSH to an acceptor molecule, thereby

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generating the dipeptide Cys-Gly. The dipeptide Cys-Gly can be further hydrolyzed by ectopeptidases to cysteine and glycine, which are subsequently taken up by cells to serve again as substrates for de novo cellular GSH synthesis [2,9,10]. The degradation of extracellular GSH can be inhibited by acivicin, an irreversible inhibitor of ␥-GT that has been used to study the role of ␥-GT in GSH homeostasis [11]. The role of GSH as an important antioxidant agent in the central nervous system has been indicated by several studies. A decrease in the tissue levels of GSH has been associated with aging and the pathogenesis of neurodegenerative diseases, including amyotrophic lateral sclerosis, Alzheimer’s disease and Parkinson’s disease [12,13]. Moreover, the levels of GSH are also decreased in the cerebrospinal fluid and prefrontal cortex of schizophrenic patients [14,15]. In line with these findings, GSH was effective against pentylenetetrazol-induced convulsions [16]. Thus, besides its antioxidant function GSH displays a neuroprotective role in the brain [17]. Several substances that exhibit neuroprotective effects have been reported to produce antidepressant effects in animal models predictive of antidepressant action and/or in clinical studies [18,19]. Considering that major depressive disorder has recently been linked to impairments in signaling pathways that regulate neuroplasticity and cell survival [20,21], the neuroprotective role of these antioxidant agents can be of pharmacological significance for the modulation of depression [22–26]. The development of an animal model of GSH deficiency, by knocking out GCL, the rate limiting enzyme in GSH synthesis, brought new insights on the importance of GSH in mood disorders [9,27]. Chronic GSH deficiency induces hyperlocomotion and higher responses to amphetamine, indicating that GSH may be an important component of bipolar disorders [9,27]. Thus, besides its antioxidant function, GSH appears also to be strongly linked with mood-related behavior. In line with this idea, the enzyme glyoxalase 1, the main pathway for disposal of the toxic aldehyde methylglyoxal [28], has been liked to anxiety-like behavior in rodents, which appears to present comorbidity with depressivelike behavior [29,30]. Since detoxification of methylglyoxal by glyoxalase 1 depends on GSH, its direct participation in these behavioral findings may not be excluded. The present study was designed to investigate whether GSH, an endogenous and neuroprotective antioxidant, produces an antidepressant-like effect in the forced swimming test (FST) and in the tail suspension test (TST), two animal models predictive for antidepressant drug activity [31,32]. Moreover, to give some insight into the possible mechanisms underlying the effect of GSH in the FST, we investigated the interference of GSH metabolism and thiol/disulfide reagents. 2. Materials and methods 2.1. Animals Swiss mice, either sex, weighing 30–40 g (55–60 days-old), were maintained at 21 ± 1 ◦ C with free access to water and food, under a 12:12 h light/dark cycle (light onset at 07.00 h). All manipulations were carried out between 09.00 h and 17.00 h, with each animal used only once. The experiments were performed after approval of the protocol by the Ethics Committee of the Institution and all efforts were made to minimize animal suffering, which are in conformity to the Guide for the Care and Use of Laboratory Animals, National Institutes of Health. 2.2. Drugs and treatment The following chemicals were used: acivicin ((␣S,5S)-␣-amino-3-chloro4,5-dihydro-5-isoxazoleacetic acid); l-buthionine-[S,R]-sulfoximine (BSO); 1chloro-2,4-dinitrobenzene; dl-dithiothreitol (DTT); 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB), reduced glutathione (GSH), bovine serum albumin; glutathione reductase (GR); nicotinamide adenine dinucleotide phosphate (reduced form, NADPH); oxidized glutathione (GSSG); tert-butyl hydroperoxide, perchloric acid; 1-chloro-2,4-dinitrobenzene; 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB) and

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (from Sigma Chemical Co., St. Louis, MO, USA). Drugs were administered by intracerebroventricularly (i.c.v.) in a constant volume of 5 ␮l/site, over 30 s, except for DTT that was administered intraperitoneally (i.p.) in a constant volume of 10 ml/kg body weight. i.c.v. injections were given under light ether anesthesia, directly into the lateral ventricle with the bregma fissure as a reference. GSH and BSO were dissolved in phosphate-buffered saline (PBS) 0.75 M, pH 7.4. All other drugs were dissolved in saline. Appropriate vehicle-treated groups were assessed simultaneously. A microsyringe (25 ␮l, Hamilton) was used to perform the i.c.v. administration of drugs. Previous to drug injection, a 26-gauge stainless-steel needle that was inserted perpendicularly 2 mm deep through the skull, following previously published methodology by Laursen and Belknap [33]. Briefly, mice were gently restrained by hand after being lightly anesthetized with ether (i.e. just that necessary for loss of the postural reflex). The sterilization of the injection site was carried out using gauze embedded in 70% ethanol. Under light anesthesia, the needle was inserted unilaterally 1 mm to the midline point equidistant from each eye, at an equal distance between the eyes and the ears and perpendicular to the plane of the skull. Drugs were injected directly into the lateral ventricle using published coordinates taken from the atlas of Franklin and Paxinos [34], taking the bregma as a reference: anterioposterior −0.1 mm; mediolateral 1 mm; and dorsoventral −3 mm. A volume of 5 ␮l was used, based on literature data, and delivered in 30 s as previously employed [35]. Mice exhibited normal behavior within 1 min after injection.

2.3. Behavioral tests The FST was carried out based on the method previously described by Porsolt et al. [31] with minor modifications. Briefly, mice were dropped individually into cylinders (height 25 cm, diameter 10 cm) containing 19 cm of water, maintained at 25 ± 1 ◦ C, and left there for 6 min. In such a situation, from which they cannot escape, animals rapidly became immobile, that is, floating in an upright position and making only small movements to keep their heads above water. The total duration of immobility was scored. The total duration of immobility induced by tail suspension was measured according to the method of Steru et al. [32]. 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. The ambulatory behavior was assessed in an open-field test as described previously [36]. The apparatus consisted of a wooden box measuring 40 cm × 60 cm × 50 cm. The floor of the arena was divided into 12 equal squares. The number of squares crossed with all paws (crossings) was counted in a 6-min session. Mice received an i.c.v. injection of GSH (dose range 100–3000 nmol/site) and the open-field test was performed after 20 min.

2.4. Pharmacological treatment To investigate the antidepressant-like effect of GSH animals received an i.c.v. injection of GSH 20 min before the FST (dose range 100–3000 nmol/site) and TST (dose range 10–1000 nmol/site). This dose-range can be taken as of pharmacological nature, considerably surpassing the physiological levels of GSH. To investigate the influence of GSH metabolism on the antidepressant-like effect of GSH, mice were pretreated with BSO (3.2 ␮mol/site, i.c.v., an inhibitor of the intracellular synthesis of GSH) or acivicin (100 nmol/site, i.c.v., an inhibitor of the degradation of extracellular GSH) and 24 h or 20 min later, respectively, animals received GSH (300 nmol/site, i.c.v., an active dose in the FST) and were tested in the FST after 20 min. In a separate series of experiments, we also investigated the possible influence of thiol/disulfide reagents in the anti-immobility effect of GSH. To this end, mice received a sub-effective dose of DTNB (0.01 nmol/site, i.c.v.), an oxidizing agent, 20 min before administration of GSH (100 nmol/site, i.c.v., a sub-active dose in the FST) and were tested in the FST 20 min later. We also tested the influence of DTNB (10 nmol/site, i.c.v., a dose that produced no effect in the FST) on the antidepressantlike effect of GSH (300 nmol/site, i.c.v., an active dose in the FST). To this end, mice were pretreated with DTNB 20 min before GSH administration. FST was carried out after 20 min. The doses of DTNB were chosen from a dose–response curve (0.01–10 nmol/site, i.c.v.) in the FST, in which this oxidizing agent was administered 20 min before the FST. The effect of DTT, a thiol reagent, on the antidepressant-like effect of GSH was also investigated. The animals were pretreated with DTT (25–100 mg/kg, i.p., doses that produced no effect in the FST) and after 20 min they received an i.c.v. injection of GSH (300 nmol/site). A further 20 min elapsed before the animals were tested in the FST. In order to investigate whether DTT would be able to prevent the antiimmobility effect of DTNB (0.1 nmol/site, i.c.v.), mice were pretreated with DTT (25 mg/kg, i.p.). Twenty min later DTNB was injected and the FST was carried out after a further 20 min.

J.M. Rosa et al. / Behavioural Brain Research 253 (2013) 165–172

Immobility time (sec)

A

Table 1 Effect of GSH administration on the number of crossings in the open-field test.

300

Treatmenta (nmol/site)

Numbers of crossings (mean ± SEM (n))

200

Vehicle GSH 100 GSH 300 GSH 3000

103.7 99.8 96.0 96.7

*** *** ***

Vehicle 100

300

1000

3000

GSH (nmol/site, i.c.v.)

B

6.1 (9) 10.9 (5) 9.1 (5) 16.3 (4)

In the TST, one-way ANOVA showed a significant effect of treatment [F(5,25) = 12.0, p < 0.001]. GSH at a dose range of 100–1000 nmol/site, i.c.v. significantly decreased the immobility time in comparison to the vehicle-treated group. Conversely, when administered at doses of 10 and 3000 nmol/site, GSH produced no effect on the immobility time in the TST (Fig. 1B). 3.2. Effect of GSH on ambulatory behavior

300

*** **

200

**

100 0

± ± ± ±

a Animals were treated with GSH (300 nmol/site, i.c.v.) or vehicle 20 min before the test.

100 0

Immobility time (sec)

167

GSH administered by i.c.v. route, at doses that produce an antidepressant-like effect in the FST (100, 300 and 3000 nmol/site, i.c.v.), did not cause significant changes in the ambulation in the open-field (p > 0.05, NS; Table 1). 3.3. Effect of BSO and acivicin on the antidepressant-like action of GSH

Vehicle 10

100

300

1000 3000

GSH (nmol/site, i.c.v.) Fig. 1. (A) Effect of administration of GSH on the immobility time in FST and (B) TST in mice. GSH was administered 20 min before the test. Values are expressed as mean + SEM (n = 5–11). **p < 0.01 and ***p < 0.001 as compared with the vehicletreated group.

Fig. 2A shows that the pretreatment of animals with BSO (3.2 ␮mol/site, 24 h before) did not significantly affect (p > 0.05, NS) the antidepressant-like effect of GSH (300 nmol/site) in the FST. In addition, acivicin (100 nmol/site) abolished the anti-immobility effect elicited by GSH (300 nmol/site) in the FST (main effect of pretreatment F(1,27) = 32.4, p < 0.001, treatment F(1,27) = 23.4, p < 0.001, pretreatment vs. treatment interaction F(1,27) = 35.1, p < 0.001) (Fig. 2B).

2.5. Biochemical analysis

3.4. Total glutathione content and antioxidant enzyme activity

Total glutathione (GSH-t) content, comprising the sum of oxidized plus reduced glutathione, was determined using the DTNB-GR recycling assay [37], as modified previously [38]. Immediately after behavioral testing, animals were decapitated and their brains removed. The hippocampus was homogenized in 0.5 M perchloric acid. The homogenates were centrifuged at 15,000 × g for 2 min at 4 ◦ C and the supernatant, after neutralization, assayed for GSH-t. Glutathione peroxidase (GPx) [39], glutathione-S-transferase (GST) [40] and GR [41] activity were measured by in brain homogenates by colorimetric standard methods and standardized to protein content. Protein content was evaluated by Coomassie Brilliant Blue method [42], using bovine serum albumin as standard.

Fig. 3A shows the hippocampal GSH-t concentration after pretreatment with BSO (3.2 ␮mol/site). Twenty-four hours after the administration of BSO, a 35% decrease in the GSH levels was observed, as compared to control animals (main effect of pretreatment F(1,31) = 31.7, p < 0.001). Since GSH (300 nmol/site, i.c.v.) was administered 20 min before measurement, it may alter GSH-t levels in the hippocampus. However, no significant alterations in GSHt levels were detected. This injection was not able to change the BSO-induced decrease in GSH-t levels (p > 0.05, NS). Fig. 3B shows that the administration of acivicin, alone or in combination with GSH, had no effect on the GSH-t concentration in the hippocampus (p > 0.05, NS). No changes were observed in the antioxidant enzyme activities (GR, GPx and GST) 60 min after the administration of GSH (p > 0.05, NS), as shown in Table 2.

2.6. Statistical analysis Comparisons between experimental and control groups were performed by one-way analysis of variance (ANOVA) (dose–response curves) or two-way ANOVA (interaction studies) followed by Tukey’s HSD post hoc test when appropriate. A value of p < 0.05 was considered to be significant.

3. Results 3.1. Effect of GSH in the FST and TST The immobility time in the FST in mice administered with GSH by i.c.v. route is shown in Fig. 1A. One-way ANOVA showed a significant effect of treatment [F(4,34) = 20.5, p < 0.001]. Post hoc analyses showed that GSH at doses of 300, 1000 and 3000 nmol/site, significantly decreased the immobility time in the FST as compared to vehicle-treated controls.

Table 2 Effect of GSH administration (300 nmol/site, i.c.v.) on GSH-related enzymes in the hippocampus of mice. Treatment

GRa

GPx

GST

Vehicle GSH

28.5 ± 1.7 30.1 ± 1.8

13.8 ± 0.1 16.3 ± 1.1

40.8 ± 1.6 42.9 ± 1.9

a Enzymatic activities in nmol/min/mg of protein (n = 12–13) were analyzed 1 h after GSH administration (300 nmol/site, i.c.v.). Values are expressed as mean ± SEM. GR, glutathione reductase; GPx, glutathione peroxidase; GST, glutathione-Stransferase.

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A 300

*** ***

200 100

GSH (µmol/g tissue)

Immobility time (sec)

A

##

***

1

+ -

+ +

+ + -

Vehicle GSH BSO

+ +

+ -

+ +

+ + -

+ +

+ -

+ +

+ + -

+ +

B 300

###

200

***

100

GSH (µmol/g tissue)

B Immobility time (sec)

2

0

0

Vehicle GSH BSO

3

2 1 0

0

+ -

+ +

+ + -

Vehicle GSH Acivicin

+ +

Fig. 2. Effect of the pretreatment of mice with (A) BSO (1.8 ␮mol/site, i.c.v.) or (B) acivicin (100 nmol/site, i.c.v.) on the GSH (300 nmol/site, i.c.v.)-induced reductions in immobility time in the FST in mice. Values are expressed as mean + SEM (n = 6–12). ***p < 0.001 compared with the vehicle-treated group; ### p < 0.001 when compared with the same group pretreated with GSH.

3.5. Effect of DTNB and DTT on the antidepressant-like action of GSH The effect of DTNB treatment on the immobility time in the FST is presented in Fig. 4. Results demonstrate that DTNB per se, given by i.c.v. route, at a dose of 0.1 nmol/site, reduced the immobility time in the FST (F(4,30) = 17.5, p < 0.001). The active dose of DTNB (0.1 nmol/site) did not cause any significant change in the locomotor activity in the open-field test (crossings: control animals = 100.2 ± 6.6 and DTNB-treated animals = 99.2 ± 6.1; n = 4; p > 0.05, NS). Fig. 5A shows the influence of pretreatment with a sub-effective dose of GSH (100 nmol/site, i.c.v.) given in combination with a subeffective dose of DTNB (0.01 nmol/site, i.c.v.) on the immobility time in the FST. A two-way ANOVA revealed a main effect of DTNB pretreatment (F(1,17) = 4.1, p > 0.05, NS); GSH treatment (F(1,17) = 27.2, p < .001); pretreatment vs. treatment interaction (F(1,17) = 11.6, p < 0.01). The antidepressant-like effect was only observed with the combined administration of GSH and DTNB. In addition, DTNB at 10 nmol/site, a dose that produced no effect in the FST (F(1,34) = 0.84, p > 0.05), was unable to abolish the anti-immobility effect elicited by GSH in the FST (main effect of treatment F(1,34) = 56.8, p < 0.001) (Fig. 5B). The results depicted in Fig. 6A show that the pretreatment of mice with DTT abolished the anti-immobility effect elicited by GSH in the FST (main effect of DTT pretreatment F(3,40) = 5.39, p < 0.01;

Fig. 3. Effect of the pretreatment of mice with (A) BSO (1.8 ␮mol/site, i.c.v.) or (B) acivicin (100 nmol/site, i.c.v.) alone or in combination with GSH (300 nmol/site, i.c.v.) on the hippocampal GSH-t levels in mice. Values are expressed as mean + SEM (n = 5–13). ***p < 0.001 as compared with the vehicle-treated group and ## p < 0.01 as compared to vehicle/GSH-treated animals.

GSH treatment F(1,40) = 5.30, p < 0.05; pretreatment vs. treatment interaction F(3,40) = 7.60, p < 0.001). Moreover, the pretreatment with DTT (Fig. 6B) also prevented the anti-immobility effect caused by DTNB in the FST (main effect of DTT pretreatment F(1,20) = 15.9, p < 0.001; DTNB treatment F(1,20) = 11.3, p < 0.01; pretreatment vs. treatment interaction F(1,20) = 19.5, p < 0.001).

Immobolity time (sec)

Vehicle GSH Acivicin

3

300 200

***

100 0

Vehicle 0.01 0.1 1 10 DTNB (nmol/site, i.c.v.)

Fig. 4. Effect of administration of DTNB on the immobility time in the FST in mice. DTNB was administered by i.c.v. route 20 min before the test. Values are expressed as mean + SEM (n = 5–12). ***p < 0.001 as compared with the vehicle-treated group.

J.M. Rosa et al. / Behavioural Brain Research 253 (2013) 165–172

300

***

200 100 0

Vehicle GSH DTNB

+ -

+ +

+ + -

+ +

Immobility time (sec)

B 300

*** ***

200 100 0

Vehicle GSH DTNB

+ -

+ +

+ + -

+ +

Fig. 5. Effect of the pretreatment of mice with DTNB on the anti-immobility effect of GSH. (A) DTNB (0.01 nmol/site, i.c.v.) potentiated the action of a sub-effective dose of GSH (100 nmol/site, i.c.v.) in the FST. (B) DTNB (10 nmol/site, i.c.v.) produced no effect on the GSH (300 nmol/site, i.c.v.)-induced reduction in immobility time in the FST in mice. Values are expressed as mean + SEM (n = 5–9). ***p < 0.001 compared with the vehicle-treated group.

4. Discussion The results show, for the first time, that GSH given centrally is effective in producing an antidepressant-like effect, when assessed in the FST, a test widely used to screen antidepressant properties of drugs [43]. The antidepressant-like action of GSH was confirmed in a second test, the TST.

Immobility time (sec)

A

The FST displays high sensitivity in detecting the action of the major classes of antidepressant drugs, including noradrenaline, reuptake inhibitors, selective serotonin reuptake inhibitors and monoamine oxidase inhibitors [31,43]. The TST, related to the FST, is used as a predictive tests for the action of antidepressant drugs [32,44]. Both tests are based on the principle that mice, exposed to an aversive situation from which there is no escape, will, after periods of agitation, cease attempting to escape. Antidepressant drugs are reported to reduce the immobility time of mice in both tests [31,32,43]. The anti-immobility effect of GSH is not associated with any motor effects, since, at doses similar to those which had a marked antidepressant-like action, locomotor activity was not affected. This indicates that a psychostimulant effect was not responsible for the decrease in the immobility time elicited by GSH. Hence, our results provide convincing evidence that GSH is able to produce a specific antidepressant-like effect in two animal models predictive of antidepressant activity. Despite the fact our experimental protocol used supraphysiological doses of GSH (i.c.v.), some preclinical and clinical findings have suggested that GSH may be involved in psychiatric diseases and stressful conditions. It was shown that repeated stress, including immobilization and cold exposure, decreased GSH content in rodent brain [45,46], a result that may be related to a neuroinflammation response to stress [47]. Furthermore, the reduction in GSH levels in the cerebral cortex of mice elicited by inescapable foot shock stress was recovered by treatment with antidepressant drugs, such as imipramine (tricyclic antidepressant), maprotiline (selective noradrenaline uptake inhibitor) and fluvoxamine (selective serotonin uptake inhibitor) [48]. A clinical study demonstrated that blood levels of GSH are within or below the lower normal range in untreated patients with bipolar disorder [49]. Related to these findings, chronic treatment with the antimanic agents lithium and valproate, at their therapeutically relevant concentrations, protected primary cultured rat cerebral cortical cells against glutamate-induced oxidative stress [50]. GSH has been pointed as an important neuroprotective agent in the effects of lithium and valproate [50]. Furthermore, glyoxalase I, an enzyme involved in the GSH metabolism, is associated with anxiety and depressive behavior in lineages selected for low and high anxiety behavior [51,52], as well as in a genomic study [53]. More recently, association between anxiety and the glyoxalase substrate methylglyoxal has been demonstrated [30]. Anxiety traits are associated with depressive-like symptoms [54]. Therefore, a growing number of evidence associates GSH metabolism to mood-related behavior highlighting the importance of studying its antidepressant potential. However, it should be noted that the effective GSH i.c.v. dose

B ###

300

###

###

***

200

100

0

Vehicle GSH DTT 25 DTT 50 DTT 100

+ -

+ + -

+ + -

+ +

+ + -

+ + -

+ + -

+ +

Immobility time (sec)

Immobility time (sec)

A

169

300

###

***

200 100 0

Vehicle DTNB DTT

+ -

+ +

+ + -

+ +

Fig. 6. Effect of pretreatment of mice with DTT (25–100 mg/kg, i.p.) on the anti-immobility effect of GSH. (A) GSH (300 nmol/site, i.c.v.)-induced reduction in immobility time in the FST in mice was reversed by DTT. (B) DTT (25 mg/kg, i.p.) reversed the DTNB (0.1 nmol/site, i.c.v.)-induced reduction in immobility time in the FST in mice. Values are expressed as mean + SEM (n = 6). **p < 0.001 compared with the vehicle-treated group; ### p < 0.001 when compared with the same group pretreated with GSH (A) or DTNB (B).

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is far from being physiological and our data should be read with caution as to translate the physiopathology of depression. It is also worth of mention that our data indicate that GSH display an oxidizing effect, possibly due to extracellular metabolism. If this effect is related to the pharmacological dose used, or to a physiological mechanism, remains to be determined. The antidepressant-like effect of GSH was not accompanied by alterations in the total GSH levels in the hippocampus and in the activity of the GSH-related enzymes GR, GPx, and GST. Precursors of GSH, displaying high membrane permeability, such as GSH-esters and NAC, are able to increase GSH levels in the brain and being a promising drug candidate for a series of diseases [55]. Nevertheless, the slow turnover rate of GSH and the need for extracellular metabolism [1,2] limits the ability of administered GSH to increase its intracellular levels. In the present study, a decrease in the levels of total GSH in the hippocampus was observed after the treatment of animals with BSO, which inhibits GCL, the rate limiting enzyme in GSH synthesis [8,56,57]. However, in spite of the decrease in the levels of GSH, BSO treatment was unable to change the antidepressant-like effect elicited by GSH in the FST. This indicates that a reduction in the intracellular levels of GSH did not prevent its antidepressant-like effect. This finding raises the possibility that the antidepressantlike effect elicited by the administration of GSH occurs through its interaction with the extracellular compartment. In line with this hypothesis, an extracellular effect of GSH was proposed in a study that reported the anticonvulsant action of GSH against pentylenetetrazol [16]. Another approach used to study the influence of GSH metabolism on the antidepressant-like effect of GSH in the FST was to test the influence of ␥-GT inhibition by administering acivicin. ␥-GT catalyses the first step in the catabolism of extracellular GSH, by cleaving the ␥-glutamyl bond between glutamate and cysteine. Consequently, ␥-GT releases the dipeptide cysteinyl-glycine, which is subsequently cleaved to cysteine and glycine by plasma membrane dipeptidase activities [8]. Moreover, the action of ␥-GT also generates ␥-glutamyl dipeptides that have been proposed as being potentially important in neurotransmission by interacting with excitatory amino acid receptors [58], mainly glutamate receptors [59]. A role for GSH metabolism through the action of ␥-GT was also associated to the hypoxia response [60], which is a possible example of ␥-GT-dependent signaling in the central nervous system. In this regard, GSH is postulated to play a role in neuronal excitability as a neuromodulator [59]. In our study, the pretreatment of mice with acivicin was able to block the antidepressant-like effect of GSH, suggesting that the GSH effect in the FST is dependent on its catabolism. Thus, one plausible possibility is that the released ␥glutamyl residues or dipeptides are the effectors of the GSH action in the FST. Some studies have indicated the possibility that GSH or a product of its extracellular metabolism interacts with receptor redox sites [61–63]. Redox modulation of receptors, such as GABA receptors [27,64], has been presumed to rely on the oxidation/reduction state of reactive cysteines. Disulfide bonds, involving cysteine residues, are implicated in redox modulation, which can be modified by chemical reduction with agents such as DTT. Conversely, oxidizing agents that promote disulfide bond formation, such as DTNB, can interfere with the thiol moiety of cysteine residues [60,64–66]. Considering that GSH may alter the function of various proteins via redox modulation [60,64,67], we investigated whether the antidepressant-like effect of GSH could be affected by thioldisulfide reagents. DTNB, a compound that is able to oxidize redox sites, produced per se a reduction in the immobility time in the FST at a dose that did not cause any alteration in the locomotor activity. The mechanism of action by which DTNB produces its

antidepressant-like effect was not studied, but this finding suggests that the oxidation of sensitive receptor redox sites produces an antidepressant-like effect in the FST. Moreover, a sub-effective dose of DTNB produced a synergistic antidepressant-like effect with a sub-effective dose of GSH. However, the pretreatment of mice with DTNB, at a dose that produced no effect per se in the FST, did not prevent the anti-immobility effect of GSH. Considering that GSH and DTNB produced a synergistic antidepressant-like effect in the FST, we may speculate that GSH produces its effect in this behavioral test by an oxidant-dependent mechanism at critical redox sites. In line with these findings, the pro-oxidative action of ␥-GT has been proposed as an important regulatory mechanism [61–63], as well as the ␥-GT product, cysteinyl-glycine [16]. The metabolites generated by GSH catabolism can lead to oxidative modifications on a variety of molecular targets, involving oxidation and/or S-thiolation of protein thiol groups. These modifications can act on redox-sensitive targets in the cell surface receptors [61,62,68–72]. The effect of the redox state on the GSH antidepressant-like action was also investigated using the reducing agent DTT. Our results further demonstrated that the modulation of redox sites has a role in the GSH antidepressant-like effect, since DTT reversed the anti-immobility effect elicited by GSH. Moreover, the pretreatment with DTT was able to reverse the reduction in the immobility time produced by an active dose of DTNB. Our data provide evidence that GSH acts most probably at extracellular critical sites to produce an antidepressant-like effect. The anti-immobility effect of DTNB in the FST, and its reversal by DTT, clearly indicates that oxidizing agents have antidepressant-like effects. These findings might indicate that GSH (or GSH-derivatives) produce its actions by oxidizing extracellular sites, like DTNB. Furthermore, the blockade of the anti-immobility effect of GSH by acivicin indicates that a GSH-metabolite produced in the extracellular milieu may be responsible for the antidepressant-like action of GSH. In conclusion, the results of our study indicate for the first time that an acute i.c.v. administration of GSH produces antidepressantlike effects in mice. However, at present it is not clear if endogenous GSH can participate in this effect, and the precise mechanisms involved in the observed antidepressant activity of GSH remains to be addressed. The experimental observations are in line with the idea that redox sites located in the extracellular environment are direct or indirect targets of thiol/disulfide reagents and, possibly, redox-active metabolites of GSH. These findings highlight a new possible role for GSH as an antidepressant, further corroborating recent trends indicating that GSH participates in the modulation of mood-related behavior. Role of funding source This work was supported by CNPq (National Council for Research Development); FINEP Research Grant “Rede Instituto Brasileiro de Neurociência” (IBN-Net #01.06.0842-00); INCTExcitotoxicity and Neuroprotection; FAPESC (Foundation for the Support of Scientific and Technological Research in the State of Santa Catarina). Conflict of interest All the authors declare to have no actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations. The work described has not been published previously (except in the form of abstract); it is not under consideration for publication elsewhere; and its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out, and if accepted,

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it will not be published elsewhere in the same form, in English or in any other language, without the written consent of the copyright holder. All the authors declare to have participated in this study, with the following participation: experimental design and analysis (all authors), performing experiments (J.M.R. and A.L.D.); methodological protocols (all authors) and article preparation (all authors). Ethical approval All procedures in this study were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the local University ethics committee, CEUA. Acknowledgements The scholarship to J.M.R., provided by Coordination of Improvement of Higher Education Personnel (CAPES), is sincerely appreciated. Alcir Luiz Dafre and Ana Lúcia S. Rodrigues are productivity research fellows of CNPq. References [1] Dringen R, Pawlowski PG, Hirrlinger J. Peroxide detoxification by brain cells. Journal of Neuroscience Research 2005;79:157–65. [2] Lewerenz J, Hewett SJ, Huang Y, Lambros M, Gout PW, Kalivas PW, et al. The cystine/glutamate antiporter system x(c)(−) in health and disease: from molecular mechanisms to novel therapeutic opportunities. Antioxidants & Redox Signaling 2013;18:522–55. [3] Anathy V, Roberson EC, Guala AS, Godburn KE, Budd RC, Janssen-Heininger YMW. Redox-based regulation of apoptosis: S-glutathionylation as a regulatory mechanism to control cell death. Antioxidants & Redox Signaling 2012;16:496–505. [4] Haddad JJ, Harb HL. l-Gamma-glutamyl-l-cysteinyl-glycine (glutathione; GSH) and GSH-related enzymes in the regulation of pro- and anti-inflammatory cytokines: a signaling transcriptional scenario for redox(y) immunologic sensor(s)? Molecular Immunology 2005;42:987–1014. [5] Hirrlinger J, Dringen R. The cytosolic redox state of astrocytes: maintenance, regulation and functional implications for metabolite trafficking. Brain Research Reviews 2010;63:177–88. [6] Lu SC. Regulation of glutathione synthesis. Molecular Aspects of Medicine 2009;30:42–59. [7] Orlowski M, Meister A. The gamma-glutamyl cycle: a possible transport system for amino acids. Proceedings of the National Academy of Sciences of the United States of America 1970;67:1248–55. [8] Griffith OW, Meister A. Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine). Journal of Biological Chemistry 1979;254:7558–60. [9] Kulak A, Cuenod M, Do KQ. Behavioral phenotyping of glutathione-deficient mice: relevance to schizophrenia and bipolar disorder. Behavioural Brain Research 2012;226:563–70. [10] Yoshiba-Suzuki S, Sagara J, Bannai S, Makino N. The dynamics of cysteine, glutathione and their disulphides in astrocyte culture medium. Journal of Biochemistry 2011;150:95–102. [11] Lantum HBM, Iyer RA, Anders MW. Acivicin-induced alterations in renal and hepatic glutathione concentrations and in gamma-glutamyltransferase activities. Biochemical Pharmacology 2004;67:1421–6. [12] Garcia-Garcia A, Zavala-Flores L, Rodriguez-Rocha H, Franco R. Thiol-redox signaling, dopaminergic cell death, and Parkinson’s disease. Antioxidants & Redox Signaling 2012;17:1764–84. [13] Pocernich CB, Butterfield DA. Elevation of glutathione as a therapeutic strategy in Alzheimer disease. Biochimica et Biophysica Acta: Molecular Basis of Disease 2012;1822:625–30. [14] Do KQ, Trabesinger AH, Kirsten-Krüger M, Lauer CJ, Dydak U, Hell D, et al. Schizophrenia: glutathione deficit in cerebrospinal fluid and prefrontal cortex in vivo. European Journal of Neuroscience 2000;12:3721–8. [15] Gawryluk JW, Wang J-F, Andreazza AC, Shao L, Young LT. Decreased levels of glutathione, the major brain antioxidant, in post-mortem prefrontal cortex from patients with psychiatric disorders. International Journal of Neuropsychopharmacology 2011;14:123–30. [16] Abe K, Nakanishi K, Saito H. The possible role of endogenous glutathione as an anticonvulsant in mice. Brain Research 2000;854:235–8. ˜ [17] Fernandez-Fernandez S, Almeida A, Bolanos JP. Antioxidant and bioenergetic coupling between neurons and astrocytes. Biochemical Journal 2012;443:3–11. [18] Maes M, Galecki P, Chang YS, Berk M. A review on the oxidative and nitrosative stress (O&NS) pathways in major depression and their possible contribution to the (neuro)degenerative processes in that illness. Progress in NeuroPsychopharmacology and Biological Psychiatry 2011;35:676–92.

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