Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 143–150

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Agmatine abolishes restraint stress-induced depressive-like behavior and hippocampal imbalance in mice

Andiara E. Freitas, Luis E.B. Bettio, Vivian B. Neis, Danúbia B. Santos, Camille M. Ribeiro, Priscila B. Rosa, Marcelo Farina, Ana Lúcia S. Rodrigues ⁎

Department of Biochemistry, Center of Biological Sciences, Universidade Federal de Santa Catarina, Campus Universitário, Trindade, 88040-900 Florianópolis, SC, Brazil article info abstract

Article history: has been recently emerged as a novel candidate to assist the conventional pharmacotherapy of depres- Received 14 October 2013 sion. The acute restraint stress (ARS) is an unavoidable stress situation that may cause depressive-like behavior in Received in revised form 2 December 2013 rodents. In this study, we investigated the potential antidepressant-like effect of agmatine (10 mg/kg, adminis- Accepted 14 December 2013 tered acutely by oral route) in the forced swimming test (FST) in non-stressed mice, as well as its ability to abolish Available online 24 December 2013 the depressive-like behavior and hippocampal antioxidant imbalance induced by ARS. Agmatine reduced the im- mobility time in the mouse FST (1–100 mg/kg) in non-stressed mice. ARS caused an increase in the immobility Keywords: Acute restraint stress time in the FST, indicative of a depressive-like behavior, as well as hippocampal peroxidation, and an in- Agmatine crease in the activity of hippocampal superoxide dismutase (SOD), (GPx) and glutathione Antioxidant reductase (GR) activities, reduced catalase (CAT) activity and increased SOD/CAT ratio, an index of pro-oxidative Depression conditions. Agmatine was effective to abolish the depressive-like behavior induced by ARS and to prevent the Forced swimming test ARS-induced and changes in SOD, GR and CAT activities and in SOD/CAT activity ratio. Hippo- campal levels of reduced glutathione (GSH) were not altered by any experimental condition. In conclusion, the present study shows that agmatine was able to abrogate the ARS-induced depressive-like behavior and the asso- ciated hippocampal imbalance observed in stressed restraint mice, suggesting that its antidepressant-like effect may be dependent on its ability to maintain the pro-/anti-oxidative homeostasis in the hippocampus. © 2013 Elsevier Inc. All rights reserved.

1. Introduction hippocampal atrophy observed in depressed individuals (Lee et al., 2002; McKinnon et al., 2009). The relationship between onset of a major depressive episode and The neurotoxic effects of glucocorticoids induced by stress could prior stressful life events has been extensively reported (Hammen be partly explained by the inflammatory and neurodegenerative et al., 2009; Kessler, 1997; Mazure, 1998). Abnormalities of the hypo- hypothesis of depression, which postulates that oxidative and thalamic pituitary adrenal (HPA) axis have been shown to play a critical nitrosative stresses contribute to neurodegenerative processes in role in development of depressive symptoms, persistence of symptoms, depression (Anisman et al., 2002; Lang and Borgwardt, 2013; Maes and recurrence of depression (Gold et al., 1988; Morris et al., 2012; et al., 2009, 2012). It is well recognized that the brain tissue is partic- Plotsky et al., 1998). The progressive HPA abnormalities caused by trau- ularly vulnerable to oxidative damage, compared with other organs, matic stress or chronic stress not only trigger behaviors and emotions due to its relatively high content of iron and peroxidizable fatty related to depression and anxiety, but also might result in prolonged acids, besides its limited antioxidant capacity (Floyd, 1999; Herbert cortisol hypersecretion that can account for neuronal death and et al., 1994). There is now ample evidence that ROS produced in the brain are deleterious to neurons since they may overwhelm the brain antioxidant defenses, thereby causing oxidative damage to , pro- teins and DNA, events that play a key role in neuronal death Abbreviations: ANOVA, analysis of variance; ARS, acute restraint stress; CAT, catalase; (Forlenza and Miller, 2006; Lang and Borgwardt, 2013; Sarandol et al., FST, forced swimming test; GSSG, glutathione disulfide; GPx, ; GR, + 2007). ; H ,hydrogenion;H2O2, ; HO•,hydroxylradical; HPA, hypothalamic pituitary adrenal; MDA, malondialdehyde; NMDA, N-methyl-D- Several studies have demonstrated that different kinds of stress, + aspartate; O2, molecular oxygen; NADP , nicotinamide adenine dinucleotide phosphate; including restraint stress, cause an impairment in the antioxidant NOS, synthase; NPSH, nonprotein ; GSH, reduced glutathione; NADPH, re- status in the brain (de Balk et al., 2010; Enache et al., 2008; García- duced nicotinamide adenine dinucleotide phosphate; SSRIs, selective reuptake in- Fernández et al., 2012; Kumar and Goyal, 2008), which could be hibitor; O •−, superoxide anion ; SOD, superoxide dismutase; TBARS, thiobarbituric 2 prevented by antidepressant drugs from distinct classes, namely acid reactive substances; TCAs, tricyclic antidepressants; TST, tail suspension test. fl ⁎ Corresponding author. Tel.: +55 48 3721 5043; fax: +55 48 37219672. imipramine and clomipramine (tricyclic antidepressants, TCAs), u- E-mail addresses: [email protected], [email protected] (A.L.S. Rodrigues). oxetine and venlafaxine (selective serotonin reuptake inhibitors,

0278-5846/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pnpbp.2013.12.012 144 A.E. Freitas et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 143–150

SSRIs), and trazodone (atypical antidepressant) (de Balk et al., 2010; groups of mice 60 min after an acute administration of agmatine by Kumar et al., 2009, 2010; Zafir and Banu, 2007; Zafiretal.,2009). gavage (1–100 mg/kg, p.o.). This experiment was carried out in order However, most of the clinically available antidepressants have only to choose the dose of agmatine to be used in subsequent experiments marginal efficacy compared with placebo (Kirsch, 2009). Therefore, that investigated the ability of agmatine to abolish the depressive-like there is a clear need for novel drugs or augmentation therapies to in- behavior and hippocampal redox impairment induced by ARS. crease the efficacy of antidepressants. In the ARS protocol, agmatine (10 mg/kg, p.o.) was administered 1 h Also known as “decarboxylated ”, agmatine is an endoge- before the ARS. The were assigned to the following groups: nous metabolite situated between the critical pathways of nitric oxide (a) unstressed/vehicle, (b) unstressed/agmatine, as the control groups; production, polyamine , and the (Reis and (c) ARS/vehicle and (d) ARS/agmatine. Number of mice per group was Regunathan, 2000). Agmatine has been proposed as a neuromodulator 7–8. A diagram of all experimental schedules is given in Fig. 1. in the central nervous system, considering that it : a) is stored in a large number of neurons with selective distribution in the brain 2.3. Acute restraint stress procedure (Otake et al., 1998); b) is released from synaptosomes in a Ca2+- dependent manner (Sastre et al., 1997); c) is enzymatically degraded After 1 h of the treatment, ARS procedure was performed by a meth- by in synaptosomes (Tabor and Tabor, 1984); d) is od described previously (Kumar and Goyal, 2008; Poleszak et al., 2006; inactivated by selective reuptake (Sastre et al., 1997); and e) has several Zafiretal.,2009) and standardized in our laboratory (Budni et al., 2013; molecular targets in the brain, namely I1R, and α2-adrenergic receptors, Moretti et al., 2013). The animals were maintained in their home cages (2+) 5HT3 receptors; inhibits membrane Ca channels, all isoforms of with free access to water and food in the period (1 h) that elapsed be- nitric oxide synthase (NOS) in the brain, and blocks N-methyl-D- tween the treatment and ARS procedure. The immobilization was aspartate (NMDA) receptors (Reis and Regunathan, 1998, 1999, 2000). applied for a period of 7 h using an individual rodent restraint device In addition, it is remarkable to note agmatine's ability to act as a made of Plexiglas fenestrate. This restrained all physical movements free radical protecting from -induced mi- without causing pain. The animals were deprived of food and water dur- tochondrial swelling, membrane potential collapse, NF-κB transloca- ing the entire period of exposure to stress. The unstressed-groups were tion, and apoptosis (Arndt et al., 2009; Battaglia et al., 2010; Condello treated with vehicle or agmatine and were kept without food and water et al., 2011). In addition, agmatine has been emerging as a putative during the entire period of exposure to stress. Forty min after ARS, the alternative therapeutic tool that couldhelptheconventionalphar- animals were released from their enclosure, submitted to the behavioral macotherapy of depression. Previous studies by our group have observations, and then to the neurochemical analysis. demonstrated that it is able to produce an antidepressant-like effect in the mouse forced swimming test (FST) and in the tail suspension 2.4. Behavioral tests test (TST), which was paralleled by modulation of monoaminergic and opioid systems, NMDA receptors and the L-arginine–NO pathway 2.4.1. Open-field test (Zomkowski et al., 2002, 2004, 2005). Of note, a recent study by To assess the effects of agmatine on locomotor activity, mice were Shopsin (2013) showed a clinical antidepressant effect of agmatine in evaluated in the open-field paradigm as previously described by depressed subjects. However, there is no study reporting the ability of Rodrigues et al. (1996). The number of squares crossed with all paws this neuromodulator to abolish depressive-like behavior induced by a (crossings) was counted in a 6 min session. The apparatus was cleaned model that reproduces symptoms of depression accompanied by an ox- with a solution of 10% ethanol between tests in order to hide idative imbalance. Therefore, the present study was aiming at investi- clues. gating the ability of agmatine to abrogate alterations in behavior and in the hippocampal cellular redox status induced by acute restraint 2.4.2. Forced swimming test (FST) stress (ARS). Mice were individually forced to swim in an open cylindrical con- tainer (diameter 10 cm, height 25 cm), containing 19 cm of water 2. Materials and methods (depth) at 25 ± 1 °C; the total amount of time each animal remained immobile during a 6-min session was recorded (in seconds) as immo- 2.1. Animals bility time, as described previously (Brocardo et al., 2008; Freitas et al., 2010). Each mouse was judged to be immobile when it ceased strug- Female Swiss mice (3 months old, 40–45 g) were maintained at gling and remained floating motionless in the water, making only constant room temperature (20–22 °C) with free access to water and those movements necessary to keep its head above water. A decrease food, under a 12:12 h light:dark cycle (lights on at 07:00 h). The cages in the duration of immobility is indicative of an antidepressant-like were placed in the experimental room 24 h before the test for acclima- effect (Porsolt et al., 1977). tization. All manipulations were carried out between 9:00 and 17:00 h, with each animal used only once. The procedures in this study were per- formed in accordance with the NIH Guide for the Care and Use of Labo- ratory Animals and approved by the local Ethics Committee. All efforts were made to minimize animal suffering and the number of animals used in the experiments.

2.2. Drugs and treatment

Agmatine (Sigma Chemical Co., St. Louis, U.S.A.) was dissolved in distilled water and was administered by oral route (p.o.) by gavage at doses of 1–100 mg/kg. The dissolution of agmatine was freshly done immediately before its administration. A control group re- ceived distilled water as vehicle. Fig. 1. Diagram of all experimental schedules. Agmatine at dose of 10 mg/kg (p.o.) was ad- ministered 1 h before the ARS procedure. The animals were submitted to immobilization In the experiments designed to study the antidepressant-like ef- for a period of 7 h. 40 min post-release from their enclosure, independent group of ani- fect of agmatine in the FST, the immobility time in the FST and the mals was submitted to the open-field, or FST, or hippocampi tissue was rapidly dissected locomotor activity in the open-field were assessed in independent and prepared for biochemical analysis. A.E. Freitas et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 143–150 145

2.5. Biochemical analysis supernatant (which was neutralized with 0.1 M NaOH). Absorbance was read in 405 nm, using GSH as standard. The values are expressed Considering that: a) the glutathione reductase (GR), glutathione as nmol NPSH/mg protein. peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD) are key endogenous antioxidant , which inhibit the formation 2.5.4. Determination of thiobarbituric acid reactive substance (TBARS) of ROS and/or promote the removal of free radicals and their precursors levels (Bilici et al., 2001; Del Río et al., 1992; Floyd, 1999; Halliwell, 2007; TBARS levels were determined in the hippocampal homogenates Kodydková et al., 2009); b) impairments in these detoxification system using the method described by Ohkawa et al. (1979), in which have been implicated in the etiology of depression (Anisman et al., malondialdehyde (MDA), an end-product of lipid peroxidation, re- 2002; Lang and Borgwardt, 2013; Leonard and Maes, 2012; Maes acts with thiobarbituric acid to form a colored complex. et al., 2009); c) lipid peroxidation is one of the major consequences of The samples were incubated at 100 °C for 60 min in acid medium free-radical-mediated injury to the brain (Halliwell and Gutteridge, containing 0.45% sodium dodecyl sulfate and 0.67% thiobarbituric acid. 1999; Niki, 2012), and could be estimated by TBARS levels, the present After centrifugation, the reaction product was determined at 532 nm study assessed these neurochemical targets. using MDA as standard.

2.5.1. Tissue preparation 2.5.5. Statistical analysis After the behavioral observations, mice were decapitated and hippo- Comparisons between experimental and control groups were campus was rapidly dissected on ice and homogenized (1:10 w/v) in performed by one-way or two-way ANOVA followed by Duncan's HEPES buffer (20 mM, pH 7.0). The tissue homogenates were centri- multiple range test, when appropriate. Pearson's correlation analysis fuged at 16,000 ×g, at 4 °C for 20 min and the supernatants obtained was performed to investigate any possible relationship between behav- were used for the determination of enzymatic activities and for the ioral and neurochemical data. P b 0.05 was considered significant. quantification of the levels of glutathione (GSH) and thiobarbituric acid reactive substances (TBARS). The protein content was quantified 3. Results according to the method described by Lowry et al. (1951), using bovine serum albumin as a standard. 3.1. Effect of an acute treatment with agmatine on the immobility time in the FST and locomotor activity in the open-field test 2.5.2. Activity of antioxidant enzymes Hippocampal glutathione reductase (GR) activity was measured The results depicted in Fig. 2A show that agmatine given by oral using an NADPH reduction assay following the protocol developed by route decreased the immobility time in the FST, a behavioral profile Carlberg and Mannervik (1985) using glutathione disulfide (GSSG) characteristic of an antidepressant-like effect. One-way ANOVA as substrate. GR activity was monitored by decreases in NADPH ab- revealed a significant effect of agmatine [F(3,24) = 8.72, P b 0.01]. sorbance at 340 nm at 37 °C in a TECAN Genios Microplate Reader Post hoc analysis indicated a significant decrease in the immobility (Tecan Group Ltd., Männedorf, Switzerland). Results were based on time elicited by agmatine at doses of 1, 10 and 100 mg/kg (P b 0.01). a molar extinction coefficient for NADPH of 6.22 × 103 M−1 cm−1. Fig. 2B shows that the administration of agmatine (dose range Hippocampal glutathione peroxidase (GPx) activity was measured 1–100 mg/kg, p.o.) produced no effect in the locomotor activity in the using an NADPH reduction assay following the technique of Wendel open-field test [F(3,24) = 0.50, P = 0.69]. (1981). Tissue supernatant (around 200 μg protein) was added to a re- action mixture containing GSH, GR and NADPH in phosphate buffer 3.2. Agmatine abolished the depressive-like behavior induced by ARS (pH 7.4). The reaction was initiated by adding tert-butyl hydroperoxide, and the absorbance decrease at 340 nm was recorded at 37 °C in a Fig. 3A shows that ARS caused a significant increase in the immobil- TECAN Genios Microplate Reader (Tecan Group Ltd., Männedorf, ity time, which is in agreement with its ability to induce depressive-like Switzerland). The activity in the absence of the samples was subtracted. behavior. The acute administration of agmatine (10 mg/kg, p.o.) was Results were based on a molar extinction coefficient for NADPH of able to prevent the depressive-like behavior induced by ARS in the 6.22 × 103 M−1 cm−1. Both GR and GPx activities were expressed as FST. Agmatine administration, in control mice, also significantly de- nmol NADPH oxidized/min/mg protein. creased the immobility time in the FST, as compared to unstressed Catalase (CAT) activity was measured by the method of Aebi (1984). mice treated with vehicle. A two-way ANOVA revealed significant The reaction was started by the addition of freshly prepared 30 mM effects of ARS [F(1,28) = 3.92, P b 0.05], treatment [F(1,28) = 49.06,

H2O2.TherateofH2O2 decomposition by catalase was measured spec- P b 0.01] and ARS × treatment interaction [F(1,28) = 6.84, P b 0.01]. trophotometrically at 240 nm and the activity was expressed Post hoc analyses indicated that agmatine treatment to stressed mice as μmol H2O2 consumed/min/mg protein. prevented the depressive-like behavior caused by ARS (P b 0.01). Superoxide dismutase (SOD) activity was assayed spectrophotomet- Fig. 3B shows that the administration of agmatine produced no effect rically as described by Misra and Fridovich (1972).Thespectrophotom- in the open-field test, indicating that a locomotor effect does not ac- eter used for these assays was a TECAN Genios Microplate Reader count for the antidepressant-like effect observed in the FST. The two- (Tecan Group Ltd., Männedorf, Switzerland). This method is based on way ANOVA revealed no significant differences of ARS [F(1,28) = the capacity of SOD to inhibit autoxidation of to adreno- 0.10, P = 0.75], treatment [F(1,28) = 0.49, P = 0.48], and ARS × chrome. The color reaction was measured at 480 nm. One unit of treatment interaction [F(1,28) = 0.002, P = 0.96]. enzyme was defined as the amount of enzyme required to inhibit the rate of epinephrine autoxidation by 50%. The SOD enzymatic activity 3.3. Agmatine abolished the ARS-induced oxidative imbalance was expressed as units (U)/mg protein. The results depicted in Fig. 4 illustrate that ARS significantly 2.5.3. Assessment of glutathione (GSH) content increased the hippocampal levels of TBARS (an indicative of lipid GSH content was measured as nonprotein thiols (NPSH) according peroxidation) and that this event was significantly blocked by to a method previously described (Ellman, 1959). Briefly, after the agmatine (10 mg/kg, p.o.) treatment; however no changes were ob- incubation, trichloroacetic acid 10% was added to the reaction medium. served in the unstressed animals. The two-way ANOVA revealed signifi- After centrifugation (5000 ×g at 4 °C for 10 min), the protein pellet cant differences of ARS [F(1,28) = 4.34, P b 0.05], treatment [F(1,28) = was discarded and free groups were determined in the clear 6.10, P b 0.01] and ARS × treatment interaction [F(1,28) = 7.84, 146 A.E. Freitas et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 143–150

Fig. 2. Effect of repeated acute administration by oral route (p.o.) of agmatine (dose range 1–100 mg/kg, p.o.) in the FST (panel A) and open-field test (panel B). Each column represents the mean + S.E.M. (n = 7–8). Statistical analysis was performed by one-way ANOVA, followed by Duncan's test. **P b 0.01 as compared with the vehicle-treated group (C).

P b 0.01]. Post hoc analyses indicated that agmatine treatment abolished ratio (Fig. 6C). The two-way ANOVA revealed significant differences of theincreaseinhippocampalTBARSlevelscausedbyARS(P b 0.01). ARS [F(1,28) = 16.00, P b 0.01], treatment [F(1,28) = 19.40, P b 0.01] Fig. 5 shows that neither ARS nor agmatine treatment altered hippo- and ARS × treatment interaction [F(1,28) = 12.21, P b 0.01]. Post hoc campal GSH (the most important non-enzymatic endogenous antioxi- analyses indicated that agmatine treatment abolished the increase in dant) levels. A two-way ANOVA revealed no significant differences of hippocampal SOD/CAT ratio caused by ARS (P b 0.01). ARS [F(1,28) = 1.91, P = 0.18], treatment [F(1,28) = 0.20, P =0.66] Finally, the effect of the treatment of ARS-mice with agmatine on and ARS × treatment interaction [F(1,28) = 0.38, P = 0.54] for GSH GPx and GR activities (important endogenous antioxidant enzymes) levels. was verified. ARS, but not vehicle or agmatine in unstressed mice, pro- As depicted in Fig. 6A, ARS caused an increase on SOD (a class of en- duced a slight increase in GPx activity (Fig. 7A). The two-way ANOVA zymes that catalyzes the reduction of superoxide to ) revealed a significant effect of ARS [F(1,28) = 6.19, P b 0.05], but no activity and this effect was abrogated by agmatine (10 mg/kg, p.o.) significant main effects of treatment [F(1,28) = 0.004, P = 0.94] and treatment, however no changes were observed in the unstressed ani- ARS × treatment interaction [F(1,28) = 0.72, P = 0.40]. Fig. 7Bshows mals. The two-way ANOVA revealed significant differences of ARS that the treatment of ARS-mice with agmatine (10 mg/kg, p.o.) was [F(1,28) = 8.64, P b 0.01], treatment [F(1,28) = 15.84, P b 0.01] and able to prevent the increase on GR activity caused by the stress pro- ARS × treatment interaction [F(1,28) = 4.97, P b 0.05]. Also, stressed cedure. It is remarkable that agmatine treatment did not induce mice presented a decreased CAT (a relevant endogenous antioxidant these changes in unstressed animals. The two-way ANOVA revealed enzyme responsible for hydrogen peroxide detoxification) activity, significant differences of ARS [F(1,28) = 9.81, P b 0.01], treatment which was prevented by the treatment with agmatine (Fig. 6B), without [F(1,28) = 6.44, P b 0.01] and ARS × treatment interaction [F(1,28) = causing significant effects in the unstressed animals. A two-way ANOVA 4.17, P b 0.05]. Post hoc analyses indicated that agmatine treatment revealed significant effects of ARS [F(1,24) = 9.90, P b 0.01] and abolished the hippocampal increase on antioxidant activities of GPX ARS × treatment interaction [F(1,24) = 4.96, P b 0.05], but no sig- and GR caused by ARS (P b 0.01). nificant main effect of treatment [F(1,24) = 2.90, P = 0.10]. Post Worth of note is the positive Pearson's correlation (Table 1)among hoc analyses indicated that agmatine treatment abolished the alteration immobility time and both TBARS (P b 0.05), GR (P b 0.05), and SOD in CAT and SOD activities induced by ARS (P b 0.01). Of note, ARS caused (P b 0.01). Also, a negative correlation was found between immobility a huge increase in the hippocampal SOD/CAT ratio (Fig. 6C), which has time and CAT (P b 0.01). It is interesting to note that a significant corre- been pointed as an index of pro-oxidative conditions (Halliwell, 2007). lation was found between TBARS and GR (P b 0.05), and between GSH Agmatine prevented the ARS effects toward hippocampal SOD/CAT with either GR (P b 0.05) or GPx (P b 0.05). A negative correlation was

Fig. 3. Effect of the treatment of ARS-mice with agmatine (10 mg/kg, p.o.) in the FST (panel A) and open-field test (panel B). Each column represents the mean + S.E.M. (n = 7–8). Sta- tistical analysis was performed by two-way ANOVA, followed by the Duncan's test. **P b 0.01 as compared with the control group (unstressed-vehicle); ##P b 0.01 as compared with the ARS-vehicle group. A.E. Freitas et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 143–150 147

associated with an oxidative imbalance, represent a novelty on the theme of antidepressant strategies. The ARS has been proposed as a model of depression induced by stress, which combines both emotional and physical components in ad- dition to affect the brain's intra-cellular redox status (Buynitsky and Mostofsky, 2009; Glavin et al., 1994; Paré and Glavin, 1986). The main improvement of using restraint is that it produces an inescapable phys- ical and mental stress which is not accompanied with a conditioned response (Jaggi et al., 2011). It is relevant to clarify that this study was performed in female mice because women are more susceptible to development of depressive disorder followed to lifetime stress events than men (Mazure and Maciejewski, 2003). Indeed, the ARS model of depression standardized previously in our laboratory (Budni et al., 2013; Moretti et al., 2013) was able to cause depressive-like behavior associated with impairment in the in vivo antioxidant defenses, provid- ing validation for the model. Our results are in line with this assumption, Fig. 4. Effect of the treatment of ARS-mice with agmatine (10 mg/kg, p.o.) on thiobarbitu- since ARS caused emotional stress, evidenced by the depressive-related ric acid reactive substances (TBARS) in the hippocampus. Each column represents the mean + S.E.M. (n = 7–8). Statistical analysis was performed by two-way ANOVA, behavior in the FST observed in animals submitted to this protocol. Of followed by the Duncan's test. **P b 0.01 as compared with the control group note, agmatine administration by oral route (p.o.) was able to prevent (unstressed-vehicle); ##P b 0.01 as compared with the ARS-vehicle group. this behavioral alteration, reinforcing the antidepressant-like effect of this neuromodulator. shown between GR and CAT (P b 0.05). Finally, GPx correlates with SOD It is well-known that the central nervous system is extremely sensi- (P b 0.05). Number of crossings did not correlate with any variable tive to peroxidative damage due to its rich content of oxidizable sub- studied (data not shown). strates, high oxygen tension and low antioxidant capacity (Metodiewa and Kośka, 2000; Zafir et al., 2009). Several studies have shown that 4. Discussion stressful life events are associated with brain oxidative damage as a con- sequence of an increase in the production of ROS, such as superoxide

Agmatinehasbeenproposedasanovelneuromodulatorinthe anion radical (O2•−), hydroxyl radical (HO•), and hydrogen peroxide central nervous system and is emerging as a novel candidate to assist (H2O2)(Anisman et al., 2002; Leonard and Maes, 2012; Maes et al., the conventional pharmacotherapy of depression. Previous findings 2009). The endogenous detoxification system involves the activity of from our group have shown that agmatine administration by intra- several antioxidant enzymes. SOD, is the first line of defense against peritoneal (i.p.) and intracerebroventricular (i.c.v.) routes produces ROS and catalyzes the dismutation of superoxide anion radical into hy- antidepressant-like effect in the FST and TST (Zomkowski et al., drogen peroxide (Singh, 1982). The H2O2 produced from O2•− can be 2002, 2004, 2005). Nevertheless, in the present study we demon- reduced to water and molecular oxygen by either catalase (CAT) (Del strated that an acute agmatine administration by oral route (p.o.) Río et al., 1992) or glutathione peroxidase (GPx) (Brigelius-Flohé, produced a significant antidepressant-like effect in the FST, a commonly 1999). The H2O2 detoxification mediated by GPx expends reduced glu- used behavioral test that predicts the efficacy of antidepressant treat- tathione (GSH), which in turn is oxidized producing glutathione disul- ment (Cryan and Holmes, 2005; Porsolt et al., 1977). Additionally, a fide (GSSG) (Flohé, 1971). The GSSG produced in GPx mediated consistent antidepressant-like activity of agmatine in mice submitted reaction can be restored to GSH through a reaction catalyzed by gluta- to the acute restrain stress, a procedure that has been extensively thione reductase (GR), at the expense of the reducing equivalents shown to cause behavioral alterations indicative of depressive-like be- from NADPH (Kirsch and De Groot, 2001). havior (Kumar and Goyal, 2008; Poleszak et al., 2006; Zafir et al., Lipid peroxidation is considered a critical mechanism of injury oc- 2009), was shown. The previous studies reporting antidepressant-like curring in cells during oxidative stress (Halliwell and Gutteridge, effects of agmatine did not use animal models of depression. In this re- 1999; Niki, 2012). An initial formation of large amounts of ROS during gard, the results presented herein, which evince the agmatine's ability stress may also initiate lipid peroxidation as demonstrated to occur in to produce an antidepressant-like effect in a model of depression brain (Hayashi et al., 2012). Emotional stress, which accompanies severe depression, may enhance lipid peroxidation (Goncharova et al., 2008) and clinical studies have directly demonstrated higher levels of MDA—an end-product of lipid peroxidation, in patients with affective disorders (Lang and Borgwardt, 2013; Ozcan et al., 2004). In addition, several studies have demonstrated that the restrained stress significant- ly elevated lipid peroxidation level in the hippocampus of rats (Abidin et al., 2004; Atif et al., 2008; de Balk et al., 2010; Fontella et al., 2005) and mice (Budni et al., 2013; García-Fernández et al., 2012; Moretti et al., 2013; Perez Nievas et al., 2011). In line with this, our results show that the ARS procedure caused a significant lipid peroxidation, as evidenced by increased amount of TBARS levels in ARS-mice, which was abolished by agmatine treatment. Thus, the beneficial effects of agmatine on behavior could be associated with its capacity to prevent the lipid peroxidative damage caused by immobilization stress. This hy- pothesis is supported by the positive correlation among immobility time and TBARS results. The glutathione (GSH) system is an important tool mediating protection against several (pro)-oxidant molecules in the brain Fig. 5. Effect of the treatment of ARS-mice with agmatine (10 mg/kg, p.o.) on glutathione levels in the hippocampus. Each column represents the mean + S.E.M. (n = 7–8). Statis- (Dringen and Hirrlinger, 2003) and alterations is this system are in- tical analysis was performed by two-way ANOVA, followed by the Duncan's test. volved in several neuropathological conditions (Lovell et al., 1998). A 148 A.E. Freitas et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 143–150

Fig. 6. Effect of the treatment of ARS-mice with agmatine (10 mg/kg, p.o.) on SOD activity (panel A), on CAT activity (panel B) and on SOD/CAT ratio in the hippocampus (panel C). Each column represents the mean + S.E.M. (n = 7–8). Statistical analysis was performed by two-way ANOVA, followed by the Duncan's test. **P b 0.01 as compared with the control group (unstressed-vehicle); ##P b 0.01 as compared with the ARS-vehicle group. recent study by Rosa et al. (2013) indicated that the central adminis- the antidepressant effect of agmatine in ARS-mice. Nevertheless, tration of GSH elicits an antidepressant-like response in the FST and considering that a positive correlation among GSH results and both TST in mice. Recent studies performed by Budni et al. (2013), Moretti GR and GPx was shown, it could be speculated that GSH is somehow et al. (2013) and Méndez-Cuesta et al. (2011), however, reported involved in the maintenance of pro/anti-oxidative homeostasis of that restraint stress in rodents did not affect GSH levels in the brain. ARS-mice hippocampus. The present study is in agreement with these findings, since neither In the present study, we identified a significant imbalance in SOD, agmatine nor ARS caused alterations in GSH levels in the hippocampus GPx, GR and CAT hippocampal activities after ARS procedure. Indeed, of mice, suggesting that this molecular target is not directly underlying Sarandol et al. (2007) have demonstrated that patients suffering from

Fig. 7. Effect of the treatment of ARS-mice with agmatine (10 mg/kg, p.o.) on GPx activity (panel A) and on GR activity (panel B) in the hippocampus. Each column represents the mean + S.E.M. (n = 7–8). Statistical analysis was performed by two-way ANOVA, followed by the Duncan's test. *P b 0.05, **P b 0.01 as compared with the control group (unstressed-vehicle); ##P b 0.01 as compared with the ARS-vehicle group. A.E. Freitas et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 143–150 149

Table 1 seem to be associated with a modulation of TBARS, SOD, GR, and CAT, Pearson's correlation among selected variables. but not directly with GSH and GPx. The role of each of these targets Measures Immobility time TBARS GSH GR GPx deserves further studies to determine their direct relation to the an- tidepressant action of agmatine. Nevertheless, the present study TBARS 0.43* GSH 0.03 0.11 delves into one of the pleiotropic effects of agmatine by highlighting GR 0.40* 0.45** 0.37* that its antidepressant-like effect in the ARS model of depression is, GPx −0.02 0.21 0.40* 0.30 at least in part, accompanied by its capability of maintaining pro-/ − − − − − CAT 0.45** 0.33 0.23 0.36* 0.16 anti-oxidant homeostasis in the hippocampus. SOD 0.50** 0.29 0.17 0.30 0.35*

Significant at *P b 0.05 or **P b 0.01. Acknowledgments major depressive disorder (MDD) presented higher MDA levels and This study was supported by the FINEP research grant “Rede SOD activity, as compared to the control group. In addition, this Instituto Brasileiro de Neurociência (IBN-Net/CNPq)”, CNPq, FAPESC, fi study found a signi cant positive correlation among the severity of CAPES/PROCAD and Núcleo de Excelência em Neurociências Aplicadas the disease and SOD activity. Similarly, Kotan et al. (2011) demon- de Santa Catarina (NENASC) Project/PRONEX Program CNPq/FAPESC strated that patients who fully met the fourth Diagnostic and Statis- (Brazil). tical Manual of Mental Disorder criteria for MDD presented increased levels of MDA and SOD activity. Furthermore, it was also demonstrat- ed that MDD patients presented higher levels of plasma GR and MDA References levels, and erythrocyte GPx and SOD than those of control (Bilici Abidin I, Yargiçoglu P, Agar A, Gümüslü S, Aydin S, Oztürk O, Sahin E. The effect of chronic et al., 2001). Increased GR activity was further found in depressive restraint stress on spatial learning and memory: relation to oxidant stress. Int. J. women (Kodydková et al., 2009). Finally, Ozcan et al. (2004) have Neurosci. 2004;114:683–99. Aebi H. Catalase in vitro. Methods Enzymol. 1984;105:121–6. shown a lower CAT activity in MDD patients. The present results concur Akpinar D, Yargiçoğlu P, Derin N, Alicigüzel Y, Ağar A. The effect of on antiox- with these clinical evidence since the ARS-mice presented a depressive- idant status and lipid peroxidation in rats exposed to chronic restraint stress. Physiol. like behavior accompanied by increased lipid peroxidation, SOD, GPx, Res. 2008;57:893–901. 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