Involvement of the serotonergic system in the anxiolytic-like effect of 2-phenylethynyl butyltellurium in mice

Behavioural Brain Research 277 (2015) 221–227

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Research report

Involvement of the serotonergic system in the anxiolytic-like effect of 2-phenylethynyl butyltellurium in mice Caroline B. Quines, Juliana T. Da Rocha, Tuane B. Sampaio, Ana Paula Pesarico, José S.S. Neto, Gilson Zeni, Cristina W. Nogueira ∗ Laboratório de Síntese, Reatividade e Avaliac¸ão Farmacológica e Toxicológica de Organocalcogênios, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, CEP 97105-900 Santa Maria, RS, Brazil

h i g h l i g h t s • PEBT treatment demonstrated an anxiolytic-like effect in different anxiety models. • PEBT reduced [3 H] 5-HT uptake. • PEBT selectively inhibited MAO-A activity in cerebral cortex.

a r t i c l e

i n f o

Article history: Received 11 March 2014 Received in revised form 29 May 2014 Accepted 30 May 2014 Available online 11 June 2014 Keywords: Anxiolytic-like Serotonin Monoamine oxidase Tellurium Organotellurium

a b s t r a c t Anxiety is a serious disorder with symptoms manifested at the psychological, behavioral, and physiological levels, accompanied by alterations in the serotonergic system and monoaminergic signaling. In this study, the anxiolytic-like effect of 2-phenylethynyl butyltellurium (PEBT), in three well-consolidated anxiety mouse models (light–dark test, novelty suppressed-feeding, elevated plus-maze), was investigated. The involvement of the serotonergic system, synaptosomal [3 H] serotonin (5-HT) uptake and monoamine oxidase (MAO A and B) activities on cerebral cortices of mice, was examined. Mice received PEBT (1 mg/kg, by intragastric route, i.g.) or canola oil (10 ml/kg, i.g.) 30 min before behavioral tests. The results showed that PEBT was effective in increasing the time spent by mice in the illuminated side on the light–dark box and in the open arms on the elevated plus-maze. PEBT decreased the latency to begin eating on the novelty suppressed-feeding test, indicating an anxiolytic-like effect of PEBT. Furthermore, PEBT reduced [3 H] 5-HT uptake and selectively inhibited MAO-A activity in cerebral cortex, suggesting the involvement of the serotonergic system in the mechanism of action of this tellurium compound. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Anxiety is a serious disorder in today’s society, manifested by disturbances of mood and emotions, as well as of thinking, behavior, and physiological activity. Studies have shown that the monoaminergic neurotransmitter, serotonin [5hydroxytryptamine (5-HT)], is involved in the pathogenesis of anxiety [1–3]. Furthermore, abnormalities in the serotonergic neurotransmission accompanied by a reduction in monoaminergic signaling [1,4] could increase the breakdown of neurotransmitters, like 5-HT, resulting in a decrease in their availability in the

∗ Corresponding author at: Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. Tel.: +55 55 3220 8140; fax: +55 55 3220 8978. E-mail address: [email protected] (C.W. Nogueira). http://dx.doi.org/10.1016/j.bbr.2014.05.071 0166-4328/© 2014 Elsevier B.V. All rights reserved.

synaptic cleft and this has been implicated in the etiology of several psychiatric disorders [5,6]. In addition, selective 5-HT reuptake inhibitors (SSRIs) have been effective as anxiolytics for long-term therapy, by inhibiting the reuptake into the presynaptic cell, resulting in an increase in the level of 5-HT in the synaptic cleft available to bind to the postsynaptic receptors [1–3]. Monoamine oxidase (MAO) is an enzyme present in mammalian tissues external at mitochondrial membrane, which is responsible for the oxidative deamination of monoamine neurotransmitters. The MAO has two isoforms, MAO-A and MAO-B, which despite catalyze the same reaction, exhibit differences in selectivity for inhibitors, and differences in the amines metabolized [7–9]. MAO-A is selectively inhibited by clorgyline and preferentially desaminates 5-HT and noradrenaline [10] and MAO-B is selectively inhibited by selegiline and metabolizes preferentially phenylethylamine [11]. In this context, the inhibition of MAO-A is associated with the

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metabolism of 5-HT, regulating its intracellular concentration in the brain. Consequently, the abnormal function of this enzyme has been implicated in the etiology and treatment of depression and anxiety disorders [12,13]. Organotellurium compounds have been the subject of research due to their pharmacological properties. Organotellurium compounds have been reported as antioxidants in several animal models of oxidative stress [14–16]. Moreover, these compounds had immunomodulatory and anti-inflammatory actions [17–19]. In addition, the vinyl alkynyl telluride class of compounds demonstrated an antidepressant-like action in mice [20]. Recently, our research group showed the antioxidant effect of different telluroacetylenes in vitro [21]. Moreover, specifically 2-phenylethynyl-butyltellurium (PEBT), a telluroacetylene compound, protected against oxidative damage caused by sodium nitroprusside in mouse brain, suggesting an antioxidant in vivo effect of this compound [22]. In addition, this compound showed low toxicity in vitro [23]. Besides, PEBT significantly ameliorated the scopolamine-induced impairment of long-term memory and A␤-induced learning deficits in mice, as indicated by a decrease in escape latency and an increase in the number of crossings over the platform location in the Morris Water Maze test. Furthermore, PEBT increased step-down latency in scopolamine-induced memory impairment in mice and A␤-treated group [24,25]. The need for the development of new therapeutic agents for treating anxiety is of great interest since there are several concerns associated with the use of current anxiolytic therapies, mainly the increased incidence of tolerance, dependence and abuse of benzodiazepines [26]. In this study a possible anxiolytic-like effect of PEBT in different tests predictive of anxiety was investigated in mice. The involvement of the serotonergic system in the anxiolytic-like effect of PEBT, by measuring [3 H] 5-HT uptake and the contribution of MAO-A and MAO-B activities in cerebral cortices of mice, were also examined. 2. Materials and methods 2.1. Chemicals PEBT (Fig. 1) was prepared according to the literature methods [27,28]. Analysis of the 1 H NMR and 13 C NMR spectra showed that the compound synthesized exhibited analytical and spectroscopic data in full agreement with its assigned structure. PEBT was diluted in canola oil. The other chemical reagents utilized for biochemical assays were obtained from Sigma Chemical (St. Louis, MO, USA). 2.2. Animals The experiments were conducted using male adult Swiss mice (25–30 g) maintained at 22–25 ◦ C with free access to water and food, under a 12:12 h light/dark cycle, with lights on at 7:00 a.m. All manipulations were carried out between 08:00 a.m. and 04:00 p.m and mice were acclimated to the behavioral room at least 2 h before the test. The animals were used according to the guidelines of the Committee on Care and Use of Experimental Animal Resources of the Federal University of Santa Maria, Brazil (#041/2012). All efforts were made to minimize animals suffering and to reduce the number of animals used in the experiments. The animals were divided into two groups: (1) control group (40 animals): mice received canola oil (vehicle) by the intragastric (i.g.)

Fig. 1. Chemical structure of 2-phenylethynyl-butyltellurium (PEBT).

route (10 ml/kg); (2) PEBT group (40 animals): mice received PEBT at a dose of 1 mg/kg (i.g.) 30 min before behavioral tests. The animals used in each behavioral test were different and all tests were carried out in different days. The number of animals per group was: 12 animals in the spontaneous locomotor activity test, 9 animals in the light dark and novelty suppressed-feeding tests and 10 animals in the elevated plus maze test. 2.3. Behavioral tests 2.3.1. Spontaneous locomotor activity The locomotor activity monitor is a clear acrylic plastic box (50 cm × 48 cm × 50 cm) with a removable plastic lid perforated with holes for ventilation. The monitor contains photocell beams and detectors that are mounted on opposite walls (2 cm above the chamber floor). General locomotor activity and the mouse position in the chamber are detected by breaks of the photocell beams, which are recorded by Software (Insight, Ribeirão Preto, SP, Brazil). Mice were placed in the center of the apparatus and allowed to freely explore the arena. Number of crossings, rearings, fecal pellets, average velocity (mm/s) and total distance traveled (mm) were recorded for a 4 min period. 2.3.2. Rotarod test The rotarod test was used to investigate motor coordination. Rotarod consisted of a wooden beam covered with masking tape (diameter, 3 cm), used to increase the roughness of the texture and thereby providing a firm grip. The rod was flanked by two cardboard plates to prevent any escape and suspended at a height of 30 cm above the mat-covered table. The mice were placed on top of the already revolving beam (10 rpm) and facing away from the investigator, in the orientation opposite to that of the beam movement in the longitudinal axis, so that forward locomotion was necessary to avoid a fall. Latencies before falling were measured for three trials, with an inter trial interval of 10 min. 2.3.3. Light–dark test (LDT) The LDT is a sensitive model to detect activity in disorders related to anxiety [29]. The apparatus is an acrylic box (46 cm × 27 cm × 30 cm) divided into light and dark chambers. The light chamber (27 cm × 27 cm) was painted white and was connected via an opening (7.5 cm × 7.5 cm) at floor level to the dark chamber (18 cm × 27 cm), which was painted black. A lamp with a 60-W white light was placed 40 cm above the light chamber. Mice were placed in the light chamber facing the opening into the dark chamber, and the following measures were recorded during a 5min trial: latency to the first transition to the dark chamber, number of zone transitions, and time spent in the light compartment. The measures were recorded manually by a human observer. 2.3.4. Novelty suppressed-feeding (NSF) The NSF is a conflict test that elicits competing motivations: the drive to eat and the fear of venturing into the center of the brightly lit arena, this is a sensitive model to detect anxiety [30]. The testing apparatus consisted of a square wooden arena (45 cm × 45 cm × 45 cm); the floor was covered with wooden bedding. The test was carried out during a 5 min period according to a previous study [31]. Twenty-four hours before behavioral testing, food was removed from the home cage. At the time of testing, a single pellet of food (regular chow) was placed in the center of the box. The mouse was placed in a corner of the box, and a stopwatch was immediately started. Latency to begin eating (defined as the mouse sitting on its haunches and biting the pellet with the use of forepaws) was used as an index of anxiety-like behavior. Then, the

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animal was transferred to the home cage and the amount of food consumed during 5 min period was measured. 2.3.5. Elevated plus-maze (EPM) This test has been widely validated to measure anxiety in rodents [32]. The apparatus consists of two elevated (26 cm high) and open arms (16 cm × 5 cm) positioned opposite to one another and separated by a central platform (5 cm × 5 cm) and two arms of the same dimension, but enclosed by walls (16 cm × 5 cm × 10 cm) forming a cross. The maze is lit by a dim light placed above the central platform. Thirty minutes after the i.g. administration of PEBT (1 mg/kg) or canola oil (vehicle), each mouse was placed at the center of the maze, facing one of the open arms. During a 5 min test period, it was recorded the number of entries either the open or enclosed arms, plus the time spent in the open arms. An entry was defined as placing all four paws within the boundaries of the arm. The following measures were obtained from the test: (a) time spent in the open arms relative to the total time spent in the plus-maze (300 s); (b) number of entries into the open arms; (c) number of entries into the closed arms; (d) number of dives. The anxiolytic effectiveness of a drug is illustrated by a significant statistical augmentation of parameters in open arms (time and/or entries) [33].

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2.4.2.2. Enzyme assay. MAO activity was determined as described by Krajl [37] with some modifications of Matsumoto et al. [38]. Aliquots of samples were incubated at 37 ◦ C for 5 min in a medium containing buffer solution (Na2 PO4 /KH2 PO4 isotonized with KCl, pH 7.4) and specific inhibitors, pargyline (a MAO-B inhibitor, 250 nM) or chlorgyline (a MAO-A inhibitor, 250 nM), at a final volume of 600 ␮l. Then kynuramine dihydrobromide (final concentration 90 ␮M to MAO-A assay and 60 ␮M to MAO-B assay) was added to the reaction mixture as substrate. Samples were then incubated at 37 ◦ C for 30 min. After incubation, the reaction was terminated by adding of 10% thichloroacetic acid. After cooling and centrifugation at 3000 × g for 15 min, an aliquot of supernatant was added to 1 M NaOH. The fluorescence intensity was detected spectrofluorimetrically with excitation at 315 nm and emission at 380 nm. The concentration of 4-hydroxyquinoline was estimated from a corresponding standard fluorescence curve of 4-hydroxyquinoline. MAO-A and MAO-B activities were expressed as nmol of 4-hydroxyquinoline formed/mg protein/min. 2.4.3. Protein determination Protein concentration was measured according to Bradford [39], using bovine serum albumin (1 mg/ml) as the standard.

2.4. Ex vivo assays 2.5. Statistical analysis To test the hypothesis that the serotonergic system plays a role in the anxiolytic-like action of PEBT, immediately after the spontaneous locomotor activity test, the animals were killed by cervical dislocation and the cerebral cortices were removed for determining synaptosomal [3 H] 5-HT uptake (3 animals per group) and MAO A and B activities (9 animals per group). 2.4.1. Synaptosomal [3 H] 5-HT uptake 2.4.1.1. Preparation of crude synaptosomes. Crude synaptosomes were obtained as described by Gray and Whittaker [34] with some modifications. The cerebral cortices were placed into ice-cold sucrose solution (0.32 M, pH 7.4), cut into small pieces and homogenized using a glass Potter-tube with a Teflon pestle (10 up and down strokes). The homogenate solution was centrifuged at 1000 × g for 10 min at 4 ◦ C in a refrigerated centrifuge. The pellet was discarded and the supernatant was subsequently centrifuged at 12,000 × g for 20 min at 4 ◦ C. The final pellet of this centrifugation was suspended in 10 volumes of ice-cold sucrose solution (0.32 M, pH 7.4) and then used as a crude synaptosome preparation in the [3 H] 5-HT uptake assay. 2.4.1.2. [3 H] 5-HT uptake assay. [3 H] 5-HT uptake into synaptosomes was carried out as described by Yura et al. [35] with some modifications. The synaptosomal suspension (100 mg of protein) was pre-incubated at 37 ◦ C for 10 min. 2.4.2. Monoamine oxidase (MAO) activity 2.4.2.1. Mitochondria preparation. A preparation of mitochondria was used for MAO assay as previously described [36]. After removal, the cerebral cortices from mice were washed in ice-cold isolation medium (pH 7.4, Na2 PO4 /KH2 PO4 isotonized with sucrose). Mitochondria were then obtained by differential centrifugation. Briefly, cerebral tissue was manually homogenized with four volumes (w/v) of the isolation medium. Then, the homogenate was centrifuged at 900 × g for 5 min at 4 ◦ C. The supernatant was centrifuged at 12,500 × g for 15 min. The mitochondria pellet was then washed once with isolation medium and recentrifuged under the same conditions. Finally, the mitochondrial pellet was reconstituted in a buffer solution (Na2 PO4 /KH2 PO4 isotonized with KCl, pH 7.4) and stored in aliquots.

All experimental results are presented as the mean ± S.E.M. comparisons between PEBT and control groups were analyzed using Student’s t-test. The values less than 0.05 (p < 0.05) were considered as statistically significant. All analyses were performed using the GraphPad software (GraphPad software, San Diego, CA, USA). Pearson’s correlation coefficient was used for the estimation of correlation between parameters analyzed. For the correlation analysis, results from all groups of animals were used. 3. Results 3.1. Effect of PEBT on spontaneous locomotor activity and rotarod test The number of crossings, rearings and fecal pellets were not altered by PEBT treatment (Table 1). By contrast, the total distance traveled and the average velocity (mm/s) (p < 0.05, Table 1) was increased in the PEBT group during the spontaneous locomotor activity. In addition, treatment with PEBT did not alter the latency to fall during the rotarod test (data not shown). 3.2. Effect of PEBT on the LDT In the LDT, PEBT at a dose of 1 mg/kg did not alter the latency for the first transition in relation to the control group (Fig. 2A). In addition, this compound increased the number of transitions between

Table 1 Spontaneous locomotor activity of mice treated with PEBT. Test parameters

Control

Number of crossing Number of rearing Velocity (mm/s) Distance (mm) Number of fecal pellets

322.3 12.20 27.44 5978 1.22

± ± ± ± ±

PEBT 29.89 1.56 1.86 492.2 0.59

388.4 15.30 34.63 7856 2.22

± ± ± ± ±

26.56 1.44 2.77* 676.3* 0.32

Data are reported as means ± S.E.M. for 12 animals per group. * p < 0.05 when compared to the control group (unpaired Student’s t test).

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A

A 250 200

15

Latency (s)

Latency (s)

20

10

150 100 50

5

0

0

*

Control

PEBT

Control

PEBT

B 0.20

Transitions (s)

20 15 10

Time in the light (s)

0.10

0.00

Control

PEBT

200

***

150 100

* Control

PEBT

Fig. 3. Effect of PEBT treatment on the latency to begin eating (A) and food consumed (B) in the NSF. Values are expressed as mean ± S.E.M. for nine animals per group. * Denotes p < 0.05 compared to the control group (unpaired Student’s t test).

Table 2 Effects of PEBT on mice submitted to the elevated plus maze test. Test parameters

Control

Open arm entries Closed arm entries Number of dives Time in open arm (s)

1.750 10.75 10.92 2.00

± ± ± ±

PEBT 0.371 1.22 1.44 0.52

1.833 9.67 9.91 7.90

± ± ± ±

0.386 0.55 0.66 2.63*

Data are reported as means ± S.E.M. for 10 animals per group. * p < 0.05 when compared to the control group (unpaired Student’s t test).

50 0

0.15

0.05

5 0

C

**

grams (g)

B

3.4. Effect of PEBT on EPM

Control

PEBT

Fig. 2. Effect of PEBT treatment on the LDT, latency for the first transition light–dark (A), number of transitions (B), time in the light (C). Values are expressed as mean ± S.E.M. for nine animals per group. ** Denotes p < 0.01 compared to the control group; *** denotes p < 0.001 compared to the control group (unpaired Student’s t test).

The compound increased the time spent in the open arms (p < 0.05, Table 2). By contrast, PEBT did not alter open and closed arm entries as well as the number of dives when compared to the control group.

3.5. Effect of PEBT on [3 H] 5-HT uptake from cerebral cortices the light/dark sides (p < 0.01, Fig. 2B). Furthermore, PEBT treatment induced a significant increment of the time spent by mice in the illuminated side (p < 0.001, Fig. 2C).

PEBT decreased [3 H] 5-HT uptake in cerebral cortices when compared to the control group (p < 0.05, Fig. 4).

3.3. Effect of PEBT on NSF test

3.6. Effect of PEBT on MAO-A and MAO-B activities from cerebral cortices

PEBT was effective in decreasing the latency to begin eating (p < 0.05, Fig. 3A) but did not alter food consumption during the test (Fig. 3B).

PEBT at a dose of 1 mg/kg selectively inhibited MAO-A activity (p < 0.05, Fig. 5A) in cerebral cortices of mice. By contrast, PEBT at the same dose did not alter MAO-B activity (Fig. 5B).

f mol [ 3 H]5-HT/mg/protn/min

C.B. Quines et al. / Behavioural Brain Research 277 (2015) 221–227

225

60

*

40

20

0

Control

PEBT

B

nmol 4-OH quinoline / mg ptn / min

A

nmol 4-OH quinoline / mg ptn / min

Fig. 4. Effect of PEBT treatment on the [3 H] 5-HT uptake in cerebral cortices. Values are expressed as mean ± S.E.M. for three animals per group. * Denotes p < 0.05 compared to the control group (unpaired Student’s t test).

2.0 1.5

*

1.0 0.5 0.0

Control

PEBT

3.0 2.5 Fig. 6. The LDT data correlate with the ex vivo assays: (A) negative correlation between the time in the light (s) and [3 H] 5-HT uptake (B) negative correlation between the time in the light (s) and MAO-A activity (Pearson’s correlation coefficient). Data are individual values for each animal of all groups.

2.0 1.5 1.0 0.5 0.0

Control

PEBT

Fig. 5. Effect of PEBT treatment on MAO-A (A) and MAO-B (B) activities in cerebral cortices. Values are expressed as mean ± S.E.M. for nine animals per group. * Denotes p < 0.05 compared to the control group (unpaired Student’s t test).

3.7. Correlation analysis between LDT and [3 H] 5-HT uptake or MAO-A activity The Pearson’s correlation analysis revealed a negative correlation between time in the light and [3 H] 5-HT uptake (r = −0.8318, p = 0.0104, Fig. 6A) and a negative correlation between time in the light and MAO-A activity (r = −0.6395, p = 0.0251, Fig. 6B). 4. Discussion The results of the present study showed that PEBT administered by intragastric route to mice produced a significant anxiolyticlike effect in three well-consolidated anxiety animal models: LDT,

NSF and EPM. Moreover, animals were more uninhibited, demonstrated by the increase in the number of transitions in the LDT, the total distance traveled and the average velocity in the spontaneous locomotor activity. However, PEBT treatment did not affect motor coordination, locomotor and exploratory behaviors. In addition to the pharmacological effect, in this study PEBT reduced [3 H] 5-HT uptake and selectively inhibited MAO-A activity in cerebral cortices. In the present study, a single PEBT administration (1 mg/kg, i.g.) produced an increase in time spent by mice in the illuminated side on the LDT and decreased the latency to begin eating on the NSF test, indicating an anxiolytic-like effect of PEBT, which was confirmed by the increase in time spent in the open arms on the EPM. Furthermore, animals treated with PEBT demonstrated an uninhibited behavior when compared to those of control group. In line that, it is important to note that PEBT did not alter the number of falls on the rotarod test. In agreement, Souza et al. [22,24] demonstrated that PEBT did not affect locomotor and exploratory behaviors of mice in the open field test. Chalcogenide compounds, as organotellurium, are versatile tools in synthetic chemistry [20,40,41]. These compounds have found such wide utility because of their effects on an extraordinary number of different reactions, including many carbon–carbon

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bond formations, under relatively mild reaction conditions. In addition, they have become attractive synthetic targets because of their chemo-, regio-, and stereoselective reactions, being used in a wide variety of functional groups, thus avoiding protection group chemistry [41]. Additionally, organotellurium compounds have been reported as pharmacological agents [20]. Accordingly, the varied pharmacological properties of these compounds have been demonstrated [42]. In fact, immunomodulatory, anti-inflammatory, antioxidant, antidepressant-like, and antitumoral properties [17,18,20,43,44] have been attributed to organotellurium compounds. Regarding the pharmacological properties of PEBT, antioxidant and nootropic activities have been reported in vivo [21,22]. In this study, PEBT was effective to decrease [3 H] 5-HT uptake in cerebral cortices of mice, which suggests a modulation of the serotonergic system by PEBT. Furthermore, PEBT showed an anxiolytic-like effect in mice, as described previously and this pharmacological effect could be associated to [3 H] 5-HT uptake. Corroborating to this, the Pearson’s coefficient revealed a negative correlation between time in the light and [3 H] 5-HT uptake. Accordingly, SSRIs are used for treatment of psychiatric disorders, since they increase extracellular 5-HT levels [1–3], which lead to the reduction in symptoms of anxiety and depression [8,9]. Furthermore, one possibility that cannot be ruled out is that PEBT might be increasing 5-HT levels by competing with [3 H] 5-HT for receptor binding sites. Monoamine neurotransmitters, such as 5-HT, are involved in anxiety disorders and play important roles in behavioral effects of anxiolytic drugs [45,46]. The MAO inhibitors are divided by their specificity for MAO-A or MAO-B isoenzyme and whether their inhibition is reversible or not [9]. The treatment response to MAO inhibitors is often superior to other drugs, and they may be effective when other treatments have fail, therefore justifying the need for the research of new drugs that act by inhibiting MAO activity [9,47]. MAO-A inhibitors are used on treatment of neuropsychiatric diseases, in view of the increase in monoamines levels in brain. In the present study, it was demonstrated the selective inhibition of MAO-A activity from cerebral cortices of mice treated with PEBT, which also could be a mechanism of action for the anxiolyticlike effect of this compound. In fact, the inhibition of MAO-A activity could be preventing the breakdown of monoamine neurotransmitters and contributing to the increase of neurotransmitters in the synaptic cleft. The Pearson’s coefficient further corroborates this hypothesis since a negative correlation between time in the light and MAO-A activity was demonstrated. Interestingly, mice treated with PEBT did not alter MAO-B activity. One possible explanation for this fact is that MAO isoenzymes have differences in the three-dimensional arrangements suggesting that the structure of the active site for both enzymes is different. This could explain the differences in both enzymes in relation to their substrate affinities and inhibitor sensitivities [9,48,49]. In this context, Tsugeno and Ito [50] demonstrated that the two forms of MAO present different amino acid residues. An amino acid residue at position 208 in MAO-A and the residue at the corresponding position 199 in MAO-B and these residues play an important role in the determination of substrate selectivity of both enzymes [50]. Corroborating to our results, it has been reported that MAO-A has more affinity for substrates with aromatic ring when compared to those with non-aromatic ring. In other words, the participation of the aromatic side chain in the substrate recognition of MAO-A suggests that the ␲–␲ interaction between aromatic ring of substrates and the enzyme plays a major part in their interaction and could explain why MAO-A has an affinity for aromatic substrates [50]. This way, one might suggest that PEBT inhibited preferentially MAO-A, because PEBT has an aromatic group in its chemical

structure. Although, a conjunctive ␲–␲ interaction of the triple bond and the aromatic ring of PEBT with a site of MAO-A enzyme cannot be ignored. In line that PEBT has an aromatic ring in its chemical structure and this feature could have facilitated the inhibition of MAO-A activity. Regarding MAO-B activity, it has been demonstrated that compounds, with a number of carbon of five to ten in the linear structure, are preferentially oxidized by MAO-B [50]. In this context, PEBT has four linear or aliphatic carbons in its chemical structure, which could help to explain why PEBT did not inhibit MAO-B. 5. Conclusion In summary, the present results demonstrated an anxiolytic-like effect of PEBT in mice, which could be related to the inhibition of [3 H] 5-HT uptake and of MAO-A activity in cerebral cortices of mice. Further studies are needed to better understand the mechanisms involved in the pharmacological effect of PEBT. Conflict of interest The authors declare that they have no conflicts of interest to disclose. Acknowledgements The financial support by the Universidade Federal de Santa Maria (UFSM), Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), andFundac¸ão de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS–CNPq/PRONEX) research grant # 10/0005-41 and FAPERGS research grant # 10/0711-6 is gratefully acknowledged. C.W.N. is recipient of CNPq fellowship. J.T.R. is recipient of FAPERGS/CAPES fellowship SPI Process # 2793-25.51/12-4. References [1] Beaudoin-Gobert M, Sgambato-Faure V. Serotonergic pharmacology in animal models: from behavioral disorders to dyskinesia. Neuropharmacology 2014;81:15–30. [2] Argyropoulos SV, Sandford JJ, Nutt DJ. The psychobiology of anxiolytic drug. Part 2: pharmacological treatments of anxiety. Pharmacol Ther 2000;3:213–27. [3] Zohar J, Westenberg HG. Anxiety disorders: a review of tricyclic antidepressants and selective serotonin reuptake inhibitors. Acta Psychiatr Scand Suppl 2000;403:39–49. [4] Meyer JH, Ginovart N, Boovariwala A, Sagrati S, Hussey D, Garcia A, et al. Elevated monoamine oxidase A levels in the brain: an explanation for the monoamine imbalance of major depression. Arch Gen Psychiatry 2006;63:1209–16. [5] Hoyer D, Hannon JP, Martin GR. Molecular, pharmacological and functional diversity of 5-HT receptors. Pharmacol Biochem Behav 2002;71:533–54. [6] Wong M, Licinio J. Research and treatment approaches to depression. Nat Ver Neurosci 2001;2:343–51. [7] Wouters J. Structural aspects of monoamine oxidase and its reversible inhibition. Curr Med Chem 1998;5:137–62. [8] Robinson DS. Monoamine oxidase inhibitors: a new generation. Gen Psychopharmacol 2006;36:124–38. [9] Costa MJJ, Moyer M, O’Reardon JP. Monoamine oxidase inhibitors: an important but underutilized treatment. Psychopharm 2012;47:10. [10] Johnston JP. Some observations upon a new inhibitor of monoamine oxidase in brain tissue. Biochem Pharmacol 1968;17:1285–97. [11] Knoll J, Magyar K. Some puzzling pharmacological effects of monoamines oxidase inhibitors. Adv Biochem Psychopharmacol 1972;5:393–408. [12] Nolen WA, Hoencamp E, Bouvy PF, Haffmans PM. Reversible monoamine oxidase – a inhibitors in resistant major depression. Clin Neuropharmacol 1993;16(Suppl 2):S69–76. [13] Yamada M, Yasuhara H. Clinical pharmacology of MAO inhibitors: safety and future. Neurotoxicology 2004;25:215–21. [14] Briviba K, Tamler R, Klotz LO, Engman L, Cotgreave IA, Sies H. Protection against organotellurium compounds against peroxynitrite-mediated oxidation and nitration reactions. Biochem Pharmacol 1998;55:817–23.

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