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Diphenyl diselenide exerts anxiolytic-like effect in Wistar rats: Putative roles of GABAA and 5HT receptors
Progress in Neuro-sychopharmacology & Biological Psychiatry 32 (2008) 1508–1515
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Progress in Neuro-Psychopharmacology & Biological Psychiatry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p n p b p
Diphenyl diselenide exerts anxiolytic-like effect in Wistar rats: Putative roles of GABAA and 5HT receptors Gabriele Ghisleni a,⁎, Vanessa Kazlauckas b, Fernanda L. Both b, Natália Pagnussat b, Sabrina Mioranzza b, João Batista T. Rocha a, Diogo O. Souza b, Lisiane O. Porciúncula b a b
Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria, RS, CEP 97105-900, Brazil Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, CEP 90035-003, Brazil
A R T I C L E
I N F O
Article history: Received 18 December 2007 Received in revised form 8 May 2008 Accepted 12 May 2008 Available online 20 May 2008 Keywords: Anxiety Diphenyl diselenide Elevated plus maze GABAA receptor Open-field arena 5HT receptors
A B S T R A C T Diphenyl diselenide [(PhSe)2] is an organoselenium compound which presents pharmacological antioxidant, anti-inflammatory, antinociceptive and antidepressant properties. The present study was designed to investigate the anxiolytic effect of (PhSe)2 in rats, employing the elevated plus maze task. The involvement of 5HT and GABA receptors in the anxiolytic-like effect was also evaluated. (PhSe)2 (5, 25 and 50 µmol/kg, i.p.) did not affect locomotor activity as evaluated in the open open-field test, and learning and memory when assessed in the inhibitory foot-shock avoidance task. However, (PhSe)2 at the 50 μmol/kg dose produced signs of an anxiolytic action, namely a decreased number of fecal boli in the open-field arena and an increased time spent in as well as an increased number of entries to the open arms of the elevated plus maze test. To evaluate the role of GABA and 5HT receptors in the anxiolytic-like effect of (PhSe)2, a selective GABAA receptor antagonist bicuculline, (0.75 mg/kg, i.p.), a non-selective 5HT2A/2C receptor antagonist, ritanserin (2 mg/kg, i.p.), a selective 5HT2A receptor antagonist, ketanserin (1 mg/kg, i.p.), and a selective 5HT1A receptor antagonist, WAY100635 (0.1 mg/kg, i.p.) were used. All the antagonists used were able to abolish the anxiolytic effect of (PhSe)2 suggesting that GABAA and 5HT receptors may play a role in the pharmacological property of this selenocompound in the central nervous system. © 2008 Elsevier Inc. All rights reserved.
1. Introduction Selenium is an essential trace element nutritionally important to mammals, with physiological roles as a structural component of several antioxidant enzymes involved in the peroxide decomposition (Rayman, 2000; Ursini and Bindoli, 1987). Studies have reported that insufficient selenium intake may affect some psychological parameters and that selenium supplementation was found to be associated with an improvement in mood and depression status (Benton, 2001; Benton and Cook, 1991). A number of novel pharmaceutical agents derived from selenium or designed to influence specific aspects of selenium metabolism are under development due to a variety of organoselenium compounds that possess pharmacological activity (Nogueira et al., 2004, 2003a,b). In fact, organoselenium compounds have provided protection against Abbreviations: ANOVA, analysis of variance; BZs, benzodiazepines; DMSO, dimethyl sulfoxide; (PhSe)2, diphenyl diselenide; GABAA, γ-amino butyric acid; 5HT2A/2C and 5HT1A, serotonin receptors; i.p., intraperitoneal; s.c., subcutaneous. ⁎ Corresponding author. Universidade Federal do Rio Grande do Sul, Instituto de Ciências Básicas da Saúde, Departamento de Bioquímica, 90035-003, Porto Alegre/RS, Brazil. Tel.: +55 51 33085557; fax: +55 51 33085540. E-mail address: [email protected] (G. Ghisleni). 0278-5846/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2008.05.008
cell damage triggered by reactive oxygen species (ROS), being effective in the treatment of brain disorders where oxidative stress is strongly implicated (Chen and Berry, 2003; Schweizer et al., 2004). Among organoselenium compounds, diphenyl diselenide [(PhSe)2] presents similar pharmacological properties as antioxidant, anti-inflammatory and antinociceptive agent to the well known compound ebselen (Barbosa et al., 2006; Ghisleni et al., 2003; Nogueira et al., 2003b; Posser et al., 2006; Rossato et al., 2002; Wilson et al., 1989). Recently, studies have demonstrated antidepressant-like and anxiolytic-like effects of (PhSe)2 in mice via different pathways such as interaction with L-arginine/NO/cGMP pathway or central monoaminergic system (Savegnago et al., 2007b, 2008). Besides its beneficial pharmacological properties, (PhSe)2 at high concentrations also has a neurotoxic potential (Meotti et al., 2003; Nogueira et al., 2004). Accordingly, (PhSe)2 can modify some parameters of the glutamatergic system, promotes seizures, accelerates the onset of pentylenetetrazol-induced seizures and leads to death, depending on the route of administration and animal species (Brito et al., 2006; Moretto et al., 2007; Nogueira et al., 2003a, 2001). Additionally to the glutamatergic neurotransmission, the GABAergic system also seems to be involved in the (PhSe)2 effects, since treatment with GABA modulators minimized or even abolished
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(PhSe)2-induced seizures (Nogueira et al., 2003a). However, it is important to mention that (PhSe)2 does not induce toxic effects at doses in which it has pharmacological effects (Borges et al., 2006; Nogueira et al., 2004; Savegnago et al., 2007a; Zasso et al., 2005). The γ-amino butyric acid (GABA) system has long been targeted in anxiety disorders by enhancing GABAA receptors function with administration of benzodiazepines (BZs) (Morris et al., 2006). However, selective and non-selective modulation of different GABAA receptor subtypes by BZs are effective as short-term anxiolytics and their clinical use is limited due to several side effects including sedation, dependence and abuse (Atack, 2005; Griebel et al., 1999; Rowlett et al., 2005; Whiting, 2006). On the other hand, selective serotonin [5-hydroxytryptamine (5-HT)] reuptake inhibitors (SSRIs) have also been effective as anxiolytics for long-term therapy (Argyropoulos et al., 2000; Zohar and Westenberg, 2000), and it has been widely accepted that the regulation of fear and anxiety is also associated with the serotoninergic system. However, according to various pharmacological and molecular criteria, mechanisms by which different 5HT receptor subtypes can affect anxiety-related behaviours are still unclear (Graeff et al., 1997; Griebel, 1996). The present study was undertaken to investigate whether treatment with (PhSe)2 could modify the behaviour of the rats in the elevated plus maze task. Moreover, taking into account the involvement of GABAergic and serotoninergic systems in the pathophysiology of anxiety, we also investigated whether the effect observed by (PhSe)2 on the elevated plus maze task could involve interaction with GABAA, 5HT2A/2C and 5HT1A receptors.
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field arena (50 × 25 × 50 cm chamber made of brown polywood with a frontal glass wall). The floor was divided into 12 equal squares by black lines. Animals were allowed to explore the arena during 5 min. The number of crossings, rearings and fecal boli were used as measurement of locomotor activity, exploratory behaviour and a sign of anxiety, respectively. 2.4. Elevated plus maze task Animals were injected with (PhSe)2 (5, 25 or 50 µmol/kg, i.p.) or vehicle and evaluated in the elevated plus maze apparatus 30 min or 24 h after injection. Another group of animals was pre-administered with bicuculline (0.75 mg/kg, i.p., a selective GABAA receptor antagonist), ritanserin (2 mg/kg, i.p., a non-selective 5HT2A/2C receptor antagonist), ketanserin (1 mg/kg, i.p., a selective 5HT2A receptor antagonist) or WAY100635 (0.1 mg/kg, s.c., a selective 5HT1A receptor antagonist) 30 min before a single dose of (PhSe)2 (50 µmol/kg, i.p.). After 24 h of drugs administration, animals were submitted to elevated plus maze session. All antagonists tested were injected single-handedly in other set of animals 30 min before the elevated plus maze session as a control. As a positive control of the task, animals received diazepam (1 mg/kg, i.p., a GABAA receptor agonist) 15 min before the session. The elevated plus maze apparatus consists of two 30×5 cm open arms and two 30×5×15 cm closed arms, with an open roof, arranged such that the two arms of each type were opposite each other. The apparatus was a 50 cm from the ground. All 5 min sessions were conducted under dim red light. The following indexes of anxiety behaviour were measured: number of entries and the time spent in open and closed arms (in seconds) and rearings.
2. Methods 2.5. Inhibitory foot-shock avoidance (IA) 2.1. Drugs (PhSe)2 was synthesized as previously described (Paulmier, 1986). Bicuculline, ritanserin, ketanserin, WAY100635 and Tween 80 were obtained from Sigma (St. Louis, MO, USA). All other chemicals were of analytical grade. (PhSe)2 was suspended in 10% Tween 80, bicuculline was dissolved in a drop of glacial acetic acid, ritanserin in 10% DMSO, WAY100635 and ketanserin in saline (0.9%) and the final volume of all solutions was adjusted with saline (0.9%). Control treatments consisted of Tween 80 (10%), DMSO (10%) or a drop of glacial acetic acid as appropriate. The rats received all drugs in a constant volume of 1 ml/kg body weight. 2.2. Animals The behavioural experiments were conducted using male adult Wistar rats (280–350 g) maintained at 22–25 °C, under a 12:12 h light/ dark cycle. They were housed in plastic cages (5 animals per cage) with tap water and commercial food ad libitum (NUVILAB CR1 with 0.05 mg/kg of selenium). The animals were acclimated for 1 h before the experiments and all manipulations were carried out between 08.00 a.m. and 04.00 p.m. The animals were firstly analysed in the open-field arena and one week later in the elevated plus maze apparatus, but each animal was used only once in each test. Another set of animals was used for the inhibitory foot-shock avoidance task and for studying the interactions among drugs in the elevated plus maze apparatus. The doses and time of treatments were chosen according to previously reported (Maciel et al., 2000; Nogueira et al., 2001). The animals were used according to the guidelines of the Committee on Care and Use of Experimental Animal Resources. All efforts were made to minimize animals suffering and to reduce the number of animals used in the experiments.
The IA apparatus was a 50 × 25 × 25 cm acrylic box whose floor consisted of parallel stainless steel bars (1 mm diameter) spaced 1 cm apart. A 7-cm wide, 2.5-cm high platform was placed against the left wall of the box. In the training session, animals were placed on the platform and the latency to step-down onto the floor with the four paws was measured with an automatic device; immediately after stepping-down animals received a 0.5 mA, 2 s foot shock. In the test sessions (performed as training sessions), carried out 1.5 h (short short-term memory, STM) and 24 h (long long-term memory, LTM) after training, no foot shock was given and the step-down latency (180 s ceiling) was taken as a measure of memory. We evaluated both short- and long-term memories, because it has been shown that both memories are processed separately in the brain and consequently many drugs are able to modulate them in a quite different way (Izquierdo et al., 2006). (PhSe)2 (5, 25 and 50 μmol/kg, i.p.), diazepam (1 mg/kg, i.p., a GABAA receptor agonist) or vehicle was administered 30 min before the training session. 2.6. Statistical analysis Data for open-field arena and elevated plus maze are shown as mean ± S.E.M. Differences among control and (PhSe)2 groups were evaluated by using one-way ANOVA followed by Newman–Keuls Multiple Comparison test and for drug interactions by using two-way
Table 1 Effects of (PhSe)2 (5, 25 and 50 µmol/kg) administered 30 min before the open-field task Groups
Crossings
Rearings
Fecal boli
2.3. Exploration of an open-field arena
Control (PhSe)2 5 (PhSe)2 25 (PhSe)2 50
63 ± 9 68 ± 7 58 ± 6 72 ± 7
24 ± 4 23 ± 3 21 ± 3 21 ± 2
4.0 ± 0.8 5.0 ± 0.6 2.6 ± 0.8 3.5 ± 0.8
Animals received a single dose of (PhSe)2 (5, 25 or 50 µmol/kg, i.p.) and 30 min or 24 h after injection they were submitted to an open-
The data are expressed as mean ± S.E.M. n = 10 animals for each group. ⁎P b 0.05 compared with respective control.
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ANOVA followed by Bonferroni post-hoc test. Data for inhibitory footshock avoidance are shown as median values (interquartile ranges) of training and test latencies to step-down on the grid. Comparisons between groups were performed using a Kruskal–Wallis analysis of variance followed by Dunns pos hoc test. Differences were considered significant for P b 0.05. 3. Results 3.1. Effect of (PhSe)2 in the open-field task (PhSe)2 in all doses administered 30 min before open-field sessions did not affect the number of crossings, rearings and fecal boli (Table 1). However, when animals were exposed to open-field 24 h after 5 and 25 µmol/kg (PhSe)2, they presented an increase in the number of rearings (F (3, 37) =4.2, P b 0.01) (Fig. 1A). As showed in Fig. 1B, the animals treated with the highest dose of (PhSe)2 presented a decrease in the number of fecal boli (F (3, 40) = 5.3, P b 0.01), that may be interpreted as a first index of anxiolytic behaviour (Commissaris et al., 1986; Ferre et al., 1995). 3.2. Anxiolytic-like effect induced by (PhSe)2 on rats evaluated in the elevated plus maze task Based on the results of Fig. 1B, we investigated the effects of (PhSe)2 (5, 25 and 50 µmol/kg, i.p.) in the elevated plus maze apparatus, which is widely used for evaluating anxiolytic/anxiogenic behaviours (Lal and Emmett-Oglesby, 1983). When (PhSe)2 was administered in all doses 30 min before the session, no effect was observed on plus maze performance (Table 2). In fact, 24 h after administration 50 µmol/kg (PhSe)2 the animals showed typical anxiolytic behaviour in the plus
Fig. 1. Effect of (PhSe)2 (5, 25 and 50 µmol/kg, i.p.) after 24 h of treatment on the openfield task. Each column represents mean ± S.E.M. from 10–15 animals for each group. (A) Data are presented as number of crossings and rearings; (B) data are presented as number of fecal boli. Statistical analysis was performed by using One-Way ANOVA followed by Newman–Keuls Multiple Comparison test. ⁎P b 0.01 denotes a significant difference from control.
Table 2 Effects of (PhSe)2 (5, 25 and 50 µmol/kg) administered 30 min before the elevated plus maze task Groups
Time spent open arms
Time spent closed arms
N° entries open arms
N° entries closed arms
Rearing
Control (PhSe)2 5 (PhSe)2 25 (PhSe)2 50
17 ± 4 9.5 ± 2 13 ± 4 14 ± 4
83 ± 4 90 ± 2 87 ± 4 86 ± 4
4 ± 0.8 3 ± 0.7 2 ± 0.6 3 ± 0.7
4.5 ± 0.5 4 ± 0.5 3±1 5±1
9 ± 1.5 8±1 10 ± 1.6 13 ± 2.5
The data are expressed as mean ± S.E.M. n = 10 animals for each group. ⁎P b 0.05 compared with respective control.
maze apparatus such as increase in the time (F (4, 69)= 5.7, P b 0.001) and number of entries in the open arms (F (4, 70)= 4.9, P b 0.001) (Fig. 2A and B). As a positive control, diazepam (1 mg/kg, i.p., a GABAA receptor agonist) was administered 15 min before the session and the animals presented classical anxiolytic behaviour characterized by an enhance in the time spent in the open arms (F (4, 69) = 5.7, P b 0.001), while there was a decrease in time spent (F (4, 70)= 4.9, P b 0.01) and number of entries (F (4, 69) = 3.2, P b 0.05) in the closed arms (Fig. 2A and B). The number of rearings analysed in the elevated plus maze was not altered for the (PhSe)2 or diazepam treatment (data not shown). Since our results point to anxiolytic-like effects of (PhSe)2, further experiments were designed to investigate the role of neurotransmitter systems strictly involved in the anxiety behaviour.
Fig. 2. Effect of (PhSe)2 (5, 25 and 50 µmol/kg, i.p.) after 24 h of treatment on the elevated plus maze task. Diazepam (1 mg/kg) was injected 15 min before the session as a positive control. Each column represents mean ± S.E.M. from 10–15 animals for each group. (A) Data are presented as percentage of time spent in the open or closed arms; (B) data are presented as number of entries in the open or closed arms. Statistical analysis was performed by using One-Way ANOVA followed by Newman–Keuls Multiple Comparison test. ⁎⁎⁎P b 0.0005, ⁎⁎P b 0.001 and ⁎P b 0.05 denotes a significant difference from control.
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3.3. Role of the GABAergic system in the anxiolytic-like effect caused by (PhSe)2 in the elevated plus maze task Fig. 3A and B show that the pre-treatment of rats with bicuculline (0.75 mg/kg, i.p., a non-competitive GABAA receptor antagonist) 30 min before the administration of a single dose of 50 µmol/kg (PhSe)2 abolishes the anxiolytic effect of (PhSe)2. Two-way ANOVA revealed a main effect of the pre-treatment (F (1, 27) = 9.8, P b 0.01), treatment (F (1, 27) = 4.7, P b 0.05), and pre-treatment × treatment interactions (F (1, 27) = 7.5, P b 0.01) to the time spent in the open and closed arms (Fig. 3A). As shown in Fig. 3B, to the number of entries in the open arms, two-way ANOVA revealed a main effect of the pretreatment (F (1, 26) = 9.8, P b 0.01), treatment (F (1, 26) = 4.2, P b 0.05) and pre-treatment × treatment interactions (F (1, 26) = 9.7, P b 0.01). The number of entries in the closed arms and rearings analysed in the elevated plus maze was not altered for the treatment with (PhSe)2 alone or in combination with bicuculline.
Fig. 4. Effects of ritanserin (2 mg/kg, i.p.) and/or (PhSe)2 (50 µmol/kg, i.p.) on the elevated plus maze task. (A) Data are presented as percentage of time spent in the open or closed arms; (B) data are presented as number of entries in the open or closed arms. Ritanserin was administered 30 min before (PhSe)2 and the elevated plus maze task was performed 24 h after (PhSe)2 administration. Each column represents mean ± S.E.M. from 10 animals for each group. Statistical analysis was performed by Two-Way ANOVA followed by Bonferroni pos hoc test. ⁎P b 0.01 when compared to vehicle-treated control; # P b 0.001 as compared to the (PhSe)2 group pretreated with vehicle.
3.4. Role of the of serotoninergic system in the anxiolytic-like effect caused by (PhSe)2 in the elevated plus maze task
Fig. 3. Effects of bicuculline (0.75 mg/kg, i.p.) and/or (PhSe)2 (50 µmol/kg, i.p.) on the elevated plus maze task. (A) Data are presented as percentage of time spent in the open or closed arms; (B) data are presented as number of entries in the open or closed arms. Bicuculline was administered 30 min before (PhSe)2 and the elevated plus maze task was performed 24 h after (PhSe)2 administration. Each column represents mean ± S.E.M. from 10 animals for each group. Statistical analysis was performed by Two-Way ANOVA followed by Bonferroni pos hoc test. ⁎P b 0.05 when compared to vehicle-treated control; # P b 0.01 as compared to the (PhSe)2 group pretreated with vehicle.
All 5HT receptor antagonists used in this work were injected 30 min before the administration of a single dose of 50 µmol/kg (PhSe)2. As shown in Fig. 4A and B, pre-treatment with ritanserin (2 mg/kg, i.p., a nonselective 5HT2A/2C receptor antagonist) prevented the effect of (PhSe)2 in the time spent in both arms and number of entries in the open arms. Twoway ANOVA revealed a main effect of the pre-treatment (F (1, 32)=15.21, Pb 0.001), treatment (F (1, 32)=9.0, Pb 0.01) and pre-treatment×treatment interactions (F (1, 32)=6.7, P b 0.01) in the time spent in the open arms and a main effect of the pre-treatment (F (1, 30)=13.5, P b 0.001), treatment (F (1, 30)=7.6, Pb 0.01) and pre-treatment×treatment interactions (F (1, 30)=5.7, Pb 0.05) in the time spent in the closed arms (Fig. 4A). In relation to the number of entries in the open arms in Fig. 4B, two-way ANOVA revealed a main effect of the pre-treatment (F (1, 34)=21.0, Pb 0.0001), treatment (F (1, 34)=9.7, Pb 0.01) and pre-treatment×treatment interactions (F (1, 34)=5.3, Pb 0.05). The results depicted in Fig. 5A show that pre-treatment of animals with ketanserin (1 mg/kg i.p., a selective 5HT2A receptor antagonist)
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Fig. 5. Effects of ketanserin (1 mg/kg, i.p.) and/or (PhSe)2 (50 µmol/kg, i.p.) on the elevated plus maze task. (A) Data are presented as percentage of time spent in the open or closed arms; (B) data are presented as number of entries in the open or closed arms. ketanserin was administered 30 min before (PhSe)2 and the elevated plus maze task was performed 24 h after (PhSe)2 administration. Each column represents mean ± S.E.M. from 10 animals for each group. Statistical analysis was performed by Two-Way ANOVA followed by Bonferroni pos hoc test. ⁎P b 0.05 when compared to vehicle-treated control; # P b 0.001 as compared to the (PhSe)2 group pretreated with vehicle.
also inhibited the anxiolytic-like effect of (PhSe)2. Two-way ANOVA revealed a main effect of the pre-treatment (F (1, 35) = 13.7, P b 0.001), treatment (F (1, 35) = 4.45, P b 0.05) and pre-treatment × treatment interactions (F (1, 35) = 4.55, P b 0.05) for the time spent in the open arms and a main effect of the pre-treatment (F (1, 35) = 14.05, P b 0.001), treatment (F (1, 35) = 4.65, P b 0.05) and pre-treatment × treatment interactions (F (1, 35) = 4.77, P b 0.05) to the time spent in closed arms. Fig. 5B shows an influence of pre-treatment with ketanserin also in the number of entries in the open arms. Two-way ANOVA shows a main effect of the pre-treatment (F (1, 33) = 17.5, P b 0.001), treatment (F (1, 33) = 8.7, P b 0.01) and pre-treatment × treatment interactions (F (1, 33) = 4.7, P b 0.05). Ketanserin pre-treatment shows no alteration in the number of closed arms entries (Fig. 5B). The anxiolytic-like effect of (PhSe)2 was also significantly prevented by the pre-treatment with WAY100635 (0.1 mg/kg s.c., a selective 5HT1A receptor antagonist) as shown in Fig. 6A. Two-way ANOVA revealed a main effect of the pre-treatment (F (1, 32)=7.4, Pb 0.01), treatment (F (1, 32)=4.3, Pb 0.05) and pre-treatment×treatment interactions (F (1, 32)= 7.5, Pb 0.01) on time spent in the open arms and a main effect of the pre-
Fig. 6. Effects of WAY100635 (0.1 mg/kg, s.c.) and/or (PhSe)2 (50 µmol/kg, i.p.) on the elevated plus maze task. (A) Data are presented as percentage of time spent in the open or closed arms; (B) data are presented as number of entries in the open or closed arms. WAY100635 was administered 30 min before (PhSe)2 and the elevated plus maze task was performed 24 h after (PhSe)2 administration. Each column represents mean ± S.E.M. from 10 animals for each group. Statistical analysis was performed by Two-Way ANOVA followed by Bonferroni pos hoc test. ⁎P b 0.05 when compared to vehicle-treated control; # P b 0.05 as compared to the (PhSe)2 group pretreated with vehicle.
Fig. 7. Effect of diazepam (1 mg/kg, i.p.) administered 30 min before training session in a step-down inhibitory avoidance task. Data are expressed as median (interquartile ranges) (in seconds) of training (black columns), 1.5 h test (gray columns) or 24 h test (white columns) session latencies from 10 animals for each group. Statistical analysis was performed by using Kruskal–Wallis followed by Dunn pos hoc test. ⁎P b 0.05 denotes a significant difference from control.
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4. Discussion
Fig. 8. Effect of (PhSe)2 (5, 25 and 50 µmol/kg, i.p.) administered 30 min before training session in a step-down inhibitory avoidance task. Latency to step-down: (A) test after 1.5 h (STM); (B) test after 24 h (LTM) of training session. Data are expressed as median (interquartile ranges) (in seconds) of training (dark columns) or test (light columns) session latencies from 10 animals for each group. Statistical analysis was performed by using Kruskal–Wallis followed by Dunn pos hoc test. ⁎P b 0.05 denotes a significant difference from control.
treatment (F (1, 32)=7.9, P b 0.01), treatment (F (1, 32)=4.5, Pb 0.05) and pre-treatment×treatment interactions (F (1, 32)=7.9, Pb 0.01) on time spent in the closed arms. In Fig. 6B, the analysis by two-way ANOVA revealed a main effect of pre-treatment (F (1, 33)=8.4, Pb 0.01), treatment (F (1, 33)=10.5, Pb 0.01), but not pre-treatment×treatment interactions (F (1, 33)=2.3, P=0.135) in the number of open arms entries. The pretreatment with WAY100635 showed no alteration in the number of closed arms entries (Fig. 6B). In all experiments carried out with (PhSe)2 and/or 5HT receptor antagonists pre-treatments, the number of rearings was not changed when analysed in the elevated plus maze task (data not shown). 3.5. Effect of (PhSe)2 in the inhibitory foot-shock avoidance task It has been reported that the effects of benzodiazepines (BZs) on acquisition of information might be partly linked to their sedative effects (Clement and Chapouthier, 1998). In Fig. 7 we confirmed data from the literature showing that diazepam (1 mg/kg, i.p., a GABAA receptor agonist) impairs memory acquisition when administered 30 min before training session in a step-down inhibitory foot-shock avoidance task (P b 0.05). Thus, (PhSe)2 (5, 25 and 50 µmol/kg i.p.,) was administered 30 min before the training session to assess possible side effects of this drug on memory in a step-down inhibitory foot-shock avoidance task. We can observe that all doses of (PhSe)2 administered did not impair the STM and LTM performance in the step-down inhibitory foot-shock avoidance task, which excludes any effect of this drug on both memory processes (Fig. 8A and B).
In the present study, systemic administration of (PhSe)2 in rats had a pronounced anxiolytic-like effect, with no alteration on locomotor and exploratory activities in the open-field arena. This parameter deserves attention in the behavioural studies concerning new drugs because alterations in the locomotor activity of the animals usually may non-specifically affect other behavioural tasks. Our results corroborate with some data reported by Machado et al. (2006), where systemic administration of a substituted (PhSe)2 induces an antipsychotic-like effect without affecting the open-field behaviour or memory in mice. In addition, classical antipsychotics have been used by clinicians in the treatment of affective disorders, anxiety, restlessness and agitation (Kaplan, 2006). More recently, Savegnago et al. (2008), demonstrated that oral administration of (PhSe)2 after 30 min produced antidepressant- and anxiolytic-like effects in mice, with no alteration on parameters of locomotor and exploratory activities. Moreover, it is important to mention that differently of Savegnago et al. (2007b, 2008) where rats and mice received oral doses of (PhSe)2, our data demonstrated a later anxiolytic-like effect of (PhSe)2. Since previous studies reported diverse toxicity potential by (PhSe)2 in different experimental models (Nogueira et al., 2003a), the anxiolytic effect showed in our work is probably due to the distinct route of administration as well the vehicle solution where (PhSe)2 is dissolved, that can be responsible to the distinctive rate of absorption and metabolism of the compound in liver. However, in this study the doses of (PhSe)2 administered did not induce toxic effects as previously demonstrated by Nogueira et al. (2003a). The first evidence that (PhSe)2 can trigger anxiolytic effect was a decrease in the number of defecation in the open-field arena. However, considering that the number of defecations usually presents a slight increase merely by exposure of animals to a new environment, this assumption was further reinforced by the observation that this compound was also able to increase the time spent in the open arms in the elevated plus maze task, which represent a strong indicative of anxiolytic effect. Although, a similar effect of (PhSe)2 was previously described in mice, the mechanisms underlying this anxiolytic effect was not evaluated yet (Savegnago et al., 2008). Thus, to better characterize the anxiolytic-like effects of (PhSe)2 by using the elevated plus maze apparatus, we also selected drugs that selectively modulate GABA and 5HT neurotransmitter systems, which are commonly involved in the physiopathology and treatment of anxiety and depressive disorders (Nutt, 2001). Although anxiety is a complex psychological and behavioural trait that can involve multiples receptors (D2, 5HT, GABA, NMDA), the mechanisms by which the neural systems can be sensitized are not fully understood. In this work our results showed that modulation of more than one neuronal system can account for the anxiolytic-like effect of (PhSe)2. Our data are in agreement with previous studies where pharmacological profile as well toxic potential of this compound occurs by modulation of different neurotransmitter systems (Nogueira et al., 2003a; Savegnago et al., 2007b, 2008). Interestingly, it has been pointed that the GABAergic system also participate of the toxic effects mediated by (PhSe)2, since seizures induced by high concentrations of this compound were prevented by diazepam, phenobarbital and muscimol (Nogueira et al., 2003a). Likewise, (PhSe)2 increased the potency of PTZinduced seizures and mortality in mice (Brito et al., 2006). Here we demonstrated the involvement of GABAergic system in the anxiolytic-like effect by (PhSe)2 since pre-treatment with bicuculline, the selective GABAA receptor antagonist, consistently reversed the anxiolytic effects of this selenium compound. Furthermore, studies have demonstrated that the control of anxiety occurs through GABAA receptors modulation, since blockage of presynaptic GABAB receptor is able to increase GABA release, which in turn affects GABAA receptors reducing anxiety (Zarrindast et al., 2001). On the other hand, abnormalities in 5HT neurotransmission have been implicated in the etiology of several psychiatric and neurological disorders as previously described to depression, anxiety and mood
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(Hoyer et al., 2002; Wong and Licinio, 2001). Moreover, the 5HT2A/2C and 5HT1A receptor subtypes are especially relevant for this discussion, given its implication in both anxiety and depression regulation (Beneytez et al., 1998; Fletcher et al., 1996), apart from the fact of they are involved on the antidepressant-like effect of (PhSe)2 (Savegnago et al., 2007b). An evidence of the involvement of 5HT2A/2C receptors in the anxiolytic-like effect of (PhSe)2 was given by the finding that pre-treatment of rats with ritanserin (a non-selective 5HT2A/2C receptor antagonist) and ketanserin (a selective 5HT2A receptor antagonist), were able to prevent the increase in the time spent and number of entries in the open arms in the elevated plus maze task. Although, there are some discrepancies whether acute administration of ritanserin may cause anxiolytic, anxiogenic or no effect at all (Barnes et al.,1992; Griebel et al.,1997; Stutzmann et al.,1991), in our study ritanserin did not affect the performance of rats in the elevated plus maze task, but precluded anxiolytic-like behaviour caused by (PhSe)2. Another interesting finding consisted on the anxiolytic-like effect of (PhSe)2 on the elevated plus maze task have has been abolished by the pre-treatment with WAY100635, a selective 5HT1A receptor antagonist. Recently, Savegnago et al. (2007b) demonstrated that the 5HT1A receptor is involved in the antidepressant effect of (PhSe)2 in the forced swimming test. Although, sufficient and significant differences have been described between anxiety and depression, supporting the view that they are independent psychiatric disorders, sharing abnormalities in the 5HT1A receptor function may explain some of the comorbidity of these disorders (Nutt and Stein, 2006). Taking into account the anxiolytic-like effect of (PhSe)2 showed here and its antidepressant-like effect previously described, we could suggest that the same neurotransmitter mechanisms can be involved in the pathophysiology of psychiatric disorders, as an important point to address the comorbid of depression and anxiety experienced by many patients (Davidson, 2001). The current study showed also that the anxiolytic-like effect produced by (PhSe)2 is comparable with diazepam, a classical anxiolytic drug. However, as BZs present common side effects as sedation and amnesia (Atack, 2005; Rudolph, 2001; Salzman, 1993), it is extremely relevant to the exploration of new therapeutic targets for treatment of anxiety disorders. As our results pointed to involvement of GABAA neurotransmission in the anxiolytic action of (PhSe)2 and by the fact that systemic administration of BZs can induce anterograde amnesia which might be partly linked to their sedative effect (Cain, 1997), we decide to investigate the action of (PhSe)2 on learning and memory task. Our data revealed absence of (PhSe)2 effects when both short- and long-term memory were evaluated, suggesting that at least in our behavioural model (PhSe)2 seems to interact with the GABAergic system in a different way than BZs. Furthermore, taking into account our results that anxiolytic-like effect of (PhSe)2 also involves the serotoninergic system it's important to note that changes in the serotoninergic transmission can interfere with learning acquisition and memory consolidation, since 5HT1 and 5HT2 receptors are present in brain structures implicated in learning and memory processes (Breese et al., 2002). 5. Conclusion The results presented here showed pharmacological evidences of (PhSe)2 anxiolytic-like effect in rats, and the absence of undesirable effects such as alterations in the locomotor activity or memory, confirming the assumption that this effect can be specific and suggesting that (PhSe)2 could be a novel potential anxiolytic drug with a prospective of minimal side effects. We also clarify here the involvement of 5HT2A/2C, 5HT1A and GABAA receptors as potential mechanisms underlying the pharmacological actions of (PhSe)2 as an anxiolytic drug. Acknowledgements This study was supported in part by the FINEP research grant “Rede Instituto Brasileiro de Neurociência (IBN-Net)” # 01.06.0842-00 and by grants from the Brazilian National Research Council CNPq, CAPES
and PRONEX. Another part of study was supported by FAPERGS (Proc. No. 0521850 to Lisiane O. Porciúncula) and Grants from L'oreal Prize “Women in Science 2006” (Lisiane O. Porciúncula). Gabriele Ghisleni is supported by a CAPES fellowship. References Argyropoulos SV, Sandford JJ, Nutt DJ. The psychobiology of anxiolytic drug. Part 2: pharmacological treatments of anxiety. Pharmacol Ther 2000;3:213–27. Atack JR. The benzodiazepine binding site of GABA(A) receptors as a target for the development of novel anxiolytics. Exp Opin Investig Drugs 2005;14:601–18. Barbosa NB, Rocha JB, Wondracek DC, Perottoni J, Zeni G, Nogueira CW. Diphenyl diselenide reduces temporarily hyperglycemia: possible relationship with oxidative stress. Chem Biol Interact 2006;163:230–8. Barnes NM, Cheng CH, Costall B, Ge J, Kelly ME, Naylor RJ. Profiles of interaction of R(+)/S (−)-zacopride and anxiolytic agents in a mouse model. Eur J Pharmacol 1992;218: 91–100. 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Progress in Neuro-Psychopharmacology & Biological Psychiatry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p n p b p
Diphenyl diselenide exerts anxiolytic-like effect in Wistar rats: Putative roles of GABAA and 5HT receptors Gabriele Ghisleni a,⁎, Vanessa Kazlauckas b, Fernanda L. Both b, Natália Pagnussat b, Sabrina Mioranzza b, João Batista T. Rocha a, Diogo O. Souza b, Lisiane O. Porciúncula b a b
Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria, RS, CEP 97105-900, Brazil Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, CEP 90035-003, Brazil
A R T I C L E
I N F O
Article history: Received 18 December 2007 Received in revised form 8 May 2008 Accepted 12 May 2008 Available online 20 May 2008 Keywords: Anxiety Diphenyl diselenide Elevated plus maze GABAA receptor Open-field arena 5HT receptors
A B S T R A C T Diphenyl diselenide [(PhSe)2] is an organoselenium compound which presents pharmacological antioxidant, anti-inflammatory, antinociceptive and antidepressant properties. The present study was designed to investigate the anxiolytic effect of (PhSe)2 in rats, employing the elevated plus maze task. The involvement of 5HT and GABA receptors in the anxiolytic-like effect was also evaluated. (PhSe)2 (5, 25 and 50 µmol/kg, i.p.) did not affect locomotor activity as evaluated in the open open-field test, and learning and memory when assessed in the inhibitory foot-shock avoidance task. However, (PhSe)2 at the 50 μmol/kg dose produced signs of an anxiolytic action, namely a decreased number of fecal boli in the open-field arena and an increased time spent in as well as an increased number of entries to the open arms of the elevated plus maze test. To evaluate the role of GABA and 5HT receptors in the anxiolytic-like effect of (PhSe)2, a selective GABAA receptor antagonist bicuculline, (0.75 mg/kg, i.p.), a non-selective 5HT2A/2C receptor antagonist, ritanserin (2 mg/kg, i.p.), a selective 5HT2A receptor antagonist, ketanserin (1 mg/kg, i.p.), and a selective 5HT1A receptor antagonist, WAY100635 (0.1 mg/kg, i.p.) were used. All the antagonists used were able to abolish the anxiolytic effect of (PhSe)2 suggesting that GABAA and 5HT receptors may play a role in the pharmacological property of this selenocompound in the central nervous system. © 2008 Elsevier Inc. All rights reserved.
1. Introduction Selenium is an essential trace element nutritionally important to mammals, with physiological roles as a structural component of several antioxidant enzymes involved in the peroxide decomposition (Rayman, 2000; Ursini and Bindoli, 1987). Studies have reported that insufficient selenium intake may affect some psychological parameters and that selenium supplementation was found to be associated with an improvement in mood and depression status (Benton, 2001; Benton and Cook, 1991). A number of novel pharmaceutical agents derived from selenium or designed to influence specific aspects of selenium metabolism are under development due to a variety of organoselenium compounds that possess pharmacological activity (Nogueira et al., 2004, 2003a,b). In fact, organoselenium compounds have provided protection against Abbreviations: ANOVA, analysis of variance; BZs, benzodiazepines; DMSO, dimethyl sulfoxide; (PhSe)2, diphenyl diselenide; GABAA, γ-amino butyric acid; 5HT2A/2C and 5HT1A, serotonin receptors; i.p., intraperitoneal; s.c., subcutaneous. ⁎ Corresponding author. Universidade Federal do Rio Grande do Sul, Instituto de Ciências Básicas da Saúde, Departamento de Bioquímica, 90035-003, Porto Alegre/RS, Brazil. Tel.: +55 51 33085557; fax: +55 51 33085540. E-mail address: [email protected] (G. Ghisleni). 0278-5846/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2008.05.008
cell damage triggered by reactive oxygen species (ROS), being effective in the treatment of brain disorders where oxidative stress is strongly implicated (Chen and Berry, 2003; Schweizer et al., 2004). Among organoselenium compounds, diphenyl diselenide [(PhSe)2] presents similar pharmacological properties as antioxidant, anti-inflammatory and antinociceptive agent to the well known compound ebselen (Barbosa et al., 2006; Ghisleni et al., 2003; Nogueira et al., 2003b; Posser et al., 2006; Rossato et al., 2002; Wilson et al., 1989). Recently, studies have demonstrated antidepressant-like and anxiolytic-like effects of (PhSe)2 in mice via different pathways such as interaction with L-arginine/NO/cGMP pathway or central monoaminergic system (Savegnago et al., 2007b, 2008). Besides its beneficial pharmacological properties, (PhSe)2 at high concentrations also has a neurotoxic potential (Meotti et al., 2003; Nogueira et al., 2004). Accordingly, (PhSe)2 can modify some parameters of the glutamatergic system, promotes seizures, accelerates the onset of pentylenetetrazol-induced seizures and leads to death, depending on the route of administration and animal species (Brito et al., 2006; Moretto et al., 2007; Nogueira et al., 2003a, 2001). Additionally to the glutamatergic neurotransmission, the GABAergic system also seems to be involved in the (PhSe)2 effects, since treatment with GABA modulators minimized or even abolished
G. Ghisleni et al. / Progress in Neuro-sychopharmacology & Biological Psychiatry 32 (2008) 1508–1515
(PhSe)2-induced seizures (Nogueira et al., 2003a). However, it is important to mention that (PhSe)2 does not induce toxic effects at doses in which it has pharmacological effects (Borges et al., 2006; Nogueira et al., 2004; Savegnago et al., 2007a; Zasso et al., 2005). The γ-amino butyric acid (GABA) system has long been targeted in anxiety disorders by enhancing GABAA receptors function with administration of benzodiazepines (BZs) (Morris et al., 2006). However, selective and non-selective modulation of different GABAA receptor subtypes by BZs are effective as short-term anxiolytics and their clinical use is limited due to several side effects including sedation, dependence and abuse (Atack, 2005; Griebel et al., 1999; Rowlett et al., 2005; Whiting, 2006). On the other hand, selective serotonin [5-hydroxytryptamine (5-HT)] reuptake inhibitors (SSRIs) have also been effective as anxiolytics for long-term therapy (Argyropoulos et al., 2000; Zohar and Westenberg, 2000), and it has been widely accepted that the regulation of fear and anxiety is also associated with the serotoninergic system. However, according to various pharmacological and molecular criteria, mechanisms by which different 5HT receptor subtypes can affect anxiety-related behaviours are still unclear (Graeff et al., 1997; Griebel, 1996). The present study was undertaken to investigate whether treatment with (PhSe)2 could modify the behaviour of the rats in the elevated plus maze task. Moreover, taking into account the involvement of GABAergic and serotoninergic systems in the pathophysiology of anxiety, we also investigated whether the effect observed by (PhSe)2 on the elevated plus maze task could involve interaction with GABAA, 5HT2A/2C and 5HT1A receptors.
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field arena (50 × 25 × 50 cm chamber made of brown polywood with a frontal glass wall). The floor was divided into 12 equal squares by black lines. Animals were allowed to explore the arena during 5 min. The number of crossings, rearings and fecal boli were used as measurement of locomotor activity, exploratory behaviour and a sign of anxiety, respectively. 2.4. Elevated plus maze task Animals were injected with (PhSe)2 (5, 25 or 50 µmol/kg, i.p.) or vehicle and evaluated in the elevated plus maze apparatus 30 min or 24 h after injection. Another group of animals was pre-administered with bicuculline (0.75 mg/kg, i.p., a selective GABAA receptor antagonist), ritanserin (2 mg/kg, i.p., a non-selective 5HT2A/2C receptor antagonist), ketanserin (1 mg/kg, i.p., a selective 5HT2A receptor antagonist) or WAY100635 (0.1 mg/kg, s.c., a selective 5HT1A receptor antagonist) 30 min before a single dose of (PhSe)2 (50 µmol/kg, i.p.). After 24 h of drugs administration, animals were submitted to elevated plus maze session. All antagonists tested were injected single-handedly in other set of animals 30 min before the elevated plus maze session as a control. As a positive control of the task, animals received diazepam (1 mg/kg, i.p., a GABAA receptor agonist) 15 min before the session. The elevated plus maze apparatus consists of two 30×5 cm open arms and two 30×5×15 cm closed arms, with an open roof, arranged such that the two arms of each type were opposite each other. The apparatus was a 50 cm from the ground. All 5 min sessions were conducted under dim red light. The following indexes of anxiety behaviour were measured: number of entries and the time spent in open and closed arms (in seconds) and rearings.
2. Methods 2.5. Inhibitory foot-shock avoidance (IA) 2.1. Drugs (PhSe)2 was synthesized as previously described (Paulmier, 1986). Bicuculline, ritanserin, ketanserin, WAY100635 and Tween 80 were obtained from Sigma (St. Louis, MO, USA). All other chemicals were of analytical grade. (PhSe)2 was suspended in 10% Tween 80, bicuculline was dissolved in a drop of glacial acetic acid, ritanserin in 10% DMSO, WAY100635 and ketanserin in saline (0.9%) and the final volume of all solutions was adjusted with saline (0.9%). Control treatments consisted of Tween 80 (10%), DMSO (10%) or a drop of glacial acetic acid as appropriate. The rats received all drugs in a constant volume of 1 ml/kg body weight. 2.2. Animals The behavioural experiments were conducted using male adult Wistar rats (280–350 g) maintained at 22–25 °C, under a 12:12 h light/ dark cycle. They were housed in plastic cages (5 animals per cage) with tap water and commercial food ad libitum (NUVILAB CR1 with 0.05 mg/kg of selenium). The animals were acclimated for 1 h before the experiments and all manipulations were carried out between 08.00 a.m. and 04.00 p.m. The animals were firstly analysed in the open-field arena and one week later in the elevated plus maze apparatus, but each animal was used only once in each test. Another set of animals was used for the inhibitory foot-shock avoidance task and for studying the interactions among drugs in the elevated plus maze apparatus. The doses and time of treatments were chosen according to previously reported (Maciel et al., 2000; Nogueira et al., 2001). The animals were used according to the guidelines of the Committee on Care and Use of Experimental Animal Resources. All efforts were made to minimize animals suffering and to reduce the number of animals used in the experiments.
The IA apparatus was a 50 × 25 × 25 cm acrylic box whose floor consisted of parallel stainless steel bars (1 mm diameter) spaced 1 cm apart. A 7-cm wide, 2.5-cm high platform was placed against the left wall of the box. In the training session, animals were placed on the platform and the latency to step-down onto the floor with the four paws was measured with an automatic device; immediately after stepping-down animals received a 0.5 mA, 2 s foot shock. In the test sessions (performed as training sessions), carried out 1.5 h (short short-term memory, STM) and 24 h (long long-term memory, LTM) after training, no foot shock was given and the step-down latency (180 s ceiling) was taken as a measure of memory. We evaluated both short- and long-term memories, because it has been shown that both memories are processed separately in the brain and consequently many drugs are able to modulate them in a quite different way (Izquierdo et al., 2006). (PhSe)2 (5, 25 and 50 μmol/kg, i.p.), diazepam (1 mg/kg, i.p., a GABAA receptor agonist) or vehicle was administered 30 min before the training session. 2.6. Statistical analysis Data for open-field arena and elevated plus maze are shown as mean ± S.E.M. Differences among control and (PhSe)2 groups were evaluated by using one-way ANOVA followed by Newman–Keuls Multiple Comparison test and for drug interactions by using two-way
Table 1 Effects of (PhSe)2 (5, 25 and 50 µmol/kg) administered 30 min before the open-field task Groups
Crossings
Rearings
Fecal boli
2.3. Exploration of an open-field arena
Control (PhSe)2 5 (PhSe)2 25 (PhSe)2 50
63 ± 9 68 ± 7 58 ± 6 72 ± 7
24 ± 4 23 ± 3 21 ± 3 21 ± 2
4.0 ± 0.8 5.0 ± 0.6 2.6 ± 0.8 3.5 ± 0.8
Animals received a single dose of (PhSe)2 (5, 25 or 50 µmol/kg, i.p.) and 30 min or 24 h after injection they were submitted to an open-
The data are expressed as mean ± S.E.M. n = 10 animals for each group. ⁎P b 0.05 compared with respective control.
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ANOVA followed by Bonferroni post-hoc test. Data for inhibitory footshock avoidance are shown as median values (interquartile ranges) of training and test latencies to step-down on the grid. Comparisons between groups were performed using a Kruskal–Wallis analysis of variance followed by Dunns pos hoc test. Differences were considered significant for P b 0.05. 3. Results 3.1. Effect of (PhSe)2 in the open-field task (PhSe)2 in all doses administered 30 min before open-field sessions did not affect the number of crossings, rearings and fecal boli (Table 1). However, when animals were exposed to open-field 24 h after 5 and 25 µmol/kg (PhSe)2, they presented an increase in the number of rearings (F (3, 37) =4.2, P b 0.01) (Fig. 1A). As showed in Fig. 1B, the animals treated with the highest dose of (PhSe)2 presented a decrease in the number of fecal boli (F (3, 40) = 5.3, P b 0.01), that may be interpreted as a first index of anxiolytic behaviour (Commissaris et al., 1986; Ferre et al., 1995). 3.2. Anxiolytic-like effect induced by (PhSe)2 on rats evaluated in the elevated plus maze task Based on the results of Fig. 1B, we investigated the effects of (PhSe)2 (5, 25 and 50 µmol/kg, i.p.) in the elevated plus maze apparatus, which is widely used for evaluating anxiolytic/anxiogenic behaviours (Lal and Emmett-Oglesby, 1983). When (PhSe)2 was administered in all doses 30 min before the session, no effect was observed on plus maze performance (Table 2). In fact, 24 h after administration 50 µmol/kg (PhSe)2 the animals showed typical anxiolytic behaviour in the plus
Fig. 1. Effect of (PhSe)2 (5, 25 and 50 µmol/kg, i.p.) after 24 h of treatment on the openfield task. Each column represents mean ± S.E.M. from 10–15 animals for each group. (A) Data are presented as number of crossings and rearings; (B) data are presented as number of fecal boli. Statistical analysis was performed by using One-Way ANOVA followed by Newman–Keuls Multiple Comparison test. ⁎P b 0.01 denotes a significant difference from control.
Table 2 Effects of (PhSe)2 (5, 25 and 50 µmol/kg) administered 30 min before the elevated plus maze task Groups
Time spent open arms
Time spent closed arms
N° entries open arms
N° entries closed arms
Rearing
Control (PhSe)2 5 (PhSe)2 25 (PhSe)2 50
17 ± 4 9.5 ± 2 13 ± 4 14 ± 4
83 ± 4 90 ± 2 87 ± 4 86 ± 4
4 ± 0.8 3 ± 0.7 2 ± 0.6 3 ± 0.7
4.5 ± 0.5 4 ± 0.5 3±1 5±1
9 ± 1.5 8±1 10 ± 1.6 13 ± 2.5
The data are expressed as mean ± S.E.M. n = 10 animals for each group. ⁎P b 0.05 compared with respective control.
maze apparatus such as increase in the time (F (4, 69)= 5.7, P b 0.001) and number of entries in the open arms (F (4, 70)= 4.9, P b 0.001) (Fig. 2A and B). As a positive control, diazepam (1 mg/kg, i.p., a GABAA receptor agonist) was administered 15 min before the session and the animals presented classical anxiolytic behaviour characterized by an enhance in the time spent in the open arms (F (4, 69) = 5.7, P b 0.001), while there was a decrease in time spent (F (4, 70)= 4.9, P b 0.01) and number of entries (F (4, 69) = 3.2, P b 0.05) in the closed arms (Fig. 2A and B). The number of rearings analysed in the elevated plus maze was not altered for the (PhSe)2 or diazepam treatment (data not shown). Since our results point to anxiolytic-like effects of (PhSe)2, further experiments were designed to investigate the role of neurotransmitter systems strictly involved in the anxiety behaviour.
Fig. 2. Effect of (PhSe)2 (5, 25 and 50 µmol/kg, i.p.) after 24 h of treatment on the elevated plus maze task. Diazepam (1 mg/kg) was injected 15 min before the session as a positive control. Each column represents mean ± S.E.M. from 10–15 animals for each group. (A) Data are presented as percentage of time spent in the open or closed arms; (B) data are presented as number of entries in the open or closed arms. Statistical analysis was performed by using One-Way ANOVA followed by Newman–Keuls Multiple Comparison test. ⁎⁎⁎P b 0.0005, ⁎⁎P b 0.001 and ⁎P b 0.05 denotes a significant difference from control.
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3.3. Role of the GABAergic system in the anxiolytic-like effect caused by (PhSe)2 in the elevated plus maze task Fig. 3A and B show that the pre-treatment of rats with bicuculline (0.75 mg/kg, i.p., a non-competitive GABAA receptor antagonist) 30 min before the administration of a single dose of 50 µmol/kg (PhSe)2 abolishes the anxiolytic effect of (PhSe)2. Two-way ANOVA revealed a main effect of the pre-treatment (F (1, 27) = 9.8, P b 0.01), treatment (F (1, 27) = 4.7, P b 0.05), and pre-treatment × treatment interactions (F (1, 27) = 7.5, P b 0.01) to the time spent in the open and closed arms (Fig. 3A). As shown in Fig. 3B, to the number of entries in the open arms, two-way ANOVA revealed a main effect of the pretreatment (F (1, 26) = 9.8, P b 0.01), treatment (F (1, 26) = 4.2, P b 0.05) and pre-treatment × treatment interactions (F (1, 26) = 9.7, P b 0.01). The number of entries in the closed arms and rearings analysed in the elevated plus maze was not altered for the treatment with (PhSe)2 alone or in combination with bicuculline.
Fig. 4. Effects of ritanserin (2 mg/kg, i.p.) and/or (PhSe)2 (50 µmol/kg, i.p.) on the elevated plus maze task. (A) Data are presented as percentage of time spent in the open or closed arms; (B) data are presented as number of entries in the open or closed arms. Ritanserin was administered 30 min before (PhSe)2 and the elevated plus maze task was performed 24 h after (PhSe)2 administration. Each column represents mean ± S.E.M. from 10 animals for each group. Statistical analysis was performed by Two-Way ANOVA followed by Bonferroni pos hoc test. ⁎P b 0.01 when compared to vehicle-treated control; # P b 0.001 as compared to the (PhSe)2 group pretreated with vehicle.
3.4. Role of the of serotoninergic system in the anxiolytic-like effect caused by (PhSe)2 in the elevated plus maze task
Fig. 3. Effects of bicuculline (0.75 mg/kg, i.p.) and/or (PhSe)2 (50 µmol/kg, i.p.) on the elevated plus maze task. (A) Data are presented as percentage of time spent in the open or closed arms; (B) data are presented as number of entries in the open or closed arms. Bicuculline was administered 30 min before (PhSe)2 and the elevated plus maze task was performed 24 h after (PhSe)2 administration. Each column represents mean ± S.E.M. from 10 animals for each group. Statistical analysis was performed by Two-Way ANOVA followed by Bonferroni pos hoc test. ⁎P b 0.05 when compared to vehicle-treated control; # P b 0.01 as compared to the (PhSe)2 group pretreated with vehicle.
All 5HT receptor antagonists used in this work were injected 30 min before the administration of a single dose of 50 µmol/kg (PhSe)2. As shown in Fig. 4A and B, pre-treatment with ritanserin (2 mg/kg, i.p., a nonselective 5HT2A/2C receptor antagonist) prevented the effect of (PhSe)2 in the time spent in both arms and number of entries in the open arms. Twoway ANOVA revealed a main effect of the pre-treatment (F (1, 32)=15.21, Pb 0.001), treatment (F (1, 32)=9.0, Pb 0.01) and pre-treatment×treatment interactions (F (1, 32)=6.7, P b 0.01) in the time spent in the open arms and a main effect of the pre-treatment (F (1, 30)=13.5, P b 0.001), treatment (F (1, 30)=7.6, Pb 0.01) and pre-treatment×treatment interactions (F (1, 30)=5.7, Pb 0.05) in the time spent in the closed arms (Fig. 4A). In relation to the number of entries in the open arms in Fig. 4B, two-way ANOVA revealed a main effect of the pre-treatment (F (1, 34)=21.0, Pb 0.0001), treatment (F (1, 34)=9.7, Pb 0.01) and pre-treatment×treatment interactions (F (1, 34)=5.3, Pb 0.05). The results depicted in Fig. 5A show that pre-treatment of animals with ketanserin (1 mg/kg i.p., a selective 5HT2A receptor antagonist)
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Fig. 5. Effects of ketanserin (1 mg/kg, i.p.) and/or (PhSe)2 (50 µmol/kg, i.p.) on the elevated plus maze task. (A) Data are presented as percentage of time spent in the open or closed arms; (B) data are presented as number of entries in the open or closed arms. ketanserin was administered 30 min before (PhSe)2 and the elevated plus maze task was performed 24 h after (PhSe)2 administration. Each column represents mean ± S.E.M. from 10 animals for each group. Statistical analysis was performed by Two-Way ANOVA followed by Bonferroni pos hoc test. ⁎P b 0.05 when compared to vehicle-treated control; # P b 0.001 as compared to the (PhSe)2 group pretreated with vehicle.
also inhibited the anxiolytic-like effect of (PhSe)2. Two-way ANOVA revealed a main effect of the pre-treatment (F (1, 35) = 13.7, P b 0.001), treatment (F (1, 35) = 4.45, P b 0.05) and pre-treatment × treatment interactions (F (1, 35) = 4.55, P b 0.05) for the time spent in the open arms and a main effect of the pre-treatment (F (1, 35) = 14.05, P b 0.001), treatment (F (1, 35) = 4.65, P b 0.05) and pre-treatment × treatment interactions (F (1, 35) = 4.77, P b 0.05) to the time spent in closed arms. Fig. 5B shows an influence of pre-treatment with ketanserin also in the number of entries in the open arms. Two-way ANOVA shows a main effect of the pre-treatment (F (1, 33) = 17.5, P b 0.001), treatment (F (1, 33) = 8.7, P b 0.01) and pre-treatment × treatment interactions (F (1, 33) = 4.7, P b 0.05). Ketanserin pre-treatment shows no alteration in the number of closed arms entries (Fig. 5B). The anxiolytic-like effect of (PhSe)2 was also significantly prevented by the pre-treatment with WAY100635 (0.1 mg/kg s.c., a selective 5HT1A receptor antagonist) as shown in Fig. 6A. Two-way ANOVA revealed a main effect of the pre-treatment (F (1, 32)=7.4, Pb 0.01), treatment (F (1, 32)=4.3, Pb 0.05) and pre-treatment×treatment interactions (F (1, 32)= 7.5, Pb 0.01) on time spent in the open arms and a main effect of the pre-
Fig. 6. Effects of WAY100635 (0.1 mg/kg, s.c.) and/or (PhSe)2 (50 µmol/kg, i.p.) on the elevated plus maze task. (A) Data are presented as percentage of time spent in the open or closed arms; (B) data are presented as number of entries in the open or closed arms. WAY100635 was administered 30 min before (PhSe)2 and the elevated plus maze task was performed 24 h after (PhSe)2 administration. Each column represents mean ± S.E.M. from 10 animals for each group. Statistical analysis was performed by Two-Way ANOVA followed by Bonferroni pos hoc test. ⁎P b 0.05 when compared to vehicle-treated control; # P b 0.05 as compared to the (PhSe)2 group pretreated with vehicle.
Fig. 7. Effect of diazepam (1 mg/kg, i.p.) administered 30 min before training session in a step-down inhibitory avoidance task. Data are expressed as median (interquartile ranges) (in seconds) of training (black columns), 1.5 h test (gray columns) or 24 h test (white columns) session latencies from 10 animals for each group. Statistical analysis was performed by using Kruskal–Wallis followed by Dunn pos hoc test. ⁎P b 0.05 denotes a significant difference from control.
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4. Discussion
Fig. 8. Effect of (PhSe)2 (5, 25 and 50 µmol/kg, i.p.) administered 30 min before training session in a step-down inhibitory avoidance task. Latency to step-down: (A) test after 1.5 h (STM); (B) test after 24 h (LTM) of training session. Data are expressed as median (interquartile ranges) (in seconds) of training (dark columns) or test (light columns) session latencies from 10 animals for each group. Statistical analysis was performed by using Kruskal–Wallis followed by Dunn pos hoc test. ⁎P b 0.05 denotes a significant difference from control.
treatment (F (1, 32)=7.9, P b 0.01), treatment (F (1, 32)=4.5, Pb 0.05) and pre-treatment×treatment interactions (F (1, 32)=7.9, Pb 0.01) on time spent in the closed arms. In Fig. 6B, the analysis by two-way ANOVA revealed a main effect of pre-treatment (F (1, 33)=8.4, Pb 0.01), treatment (F (1, 33)=10.5, Pb 0.01), but not pre-treatment×treatment interactions (F (1, 33)=2.3, P=0.135) in the number of open arms entries. The pretreatment with WAY100635 showed no alteration in the number of closed arms entries (Fig. 6B). In all experiments carried out with (PhSe)2 and/or 5HT receptor antagonists pre-treatments, the number of rearings was not changed when analysed in the elevated plus maze task (data not shown). 3.5. Effect of (PhSe)2 in the inhibitory foot-shock avoidance task It has been reported that the effects of benzodiazepines (BZs) on acquisition of information might be partly linked to their sedative effects (Clement and Chapouthier, 1998). In Fig. 7 we confirmed data from the literature showing that diazepam (1 mg/kg, i.p., a GABAA receptor agonist) impairs memory acquisition when administered 30 min before training session in a step-down inhibitory foot-shock avoidance task (P b 0.05). Thus, (PhSe)2 (5, 25 and 50 µmol/kg i.p.,) was administered 30 min before the training session to assess possible side effects of this drug on memory in a step-down inhibitory foot-shock avoidance task. We can observe that all doses of (PhSe)2 administered did not impair the STM and LTM performance in the step-down inhibitory foot-shock avoidance task, which excludes any effect of this drug on both memory processes (Fig. 8A and B).
In the present study, systemic administration of (PhSe)2 in rats had a pronounced anxiolytic-like effect, with no alteration on locomotor and exploratory activities in the open-field arena. This parameter deserves attention in the behavioural studies concerning new drugs because alterations in the locomotor activity of the animals usually may non-specifically affect other behavioural tasks. Our results corroborate with some data reported by Machado et al. (2006), where systemic administration of a substituted (PhSe)2 induces an antipsychotic-like effect without affecting the open-field behaviour or memory in mice. In addition, classical antipsychotics have been used by clinicians in the treatment of affective disorders, anxiety, restlessness and agitation (Kaplan, 2006). More recently, Savegnago et al. (2008), demonstrated that oral administration of (PhSe)2 after 30 min produced antidepressant- and anxiolytic-like effects in mice, with no alteration on parameters of locomotor and exploratory activities. Moreover, it is important to mention that differently of Savegnago et al. (2007b, 2008) where rats and mice received oral doses of (PhSe)2, our data demonstrated a later anxiolytic-like effect of (PhSe)2. Since previous studies reported diverse toxicity potential by (PhSe)2 in different experimental models (Nogueira et al., 2003a), the anxiolytic effect showed in our work is probably due to the distinct route of administration as well the vehicle solution where (PhSe)2 is dissolved, that can be responsible to the distinctive rate of absorption and metabolism of the compound in liver. However, in this study the doses of (PhSe)2 administered did not induce toxic effects as previously demonstrated by Nogueira et al. (2003a). The first evidence that (PhSe)2 can trigger anxiolytic effect was a decrease in the number of defecation in the open-field arena. However, considering that the number of defecations usually presents a slight increase merely by exposure of animals to a new environment, this assumption was further reinforced by the observation that this compound was also able to increase the time spent in the open arms in the elevated plus maze task, which represent a strong indicative of anxiolytic effect. Although, a similar effect of (PhSe)2 was previously described in mice, the mechanisms underlying this anxiolytic effect was not evaluated yet (Savegnago et al., 2008). Thus, to better characterize the anxiolytic-like effects of (PhSe)2 by using the elevated plus maze apparatus, we also selected drugs that selectively modulate GABA and 5HT neurotransmitter systems, which are commonly involved in the physiopathology and treatment of anxiety and depressive disorders (Nutt, 2001). Although anxiety is a complex psychological and behavioural trait that can involve multiples receptors (D2, 5HT, GABA, NMDA), the mechanisms by which the neural systems can be sensitized are not fully understood. In this work our results showed that modulation of more than one neuronal system can account for the anxiolytic-like effect of (PhSe)2. Our data are in agreement with previous studies where pharmacological profile as well toxic potential of this compound occurs by modulation of different neurotransmitter systems (Nogueira et al., 2003a; Savegnago et al., 2007b, 2008). Interestingly, it has been pointed that the GABAergic system also participate of the toxic effects mediated by (PhSe)2, since seizures induced by high concentrations of this compound were prevented by diazepam, phenobarbital and muscimol (Nogueira et al., 2003a). Likewise, (PhSe)2 increased the potency of PTZinduced seizures and mortality in mice (Brito et al., 2006). Here we demonstrated the involvement of GABAergic system in the anxiolytic-like effect by (PhSe)2 since pre-treatment with bicuculline, the selective GABAA receptor antagonist, consistently reversed the anxiolytic effects of this selenium compound. Furthermore, studies have demonstrated that the control of anxiety occurs through GABAA receptors modulation, since blockage of presynaptic GABAB receptor is able to increase GABA release, which in turn affects GABAA receptors reducing anxiety (Zarrindast et al., 2001). On the other hand, abnormalities in 5HT neurotransmission have been implicated in the etiology of several psychiatric and neurological disorders as previously described to depression, anxiety and mood
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(Hoyer et al., 2002; Wong and Licinio, 2001). Moreover, the 5HT2A/2C and 5HT1A receptor subtypes are especially relevant for this discussion, given its implication in both anxiety and depression regulation (Beneytez et al., 1998; Fletcher et al., 1996), apart from the fact of they are involved on the antidepressant-like effect of (PhSe)2 (Savegnago et al., 2007b). An evidence of the involvement of 5HT2A/2C receptors in the anxiolytic-like effect of (PhSe)2 was given by the finding that pre-treatment of rats with ritanserin (a non-selective 5HT2A/2C receptor antagonist) and ketanserin (a selective 5HT2A receptor antagonist), were able to prevent the increase in the time spent and number of entries in the open arms in the elevated plus maze task. Although, there are some discrepancies whether acute administration of ritanserin may cause anxiolytic, anxiogenic or no effect at all (Barnes et al.,1992; Griebel et al.,1997; Stutzmann et al.,1991), in our study ritanserin did not affect the performance of rats in the elevated plus maze task, but precluded anxiolytic-like behaviour caused by (PhSe)2. Another interesting finding consisted on the anxiolytic-like effect of (PhSe)2 on the elevated plus maze task have has been abolished by the pre-treatment with WAY100635, a selective 5HT1A receptor antagonist. Recently, Savegnago et al. (2007b) demonstrated that the 5HT1A receptor is involved in the antidepressant effect of (PhSe)2 in the forced swimming test. Although, sufficient and significant differences have been described between anxiety and depression, supporting the view that they are independent psychiatric disorders, sharing abnormalities in the 5HT1A receptor function may explain some of the comorbidity of these disorders (Nutt and Stein, 2006). Taking into account the anxiolytic-like effect of (PhSe)2 showed here and its antidepressant-like effect previously described, we could suggest that the same neurotransmitter mechanisms can be involved in the pathophysiology of psychiatric disorders, as an important point to address the comorbid of depression and anxiety experienced by many patients (Davidson, 2001). The current study showed also that the anxiolytic-like effect produced by (PhSe)2 is comparable with diazepam, a classical anxiolytic drug. However, as BZs present common side effects as sedation and amnesia (Atack, 2005; Rudolph, 2001; Salzman, 1993), it is extremely relevant to the exploration of new therapeutic targets for treatment of anxiety disorders. As our results pointed to involvement of GABAA neurotransmission in the anxiolytic action of (PhSe)2 and by the fact that systemic administration of BZs can induce anterograde amnesia which might be partly linked to their sedative effect (Cain, 1997), we decide to investigate the action of (PhSe)2 on learning and memory task. Our data revealed absence of (PhSe)2 effects when both short- and long-term memory were evaluated, suggesting that at least in our behavioural model (PhSe)2 seems to interact with the GABAergic system in a different way than BZs. Furthermore, taking into account our results that anxiolytic-like effect of (PhSe)2 also involves the serotoninergic system it's important to note that changes in the serotoninergic transmission can interfere with learning acquisition and memory consolidation, since 5HT1 and 5HT2 receptors are present in brain structures implicated in learning and memory processes (Breese et al., 2002). 5. Conclusion The results presented here showed pharmacological evidences of (PhSe)2 anxiolytic-like effect in rats, and the absence of undesirable effects such as alterations in the locomotor activity or memory, confirming the assumption that this effect can be specific and suggesting that (PhSe)2 could be a novel potential anxiolytic drug with a prospective of minimal side effects. We also clarify here the involvement of 5HT2A/2C, 5HT1A and GABAA receptors as potential mechanisms underlying the pharmacological actions of (PhSe)2 as an anxiolytic drug. Acknowledgements This study was supported in part by the FINEP research grant “Rede Instituto Brasileiro de Neurociência (IBN-Net)” # 01.06.0842-00 and by grants from the Brazilian National Research Council CNPq, CAPES
and PRONEX. Another part of study was supported by FAPERGS (Proc. No. 0521850 to Lisiane O. Porciúncula) and Grants from L'oreal Prize “Women in Science 2006” (Lisiane O. Porciúncula). Gabriele Ghisleni is supported by a CAPES fellowship. References Argyropoulos SV, Sandford JJ, Nutt DJ. The psychobiology of anxiolytic drug. Part 2: pharmacological treatments of anxiety. Pharmacol Ther 2000;3:213–27. Atack JR. The benzodiazepine binding site of GABA(A) receptors as a target for the development of novel anxiolytics. Exp Opin Investig Drugs 2005;14:601–18. Barbosa NB, Rocha JB, Wondracek DC, Perottoni J, Zeni G, Nogueira CW. Diphenyl diselenide reduces temporarily hyperglycemia: possible relationship with oxidative stress. Chem Biol Interact 2006;163:230–8. Barnes NM, Cheng CH, Costall B, Ge J, Kelly ME, Naylor RJ. Profiles of interaction of R(+)/S (−)-zacopride and anxiolytic agents in a mouse model. Eur J Pharmacol 1992;218: 91–100. 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