Riluzole produces distinct anxiolytic-like effects in rats without the adverse effects associated with benzodiazepines

Neuropharmacology 62 (2012) 2489e2498

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Riluzole produces distinct anxiolytic-like effects in rats without the adverse effects associated with benzodiazepines Azusa Sugiyama a, b, Akiyoshi Saitoh a, *, Takashi Iwai a, Kou Takahashi a, Misa Yamada a, Sachie Sasaki-Hamada b, Jun-Ichiro Oka b, Masatoshi Inagaki a, Mitsuhiko Yamada a a

Department of Neuropsychopharmacology, National Institute of Mental Health, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashimachi, Kodaira, Tokyo 187-8553, Japan Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba 278-8510, Japan

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 August 2011 Received in revised form 9 February 2012 Accepted 12 February 2012

In this study, we investigated the anxiolytic-like effect of riluzole using three different innate anxiety models in rats. In the elevated plus-maze test, riluzole significantly increased the time spent in, and entries into, the open arm after 60 min administration. This finding was supported by results obtained from light/dark and open-field tests. The magnitude of the anxiolytic-like effects of riluzole in each of the behavioral models was similar to those produced by a benzodiazepine, diazepam, suggesting that riluzole has a robust anxiolytic-like activity in rats. To clarify the involvement of sodium channels in this anxiolytic activity, we examined the effect of a co-administered sodium channel activator, veratrine. The anxiolytic-like action of riluzole was diminished by veratrine in the elevated plus-maze, light/dark and open-field tests. Based on these results, it is suggested that the anxiolytic mechanism of riluzole is clearly distinct from that of diazepam. In addition, to examine whether riluzole directly and non-selectively affected the GABAA-benzodiazepine receptor complex, we performed three behavioral tests (footprint analysis, Y-maze test and the ethanol-induced sleeping time test) that are closely related to the GABAAbenzodiazepine pathways. In contrast to diazepam, riluzole produced no significant effects in these tests. Here, we provide the first report demonstrating that riluzole produces distinct anxiolytic-like effects in rats without the adverse effects associated with benzodiazepines. Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.

Keywords: Glutamate Veratrine GABA Behavioral Elevated plus-maze

1. Introduction Anxiety disorders are common psychiatric problems, for the treatment of which benzodiazepine anxiolytics have been widely used. Although these drugs are relatively safe, they produce many undesirable side effects via the GABA-benzodiazepine receptor complex, such as sedation, muscle relaxation, motor coordination deficits, memory/cognitive dysfunctions, interactions with ethanol/ barbiturates, and dependency/abuse liability (Lister, 1985; Thiébot, 1985; Venault et al., 1986; Woods et al., 1992). These side effects limit their clinical usefulness (Greenblatt and Shader, 1978) and there is a need to identify novel therapeutic targets for anxiety disorders (Kent et al., 2002). Growing evidence suggests that sodium channel blockers are effective as a non-benzodiazepine treatment of anxiety disorders.

Abbreviations: CMC, carboxymethyl cellulose. * Corresponding author. Tel.: þ81 42 346 1987; fax: þ81 42 346 1994. E-mail address: [email protected] (A. Saitoh).

For example, lamotrigine, which blocks a voltage-sensitive sodium channel, was found to be effective in treating post-traumatic stress disorder in a small placebo-controlled trial (Hertzberg et al., 1999). Interestingly, although in open-label trials, another voltagesensitive sodium channel blocker, riluzole, has recently been suggested to reduce symptoms of obsessive-compulsive disorder (Coric et al., 2003, 2005; Pittenger et al., 2008; Grant et al., 2007) and generalized anxiety disorder (Mathew et al., 2005). In addition, it was reported that anxiety ratings were improved in depressed patients treated with riluzole monotherapy (Zarate et al., 2004) or augmentation therapy (Sanacora et al., 2007; Coric et al., 2005). Although definitive randomized double-blind placebo-controlled trial data are not yet available, these early clinical results suggest that a voltage-sensitive sodium channel blocker could be effective in the treatment of pathological anxiety. Two previous studies reported that lamotorigine and riluzole showed anti-conflict effects in rats undergoing the conditioned emotional response test (Mirza et al., 2005; Munro et al., 2007). Interestingly, the anti-conflict effect of lamotrigine was diminished by the sodium channel activator, veratrine (Mirza et al., 2005).

0028-3908/$ e see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2012.02.012

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These anti-conflict effects may suggest possible anxiolytic-like effects of these drugs, but were observed only with relatively high doses, which also caused sedation in rats (Mirza et al., 2005; Munro et al., 2007). In addition, another study using a different type of conditioned conflict test showed that riluzole itself had no anticonflict effect at doses of 2 and 4 mg/kg in rats (Stutzmann et al., 1989). The olfactory bulbectomized (OBX) rat model exhibits hyperemotional responses that may mimic anxiety, aggression and irritability found in depressed patients. We recently reported that riluzole rapidly attenuates these hyperemotional responses (Takahashi et al., 2011), further suggesting the possible use of riluzole in combating the symptoms of anxiety and depression. Another study using a different type of conditioned conflict test showed that riluzole was able to diminish the pro-conflict effect of the b-carboline derivative, FG7142, an inverse agonist for the benzodiazepine receptor (Stutzmann et al., 1989). However, it is still unclear whether the anti-conflict effect of riluzole occurs directly and non-selectively via the GABAA-benzodiazepine receptor complex. The precise molecular mechanisms of the putative anxiolytic-like effects of riluzole are yet to be determined. In the present study, we investigated the anxiolytic-like effects of riluzole, initially using rats in several different innate anxiety models, such as the elevated plus-maze, light/dark and open-field tests, which are not conditioned conflict procedures. The elevated plus-maze test is the most widely used animal model of anxiety, and is based on a conflict between the tendency of rodents to explore a novel environment and the aversive properties of the open arms (Pellow et al., 1985). The light/dark test and open-field test are popular unconditioned anxiety models, which are based on an innate aversion to mild stressors such as a novel environment and/or a bright light (Crawley, 1981; Wilson et al., 1976; Treit and Fundytus, 1988). To clarify the involvement of sodium channels in the anxiolytic-like effects of riluzole, we examined the effect of coadministration of the sodium channel activator, veratrine. Finally, to examine whether riluzole directly and non-selectively affects the GABAA-benzodiazepine receptor complex, we performed behavioral tests that are closely related to the GABAAbenzodiazepine pathways. The Y-maze test was performed to evaluate learning and memory dysfunction (Izquierdo et al., 1990). The ethanol-induced sleeping time test was carried out to evaluate the alcohol interaction (Ticku, 1989), and the footprint test was performed to evaluate motor coordination deficits (Barlow et al., 1996; Teunissen et al., 2001; Carter et al., 2001). Here, we provide the first report demonstrating that riluzole has an anxiolytic-like effect in several different behavioral models in rats that are not conditioned conflict procedures. 2. Material and methods 2.1. Animals Male Wistar rats were used for behavioral experiments (age: 9e12 weeks, SLC, Shizuoka, Japan). These had free access to food and water in an animal room maintained at 23  1  C with a 12 h lightedark cycle (lights were automatically switched on at 8:00 am), and were kept in this environment for at least 2 weeks prior to study. The study protocol was in accordance with a protocol approved by the Institutional Animal Care and Use Committee of the National Center of Neurology and Psychiatry (Approval No. 2010001). 2.2. Drugs The drugs used in the present study were riluzole (RILUTEKÒ 50 mg Tablets, Sanofi Aventis K.K., Japan), diazepam (Sigma Chemical Co., St. Louis, MO, USA) and veratrine hydrochloride (Sigma Chemical Co., St. Louis, MO, USA). Diazepam was used as a positive control anxiolytic drug. Veratrine, the sodium channel activator, was used to clarify the involvement of sodium channels in the anxiolytic-like effects of riluzole. The doses of riluzole, diazepam and veratrine used in the present study

were calculated based on the free base. Riluzole tablets were crushed and suspended uniformly in 0.5% carboxymethyl cellulose (CMC). Diazepam was also dissolved in CMC. Veratrine hydrochloride was dissolved in saline. 2.3. Behavioral studies 2.3.1. Elevated plus-maze test The elevated plus-maze test was performed using a modification of the procedure described in our previous report (Saitoh et al., 2004). The elevated plus-maze apparatus was made of PlexiglasÒ and consisted of four arms set in a cross pattern from a neutral central square. Two opposite arms were delimited by vertical walls (closed arms, 48  12  40 cm), whereas the two other opposite arms had unprotected edges (open arms, 48  12 cm). The maze was elevated 60 cm above the ground and placed in indirect light (150 Lux). Animals were allowed at least 2 h for adaptation to the new environment before drug administration. All rats used for the elevated plus-maze tests were treated only once. In dose-dependence experiments, riluzole (within the dose range of 0.3e10 mg/kg) or diazepam (1 mg/kg) was orally administered 60 min prior to study. The dose of diazepam was determined by a preliminary study in the elevated plusmaze test (data not shown). In time-dependence experiments, riluzole (3 mg/kg) was administered orally 30, 60 and 120 min before the elevated plus-maze test. Veratrine (0.1 mg/kg, s.c.) was co-administered with riluzole (3 mg/kg, p.o.) or diazepam (1 mg/kg, p.o.) to clarify the involvement of sodium channels in the anxiolytic-like effects of riluzole. This dose of veratrine was derived from a previously published study (Mirza et al., 2005). At the beginning of the 5 min test session, each rat was placed in the central neutral zone, facing one of the open arms. The total number of visits to the closed and open arms and the cumulative time spent (% time spent open arm) and visits (% open arm entry) in the open arms were then observed on a monitor through a video camera system. An arm visit was recorded when the rat moved half of its body into the arm. After removal of each animal, the apparatus was cleaned. 2.3.2. Light/dark test One week after the elevated plus-maze tests performed for the veratrine experiments, the light/dark tests were done with the same rats using a modification of the procedure described in the previous report (Forestiero et al., 2006). The light box apparatus consisted of a PlexiglasÒ apparatus with two compartments of equal size (27  23  27 cm), one light and one dark. The floors in each compartment were connected via a small opening (10  10 cm) enabling transition between the compartments. The box was elevated 70 cm above the floor and placed in indirect light (60 Lux). Animals were allowed at least 2 h for adaptation to the new environment before drug administration. Riluzole (3 mg/kg) or diazepam (1 mg/kg) was administered orally 60 min before the light/dark test. Where appropriate, veratrine (0.1 mg/kg, s.c.) was co-administered with riluzole or diazepam to clarify the involvement of sodium channels in the anxiolytic-like effects of riluzole. At the 5 min test session, each rat was placed in the dark box for 1 min. Two behaviors were noted: (I) the time spent in the light box and (II) the number of line crossings in the light and dark boxes. After removal of each animal, the apparatus was cleaned. 2.3.3. Open-field test The open-field test was performed immediately after the light/dark test, using a modification of the procedure described in our previous report (Saitoh et al., 2004). The open-field apparatus consisted of a square area (90  90 cm) with opaque walls 30 cm high and placed in indirect light (60 Lux). The floor was divided by lines into 36 equal squares. The rats were placed in a corner of the open-field facing the opaque walls. The time spent in the center area (60  60 cm), latency to the center area and the number of line crossings was then observed for 5 min on a monitor through a video camera system. After removal of each animal, the apparatus was cleaned. 2.3.4. Y-maze test The Y-maze test was performed 2e3 days after the open-field test, using a modification of previously published procedures (Saitoh et al., 2006; Sarter et al., 1988). The test apparatus was made of black-painted wood and consisted of three arms positioned at equal angles. Each arm was 35 cm long, 25 cm high walls, and 10 cm wide, and illuminated by indirect light (60 Lux). Animals were allowed at least 2 h for adaptation to the new environment before drug administration. Riluzole (3 mg/kg) or diazepam (1 mg/kg) was administered orally 60 min before the Y-maze test. Each rat was placed at the end of one arm and allowed to move freely through the Y-maze during an 8 min session. An arm visit was recorded when the rat moved half of its body into the arm. After the testing period, we calculated the maximum number of alternations for each rat (an alternation being entry into each of the three arms sequentially without revisiting any of them). This theoretical maximum is equal to two less than the total number of times a rat entered any arm. We also calculated the percent alternation according to the following formula: (actual alternations/maximum possible alternations)  100. For example, if the three arms

A. Sugiyama et al. / Neuropharmacology 62 (2012) 2489e2498

Fig. 1. Anxiolytic-like effects of riluzole in the rat elevated plus-maze model. Panel A shows the percentage of time spent in the open arms. Panel B shows the percentages of entries into the open arms. Diazepam was used as an anxiolytic positive control drug. Riluzole (0.3, 1, 3, and 10 mg/kg), diazepam (1 mg/kg) and CMC were administered 60 min before the test. Each column represents the mean  SEM. The statistical significance of differences among riluzole treatment groups was assessed with oneway ANOVA. Post-hoc individual group comparisons were made with Bonferroni’s test. The statistical analysis for diazepam experiments performed with Student’s t test. Columns are in the following order: CMC (n ¼ 10); riluzole 0.3 mg/kg (n ¼ 7), 1 mg/kg (n ¼ 10), 3 mg/kg (n ¼ 10), 10 mg/kg (n ¼ 8), CMC (n ¼ 5), diazepam 1 mg/kg (n ¼ 5). Totally 55 rats were used. Statistical significance is denoted by * and ** (p < 0.05 and p < 0.01, respectively, vs. CMC-treated rats). are designated A, B, and C, and the rat makes ten individual arm entries in the sequence ACBABACBAB, there would be eight (i.e. 10  2) possible triplets of arm entries (ACB, CBA, BAB etc.). Valid alternations would be ABC, ACB, BAC, BCA, CAB and CBA, whereas any other combination will involve re-entry into an arm before visiting all three in sequence and is not a valid alternation. In this example, the rat’s performance is 62.5%, because it makes five alternations (ACB, CBA, BAC, ACB, and CBA again) out of a maximum of eight. BAB (2) and ABA are not valid alternations because the rat did not visit C before returning to A or B. These behaviors were recorded using a digital video camera on the ceiling of the soundproof room. After removal of each animal, the Y-maze was thoroughly cleaned. Table 1 Effects of riluzole and diazepam on total arm entries in the rat elevated plus-maze model. Drugs (mg/kg)

Number of animals

CMC Riluzole 0.3 1 3 10 CMC Diazepam 1

10 7 10 10 8 5 5

Total arm entry counts 8.3  0.4 6.3 8.6 8.3 8.9 11.2

    

0.8 0.9 1.0 1.3 1.0

15.4  2.2

Diazepam was used as an anxiolytic positive control, and CMC was used as a vehicle control. Riluzole (0.1, 1, 3, and 10 mg/kg), diazepam (1 mg/kg) and CMC were administered 60 min before the test. Data are expressed as mean  SEM.

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Fig. 2. Anxiolytic-like effects of riluzole in the rat elevated plus-maze model. Panel A shows the percentage of time spent in the open arms. Panel B shows the percentage of entries into the open arms. CMC was administered at 60 min prior to the test, while riluzole (3 mg/kg) was administered 30, 60, and 120 min before the test. The statistical significance of differences among riluzole treatment groups was assessed with one-way ANOVA. Post-hoc individual group comparisons were made with Bonferroni’s test. Columns represent the mean  SEM, in the following order: CMC (n ¼ 8), riluzole 30 min (n ¼ 7), 60 min (n ¼ 8), 120 min (n ¼ 6). Totally 29 rats were used. Statistical significance is denoted by * and ** (p < 0.05 and p < 0.01, respectively, vs. CMC-treated rats).

2.3.5. Ethanol-induced sleeping test The ethanol-induced sleeping test was performed independently from other behavioral experiments, using a modification of the procedure described in a previous report (Kita et al., 2004). Animals were allowed at least 2 h for adaptation to the new environment before drug administration. Rats received an intraperitoneal injection of ethanol (2.5 g/kg in a volume of 20 ml/kg body weight) at 60 min after the oral administration of riluzole (3 mg/kg) or diazepam (1 mg/kg). After injection, rats were placed individually into new plastic cages with sawdust. The duration of loss of righting reflex was recorded as sleeping time. Table 2 Effects of riluzole on the total arm entries in the rat elevated plus-maze model. Drugs

Time after administration (min)

Number of animals

Total arm entry counts

CMC Riluzole

60 30 60 120

8 7 8 6

11.8 15.6 12.1 13.8

   

1.3 1.0 1.0 1.0

CMC was administered 60 min prior to the test, while riluzole (3 mg/kg) was administered 30, 60 and 120 min before the test. Data are expressed as mean  SEM.

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A. Sugiyama et al. / Neuropharmacology 62 (2012) 2489e2498 orally 60 min before the footprint test. At the beginning of the test, the feet of each rat were stained with black ink and the animal was placed in the central neutral zone, facing the enclosed arm, which was carpeted with white paper for the recording. Rats were then allowed to walk down the enclosed arm. A number of different parameters of gait can be analyzed simply and effectively using footprint patterns (Carter et al., 2001). The following parameters were measured: A) total spreading of toes (distance between toes 1 and 5); B) intermediary spreading of toes (distance between toes 2 and 4); C) step width; D) step length; E) foot length; and F) angle of foot axis relative to the canal length and area touched per foot. After removal of each animal, the apparatus was cleaned. 2.4. Data analysis Data are expressed as the mean  S.E.M. Statistical significance of differences between two groups was assessed by Student’s t test. Statistical significance of differences among groups was assessed by one-way factorial analysis of variance (ANOVA). Post-hoc individual group comparisons were made using the Bonferroni’s test for multiple comparisons. All statistical analyses were performed using Graphpad PrismÒ (GraphpadÒ Software Inc., San Diego, CA, USA). P-values less than 0.05 were considered significant.

3. Results 3.1. Elevated plus-maze test The oral administration of riluzole significantly increased the percentage of time spent in the open arms at the doses of 1 mg/kg

Fig. 3. Effect of a sodium channel activator, veratrine, on riluzole-induced anxiolyticlike effects in the rat elevated plus-maze model. Panel A shows the percentage of time spent in the open arms. Panel B shows the percentage of entries into the open arms. Diazepam was used as a positive control anxiolytic drug. Riluzole (3 mg/kg), diazepam (1 mg/kg) and CMC were administered orally 60 min before the test. Veratrine (0.1 mg/ kg) was co-administered with riluzole and diazepam. The statistical significance of differences among riluzole treatment groups was assessed with one-way ANOVA. Posthoc individual group comparisons were made with Bonferroni’s test. Each column represents the mean  SEM, n ¼ 6 rats/group. Totally 36 rats were used. **p < 0.01 vs. CMC-treated rats, ##p < 0.01 vs. riluzole-treated rats. 2.3.6. Footprint test The footprint test was performed independently from other behavioral experiments, using a modification of a previously published procedure (Teunissen et al., 2001). We used a part of the elevated plus-maze enclosed PlexiglasÒ arm (48  12  40 cm) for the test apparatus. Animals were allowed at least 2 h for adaptation to the new environment before drug administration. Riluzole (3 mg/kg) or diazepam (1 mg/kg) was administered Table 3 Effect of the sodium channel activator, veratrine, on total arm entries in the rat elevated plus-maze model. Drugs (mg/kg, p.o.) Riluzole CMC 3 3 CMC Diazepam 1 1

Veratrine (mg/kg, s.c.)

Number of animals

Total arm entry counts

Saline Saline 0.1 0.1

6 6 6 6

7.5 13.3 8.0 4.3

Saline 0.1

6 6

   

1.4 3.2 2.1 0.8

7.0  2.1 9.8  2.2

Diazepam was used as an anxiolytic positive control. Riluzole (3 mg/kg), diazepam (1 mg/kg) and CMC were administered orally 60 min before the test. Veratrine (0.1 mg/kg) was co-administered with riluzole and diazepam. Data are expressed as mean  SEM.

Fig. 4. Anxiolytic-like effects of riluzole in the rat light/dark test. Panel A shows the time spent in the light box. Panel B shows the total entries into the light box. Diazepam was used as an anxiolytic positive control drug. Riluzole (3 mg/kg), diazepam (1 mg/ kg) and CMC were administered 60 min before the test. A sodium channel activator, veratrine (0.1 mg/kg) was co-administered with riluzole and diazepam. The statistical significance of differences among riluzole treatment groups was assessed with oneway ANOVA. Post-hoc individual group comparisons were made with Bonferroni’s test. Each column represents the mean  SEM, n ¼ 6 rats/group. Totally 36 rats were used. *p < 0.05 and **p < 0.01 vs. CMC-treated rats, #p < 0.05 and ##p < 0.01 vs. riluzole-treated rats.

A. Sugiyama et al. / Neuropharmacology 62 (2012) 2489e2498

and 3 mg/kg at 60 min after administration (one-way ANOVA: F4,40 ¼ 5.133, p < 0.01, Fig. 1A). There were no significant differences between 10 mg/kg and 3 mg/kg treatment groups (Fig. 1A). Riluzole at a dose of 3 mg/kg also significantly increased the percentage of open arm entries (one-way ANOVA: F4,40 ¼ 5.119, p < 0.01, Fig. 1B). In addition, diazepam (1 mg/kg) produced a significant increase in the percentage of time spent in the open arms 60 min after administration (Student’s t test: t(8) ¼ 2.652, p < 0.05, Fig. 1A). Diazepam at a dose of 1 mg/kg did not produce the significant effects in the percentage of open arm entries (Fig. 1B). Neither riluzole nor diazepam had a significant effect on total arm entries at 60 min after administration when compared with the control group (Table 1). As shown in Fig. 2A and B, riluzole (3 mg/kg) produced a significant increase in the percentage of time spent in the open arms (one-way ANOVA: F3,25 ¼ 15.96, p < 0.01, Fig. 2A) and in the total number of open arm entries (one-way ANOVA: F3,25 ¼ 7.430, p < 0.01, Fig. 2B) at 30, 60 and 120 min after administration. The peak effect for time spent in the open arm occurred at 60 min after riluzole administration (3 mg/kg) (Fig. 2A). However, riluzole (3 mg/kg) had no effect on total arm entry counts 30e120 min after administration compared to CMC (control) (Table 2). We examined the effects of veratrine on riluzole-induced anxiolytic-like effects. As shown in Fig. 3, veratrine (0.1 mg/kg) alone had no significant effect on either time spent on the open arms (Fig. 3A) and or open arm entries (Fig. 3B). Interestingly, veratrine (0.1 mg/kg) significantly reduced the riluzole (3 mg/kg)-induced increases in the percentage of time spent in the open arms (oneway ANOVA: F5,30 ¼ 43.98, p < 0.01, Fig. 3A), and caused a nonsignificant reduction in the riluzole (3 mg/kg)-induced increase in the percentage of open arm entries (Fig. 3B). In contrast, veratrine

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(0.1 mg/kg) had no significant effect on diazepam (1 mg/kg)induced increases in either the percentage of time spent in the open arms (Fig. 3A) or the number of open arm entries (Fig. 3B). Veratrine (0.1 mg/kg) had no significant effect on the total arm entry counts in either the riluzole or diazepam treatment groups (Table 3). 3.2. Light/dark test Riluzole (3 mg/kg) significantly increased the time spent in the light box (one-way ANOVA: F5,30 ¼ 8.356, p < 0.01, Fig. 4A) and total light box entries (one-way ANOVA: F5,30 ¼ 66.76, p < 0.01, Fig. 4B) at 60 min after administration. Similarly, diazepam (1 mg/kg) also significantly increased both of these parameters (Fig. 4A and B) at the same time point. We examined the effects of veratrine on riluzole-induced anxiolytic-like effects. Veratrine (0.1 mg/kg) alone did not produce any effect on the time spent in the light box (Fig. 4A) and total light box entries (Fig. 4B), when compared with control groups. Interestingly, the anxiolytic-like effects of riluzole were diminished by veratrine (0.1 mg/kg). In contrast, veratrine (0.1 mg/ kg) had no significant effect on diazepam (1 mg/kg)-induced increases in these parameters (Fig. 4A and B). 3.3. Open-field test Riluzole (3 mg/kg) significantly increased the time spent in the center area at 60 min after administration, as did diazepam (1 mg/ kg) (one-way ANOVA: F5,30 ¼ 5.680, p < 0.01, Fig. 5A). Veratrine (0.1 mg/kg) alone had no effect on the amount of time spent in the center area, when compared with control group, but significantly

Fig. 5. Anxiolytic-like effects of riluzole in the rat open-field test. Panel A shows time spent in the center area. Panel B shows the latency to the center area. Diazepam was used as an anxiolytic positive control. The open-field test was performed immediately after the light/dark test (Fig. 4.). A sodium channel activator, veratrine (0.1 mg/kg) was co-administered with riluzole (3 mg/kg), diazepam (1 mg/kg) and CMC. The statistical significance of differences among riluzole treatment groups was assessed with one-way ANOVA. Post-hoc individual group comparisons were made with Bonferroni’s test. Each column represents the mean  SEM, n ¼ 6 rats/group. Totally 36 rats were used. *p < 0.05 and **p < 0.01 vs. CMC-treated rats.

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reduced the riluzole (3 mg/kg)-induced increases in this parameter (Fig. 5A). Conversely, veratrine (0.1 mg/kg) had no significant effect on diazepam (1 mg/kg)-induced increases in time spent in the center area (Fig. 5A). As shown in Fig. 5B and C, riluzole, veratrine and diazepam had no significant effect on either latency to the center area or the total crossing counts between individual treatment groups (compared to control). 3.4. Y-maze test Riluzole (3 mg/kg) caused no significant change in the percentage of spontaneous alternation behaviors when compared with the control group (Fig. 6A). In contrast to riluzole, diazepam (1 mg/kg) significantly decreased the percentage of spontaneous alternation behaviors compared to control (one-way ANOVA: F2,13 ¼ 72.90, p < 0.01, Fig. 6A). Total arm entry counts were slightly, but not significantly, increased by riluzole (3 mg/kg) and diazepam (1 mg/kg) (Fig. 6B). 3.5. Ethanol-induced sleeping time Ethanol-induced sleeping time was not affected by riluzole (3 mg/kg), but was significantly prolonged by diazepam (1 mg/kg) compared with the control group (one-way ANOVA: F2,15 ¼ 16.11, p < 0.01, Fig. 7). 3.6. Footprint test Representative footprint patterns of treated rats are shown in Fig. 8. Compared to control, riluzole (3 mg/kg) produced no significant changes in any of the footprint parameters of rats, whereas diazepam (1 mg/kg) produced a significant increase in foot angle to walking direction (one-way ANOVA: F2,18 ¼ 22.88, p < 0.01, Fig. 9F) and (although not significantly) the step length (Fig. 9C). There were no changes in spreading of inner toes (Fig. 9A), total spreading of toes (Fig. 9B), step width (Fig. 9D) and foot length (Fig. 9E) between the CMC, riluzole and diazepam treatment groups. 4. Discussion 4.1. Anxiolytic-like effects of riluzole in innate animal models There is currently no published evidence of an anxiolytic-like effect of riluzole when using innate anxiety models in rodents. Interestingly, in this study, we first found that, in rats, riluzole has anxiolytic-like effects in several different innate behavioral models that are not conditioned conflict procedures. We clearly demonstrated that riluzole produces dose- and time-dependent anxiolytic-like effects in the elevated plus-maze test, a finding supported by results from the light/dark and open-field tests. The magnitude of the anxiolytic-like effects of riluzole (3 mg/kg) in each of the behavioral models was similar to that produced by the benzodiazepine, diazepam (1 mg/kg). In addition, the dose of riluzole (3 mg/kg) used in this study produced no sedative effects in rats. Previously, it was reported that the sodium channel blocker, lamotrigine (10 mg/kg), produced a significant increase in open arm entries in the elevated plus-maze test (Foreman et al., 2009). Our data support the hypothesis that blockers of voltage-activated sodium channels are putative novel therapeutic agents for anxiety disorders. A clinical trial to examine the efficacy of riluzole as a nonbenzodiazepine anxiolytic in patients may be warranted on this evidence. The effects of riluzole at the higher dose of 10 mg/kg may be partially affected by sedation. Indeed, a previous study indicated

Fig. 6. Effects of riluzole in the rat Y-maze test. Panel A shows the percentage of spontaneous alternations. Panel B shows the total arm entries. Diazepam was used as an anxiolytic positive control. Riluzole (3 mg/kg), diazepam (1 mg/kg) and CMC were administered 60 min before the test. The statistical significance of differences among riluzole treatment groups was assessed with one-way ANOVA. Post-hoc individual group comparisons were made with Bonferroni’s test. Each column represents the mean  SEM. (CMC, n ¼ 10; riluzole, n ¼ 9; Diazepam, n ¼ 8). Totally 27 rats were used. **p < 0.01 vs. CMC-treated rats.

that riluzole 10 mg/kg (i.p.) decreased the spontaneous locomotor activity in rats (Lourenço Da Silva et al., 2003). In addition, we also reported that oral administration of riluzole (10 mg/kg) significantly suppressed the hyperemotional behaviors in olfactory bulbectomized rat model (Takahashi et al., 2011). On the other hand, there was no apparent sedative effect of riluzole (10 mg/kg) in our

Fig. 7. Effects of riluzole on ethanol-induced sleeping time in rats. Diazepam was used as a positive control. Riluzole (3 mg/kg), diazepam (1 mg/kg) and CMC were administered 60 min before the ethanol injection (2.5 g/kg). The statistical significance of differences among riluzole treatment groups was assessed with one-way ANOVA. Posthoc individual group comparisons were made with Bonferroni’s test. Each column represents the mean  SEM, n ¼ 6 rats/group. Totally 18 rats were used. **p < 0.01 vs. CMC-treated rats.

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Fig. 8. Representative footprint pattern of treated rats. The following parameters were measured: A, total spreading distance between toes 1 and 5; B, intermediary spreading of toes (distance between toes 2 and 4); C, step width; D, step length; E, foot length; F, angle of foot axis relative to the canal length and area touched per foot.

experiment, when we used the total number of entry counts as an index of locomotor activity. And there were no significant differences between 10 mg/kg and 3 mg/kg treatment groups in the present study. Together, it is difficult to distinguish sedation from anxiolytic-like effects of riluzole at the dose of 10 mg/kg. 4.2. The effects of veratrine on the anxiolytic-like effects of riluzole The involvement of sodium channels in the anxiolytic-like effects of riluzole have not been reported until now. Interestingly, we found that the effects of riluzole in the elevated plus-maze, light/dark and open-field tests were diminished by veratrine, whereas veratrine alone had no significant effects in these models. Notably, veratrine also has no significant effect on the anxiolytic effects of diazepam. These results suggest that the anxiolytic mechanism of riluzole is clearly distinct from that of diazepam. It is well known that voltage-activated sodium channels mediate action potential invasion of the synaptic terminal, and thereby modulate glutamatergic neurotransmission. It has been shown that veratrine elicits glutamate release in rat prefrontal cortex (Waldmeier et al., 1996; Go1embiowska and Dziubina, 2000), and lamotrigine suppresses this action (Waldmeier et al., 1996). It was previously reported that riluzole, the only drug approved for the treatment of amyotrophic lateral sclerosis (ALS), also decreases extracellular glutamate levels. Several studies have reported that riluzole inhibits the release of glutamate from cultured neurons and from brain slices (Doble et al., 1992; Martin et al., 1993; Prakriya and Mennerick, 2000). These effects were suggested to have been mediated by a pertussis toxin-sensitive G protein signaling pathway (Wang et al., 2004). In addition, riluzole at doses of 2 and 4 mg/kg decreases extracellular glutamate levels in the ventral posterolateral thalamic nucleus and hippocampus in some experimental rat models (Abarca et al., 2000; Pena and Tapia, 2000).

Riluzole has been shown to potentiate glutamate uptake (Frizzo et al., 2004; Fumagalli et al., 2008). Also several investigations suggested that riluzole produces glutamate inhibitory effects through blockade of voltage-activated sodium channels (Benoit and Escande, 1991; Hebert et al., 1994; Stefani et al., 1997; Song et al., 1997; Zona et al., 1998; Urbani and Belluzzi, 2000; Prakriya and Mennerick, 2000). Diminished glutamate release would prevent the excitotoxicity that is thought to cause the death of motor neurons in ALS (Risterucci et al., 2006). However, other evidence reports no significant effect of riluzole (2 or 8 mg/kg) on hippocampal glutamate release in a rabbit cerebral ischemia model (Kwon et al., 1998). The precise molecular mechanisms of the anxiolytic-like effects of riluzole are still unclear. Further in vivo behavioral studies are necessary to delineate these mechanisms. 4.3. GABAA-benzodiazepine receptor complex and anxiolytic-like effects of riluzole The GABAA receptor, an active site for the benzodiazepines, plays an important role in the pathophysiology of learning and memory dysfunctions (Izquierdo et al., 1990; Woods et al., 1992). Amnesia is a classical side effect of benzodiazepines, and is seen with sub-ataxic doses in both humans (Lister, 1985) and animals (Thiébot, 1985; Venault et al., 1986). It has been reported that diazepam decreases the spontaneous alternation performance in the Y-maze test (Cahill et al., 1986; Maurice et al., 1994). In the present study, we also obtained similar results in rats treated with a dose of diazepam that was also shown to produce anxiolytic-like effects. In contrast, riluzole had no effects on the spontaneous alternation performance. Our results suggest that riluzole, at doses that produce anxiolytic effects, does not affect the GABAA-benzodiazepine receptor complex in the rat brain regions responsible for learning and memory, and thus does not produce amnesia.

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Fig. 9. Effects of riluzole on the footprint pattern in rats. Diazepam was used as a positive control. Riluzole (3 mg/kg), diazepam (1 mg/kg) and CMC were administered 60 min before the test. The following parameters were measured: A, total spreading distance between toes 1 and 5; B, intermediary spreading of toes (distance between toes 2 and 4); C, step width; D, step length; E, foot length; F, angle of foot axis relative to the canal length and area touched per foot. The statistical significance of differences among riluzole treatment groups was assessed with one-way ANOVA. Post-hoc individual group comparisons were made with Bonferroni’s test. Each column represents the mean  SEM, n ¼ 7 rats/group. Totally 21 rats were used. **p < 0.01 vs. CMC-treated rats.

Another adverse effect of benzodiazepine drugs is their propensity to interact with ethanol, producing marked decrements in psychomotor performance in both animals and humans (Chan, 1984). The GABAA receptor plays an important role in the pathophysiology of this interaction (Ticku, 1989; Woods et al., 1992). In the present study, diazepam prolonged the ethanol-induced sleeping time in rats. In contrast, riluzole had no such effect. Our results suggest that riluzole does not affect the GABAA-benzodiazepine receptor complex in the rat brain regions responsible for this benzodiazepine side effect and thus, at concentrations that produce anxiolytic-like effects, does not cause this ethanol interaction. Footprint analysis was performed to evaluate motor coordination deficits in rats after riluzole or diazepam treatment. This analysis is well established and widely used for measuring motor coordination and balance in rodents (Carter et al., 2001). In addition to its roles described above, the GABAA receptor plays an important

role in the pathophysiology of these motor coordination deficits (Woods et al., 1992). It has been suggested that abnormal locomotor functions were represented by various parameters, such as a step length and the foot angle in the footprint test (Barlow et al., 1996; Teunissen et al., 2001). In the present study, therapeutic doses of diazepam significantly increased the foot angle and step length in rats, whereas riluzole had no such effects, suggesting that riluzole does not act in the rat brain region(s) responsible for motor coordination. Therefore, at doses that cause anxiolytic-like effects, riluzole does not produce motor coordination deficits. 5. Conclusions In this study, riluzole produced robust anxiolytic-like effects in rats. Based on these results, it is suggested that voltage-activated sodium channels play some important roles in these anxiolytic-

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like effects. Interestingly, riluzole does not appear to affect the GABAA-benzodiazepine receptor complex directly and nonselectively in the rat brain. As a result, riluzole does not seem to cause the adverse effects associated with benzodiazepines, such as amnesia, ethanol interaction, and motor coordination deficits, at the doses that produce anxiolytic-like effects, and may thus represent a putative novel therapy for anxiety disorders. Acknowledgments This work was supported, in part, by research grants from Health Science Research Grants from the Japanese Ministry of Health, Labour, and Welfare and the Japan Foundation for Neuroscience and Mental Health. This manuscript was thoroughly checked for English grammar and spelling by a professional scientific editing company (Edanz Editing; http://edanzediting.co. jp/about english.html). We are entirely responsible for the scientific content of this manuscript. References Abarca, C., Silva, E., Sepulveda, M.J., Oliva, P., Contreras, E., 2000. Neurochemical changes after morphine, dizocilpine or riluzole in the ventral posterolateral thalamic nuclei of rats with hyperalgesia. Eur. J. Pharmacol. 403, 67e74. Barlow, C., Hirotsune, S., Paylor, R., Liyanage, M., Eckhaus, M., Collins, F., Shiloh, Y., Crawley, J.N., Ried, T., Tagle, D., Wynshaw-Boris, A., 1996. Atm-deficient mice: a paradigm of ataxia telangiectasia. Cell 86, 159e171. Benoit, E., Escande, D., 1991. Riluzole specifically blocks inactivated Na channels in myelinated nerve fibre. Pflugers Arch. 419, 603e609. Cahill, L., Brioni, J., Izquierdo, I., 1986. Retrograde memory enhancement by diazepam: its relation to anterograde amnesia, and some clinical implications. Psychopharmacology (Berl) 90, 554e556. Carter, R.J., Morton, J., Dunnett, S.B., 2001. Motor coordination and balance in rodents. Curr. Protoc. Neurosci. (Chapter 8), Unit 8.12. Chan, A.W., 1984. Effects of combined alcohol and benzodiazepine: a review. Drug Alcohol Depend. 13, 315e341. Coric, V., Milanovic, S., Wasylink, S., Patel, P., Malison, R., Krystal, J.H., 2003. Beneficial effects of the antiglutamatergic agent riluzole in a patient diagnosed with obsessive-compulsive disorder and major depressive disorder. Psychopharmacology (Berl) 167, 219e220. Coric, V., Taskiran, S., Pittenger, C., Wasylink, S., Mathalon, D.H., Valentine, G., Saksa, J., Wu, Y.T., Gueorguieva, R., Sanacora, G., Malison, R.T., Krystal, J.H., 2005. Riluzole augmentation in treatment-resistant obsessive-compulsive disorder: an open-label trial. Biol. Psychiatry 58, 424e428. Crawley, J.N., 1981. Neuropharmacologic specificity of a simple animal model for the behavioral actions of benzodiazepines. Pharmacol. Biochem. Behav. 15, 695e699. Doble, A., Hubert, J.P., Blanchard, J.C., 1992. Pertussis toxin pretreatment abolishes the inhibitory effect of riluzole and carbachol on D-[3H]aspartate release from cultured cerebellar granule cells. Neurosci. Lett. 140, 251e254. Foreman, M.M., Hanania, T., Eller, M., 2009. Anxiolytic effects of lamotrigine and JZP-4 in the elevated plus maze and in the four plate conflict test. Eur. J. Pharmacol. 602, 316e320. Forestiero, D., Manfrim, C.M., Guimarães, F.S., de Oliveira, R.M., 2006. Anxiolytic-like effects induced by nitric oxide synthase inhibitors microinjected into the medial amygdala of rats. Psychopharmacology (Berl) 184, 166e172. Fumagalli, E., Funicello, M., Rauen, T., Gobbi, M., Mennini, T., 2008. Riluzole enhances the activity of glutamate transporters GLAST, GLT1 and EAAC1. Eur. J. Pharmacol. 578, 171e176. Frizzo, M.E., Dall’Onder, L.P., Dalcin, K.B., Souza, D.O., 2004. Riluzole enhances glutamate uptake in rat astrocyte cultures. Cell Mol. Neurobiol. 24, 123e128. Go1embiowska, K., Dziubina, A., 2000. Effect of acute and chronic administration of citalopram on glutamate and aspartate release in the rat prefrontal cortex. Pol J. Pharmacol. 52, 441e448. Grant, P., Lougee, L., Hirschtritt, M., Swedo, S.E., 2007. An open-label trial of riluzole, a glutamate antagonist, in children with treatment-resistant obsessivecompulsive disorder. J. Child. Adolesc. Psychopharmacol. 17, 761e767. Greenblatt, D.J., Shader, R.I., 1978. Dependence, tolerance, and addiction to benzodiazepines: clinical and pharmacokinetic considerations. Drug Metab. Rev. 8, 13e28. Hebert, T., Drapeau, P., Pradier, L., Dunn, R.J., 1994. Block of the rat brain IIA sodium channel alpha subunit by the neuroprotective drug riluzole. Mol. Pharmacol. 45, 1055e1060. Hertzberg, M.A., Butterfield, M.I., Feldman, M.E., Beckham, J.C., Sutherland, S.M., Connor, K.M., Davidson, J.R., 1999. A preliminary study of lamotrigine for the treatment of posttraumatic stress disorder. Biol. Psychiatry 45, 1226e1229. Izquierdo, I., Da Cunha, C., Huang, C.H., Walz, R., Wolfman, C., Medina, J.H., 1990. Post-training down-regulation of memory consolidation by a GABA-A

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