A combined marble burying–locomotor activity test in mice: A practical screening test with sensitivity to different classes of anxiolytics and antidepressants

European Journal of Pharmacology 547 (2006) 106 – 115 www.elsevier.com/locate/ejphar

A combined marble burying–locomotor activity test in mice: A practical screening test with sensitivity to different classes of anxiolytics and antidepressants Laurent B. Nicolas, Yeter Kolb, Eric P.M. Prinssen ⁎ CNS Research, F. Hoffmann-La Roche Ltd. CH-4070 Basel, Switzerland Received 10 March 2006; received in revised form 13 July 2006; accepted 17 July 2006 Available online 25 July 2006

Abstract Over the last decades, the inhibition of spontaneous burying of glass marbles by mice has been used as an index of anxiolytic drug action in the socalled marble burying test. Indeed, acute administration of rapid-onset (e.g. diazepam) and slow-onset (e.g. fluoxetine) anxiolytics inhibit marble burying. However, non-anxiolytic compounds such as classical antipsychotics also reduce marble burying thus suggesting that the predictive validity of this procedure for anxiety may be limited. In the present study, after having selected a strain of mice (C57BL/6J) that showed spontaneous avoidance of glass marbles, we tried to improve the predictive validity of the marble burying test for anxiety by measuring locomotor activity during the marble burying test and – if needed – in control experiments by using a videotracking system. Twenty-four reference compounds were tested including anxiolytics, anxiogenics, antidepressants, antipsychotics and other classes. By comparing marble burying scores with locomotor measures, we found that, based on our criteria, most of the anxiolytics and antidepressants selectively inhibited marble burying in contrast to most of the other compounds (e.g. haloperidol, morphine). Two putative anxiolytics, i.e. the nociceptin orphanin FQ peptide receptor agonist Ro 64-6198 and the metabotropic glutamate 5 receptor antagonist 2-methyl-6-(phenylethynyl)pyridine, also showed a selective profile. We propose this modified procedure, requiring only a limited number of animals, as a valuable screening test for the detection of compounds having anxiolytic effects. © 2006 Elsevier B.V. All rights reserved. Keywords: Marble burying; Anxiety; Anxiolytics; Predictive validity; (Mice)

1. Introduction In both natural and laboratory conditions, rats and mice spontaneously use available bedding material to bury unpleasant sources of discomfort present in their home environment (Archer et al., 1987). Burying behavior consists in forwardshoving the diggable material over the source of aversion using the snout and forepaws in order to avoid and protect from the localized threat (Poling et al., 1981). This characteristic behavior, which is usually directed toward several classes of harmful and noxious objects such as food associated with unpleasant tasting (Wilkie et al., 1979), small predators such as scorpions (Londei et al., 1998), dead conspecifics or electrified prod (Treit, ⁎ Corresponding author. Behavioral Pharmacology, F. Hoffmann-La Roche Ltd. Department PRBD-N, Bldg. 72/149 CH-4070 Basel, Switzerland. Tel.: +41 61 68 87056; fax: +41 61 68 81895. E-mail address: [email protected] (E.P.M. Prinssen). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.07.015

1990), is described as a defensive behavior reflecting the anxiety state of animals. However, the observation that mice and rats, when placed in a cage containing diggable substrate, spontaneously bury harmless objects, such as glass marbles (Archer et al., 1987; Broekkamp et al., 1986; Gyertyan, 1995; Poling et al., 1981) questioned the defensive nature of such behavior. However, whereas the defensive nature of marble burying behavior is still actively debated, the mouse marble burying test has been used as a screening model for the detection of anxiolytics. Indeed, benzodiazepine receptor agonists such as diazepam or chlordiazepoxide (Archer et al., 1987; Broekkamp et al., 1986; Gyertyan, 1995) decrease the number of marbles buried. In addition, acute administration of certain classes of antidepressants (for review see Borsini et al., 2002; De Boer and Koolhaas, 2003) like selective serotonin reuptake inhibitors (SSRIs) (Ichimaru et al., 1995; Martin et al., 1998; Njung'e and Handley, 1991a), serotonin and noradrenaline reuptake inhibitors (SNRIs) (Millan et al., 2001) and tricyclic antidepressants

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(TCAs) (Ichimaru et al., 1995; Millan et al., 2001) has been shown to dose-dependently inhibit marble burying in mice. In the clinic, these classes of antidepressants also show anxiolytic effects, but only after chronic treatment (Bandelow et al., 2002). In addition to sensitivity to a wide range of clinically used anxiolytic agents, marble burying can be reduced by molecules such as metabotropic glutamate 5 receptor antagonists or nonpeptidergic neurokinin receptor antagonists, compounds having anxiolytic-like properties in classical anxiety tests in rodents (Millan et al., 2002; Spooren et al., 2000). Even though the foregoing suggests that the marble burying test may have a good predictive validity for anxiety, burying behavior is also reduced by other classes of compounds such as classical antipsychotics (Broekkamp et al., 1986) suggesting that marble burying alone has limited predictive validity for anxiety. To overcome this issue and try to differentiate anxiolytic effects from unspecific effects of compounds, Broekkamp et al. (1986) compared effects on marble burying and grooming in separate experiments in mice. They showed that anxiolytics, but not antipsychotics, selectively reduced marble burying without affecting grooming. On the other hand, the antidepressants imipramine and mianserin failed to show any specific effects since they reduced the burying score only at doses that also affected grooming. An alternative procedure was proposed by Njung'e and Handley (1991b) who measured locomotor activity in separate experiments. They showed that, in contrast to yohimbine, both diazepam and the SSRI zimelidine could reduce marble burying at doses that did not affect locomotor activity. In other studies, horizontal activity was measured during marble burying (Archer et al., 1987; Ichimaru et al., 1995). In the first of these studies diazepam reduced marble burying and locomotor activity at similar doses. In the second, Ichimaru et al. (1995) found selective effects of the antidepressants clomipramine and fluvoxamine whereas desipramine did not reduce marble burying. Taken together these modified procedures suggest that it is possible to improve the predictive validity of the marble burying test. In the present study, we tried to improve the predictive validity of the marble burying test in a slightly different manner. First, we selected a mouse strain that showed high marble burying and which avoided marbles when given the chance. Second, we used a videotracking system to measure locomotor activity during the marble burying test. This allowed us to examine whether a compound affected marble burying but not locomotor activity, i.e. a selective effect on marble burying. In case locomotor activity during the marble burying test was reduced, additional tests were performed in a test box without marbles or sawdust (spontaneous locomotor activity). The latter was measured only for doses that significantly decreased both burying behavior and locomotor activity in order to limit the number of animals used and to determine whether hypolocomotor effects could underlie the effects on marble burying. To validate this new paradigm as a screening test for anxiolytics, we tested a variety of anxiolytics, anxiogenics, antidepressants (i.e. those with reported anxiolytic effects after chronic treatment) and several miscellaneous compounds. In addition, two compounds acting on novel targets (the metabotropic glutamate 5 receptor antagonist 2-methyl-6-(phenylethynyl)pyridine (MTEP) (Busse et al., 2004) and the nociceptin

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orphanin FQ peptide receptor agonist Ro 64-6198 (Jenck et al., 2000)) were examined. Part of this work was published before in an abstract form (Prinssen et al., 2005). 2. Materials and methods 2.1. Animals Experiments were performed with male C57BL/6J, CBA/J and BALB/c mice (RCC, Füllinsdorf, CH-4414 Switzerland) weighing between 25 and 30 g. Animals were housed under standard maintenance conditions (12:12 h light–dark cycle, lights on at 6:00 AM; 21–23 °C; 55–65% relative humidity). Food and water were given ad libitum. In general, animals were group-housed (5/cage) in transparent polycarbonate cages (type 3, length 42 × width 46 × height 15 cm) with a thin layer of sawdust bedding for 5– 7 days. One day prior to behavioral testing, mice were singlehoused in a type 2 cage (length 26 × width 21 × height 15 cm) on a thick layer (5 cm) of sawdust. For pharmacological validation of the MBT, performed in C57BL/6J mice, some of the compounds (i.e. alprazolam, tiagabine, fenobam, buspirone, citalopram, phenelzine, chlorpromazine and atropine) were tested in mice that were single-housed in a type 2 cage upon arrival (i.e. 5–7 days before test). This was made necessary due to an increase in aggressive behaviors in group-housed animals, leading to the death of one animal of the group. We believe that this change in housing duration did not markedly affect our results for several reasons: 1) there was no systematic difference in the baseline marble burying and locomotor activity during the marble burying test between animals single-housed for 1 day and those single-housed for 5– 7 days (Table 1) we repeated the dose–response for diazepam in animals that were single-housed upon arrival (data not shown) and found results similar to those isolated for 1 day (Fig. 2) singlehoused mice show clear differences in neurochemical measures or drug sensitivity only after several weeks of isolation (Guidotti et al., 2001; Pinna et al., 2003, 2006; Rilke et al., 1998; Welch and Welch, 1968). All compounds in the spontaneous locomotor activity experiments were tested in animals that were single-housed upon arrival. For each test, animals were used only once. The present procedure received prior approval from local committee based on adherence to Swiss federal regulations and guidelines on animal experimentation provided by the Swiss Academy of Sciences and Swiss Academy of Medical Sciences (1995). 2.2. Behavioral procedures All behavioral tests were performed under a dim white ceiling light (15 lx). 2.2.1. Marble burying test In the standard marble burying condition and for strain comparisons, 12 glass marbles were evenly spaced in the home cage in the presence of the mouse. Similarly, in the two-zones marble burying condition, 8 glass marbles were evenly spaced but only on one half of the box. After 30 min (strain comparisons and two-zones condition) or 15 min (standard condition) the number of marbles at least two-thirds covered by

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Table 1 Vehicle group values for marble burying, locomotor activity during the marble burying test and spontaneous locomotor activity Drugs

Marble burying

Locomotor activity (cm)

Spontaneous locomotor activity (cm)

Alprazolam Diazepam Chlordiazepoxide Tiagabine Pregabalin Fenobam Buspirone MTEP Ro 64-6198 Paroxetine Fluoxetine

10.2 ± 0.73 11.1 ± 0.30 10.5 ± 0.87 8.7 ± 0.88 9.4 ± 0.72 8.7 ± 0.88 10.2 ± 0.73 9.7 ± 0.88 7.0 ± 0.85 9.2 ± 1.10 10.5 ± 0.87

2482 ± 312.7 2208 ± 230.4 1567 ± 168.0 2626 ± 329.6 3258 ± 680.6 2626 ± 329.6 2482 ± 312.7 2303 ± 318.3 1291 ± 209.3 2149 ± 210.7 1567 ± 168.0

Citalopram Duloxetine Clomipramine

10.6 ± 0.42 8.2 ± 1.57 8.5 ± 0.46

2976 ± 338.7 1578 ± 217.7 2039 ± 262.6

Desipramine Phenelzine Yohimbine

6.7 ± 0.96 9.1 ± 0.88 9.2 ± 1.10

2472 ± 473.3 2490 ± 326.5 2149 ± 210.7

FG 7142 mCPP Haloperidol

7.0 ± 0.85 9.7 ± 1.11 9.7 ± 1.11

1291 ± 209.3 1991 ± 355.8 1991 ± 355.8

9.2 ± 0.70 9.5 ± 0.42 10.7 ± 0.45 10.7 ± 0.45

2784 ± 316.4 3370 ± 260.7 2132 ± 179.7 2132 ± 179.7

2478 ± 193.6 (1) nt 1969 ± 158.9 (100) 2576 ± 211.0 (3; 10) nt nt 2943 ± 312.0 (10; 30) nt 2478 ± 193.6 (3) 2387 ± 373.2 (3) 1805 ± 125.9 (10); 1969 ± 158.9 (30) 2336 ± 270.8 (30) 2092 ± 317.7 (30) 2092 ± 317.7 (10); 1969 ± 158.9 (30) nt nt 1969 ± 158.9 (3); 2387 ± 373.2 (10) nt 2387 ± 373.2 (3) 1969 ± 158.9 (0.3); 2387 ± 373.2 (1) 2789 ± 446.4 (3) nt nt nt

Chlorpromazine Atropine D-Amphetamine Morphine

Vehicle values expressed as mean ± SEM; nt, not tested. For spontaneous locomotor activity, each value corresponds to the vehicle control value for the dose(s) shown between brackets in mg/kg.

sawdust was counted. Note that the duration of the test in the standard condition was limited to 15 min because pilot studies showed that the number of marbles buried after this period was nearly maximal (cf. results). Simultaneously, the locomotor activity, expressed as distance traveled in cm, was recorded by a videotracking system (Videotrack, View Point-Behavior Technology, France). In the two-zones condition, the videotracking system allowed the measurement of the time spent on the side with or without marbles. During the pretreatment time period of 30 min for all tested compounds (except D-amphetamine: 10 min), test mice were placed back in their home cage. 2.2.2. Spontaneous locomotor activity measures Mice treated with either vehicle or selected compounds were placed in an empty cage (type 2; see above) and spontaneous locomotor activity (in cm) was recorded by the videotracking system for 15 min. During the pretreatment time period of 30 min test mice were placed back in their home cage. 2.3. Drugs Alprazolam, diazepam, chlordiazepoxide HCl, pregabalin, fluoxetine HCl, desipramine HCl, buspirone, chlorpromazine HCl, morphine, fenobam, 2-methyl-6-(phenylethynyl)pyridine (MTEP) HCl, Ro 64-6198 HCl, (synthesized at F. Hoffmann-La

Roche Ltd.), tiagabine HCl (Sequoia Research Products Ltd.), duloxetine HCl (extracted from commercially available capsules at F. Hoffmann-La Roche Ltd.), clomipramine, D-amphetamine sulfate, atropine, yohimbine HCl, FG 7142, meta-chlorophenylpiperazine (mCPP), phenelzine (Sigma-Aldrich), paroxetine HCl and citalopram HCl (TRC inc.) and haloperidol (Janssen-Cilag, Switzerland) were examined. All compounds were administered i. p. in 0.3% (weight/volume) Tween-80 in physiological saline (0.9%) in a volume of 10 ml/kg body weight. Doses are expressed as free base, alprazolam (0.03, 0.1, 0.3, 1 mg/kg), diazepam (1, 3, 10 mg/kg), chlordiazepoxide (10, 30, 100 mg/kg), tiagabine (1, 3, 10 mg/kg), pregabalin (3, 10, 30, 100 mg/kg), fenobam (30, 100, 300 mg/kg), buspirone (1, 3, 10, 30 mg/kg), paroxetine (0.1, 0.3, 1, 3 mg/kg), fluoxetine (3, 10, 30 mg/kg), citalopram (0.3, 1, 3, 10, 30 mg/kg), duloxetine (1, 3, 10, 30 mg/kg), clomipramine (3, 10, 30 mg/kg), desipramine (1, 3, 10, 30 mg/kg), phenelzine (3, 10, 30, 100 mg/kg), yohimbine (0.3, 1, 3, 10 mg/kg), FG 7142 (1, 3, 10, 30 mg/kg), mCPP (0.3, 1, 3 mg/kg), haloperidol (0.03, 0.1, 0.3, 1 mg/kg), chlorpromazine (0.3, 1, 3 mg/kg), atropine (1, 3, 10 mg/ kg), D-amphetamine (0.1, 0.3, 1, 3 mg/kg), morphine (0.3, 1, 3, 10 mg/kg), MTEP (1, 3, 10, 30 mg/kg), Ro 64-6198 (0.1, 0.3, 1, 3 mg/kg). 2.4. Statistical analysis All data were expressed as mean ± SEM (n = 7–10 per group). The number of marbles buried, locomotor activity during the marble burying test, and time spent in different zones of the test box, were analyzed by one-way analysis of variance (ANOVA). Except for strain comparisons for which a Newman–Keuls test was used as a post-hoc test, planned Dunnett's tests were used to determine significant differences between drug-treated groups and vehicle groups (1-tailed for marble burying and 2-tailed for locomotor activity during marble burying). Spontaneous locomotor activity was analyzed by t-tests (2-tailed). Significance was set at P b 0.05. 3. Results Note that in this section we always refer to the locomotor activity measured during the marble burying test as “locomotor activity”, and to the locomotor activity measured in control experiments as “spontaneous locomotor activity”. 3.1. Strain comparison As shown in Fig. 1A, in the standard marble burying condition, there was an overall strain effect on marble burying [F (2,21) = 16.8, P b 0.001]. CBA/J and BALB/c mice buried significantly less marbles (P b 0.01 and P b 0.001, respectively) than C57BL/6J. In addition, BALB/c mice buried significantly less marbles than CBA/J (P b 0.05). The two strains showing higher burying scores, C57BL/6J and CBA/J mice, were selected for further investigations in the two-zone condition. In this condition, the number of marbles buried was not different between both strains (Fig. 1B). However, the time spent in the zone containing marbles, compared to the time spent in the

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Fig. 1. Strain comparison on number of marbles buried: (A) in standard condition; (B) in two-zones condition. Strain effect on time spent on side with or without marbles in the two-zones condition: (C) in C57BL/6J; (D) in CBA/J. Data represent mean ± SEM (n = 8–10). ⁎P b 0.05, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 compared to C57BL/ 6J mice, Newman–Keuls test; #P b 0.05, ##P b 0.01, ###P b 0.001 compared to CBA/J mice, Newman–Keuls test. +P b 0.05, ++P b 0.01, +++P b 0.001, t-test.

zone without marbles, was significantly lower (P b 0.01) in C57BL/6J (Fig. 1C), whereas in CBA/J mice no such difference was observed (Fig. 1D). Whereas CBA/J mice spent about 50% of total time on the side containing marbles, C57BL/6J spent only 25% of the test duration on this side. Based on these results, C57BL/6J mice were used for the pharmacological studies.

3.2. Effects of compounds of different classes on marble burying, locomotor activity and spontaneous locomotor activity Data are expressed as percentage of vehicle group, but the mean values for vehicle groups are given in the Table 1. The overall mean for all the vehicle groups was 9.4 ± 0.18 for marble burying score (n = 189), 2253 ± 70 (cm) for locomotor activity

Fig. 2. Effects of anxiolytics on marble burying, locomotor activity during the marble burying test and spontaneous locomotor activity. Data represent mean ± S.E.M. expressed as percentage of the vehicle group (n = 7–8). For marble burying and locomotor activity, ⁎P b 0.05, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 significantly different from vehicle group, Dunnett's test (1-tailed for marble burying, 2-tailed for locomotor activity). For spontaneous locomotor activity, +P b 0.05, ++P b 0.01, +++P b 0.001 significantly different from vehicle group, t-test (2-tailed). Filled circles indicate numbers of marbles buried or locomotor activity during the marble burying test. Open triangles indicate spontaneous locomotor activity.

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(n = 189) and 2257 ± 76 (cm) for spontaneous locomotor activity (n = 112), expressed as mean ± S.E.M.. 3.2.1. Anxiolytics As shown in Fig. 2, alprazolam, diazepam and chlordiazepoxide significantly reduced the number of marbles buried [F(4,35) = 25.6, P b 0.001, F(4,35) = 8.7, P b 0.001 and F(3, 26) =7.7, P b 0.001, respectively] and significance was reached for the doses of 0.3 and 1 mg/kg of alprazolam, 3 and 10 mg/kg of diazepam and 30 and 100 mg/kg of chlordiazepoxide. Whereas diazepam had no significant effect on locomotor activity [F(4,35) = 1.6, P = 0.20], chlordiazepoxide and alprazolam significantly modified this parameter [F(4,35) = 10.4, P b 0.001 and F(3,26) = 4.9, P b 0.01, respectively] and activity was significantly decreased for the dose of 1 mg/kg of alprazolam and showed a trend to a significant decrease (P = 0.07) for the highest dose of chlordiazepoxide. Alprazolam significantly reduced spontaneous locomotor activity at 1 mg/kg (P b 0.001). In contrast, chlordiazepoxide did not affect spontaneous locomotor activity at 100 mg/kg (P = 0.11). Tiagabine significantly reduced marble burying and locomotor activity [F (3,28) = 30.9, P b 0.001 and F(3,28) = 10.78, P b 0.001, respectively] and significance was reached at 3 and 10 mg/kg for both parameters. At 10 mg/kg (P b 0.001), but not 3 mg/kg (P = 0.3), tiagabine significantly decreased spontaneous locomotor activity. Pregabalin did not affect marble burying [F(4,32) = 0.6, P = 0.7] and locomotor activity [F(4,32) = 0.75, P = 0.6]. Fenobam dosedependently decreased marble burying [F(3,27) = 9.7, P b 0.001] and significance was reached for the dose of 300 mg/kg. Locomotor activity was significantly increased by fenobam [F (3,27) = 3.37, P b 0.05], this was due to a significant effect of the lowest dose tested. Buspirone significantly reduced marble burying [F(4,35) = 40.7, P b 0.001] and significance was reached at 3, 10 and 30 mg/kg. Locomotor activity was also significantly reduced [F(4,35) = 13.6, P b 0.001] and doses of 10 and 30 mg/kg

were different from the vehicle group. For these two highest doses, spontaneous locomotor activity was also reduced (P b 0.001). 3.2.2. Antidepressants The 3 SSRIs, paroxetine, fluoxetine and citalopram, dosedependently reduced marble burying [F(4,35) = 35.56, P b 0.001, F(3,28) = 40, P b 0.001 and F(5,42)= 8.4, P b 0.001, respectively] (Fig. 3). Significance was reached for the doses of 0.3, 1, 3 mg/kg for paroxetine, 10 and 30 mg/kg for fluoxetine and 3, 10, 30 mg/ kg for citalopram. Whereas locomotor activity was significantly altered by paroxetine [F(4,35) = 3.9, P b 0.01] at all doses tested, spontaneous locomotor activity was not affected at the dose of 3 mg/kg (P = 0.21). Fluoxetine significantly decreased locomotor activity [F(3.28) = 16.2, P b 0.001] for the doses of 10 and 30 mg/ kg and spontaneous locomotor activity was decreased at 30 mg/kg (P b 0.001) but not 10 mg/kg (P = 0.47). Citalopram reduced locomotor activity [F(5,42) = 2.5, P b 0.05] with significant effects at 3 and 30 mg/kg. In contrast, spontaneous locomotor activity was increased by the dose of 30 mg/kg (P b 0.05). Duloxetine significantly reduced marble burying and locomotor activity [F (4,35) = 9.9, P b 0.001 and F(4,35) = 11.15, P b 0.001]. Significance was reached at 10 and 30 mg/kg for marble burying and 3, 10 and 30 mg/kg for locomotor activity. However, spontaneous locomotor activity was not significantly affected by the dose of 30 mg/kg (P = 0.7). As shown in Fig. 3, clomipramine, but not desipramine, significantly reduced marble burying [F(3,28) = 17.0, P b 0.001 and F(4,35) = 0.22, P = 0.93] and locomotor activity [F(3,28) = 17.8, P b 0.001 and F(4,35) = 0.31, P = 0.65, respectively]. For clomipramine, significance was reached at 10 and 30 mg/kg for marble burying and at 3, 10 and 30 mg/kg for locomotor activity. Spontaneous locomotor activity was decreased at 30 mg/kg (P b 0.001) but not 10 mg/kg (P = 0.15). Phenelzine decreased marble burying [F(4,35) = 10.2, P b 0.001]

Fig. 3. Effects of antidepressants on marble burying, locomotor activity during the marble burying test and spontaneous locomotor activity. Effects of SSRIs (paroxetine, fluoxetine, citalopram), SNRI (duloxetine), TCAs (clomipramine, desipramine) and an MAOI (phenelzine). Data represent mean ± S.E.M. expressed as percentage of the vehicle group (n = 7–8). For marble burying and locomotor activity, ⁎P b 0.05, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 significantly different from vehicle group, Dunnett's test (1-tailed for marble burying, 2-tailed for locomotor activity). For spontaneous locomotor activity, +P b 0.05, ++P b 0.01, +++P b 0.001 significantly different from vehicle group, t-test (2-tailed). Filled circles indicate numbers of marbles buried or locomotor activity during the marble burying test. Open triangles indicate spontaneous locomotor activity.

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Fig. 4. Effects of miscellaneous compounds on marble burying, locomotor activity during the marble burying test and spontaneous locomotor activity. Effects of anxiogenics (yohimbine, FG 7142, mCPP), antipsychotics (haloperidol, chlorpromazine), atropine, D-amphetamine and morphine. Data represent mean ± S.E.M. expressed as percentage of the vehicle group (n = 7–8). For marble burying and locomotor activity, ⁎P b 0.05, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 significantly different from vehicle group, Dunnett's test (1-tailed for marble burying, 2-tailed for locomotor activity). For spontaneous locomotor activity, +P b 0.05, ++P b 0.01, +++P b 0.001 significantly different from vehicle group, t-test (2-tailed). Filled circles indicate numbers of marbles buried or locomotor activity during the marble burying test. Open triangles indicate spontaneous locomotor activity.

but not locomotor activity [F(4,35) = 0.9, P = 0.46], reaching significance at 100 mg/kg. 3.2.3. Miscellaneous compounds As reported in Fig. 4, yohimbine, haloperidol and chlorpromazine significantly decreased marble burying [F(4,35) = 15.5, P b 0.001, F(4,35) = 7.57, P b 0.001 and F(3,28) = 6.7, P b 0.01, respectively] and locomotor activity [F(4,35) = 24.8, P b 0.001, F (4,35)= 15.63, P b 0.001 and F(3,28) = 6.2, P b 0.01, respectively]. Spontaneous locomotor activity was significantly reduced at doses that reduced marble burying and locomotor activity, i.e. 3 and 10 mg/kg for yohimbine, 0.3 and 1 mg/kg for haloperidol, 3 mg/kg for chlorpromazine (P b 0.001 for all comparisons). Atropine significantly decreased marble burying [F(3,28)= 9.4, P b 0.001] reaching significance only for the highest dose. However, it did not affect locomotor activity [F(3,28) = 1.57, P = 0.22]. Whereas FG 7142 did not significantly inhibit marble burying [F (4,35) = 0.95, P = 0.44], it significantly increased locomotor activity [F(4,35) = 3.35, P b 0.05] reaching significance for the dose of 10 mg/kg. In contrast to FG 7142, D-amphetamine and morphine reduced marble burying [F(4,35) = 12.64, P b 0.001 and F(4,35) = 15.6, P b 0.001], and doses of 1 and 3 mg/kg for Damphetamine and of 10 mg/kg for morphine differed significantly from vehicle groups (Fig. 4). Both compounds also significantly increased locomotor activity [F(4,35) = 8.67, P b 0.001 and F (4,32)= 3.18, P b 0.05] and significance was reached for the highest doses tested, i.e. 3 mg/kg for D-amphetamine and 10 mg/ kg for morphine. mCPP induced a significant reduction of marble burying [F(3,25) = 19.18, P b 0.001] and locomotor activity [F (3,25)= 6.6, P b 0.01]. Post-hoc tests showed that doses of 0.3, 1 and 3 mg/kg for marble burying and doses of 1 and 3 mg/kg for locomotor activity differed from vehicle. In contrast, spontaneous locomotor activity was not affected by mCPP at 3 mg/kg (P = 0.99).

3.2.4. Putative anxiolytics As shown in Fig. 5, MTEP significantly decreased marble burying [F(4,35) = 3.3, P b 0.05] and significance was reached at 30 mg/kg. In contrast, it did not affect locomotor activity [F (4,35) = 0.37, P = 0.82]. Ro 64-6198 significantly reduced

Fig. 5. Effects of the putative anxiolytics MTEP and Ro 64-6198 on marble burying, locomotor activity during the marble burying test and spontaneous locomotor activity. Data represent mean ± S.E.M. expressed as percentage of the vehicle group (n = 7–8). For marble burying and locomotor activity, ⁎P b 0.05, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 significantly different from vehicle group, Dunnett's test (1-tailed for marble burying, 2-tailed for locomotor activity). For spontaneous locomotor activity, +P b 0.05, ++P b 0.01, +++P b 0.001 significantly different from vehicle group, t-test (2-tailed). Filled circles indicate numbers of marbles buried or locomotor activity during the marble burying test. Open triangles indicate spontaneous locomotor activity.

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Table 2 Minimal effective doses of compounds to inhibit marble burying, locomotor activity during the marble burying test and spontaneous locomotor activity Drugs

Alprazolam Diazepam Chlordiazepoxide Tiagabine Pregabalin Fenobam Buspirone MTEP Ro 64-6198 Paroxetine Fluoxetine Citalopram Duloxetine Clomipramine Desipramine Phenelzine Yohimbine FG 7142 mCPP Haloperidol Chlorpromazine Atropine D-Amphetamine Morphine

Marble burying MED

Locomotor activity MED

Spontaneous locomotor activity MED

Ratio MEDs

Ratio MEDs

Locomotor activity/ marble burying

Spontaneous locomotor activity/marble burying

0.3 3 30 3 N100 300 3 30 1 0.3 10 3 10 10 N30 100 3 N30 ≤0.3 0.3 3 10 1 10

1 N30 N100a 3 N100 N300 10 N30 3 ≤0.1 10 3 3 3 N30 N100 1 N30 1 0.3 3 N10 N3c N10c

≤1 nt N100 10 nt nt ≤10 nt N3 N3 30 N30b N30 30 nt nt ≤3 nt N3 ≤0.3 ≤3 nt nt nt

3 N10 N3 1 na N1 3 N1 3 ≤0.3 1 1 0.3 0.3 na N1 0.3 na ≥3 1 1 N1 N3 N1

≤3 na N3 3 na na ≤3 na N3 N10 3 N10 N3 3 na na ≤1 na N10 ≤1 ≤1 na na na

Anxiolytic profile

+ + + + − + + + + + + + + + − + − − + − − + −c −c

Minimal effective dose (MED), expressed in mg/kg; aTendency towards a decrease in locomotor activity (P = 0.07); bIncrease in spontaneous locomotor activity, but not locomotor activity during the marble burying test, was observed; cIncrease in locomotor activity during the marble burying test was observed; +, anxiolytic-like effect, i.e. selective effect on marble burying with respect to locomotor activity during the marble burying test or spontaneous locomotor activity (ratios N1) and no increase in locomotor activity during the marble burying test at high doses; −, unselective effect on marble burying with respect to locomotor activity during the marble burying test and spontaneous locomotor activity (ratios ≤ 1) or increase in locomotor activity during the marble burying test at high doses; nt, not tested; na, not applicable.

marble burying [F(4,34) = 4.7, P b 0.01] and doses of 1 and 3 mg/kg were significantly different from the vehicle group. There was an overall effect on locomotor activity [F(4,34) = 7.0, P b 0.001] and significance was reached for the dose of 3 mg/kg. In contrast, at this dose, spontaneous locomotor activity was not significantly affected (P = 0.4). 3.2.5. Minimal effective dose comparisons As shown in Table 2, comparisons of minimal effective doses showed that, except for pregabalin which was inactive in the marble burying test, most of the anxiolytics (5 out of 6) were characterized by a selective inhibition of marble burying with respect to locomotor activity (ratio of locomotor activity/marble burying N 1). The same was true for the putative anxiolytics MTEP and Ro 64-6198. Most of the antidepressants (5 out of 7) were characterized by a selective inhibition of marble burying with respect to spontaneous locomotor activity (ratio spontaneous locomotor activity/marble burying N 1) but not with respect to locomotor activity (locomotor activity/marble burying ≤ 1). The other miscellaneous compounds showed a large variety of profiles, mostly different from the above described profiles. For example, haloperidol, chlorpromazine, and yohimbine, showed ratios spontaneous locomotor activity/marble burying that were ≤ 1. Morphine and D-amphetamine had a different profile since

they increased locomotor activity at doses that inhibited marble burying. mCPP showed a selective inhibition of marble burying with respect to spontaneous locomotor activity (ratio spontaneous locomotor activity/marble burying N 1) but not with respect to locomotor activity (locomotor activity/marble burying = 1). Atropine showed selective inhibition of marble burying with respect to locomotor activity (ratio locomotor activity/marble burying N 1). 4. Discussion In the present study, we tried to improve the predictive validity of the marble burying test for anxiety (1) by selecting a strain of mice (C57BL/6J) that showed spontaneous avoidance of glass marbles, and (2) by measuring locomotor activity with a videotracking system both during the marble burying test and, if needed, in control experiments (spontaneous locomotor activity). Using this protocol and by comparing marble burying scores with locomotor activity measures, we showed that most of the anxiolytics and antidepressants inhibited marble burying at doses that did not affect locomotor activity during the marble burying test or spontaneous locomotor activity, whereas most of the non-anxiolytics either did not show such a separation or increased locomotor activity during the marble burying test. As

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summarized in Table 2, this modified paradigm clearly improves the predictive validity for anxiety as compared to marble burying alone. In the two-zones marble burying procedure, in which 8 glass marbles were present in one half of the box, C57BL/6J mice displayed marked avoidance of the zone containing marbles, whereas CBA/J conspecifics did not. In that respect, our data in CBA/J mice are in line with other studies showing that Swiss and MF1 mice as well as Hooded rats did not avoid the marbles (Gyertyan, 1995; Njung'e and Handley, 1991b; Poling et al., 1981). Our data with C57BL/6J are the first to clearly demonstrate avoidance of glass marbles in a mouse strain. However, to which extent marbles may be considered as objects inducing expression of defensive behaviors commonly related to fear and anxiety in rodents (Gray and McNaughton, 2000) remains to be determined. The present results also showed that C57BL/6J mice buried more marbles than CBA/J and BALB/c mice when tested in a standard marble burying test. Taken together, this high burying score together with the marble-induced avoidance observed in C57BL/6J mice (apparent face validity for anxiety) made us select this strain to re-examine the predictive validity of the marble burying test for anxiety. Using C57BL/6J mice in the standard procedure, we showed that, out of 24 compounds tested, 21 decreased marble burying. This effect of alprazolam, diazepam and chlordiazepoxide was in agreement with the previous findings (Broekkamp et al., 1986; Spooren et al., 2000). Similarly, as reported in literature (Ichimaru et al., 1995; Njung'e and Handley, 1991a), marble burying was decreased by the 5HT1A agonist buspirone. Our data are the first to demonstrate an effect of fenobam, MTEP, tiagabine and Ro 64-6198 on marble burying. Among all the anxiolytics tested, pregabalin was the only one which did not inhibit marble burying. As expected (for review, cf. Borsini et al., 2002), marble burying was reduced by acute administration of different classes of antidepressants with slow-onset anxiolytic properties in the clinic, such as SSRIs (citalopram, paroxetine, fluoxetine), an SNRI (duloxetine), an TCA (clomipramine) and an MAOI (phenelzine). In contrast, the TCA desipramine was ineffective on marble burying in line with a reported study (Ichimaru et al., 1995). Our data also confirmed that miscellaneous compounds such as chlorpromazine, haloperidol, atropine, mCPP and yohimbine can reduce marble burying (Broekkamp et al., 1986; Njung'e and Handley, 1991a,b). As further new findings, we found that morphine and D-amphetamine, but not FG 7142, decreased marble burying. Taken together, these data show that marble burying under our conditions is not only sensitive to anxiolytics and antidepressants but also to certain classes of non-anxiolytic compounds and suggest that marble burying alone has limited predictive validity for anxiety. The combination of marble burying and simultaneous measurements of locomotor activity allowed us to improve the predictive validity. That is, most of the anxiolytics and antidepressants (i.e. those with reported anxiolytic effects after chronic treatment) produced a selective inhibition of marble burying either with respect to locomotor activity during the marble burying test or spontaneous locomotor activity. Impor-

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tantly, the majority of the miscellaneous compounds showed several profiles that were mostly different from that of anxiolytics, the exception being mCPP and atropine which will be discussed below. The profile of chlordiazepoxide and diazepam was consistent with the previous marble burying studies showing selective effects of benzodiazepine receptor agonists (Broekkamp et al., 1986; Njung'e and Handley, 1991b; but see Archer et al., 1987; Ichimaru et al., 1995). The weak separation between marble burying and locomotor activity during the marble burying test following alprazolam is in line with its more marked sedative properties as compared to diazepam in rodents after acute treatment (Soderpalm et al., 1989). Even though we found a selective effect of buspirone on marble burying (based on MED comparisons), there was a clear overlap between the observed inhibition of marble burying and locomotor activity during the test in agreement with the previous reported findings (De Boer and Koolhaas, 2003; Ichimaru et al., 1995; Njung'e and Handley, 1991a). In accordance with literature, SSRIs, SNRIs or the TCA clomipramine decreased marble burying at doses that did not affect spontaneous locomotor activity (Ichimaru et al., 1995; Njung'e and Handley, 1991a,b). In agreement with Broekkamp et al. (1986), chlorpromazine and haloperidol did not show any selective effects in our paradigm whereas atropine showed a selective profile in both tests. Similar to the reported studies with yohimbine (Njung'e and Handley, 1991b) and mCPP (Njung'e and Handley, 1991a), we found that yohimbine had no selective profile in the current paradigm whereas mCPP selectively inhibited marble burying as compared to spontaneous locomotor activity. Taken together this modified procedure shows an improved predictive validity for anxiety when compared to marble burying alone (cf. Table 2). The present results also show some limitations in the predictive validity of this procedure for anxiety. First, atropine and mCPP showed anxiolytic-like profiles. This was surprising since mCPP is commonly described as an anxiogenic both in man and in animal studies (Graeff, 2002). However anxiolyticlike effects of mCPP have already been described in other paradigms such as the periaqueductal gray-stimulation model (Jenck et al., 1998). In this study, authors suggested that these anxiolytic-like effects may be due to the 5HT2C receptor agonist properties of mCPP. This may also be the case in our procedure since it was already shown that 5HT2C receptor agonists were effective in reducing marble burying (Jenck et al., 1998). The anxiolytic-like profile of atropine is of interest in that this compound may have calming properties in patients having to undergo surgery (Kirvela and Kanto, 1992). Also, the effects of anticholinergic agents in preclinical anxiety tests are far from clear. Indeed, whereas anticholinergic drugs reduced anxietyrelated behaviors in rat exposed to threatening stimuli (Mollenauer et al., 1976), they showed anxiogenic-like properties in the mouse elevated plus maze (Rodgers and Cole, 1995; Zarrindast et al., 2000). Second, desipramine and pregabalin were false negatives in the present procedure. The lack of effect of desipramine suggests that our paradigm is not sensitive to compounds acting mainly at the norepinephrine system in contrast with compounds acting on serotonin. The lack of effect of pregabalin suggests that the mouse marble burying test, in contrast to the rat elevated plus

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maze or the conflict test (Field et al., 2001), may not be sensitive to compounds acting at voltage-dependent calcium channels (Lauria-Horner and Pohl, 2003; Stahl, 2004). However, it cannot be excluded that strain-specific pharmacodynamic and/or pharmacokinetic properties play a role. Note that in spite of these limitations, 14 out of 16 of the anxiolytics and antidepressants tested in this procedure showed a selective anxiolytic profile following an a posteriori definition of selectivity (i.e. selective effect on marble burying with respect to locomotor activity during the marble burying test or spontaneous locomotor activity and no increase in locomotor activity during the marble burying test at high doses, cf. Table 2), whereas only 1 out of 7 of the miscellaneous compounds, excluding atropine (see above), showed an anxiolytic-like profile. In addition, our data suggest that this paradigm is sensitive to putative anxiolytics, i.e. the NOP receptor agonist Ro 64-6198 and the metabotropic glutamate 5 receptor antagonist MTEP. The latter was not unexpected since the atypical anxiolytic fenobam, which was recently reported to be a selective metabotropic glutamate 5 receptor antagonist (Porter et al., 2005), showed a selective anxiolytic profile in the present study. In conclusion, the present results show that combining the marble burying test with measures of locomotor activity improves the predictive validity of the test for anxiety. Also, our procedure allows to limit the number of animals used and therefore may be used as a valuable screening test for the detection of compounds having anxiolytic effects. Acknowledgements We thank Drs Joe Wettstein and James Martin for helpful discussions, Dr Robert Weikert for providing us with the extracted compounds, and Aurelie Boucher for her help in establishing early versions of the test paradigm. References Archer, T., Fredriksson, A., Lewander, T., Soderberg, U., 1987. Marble burying and spontaneous motor activity in mice: interactions over days and the effect of diazepam. Scand. J. Psychol. 28, 242–249. Bandelow, B., Zohar, J., Hollander, E., Kasper, S., Moller, H.J., 2002. World Federation of Societies of Biological Psychiatry (WFSBP) guidelines for the pharmacological treatment of anxiety, obsessive–compulsive and posttraumatic stress disorders. World J. Biol. Psychiatry 3, 171–199. Borsini, F., Podhorna, J., Marazziti, D., 2002. Do animal models of anxiety predict anxiolytic-like effects of antidepressants? Psychopharmacology 163, 121–141. Broekkamp, C.L., Rijk, H.W., Joly-Gelouin, D., Lloyd, K.L., 1986. Major tranquillizers can be distinguished from minor tranquillizers on the basis of effects on marble burying and swim-induced grooming in mice. Eur. J. Pharmacol. 126, 223–229. Busse, C.S., Brodkin, J., Tattersall, D., Anderson, J.J., Warren, N., Tehrani, L., Bristow, L.J., Varney, M.A., Cosford, N.D., 2004. The behavioral profile of the potent and selective mGlu5 receptor antagonist 3-[(2-methyl-1,3-thiazol4-yl)ethynyl]pyridine (MTEP) in rodent models of anxiety. Neuropsychopharmacology 29, 1971–1979. De Boer, S.F., Koolhaas, J.M., 2003. Defensive burying in rodents: ethology, neurobiology and psychopharmacology. Eur. J. Pharmacol. 463, 145–161. Field, M.J., Oles, R.J., Singh, L., 2001. Pregabalin may represent a novel class of anxiolytic agents with a broad spectrum of activity. Br. J. Pharmacol. 132, 1–4. Graeff, F.G., 2002. On serotonin and experimental anxiety. Psychopharmacology 163, 467–476.

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