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

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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 so-called 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.

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 forward-shoving 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, 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 (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 non-peptidergic 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 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).

Section snippets

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

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”.

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

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.

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