Factors controlling measures of anxiety and responses to novelty in the mouse

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1 Behavioural Brain Research 125 (2001) Factors controlling measures of anxiety and responses to novelty in the mouse Sandra E. File * Psychopharmacology Research Unit, Centre for Neuroscience, GKT School of Biomedical Sciences, King s College London, Hodgkin Building, Guy s Campus, London SE1 1UL, UK Received 25 May 2000; accepted 25 January 2001 Abstract This review focuses on factors influencing behaviour in the elevated plus-maze, the holeboard and the social transmission of food preference. The elevated plus-maze provides independent measures of anxiety (percentage of time spent on open arms) and activity (number of closed arm entries) and can be used in both males and females. Important sex differences emerge in factor loadings, and, whereas in males, anxiety is the primary factor, in females it is activity. On trial 2 in the plus-maze, the nature of the anxiety state is changed and thus this maze can be used to screen for possible genetic alterations in two distinct anxiety states. The holeboard provides independent measures of exploration and locomotor activity and habituation between sessions provides a useful measure of learning. Mice display neophobia and avoid novel foods, but information about their safety can be socially transmitted. A mouse that has sampled a novel food will be actively sniffed by others on its return to the colony. It is important to control for possible changes in social investigation, neophobia, olfactory sensitivity, anxiety and exploration, before it is concluded that a changed performance in this task is due to changes in learning Elsevier Science B.V. All rights reserved. Keywords: Anxiety; Phobia; Exploration; Motor activity; Learning 1. Introduction * Tel.: ; fax: address: sandra.file@kcl.ac.uk (S.E. File). This review focuses on tests of anxiety and responses to novelty, with detailed consideration of the elevated plus-maze test of anxiety, the use of the holeboard to measure exploration and motor activity and the social transmission of information about cues relating to novel food. Two main tests of anxiety have been developed for mice: the light dark exploration test [7] and the elevated plus-maze [30]. The light dark exploration test is heavily dependent on locomotor activity and, therefore, changes in this test may not necessarily be due to changes in anxiety. Thus, when this test is used, careful controls for possible changes in locomotor activity and light sensitivity should be included. Other tests have been used to measure anxiety, but these are even more contaminated by changes in exploration and locomotor activity. The punished four-plate test introduced by Boissier et al. [4] produces many false positives and even the anxiolytic benzodiazepines produce increases in both punished and unpunished crossings, [42] precluding a specific interpretation with respect to anxiety. The use of the open field has been criticised [1,16] and its main problem is that the measures are confounded and reflect changes in activity, exploration and anxiety. However, the Digiscan open field measure of locomotion has been recommended for an initial screen [9] in the behavioural phenotyping of transgenic and knockout mice. Differences among inbred mouse strains in open field activity depend on both the apparatus and the test condition, [6] but, in general, C57 strains of mice show high levels of locomotion and DBA/1 and BALB/c strains show low levels. It is important to note that some strain differences in open field locomotor activity have been found to be age-dependent [28]. In general, females show higher levels of locomotor activity, but the sex difference is dependent on strain and on early experience [1,2]. In male rats, locomotor activity in the central portion of the open /01/$ - see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S (01)

2 152 S.E. File / Beha ioural Brain Research 125 (2001) field is a parameter that loads on the same factor as the anxiety factor in the plus-maze, whereas in females it does not, and loads instead on the activity factor [15]. It is not known whether this will also be found in mice and, if so, whether it will apply to all strains. However, the finding does have potentially important implications for studies using genetically modified mice, where groups are often composed of both males and females, since it means that the same parameter is actually a measure of two quite distinct things in the two sexes. For an extended discussion of the importance of conceptually, and wherever possible, practically separating measures of anxiety, exploration and locomotor activity, see [15]. For a discussion of the importance of careful background strain selection with regard to the level of open field activity, see [8]. Due to the problems in interpreting the behavioural measures from the open field, the use of a specific test of anxiety is strongly advised and the holeboard test is recommended as a test that can provide independent measures of exploration and motor activity [12,16]. The sections on the plus-maze and holeboard will include discussion of the results of factor analysis studies, which provide useful data on the best behavioural parameters in each test. A brief outline of some of the most pertinent methodological issues will be given and illustrative data will be provided from experiments with genetically modified animals. The final section will describe an ethologically based test of learning, the social transmission of food preference [46] and the control experiments that should be conducted before conclusions are drawn that a genetic modification has changed learning, rather than indirectly changing performance. This will be illustrated by data from transgenic mice. 2. Elevated plus-maze trial 1 naïve animals The elevated plus-maze is in the form of a plus, with two open, elevated arms facing opposite to each other and separated by a central square, and two arms of the same dimensions, but enclosed by walls. The maze is raised off the ground so that the open arms combine elements of unfamiliarity, openness and elevation. The elevated plus-maze was extensively validated as a test of anxiety in the rat, using both physiological and pharmacological measures [35] and was then developed for use in the mouse [30]. For a detailed step-by-step protocol of this test see [20]. The measures of anxiety are the percentage of open arm entries and the percentage (%) of time spent on the open arms, both expressed asa%ofthetotal entries onto, or time spent on, the open and closed arms [30,35]. It is important to note this methodological detail. The percentages are not expressed with respect to the test duration (which is usually 5 min), because the time spent in the central square can vary between groups (e.g. drug treatment or genetic modification). It is not really known what this parameter reflects, but it has been suggested that it may relate to decision making and/or risk assessment [10,38,40,45]. Lister [30] showed that the behavioural parameters in the mouse plus-maze provided measures of two independent factors, one reflecting anxiety and one reflecting motor activity. A factor loading of unity means that an individual parameter is a perfect measure of the underlying factor, whereas a loading of 0.3 suggests that the parameter is a poor measure. The % of open arm entries and the % of time spent on the open arms both had loadings of 0.95, which means that they are extremely good measures of the state of anxiety generated by this test. In contrast, the total arm entries, the measure of activity that was originally proposed, had a loading of only 0.67 on the activity factor. It also carried a loading of 0.42 on the anxiety factor. This means that it is a contaminated measure and that changes in this parameter could reflect changes in anxiety or in activity. In later factor analyses of data from rats [14,18] and mice [38], the same factor structure was confirmed and it was found that the number of closed arm entries provided a better measure of motor activity. This was because it had a higher loading on the activity factor, but more importantly, because it did not also load on the anxiety factor (see Table 1). As well as being maintained across species, the same factor struc- Table 1 Orthogonal factor loadings ( 0.4) of parameters from the plus-maze in mice and rats Behavioral parameter Mice Rats Factor 1 (anxiety) Factor 2 (activity) Factor 1 (anxiety) Factor 2 (activity) % Open arm entries % Time open arms Total arm entries Closed arm entries er 0.98 Data reproduced with permission from Rodgers and Johnson [38] and Fernandes and File [14].

3 S.E. File / Beha ioural Brain Research 125 (2001) ture has also been found in several mouse strains [13,30,38]. Thus, it is likely to apply to the background strains used for genetic manipulations. Whilst it is always a possibility that a genetic modification will change this factor structure, this is probably not a major worry. However, there is one genetic difference that does change the factor structure of the plus-maze parameters. The plus-maze has been used in both male and female rats and mice [29,37], but a recent factor analysis in rats revealed a major sex difference in the relative importance of the two underlying factors [15]. The same two underlying factors of anxiety and motor activity emerged for males and females, but the relative importance of the two factors was different. For the males, the strongest factor was anxiety, with motor activity being relatively unimportant. For the females, the situation was reversed, with activity being the more important factor. This has important implications for experiments, since females may be less sensitive to manipulations that change anxiety in this test and more sensitive to those that influence activity. For studies of genetically modified mice, where animal numbers are limited and groups may comprise both males and females there could be a loss of sensitivity to genetic effects. This may not necessarily occur and in one study on Fyn-kinase deficient mice, there was a more marked increase in anxiety in the females than in the males [33]. It would be important to determine whether the same sex differences in factor structure that are found in rats also apply in mice. Certainly, the general finding in rats that males demonstrate higher levels of anxiety/fear than females [36] might be partly due to different underlying factor structures in the sexes, which might in turn be a consequence of the fact that most tests were developed and validated on males. This gender bias in animal tests could have important implications for both fundamental research and drug discovery. 3. Trial 2 and repeated testing The scores of rats can remain constant on repeated exposures to the plus-maze [35,45] and it was, therefore, originally thought that it would be possible to test animals on more than one occasion. In mice, however, the open arm entries have frequently been found to decrease with repeated testing [13,37,39], perhaps indicating increased anxiety. Also, Lister [30] found that whereas naïve mice, tested for the first time in the plus-maze, responded with a clear increase in the % of open arm entries and the % time spent on the open arms when treated with a benzodiazepine, those tested on trial 2 failed to respond. This insensitivity to anxiolytic drugs on trial 2 has been confirmed by other laboratories and applies to both mice [39] and rats [17,24,25]. File [19] found that the measures of anxiety derived from trials 1 and 2 in the plus-maze load on two independent factors, i.e. two distinct states of anxiety are generated on the two trials and this has been confirmed in mice [27]. It seems that it is the open aspect of the arms that is the most important aspect controlling behaviour on trial 1 [44], whereas it is the elevation that is most important on trial 2 [14]. This has two implications for genetic research. First, it means that the same apparatus can be used to assess changes in two different states of anxiety and it is quite likely that a change may be detected in one kind of anxiety, but not the other. Secondly, it means that care must be exercised, if the mice are retested and that it is not possible to test on one occasion, say undrugged, and on a second trial after drug treatment. In the plus-maze, the desire to explore a novel environment acts as a positive motivation to enter the open arms, whereas the aversive aspects of the arm act as negative motivation. The plus-maze test has often been used immediately after the holeboard test [30,45], but there is evidence that prior apparatus exposure can have opposite effects in different mouse strains [37]. If the animals have been exposed to a series of tests before the plus-maze, this test may not then offer sufficient novelty to stimulate exploration. The animals will show very low levels of entries into both open and closed arms. This was found when we were testing Thy-1 null mice and their wild-type controls, and those who had extensive test experience showed very low levels of activity in the plus-maze (File and Morris, unpublished data). It is, therefore, recommended that this test be conducted early in a battery of tests [41]. 4. Holeboard test The holeboard test was introduced by Boissier and Simon [3], who claimed that head-dipping provided a measure of exploration that was distinct from motor activity. In their original version, a 40-cm square floor had 16 equally spaced holes of 3 cm diameter and was mounted on four 25 cm legs. In order to validate this test and provide more independent measures of exploration and motor activity, File and Wardill [22] modified the apparatus so there were only four holes. This was because with 16 holes, the animal would be unable to discriminate between the holes and the density was such that it was impossible for the mouse to move around without coming in contact with a hole. Two criteria were used to establish that head-dipping reflected exploration. Firstly, it should reflect any novel aspects of the environment. This was shown by a longer duration of head-dipping on initial exposure, if objects were placed under the holes, and also on a second exposure, if objects were introduced for the first time. Secondly, exposure to the stimulus complex should result in information storage, which was shown by habituation on re-exposure to the

4 154 S.E. File / Beha ioural Brain Research 125 (2001) Table 2 Orthogonal factor loadings of parameters from the holeboard test in male and female rats Behavioral parameter Males Females Factor 1 (exploration) Factor 2 (activity) Factor 1 (exploration) Factor 2 (activity) Number of head-dips Time spent head-dipping Locomotor activity Rears Data from Fernandes et al. [15]. holeboard. A comparison was made between scores in the 16 and 4 hole versions of the apparatus and, for mice, whilst there was a significant correlation between locomotor activity in the two holeboards, there was no correlation between head-dipping [22]. Thus, the 16- hole version would seem to provide only a measure of general activity and it is necessary to use the 4-hole version to obtain a measure of directed exploration. A detailed characterisation of six inbred strains of mouse has also shown a clear dissociation between measures of locomotor activity and exploration, with for example DBA/2 mice showing high levels of locomotor activity, but low levels of holeboard exploration [41]. Two aspects of the reliability of the holeboard were also assessed: the similarity between scores of different samples of the same population on their first exposure to the apparatus, and the test retest reliability [23]. All animals showed good test retest reliability, but whereas groups of male mice did not differ in their initial scores, the groups of female mice did differ significantly. This may well be because the female mice were not matched for their phase of the oestral cycle, but does provide a note of caution when testing female mice. However, factor analysis has shown that in both males and females head-dipping reflects exploration, and locomotor activity and rears in the holeboard are measures of an independent factor of activity, see Table 2. Unlike the plus-maze, the factor structure is the same in both sexes with the stronger factor being exploration. Habituation of the response to novelty is a basic form of learning displayed by many species. Genetic manipulations could disrupt the rate of habituation to novelty, which could indirectly impair performance in other tests, such as maze learning. By exposing mice to the holeboard over successive trials, it is possible to obtain a measure of habituation to novelty (see Fig. 6). 1. These mice showed striking reductions in anxiety, compared with their normal littermates, as reflected in increases in the % of open arm entries and the % of time spent on open arms, see Fig. 1. As it can be seen, these changes were found as early as 6 weeks of age. These increases were not the result of a non-specific increase in activity, since the closed arm entries were unchanged until 10 weeks of age, when they decreased, along with a general reduction of motor activity, as also measured in the holeboard. This was also the age at which the movement disorders characteristic of Huntington s disease started to be apparent. However, since entries into the open arms are motivated by exploration, as well as by fear, it was important to check that 5. Huntington s disease transgenic mice Mice that were transgenic for a human genomic fragment that contains HD promoter elements, exon 1 of the HD gene and a portion of intron 2 [11] were recently tested in the plus-maze and holeboard [21] Fig. Fig. 1. Mean ( S.E.M.) percentage of open arm entries (top panel) and percentage of time spent on open arms (lower panel) by mice transgenic for Huntington s disease (H) and their litter-mate controls, tested in the elevated plus-maze at 6, 8, 10 and 12 weeks of age. *P 0.01; **P 0.001; ***P compared with age-matched control. Data from File et al. [21].

5 S.E. File / Beha ioural Brain Research 125 (2001) Fig. 2. Mean ( ) S.E.M. number of head-dips made by mice transgenic for Huntington s disease (H) and their litter-mate controls, tested in the holeboard at 6, 8, 10 and 12 weeks of age. *P 0.05 compared with age-matched control. Data from File et al. [21]. tion that a novel food is safe does not depend solely on an individual s experience. If a mouse has sampled a novel food, on its return to the colony, it will be extensively investigated by the other members of the colony. This social investigation results in other mice preferentially selecting food with the same scent, rather than other novel foods. The possibility of sex differences in social learning has been insufficiently investigated, but for a discussion of this, see [5]. Thy-1 is a cell adhesion molecule and it is thought that the Thy-1 protein may regulate neuronal plasticity in the astrocyte-dominated grey-matter areas of the brain [43]. Mice in which the Thy-1 gene had been inactivated show excessive GABAergic inhibition in the dentate gyrus [26,34]. These mice showed striking impairments in the test using socially transmitted food cues, regardless of their background strain of C57Bl6 or 129/Sv/Ev [32], and see Fig. 4. However, before it was concluded that the deficit related specifically to learning in this task, a number of control experiments were conducted. First of all, it was necessary to determine whether or not the Thy-1 null mice displayed normal levels of neophobia. This was established by showing that they ate significantly more of their normal food than food that had been flavoured with a novel scent (Fig. 5a). It was also necessary to show that the deficit was not due to a reduced interaction with the mouse that had earlier sampled the novel food and Fig. 5b shows that this was normal in the thy-1 null mice. It was also important to establish that the knock-out mice could distinguish between the novel food odours and their ability to do this was shown by their normal acquisition of a T- maze, using the food odours as cues [32]. Finally, we used the elevated plus-maze to determine whether there were any differences between the null mice and their wild-type controls in their levels of anxiety. Greater anxiety could lead to greater avoidthe HD transgenic mice did not have changes in exploration that could account for these changes observed in the plus-maze. It can be seen from Fig. 2 that there was no evidence of increased exploration in these mice and, on the contrary, they showed reduced exploration (a decreased number of head-dips) from 8 weeks of age. Finally, it was important to check that the differences in the plus-maze that appeared in the transgenic mice were not due to differences in the background strains. The transgenic mice were generated by microinjection into single cell embryos from CBA C57BL/F1 donors and thus there was the possibility that the transgene had integrated close to a gene that was dominant for anxiety. Strain differences were found in the plus-maze, but these were far less striking than in the transgenic mice. Thus they were significantly different for % open arm entries at P for the transgenic mice and did not reach significance for the strain differences. For % time on the open arms, the differences were more marked and reached higher significance in the transgenic mice than for the strain differences (P 0.01 compared with P 0.05), see Fig. 3. Furthermore, in the CBA mice, the decreased anxiety in the plus-maze was accompanied by increased holeboard exploration, and was, therefore, a less specific behavioural pattern than that seen in the Huntington transgenic mice [21]. 6. Social transmission of food cues This test is based on the normal ability of mice in a colony to learn from each other which novel foods are safe to eat [31,46] Mice, like other rodents, display neophobia and will normally avoid novel foods. Indeed, if a rodent experiences aversive consequences after sampling a novel food, it will display long-lasting avoidance of that food. This is the basis of the widely used conditioned taste aversion paradigm. The recogni- Fig. 3. Mean ( ) S.E.M. percentage of open arm entries (top panel) and percentage of time spent on open arms (lower panel) by C57 and CBA mice, tested in the elevated plus-maze at 10 weeks of age. *P 0.05 compared with C57 strain. Data from File et al. [21].

6 156 S.E. File / Beha ioural Brain Research 125 (2001) Fig. 6. Mean ( ) S.E.M. number of head-dips made over three trials in the holeboard by Thy-1 null mice (Null) and their wild-type controls (WT). ****P significant between-trial habituation. Data from Mayeux-Portas et al. [32]. Fig. 4. Mean ( ) S.E.M. amount (g) of cued (black columns) and uncued (clear columns) novel food eaten by Thy-1 null mice (Null) and their wild-type controls (WT) in the test of social transmission of food preference. **P 0.01 comparing WT and Null mice on cued food. Data from Mayeux-Portas et al. [32]. ance of all novel foods and hence the social learning might not be expressed. This could lead to an erroneous conclusion that the learning, rather than the performance, of the task was impaired. There were no differences in anxiety, as measured in the plus-maze nor in activity in this test (number of closed arm entries) or in the holeboard (locomotor activity measured by infrared beam breaks). This is also important, since changes in overall activity or exploration of the test box could affect performance in the food preference test. Fig. 6 shows that the null mice showed a normal response to novelty (trial 1) and normal habituation across three successive trials. Thus, their overall response to a novel situation, and their ability to adapt to it was normal. Therefore, in summary the impaired performance in the social transmission test can be attributed to a learning impairment and non-specific changes that could impair performance can be excluded Figs. 5 and 6. Fig. 5. (a) Left panel: mean ( ) S.E.M. amount of normal diet (solid columns) and novel food (clear columns) eaten by Thy-1 null mice (Null) and their wild-type controls (WT) in the test of neophobia. **P 0.01 comparing normal diet and novel food. (b) Right panel: mean ( ) S.E.M. time spent interacting with the mouse that had sampled novel food, in the test for social transmission of food preference. Data from Mayeux-Portas et al. [32]. 7. Conclusions Changes in anxiety and/or responses to novelty might arise from a wide variety of genetic manipulations. Indeed, the most striking change that has been produced in anxiety is that seen in the Huntington transgenic mice, which was completely unexpected. Changes in anxiety, exploration and locomotor activity might influence behaviour in a number of other tests, e.g. tests of learning such as the Morris water maze. It is, therefore, important to include measures of this as part of a behavioural screening battery. The use of non-specific tests, such as the open field, where the behavioural parameters are confounded because they are influenced by several factors, should be avoided. Acknowledgements I am grateful to Nick Jarrett for his assistance with the figures. References [1] Archer J. Tests for emotionality in mice and rats: a review. Anim Behav 1973;21: [2] Archer J. Rodent sex differences in emotional and related behaviour. Behav Biol 1975;14: [3] Boissier JR, Simon P, Aron C. A new method for rapid screening of minor tranquilizers in mice. Eur J Pharmacol 1968;4: [4] Boissier JR, Simon P. La reaction d exploration chez la souris. Therapie 1962;17: [5] Choleris E, Kavaliers M. Social learning in animals: sex differences and neurobiological analysis. Pharmacol Biochem Behav 1999;64: [6] Crabbe JC. Genetic differences in locomotor activity in mice. Pharmacol Biochem Behav 1986;25: [7] Crawley JN. Neuropharmacological specificity of a simple animal model for the behavioral actions of benzodiazepines. Pharmacol Biochem Behav 1981;15:695 9.

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