R.J. Rodgers

Factor analysis of spatiotemporal and ethological measures in the murine elevated plus-maze test of anxiety

Recent research employing the elevated plus-maze to assess anxiety in rodents has incorporated a variety of behavioral elements in addition to the standard parameters of entries onto and time spent in the aversive open arms. In the present study, we have used a large database comprising the behavioral profiles of 90 undrugged mice to examine the relationship between the standard spatiotemporal measures and a range of specific behaviors related to the defensive repertoire of the mouse. A factor analysis applied to the standard measures revealed two factors related to anxiety and locomotor activity. The simple addition of center time (an infrequently recorded measure) to the analysis yielded a third factor, most probably related to decision making. A large-scale factor analysis applied to all measures further confirmed the existence of factors related to anxiety, locomotor activity, and decision making, and revealed three further factors thought to represent risk assessment, vertical activity, and exploratory behavior. Thus, the inclusion of ethological measures not only confirmed prior knowledge based on a very limited range of measures, but also demonstrated the existence of additional behavioral dimensions. The potential applications of this knowledge are discussed.

Anxiety, defence and the elevated plus-maze

The elevated plus-maze test has been in use as a rodent model of anxiety for a decade, and is representative of those tests that are based upon the study of spontaneous behaviour patterns and which have high ecological validity. The origins of the test in studies of the relationship between exploration and fear are reviewed, and attention is drawn to the distinct possibility that variation in the pharmacosensitivity of the procedure may be attributable to often extreme methodological variation between laboratories. In considering further this issue, attention is also drawn to the need to collect data under constant test conditions and to provide the minimum database necessary to reach conclusions regarding the behavioural specificity of drug action. Recent research, which has extended the conventional plus-maze scoring technique to include specific behavioural acts and postures (in particular, those relating to defensive behaviour), is described. The value of such an ethological approach to the plus-maze is then exemplified with original data that demonstrate behaviourally selective, anti-anxiety effects of the GABAA receptor agonist, muscimol (0.125-1.0 mg/kg). It is concluded that, when used appropriately, the elevated plus-maze test can be a very valuable tool in drug screening and in the study of the neurobiology of anxiety and defence. More attention to behaviour and somewhat less emphasis on test simplicity and convenience would seem to be warranted.

Influence of social isolation, gender, strain, and prior novelty on plus-maze behaviour in mice

Behavioural baseline is a critical determinant of response to drugs and other manipulations. In the present study, the influence of several organismic and procedural variables on basal plus-maze profiles in mice were examined. The methodology incorporated traditional behavioural parameters as well as novel measures derived from ethological analysis. Experiment 1 showed that social isolation for 1-3 weeks enhanced aggression in male DBA/2 mice but did not substantially alter their behaviour on the maze. A reduction in stretch attend postures did, however, suggest a minor reduction in anxiety in socially isolated animals. In Experiment 2, males of both DBA/2 and T1 strains exhibited higher levels of general activity on the maze than their female counterparts. Although additional evidence suggested that DBA/2 (but not T1) females were less anxious than males, no major sex differences were noted. Experiment 3 revealed a significant strain difference in plus-maze profiles, with T1 males showing a lower basal level of anxiety than DBA/2 males. This study also demonstrated that DBA/2 and T1 males react very differently to prior novelty experience, with enhanced anxiety evident in the former and reduced anxiety in the latter. Together, these findings point to a range of organismic and procedural variables that may account for inconsistencies in the literature on the elevated plus-maze.

Corticosterone response to the plus-maze: High correlation with risk assessment in rats and mice

Exposure to the elevated plus-maze induces behavioural and physiological effects in rodents consistent with fear/anxiety. Maze-naive animals display high levels of risk assessment towards the open arms, and explore these areas less extensively than other parts of the maze while, immediately following the test, pain latencies, skin conductance levels, and plasma corticosterone titres (CORT) are significantly elevated. Although previous research has suggested a link between the plasma CORT response and open-arm exploration, significant elevations in CORT have also been found with restricted exposure to the closed arms. The present study employed ethological measures in an attempt to further characterise the relationship between behavioural and CORT responses to this widely used animal model of anxiety. Our results confirm that, relative to home-cage controls, 5-min exposure to the plus-maze significantly increases plasma CORT levels in test-naive male Wistar rats and male Swiss-Webster mice. Furthermore, in both species, the CORT response was found to be highly correlated with measures of risk assessment (mice: rs = +0.87; rats: rs = +0.58), but not with measures of open-arm activity (entries, time), general locomotor activity, rearing, or head dipping. Findings are discussed in relation to the functional significance of risk assessment in potentially dangerous situations and the potential involvement of glucocorticoids in this process. All rights reserved.

Influence of prior maze experience on behaviour and response to diazepam in the elevated plus-maze and light/dark tests of anxiety in mice

A single prior undrugged exposure to the elevated plus-maze has been reported to reduce open arm activity on retest and to attenuate/abolish the anxiolytic response to benzodiazepines at retest intervals ranging from 48 h to 14 days. The present study was designed to examine the generality of these findings by comparing the effects of prior maze experience on baseline behaviour and response to diazepam in two murine models of anxiety. Parallel experiments were conducted in which DBA/2 mice were exposed/not exposed to the plus-maze, treated daily with saline or diazepam (2–4 mg/kg daily for 8 days) and then tested on either the elevated plus-maze or in the light/dark test of exploration. Results show that, in both tests, diazepam reduced behavioural indices of anxiety in maze-naive mice only. However, interpretation of this apparent loss of diazepam efficacy is at least partially confounded by the observation that maze experience per se altered baseline behaviour in both procedures,reducing open arm activity in the plus-maze andincreasing light compartment activity in the light/dark test. The apparent elimination of an anxiolytic response to diazepam in two animal models of anxiety by prior plus-maze experience is discussed in relation to experience-related baseline shifts in behaviour.

Anxiety enhancement in the murine elevated plus maze by immediate prior exposure to social stressors

Anxiety has been implicated in the acute nonopioid analgesic reaction seen in defeated mice. In the present study, behavioural responses to the elevated plus-maze test were examined in male DBA/2 mice immediately following defeat by an experienced aggressive conspecific. Compared to home-cage controls, defeat reduced total arm entries and rearing, although anxiety enhancement was indicated by decreases in percent open-arm entries and percent time spent on the open arms. These effects were accompanied by significant increases in nonexploratory behaviour (movement arrest and grooming) and risk assessment (closed arm returns, protected head dipping, and stretch-attend postures). This anxiogenic effect of social defeat was partially replicated in mice merely exposed to the scent of an aggressive male conspecific. The specificity of present findings to socially relevant stressors was confirmed by the general lack of effect on plus-maze behaviour of prior exposure to a novel cage or to interaction with a nonaggressive male. Present results are not only consistent with the anxiety hypothesis of defeat analgesia but also show that the elevated plus-maze test is sensitive to alterations in anxiety produced by ecologically relevant stimuli. Possible implications for pharmacological studies are discussed.

Responses of Swiss-Webster mice to repeated plus-maze experience : Further evidence for a qualitative shift in emotional state?

Behavioral, endocrinological, and pharmacological data suggest that the emotional response of rodents to the elevated plus-maze alters as a function of prior test experience. In the present study, 74 intact male Swiss-Webster mice were exposed to the plus-maze for 5 min on each of 3 consecutive days, with all test sessions recorded on videotape. Behavior patterns for each trial were scored using ethological analysis software and the resultant database subjected to a number of statistical treatments. Analysis of full session profiles (i.e., 5 min total scores) showed that a single prior undrugged experience of the maze increases behavioral indices of anxiety and that these alterations are either maintained or further enhanced on subsequent trials. Furthermore, the behavioral profile evident by trial 3 was largely unchanged when animals were reexposed to the maze 10 days later. More detailed (i.e., min by min) examination of behavior patterns within and between trials demonstrated that unambiguous open arm avoidance is acquired by the third minute of trial 1, and that the behavioral profile evident by the end of trial 1 is (a) markedly different to that seen at the beginning of that trial, and (b) generally maintained or even accentuated on trials 2 and 3. The implied impact of prior test experience on future behavioral strategy in the maze was strongly supported by a series of factor analyses. Thus, while the factor associations of vertical activity and directed exploration remained constant across trials, trial 2 and 3 anxiety measures loaded on a separate factor to that loading trial 1 anxiety measures. A similar trial 1 vs. trials 2 and 3 dissociation was observed for measures of locomotor activity. Although the present findings are consistent with the proposal that prior test experience produces a qualitative shift in emotional response to the elevated plus-maze, the precise basis for this change as well as its full significance for our understanding of anxiety-related processes remain to be determined.

Ethological evaluation of the effects of acute and chronic buspirone treatment in the murine elevated plus-maze test: comparison with haloperidol

Buspirone is renowned for its highly inconsistent effects in animal models of anxiety. In the present study, the effects of acute (0.63–5.0 mg/kg) and chronic (1.25–5.0 mg/kg, daily, 15 days) buspirone treatment on the behaviour of mice in the elevated plus-maze test were assessed using a recently developed ethological scoring method. On acute administration, a selective reduction in risk assessment behaviours was observed at 1.25 mg/kg; these mild anxiolytic-like effects were maintained at higher doses (2.5–5.0 mg/kg) which also reduced measures of general activity. Similar, though more potent, effects were observed with chronic administration; the lowest dose tested (1.25 mg/kg) reduced open arm entries and total stretch attend postures while higher doses profoundly reduced all major indices of anxiety (traditional and novel) and, concomitantly, suppressed total entries and rearing. Acute administration of haloperidol (0.0125–0.1 mg/kg) appeared to mimic the behavioural suppressant effects of buspirone without selectively affecting anxiety-related measures at any dose. It is suggested that the anti-anxiety and behavioural suppressant profile of buspirone may reflect combined action at 5-HT1A and D2 receptors, respectively. Results are discussed in relation to the utility of risk assessment as a sensitive index of anxiety in models based upon unconditioned behaviour.

GABAergic influences on plus-maze behaviour in mice.

Abstract Manipulations of GABA function have been found to produce highly variable effects in animal models of anxiety. In the present series, an ethological version of the murine elevated plus-maze was used to examine in detail the behavioural profiles of diazepam (1.5 mg/kg; positive control) and a range of GABA-related compounds: valproic acid (100–400 mg/kg), No-711 (1.25–10.0 mg/kg), muscimol (0.5–3.0 mg/ kg), (+)bicuculline (4.0–8.0 mg/kg), picrotoxin (0.25– 2.0 mg/kg), R(+)baclofen (0.375–3.0 mg/kg) and CGP 35348 (25–200 mg/kg). On both conventional and ethological indices, results confirmed the anxiolytic profile of diazepam under present test conditions, and revealed substantially similar effects for the GABA-T inhibitor, valproic acid (100–400 mg/kg), and the GABAA receptor agonist, muscimol (2 mg/kg). The GABA reuptake inhibitor, No-711, produced weak anxiolytic-like effects at low doses (1.25–2.5 mg/kg) but disrupted behaviour at the highest dose tested (10 mg/ kg). Although the GABAA receptor antagonists, (+)bicuculline and picrotoxin, produced changes indicative of anxiety enhancement, concomitant behavioural suppression was evident at high doses (8 mg/kg and 1–2 mg/kg, respectively). Further studies suggested that the effects observed with bicuculline may be mediated by an active metabolite, such as bicucine. In contrast to the effects of valproic acid and direct GABAA receptor manipulations, the GABAB receptor agonist, R(+) baclofen, non-specifically disrupted behaviour at the highest dose tested (3 mg/kg) while the GABAB receptor antagonist, CGP 35348, was inactive over the dose range studied. Although present data confirm the sensitivity of the plus-maze to agents which modify GABAA receptor function, further studies will be required in order more fully to characterize this relationship.

Effects of diazepam on behavioural and antinociceptive responses to the elevated plus-maze in male mice depend upon treatment regimen and prior maze experience

Recent studies have shown that brief exposure to an elevated plus-maze (EPM) produces non-opioid antinociception in male mice. The present experiments were designed to assess the effects of diazepam on this phenomenon. When acutely administered, low doses (0.5–1.0 mg/kg) of diazepam failed to produce an anxiolytic profile and exerted rather inconsistent effects on EPM-induced elevations in tail-flick latencies. In EPM-experienced mice, chronic treatment with higher doses of diazepam (2–4 mg/kg, 8 days) produced a weak anxiolytic action and inhibited the early phase of EPM antinociception only. However, in EPM-naive mice, 8-day diazepam pretreatment exerted a marked anxiolytic effect and completely eliminated the antinociceptive response to the maze. Together, these data support the view that anxiety is a key factor in certain forms of adaptive pain inhibition and suggest a possible mediational role for benzodiazepine receptors. Our findings also show that prior exposure to the EPM, rather than chronic handling/injection, greatly reduces the anti-anxiety effect of diazepam. Furthermore, since re-exposure to the maze, perse, decreased time spent on the open arms and central platform, a shift in behavioural baseline (“retest anxiogenesis”) may have contributed to the weak behavioural effects of diazepam in test-experienced animals. Importantly, as chronic treatment with diazepam did not influence this anxiogenic-like retest profile, our data suggest that a single prior experience of the EPM may radically alter the nature of the anxiety reaction provoked by this test.