Jon K. Shepherd

Behavioural and pharmacological characterisation of the elevated “zero-maze” as an animal model of anxiety

The elevated “zero-maze” is a modification of the elevated plus-maze model of anxiety in rats which incorporates both traditional and novel ethological measures in the analysis of drug effects. The novel design comprises an elevated annular platform with two opposite enclosed quadrants and two open, removing any ambiguity in interpretation of time spent on the central square of the traditional design and allowing uninterrupted exploration. Using this model, the reference benzodiazepine anxiolytics, diazepam (0.125–0.5 mg/kg) and chlordiazepoxide (0.5–2.0 mg/kg) significantly increased the percentage of time spent in the open quadrants (% TO) and the frequency of head dips over the edge of the platform (HDIPS), and reduced the frequency of stretched attend postures (SAP) from the closed to open quadrants. In contrast, the anxiogenic drugm-chlorophenyl-piperazine (mCPP; 0.25–1.0 mg/kg) induced the opposite effects, decreasing %TO and HDIPS, and increasing SAP. The 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 0.001–0.1 mg/kg) had no effects on either %TO or HDIPS, but did decrease SAP at 0.01 mg/kg although not at higher or lower doses. Similarly, the 5-HT3 receptor antagonist, ondansetron (0.0001–1.0 mg/kg) decreased SAP and increased %TO at 0.01 mg/kg, but not at other doses. The present data suggest that a combination of the novel “zero-maze” design and a detailed ethological analysis provides a sensitive model for the detection of anxiolytic/anxiogenic drug action.

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.

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.

Behavioural and pharmacological characterisation of the canopy stretched attend posture test as a model of anxiety in mice and rats

Abstract The behavioural element, stretched attend posture (SAP), is an important component of the “risk-assessment” repertoire of defensive behaviour in rodents. The present experimental paradigm was devised as a novel and simple method of eliciting high levels of SAP in mice and rats. The SAP test apparatus comprised an elevated black Perspex circular platform. A smaller clear red Perspex circular “Canopy” was supported directly above the platform by a central pillar, thus dividing the platform into an inner, dimly lit covered zone and an outer, brightly lit exposed zone. In both the rat and mouse version of this model, vehicle-treated animals exhibited a marked preference for exploring the covered zone and also exhibited high baseline levels of SAP, particularly at the covered zone boundary whilst they investigated the exposed zone. In the mouse SAP test, the benzodiazepine receptor agonists, diazepam (0.5 mg/kg SC) and chlordiazepoxide (2 mg/kg SC), and the 5-HT1A receptor agonists, buspirone (1 and 3 mg/kg SC), ipsapirone (3 mg/kg SC) and 8-OH-DPAT (0.2 mg/kg SC), all significantly decreased the frequency of SAP without impairing motor activity. In the rat SAP test, diazepam (0.5 mg/kg SC) significantly decreased, whilst the anxiogenic 5-HT2C/1B receptor agonist, mCPP (0.25 and 0.5 mg/kg SC), significantly increased, the frequency of SAP. Ipsapirone (3 mg/kg SC) induced a non-specific behavioural inhibition. These data suggest that the “Canopy” SAP test is a useful paradigm to investigate risk assessment behaviour in both rats and mice, and may provide a sensitive novel rodent model of anxiety.

Attenuation of antipredator defensive behavior in rats following chronic treatment with imipramine

The anxiety/defense test battery has been developed to assess defensive reactions in rats to situations associated with a natural predator, the domestic cat. This comprises three paradigms designed to study the effects of cat exposure on general activity and location with respect to the cat (proxemic avoidance), the effects of cat exposure on non-defensive consummatory behavior, and the behavioral response to cat odor. In the present study subjects were exposed to 21 days pretreatment with imipramine (0, 5, and 15 mg/kg), before being assessed in the three experimental paradigms (carried out over a total period of 7 days). Imipramine treatment was maintained on a daily basis during the 7 days taken to complete the series of tests. The data indicated a behaviorally specific profile consistent with anxiety/fear reduction, but not with sedation, in three different paradigms, following treatment with 15 mg/kg imipramine. These behavioral changes included a reduction in freezing, proxemic avoidance and a disinhibition of suppressed feeding in response to cat presentation. Similarly, imipramine treatment (15 mg/kg) significantly reduced behaviors associated with risk assessment (e.g. flat back approach, stretch attend) during presentation of a cat odor stimulus. This behavioral profile suggests that chronic pretreatment with imipramine produces an attenuation of antipredator defensive behavior.

An ethopharmacological analysis of the behavioral effects of 8-OH-DPAT

Several behaviors associated with the serotonin syndrome have been reported in rats following administration of the 5-HT1A receptor agonist 8-OH-DPAT. The present investigation approached this phenomenon from an ethopharmacological perspective, and provided a detailed temporal analysis of the behavioral effects of this compound over a 2-h period, in both male and female rats in the home cage. In addition, in order to further characterize the nature of the forepaw-treading and locomotor elements, and assess the extent of influence of the physical characteristics of the test arena, this study provided a detailed analysis of these behaviors in both the home cage and a large oval runway. In the initial analysis, the data indicate a distinct chronological sequence of effects following 8-OH-DPAT treatment. For example, “flat back” activity and lower lip retraction were apparent within a few minutes post-injection, the former dissipating after about 30 min and being replaced as the prepotent response by a more general (curved back) locomotor enhancement, while the latter effect remained throughout the 2-h test period. Interestingly, there were reliable gender differences in terms of the onset and disappearance of several behavioral components, with females generally being more rapidly affected, but recovering earlier than males. The detailed analysis of locomotor activity and forepaw treading would suggest that the locomotor syndrome primarily involves forward movement, heavily guided by the physical environment. Furthermore, forepaw-treading would seem only to occur when an animal reaches a barrier and forward movement is briefly interrupted, as no reliable incidence of this behavior was observed in the open area of the test area. Together, these findings provide further characterization of the behavioral syndrome induced by 8-OH-DPAT, and indicate the importance of time post-administration, gender of the subject, and the physical characteristics of the test environment, when considering stereotypical drug effects.

Effects of the selective angiotensin II receptor antagonists losartan and PD123177 in animal models of anxiety and memory

There is increasing interest in the potential functional role of the octapeptide angiotensin II (AII) in psychiatric and cognitive disorders. The novel angiotensin II (AII) receptor antagonists, losartan and PD123177, selective for the AT1 and AT2 receptor subtypes respectively, constitute important pharmacological tools for the assessment of the behavioural consequences of modulation of AII function. The present series of studies investigated the effects of each compound in two animal models of anxiety, the rat elevated zero-maze and mouse light/dark box, and two models of working memory in the rat, the operant delayed matching to position (DMTP) task and the spatial reinforced alternation test in the T-maze. Our data indicate that both compounds (0.01–10 mg/kg SC) were without significant effect in any of the behavioural assays. Using the present methods and strains of laboratory rodents, these findings provide no support for the involvement of AII receptor function in the mediation of anxiety or working memory.

8-OH-DPAT specifically enhances feeding behaviour in mice: evidence from behavioural competition

The behavioural specificity of the hyperphagic effects of 8-OH-DPAT is a controversial issue. The present study addressed this question through the introduction of behavioural competition. Feeding behaviour in male mice was assessed under both basal (free-feeding) and social conflict conditions. Since, in the latter condition, defence and escape are prepotent responses, elicitation of feeding would be indicative of a specific treatment effect on mechanisms controlling food intake. Results showed that 8-OH-DPAT enhanced basal feeding duration (at doses of 0.05–0.50 mg/kg) and also elicited feeding in intruder mice during encounters with aggressive resident conspecifics (at doses of 0.10–0.50 mg/kg). As the 5-HT3 antagonist GR 38032F (1.0–2.0 mg/kg) enhanced feeding only under basal conditions, the effect of 8-OH-DPAT cannot readily be attributed to anxiety reduction. Finally, diazepam (1.0–2.0 mg/kg) produced a similar profile to that of 8-OH-DPAT, suggesting that the hyperphagic effects of the 5-HT1A agonist are not pharmacologically specific.

Morphine attenuates antipredator ultrasonic vocalizations in mixed-sex rat colonies

Mixed-sex groups of laboratory rats living in a visible burrow system (VBS) emit 18-27 kHz ultrasound and retreat to the burrow when a cat is placed in the open area of the VBS. The total duration of ultrasonic vocalizations was reliably reduced by pretreatment with 5 mg/kg morphine. In a subsequent study using male-female colony pairs, presentation of a cat to individual rats in the absence of their colony mate indicated significant gender differences in base frequency, degree of emission, and characteristics of pulses elicited. Specifically, females showed a greater number and duration of vocalizations, of higher frequency (kHz), and with shorter individual pulse durations than males. In the same study, morphine (5 mg/kg) produced a general decrease in the level of ultrasonic emissions in both sexes, reduced the mean base frequency (kHz), and increased the mean duration of individual pulses. These data suggest that endogenous opioid mechanisms may be involved in the mediation of ultrasonic vocalization in response to a predator, and are discussed with reference to known involvement of such systems in defensive responding.

Stereospecific inhibition of non-opioid defeat analgesia in male mice by MDL 72832, a selective 5-HT1A receptor agonist

The effects of MDL 72832, a potent and stereoselective ligand for 5-HT1A sites, on basal nociception and non-opioid defeat analgesia in male mice were examined. Neither (+)- nor (-)-MDL 72832 significantly altered basal tail-flick latencies. In contrast, (-)-MDL 72832 potently inhibited defeat analgesia (0.1-0.5 mg/kg i.p.), with similar effects produced by (+)-MDL 72832 only at substantially higher doses (3.0-5.0 mg/kg i.p.). These data clearly demonstrate a stereoselective action of this 5-HT1A ligand on non-opioid defeat analgesia.