Amygdaloid involvement in the defensive behavior of mice exposed to the open elevated plus-maze.

Previous studies have shown that the exposure to an open elevated plus maze (oEPM, an EPM with all four open arms) elicits fear/anxiety-related responses in laboratory rodents. However, very little is known about the underlying neural substrates of these defensive behaviors. Accordingly, the present study investigated the effects of chemical inactivation of the amygdala [through local injection of cobalt chloride (CoCl2: a nonspecific synaptic blocker)] on the behavior of oEPM-exposed mice. In a second experiment, the pattern of activation of the basolateral (BLA) and central (CeA) nuclei of the amygdala was assessed through quantification of Fos protein expression in mice subjected to one of several behavioral manipulations. To avoid the confound of acute handling stress, 4 independent groups of mice were habituated daily for 10days to an enclosed EPM (eEPM) and, on day 11 prior to immunohistochemistry, were either taken directly from their home cage (control) or individually exposed for 10min to a new clean holding cage (novelty), an eEPM, or the oEPM. An additional group of mice (maze-naïve) was not subjected to either the habituation or exposure phase but were simply chosen at random from their home cages to undergo an identical immunohistochemistry procedure. Results showed that amygdala inactivation produced an anxiolytic-like profile comprising reductions in time spent in the proximal portions of the open arms and total stretched attend postures (SAP) as well as increases in time spent in the distal portions of the open arms and total head-dipping. Moreover, Fos-positive labeled cells were bilaterally increased in the amygdaloid complex, particularly in the BLA, of oEPM-exposed animals compared to all other groups. These results suggest that the amygdala (in particular, its BLA nucleus) plays a key role in the modulation of defensive behaviors in oEPM-exposed mice.

No man is an island. A personal tribute to Bob Blanchard and ethoexperimental approaches to the study of behaviour.

I first met Bob Blanchard at an international conference in Paris some 40 years ago. We collaborated intensively during the late 1980s/early 1990s on the ethopharmacology of antipredator defence in wild and laboratory rats, and remained good friends until his untimely passing in November 2013. Bob will undoubtedly be remembered as one of the most influential behavioural neuroscientists of the 20th century and, with Caroline, the most eloquent advocate of ethoexperimental approaches to the study of behaviour. In this brief trip down memory lane, I describe when and where Bob and I first met and how, over a lengthy period, he directly and indirectly helped shape my own research career. His profound influence in this regard is illustrated by reference to not only our collaborative research on antipredator behaviour but also my other work on the ethopharmacology of agonistic behaviour, social conflict analgesia, anxiety, and appetite. The element common to all of this work has been ethoexperimental analysis and, for teaching me the true value of this approach, I shall always remain indebted to the big man. Literally and figuratively, Bob was most certainly larger than life.

Ethopharmacological analysis of the open elevated plus-maze in mice.

Exposure of rodents to an open elevated plus-maze (oEPM) elicits antinociception and increases plasma corticosterone levels. However, no studies have yet assessed the defensive behaviour repertoire of animals in this modified test. In Experiment 1, factor analysis was employed to characterise the behavioural profile of mice exposed to the oEPM. Experiments 2 and 3 assessed the effects of acute alprazolam (0.5-1.5mg/kg; diazepam 0.5-1.5mg/kg), pentylenetetrazole (10.0-30.0mg/kg), yohimbine (2.0-6.0mg/kg), mCPP (0.3-3.0mg/kg), and acute and chronic fluoxetine (10.0-30.0mg/kg) and imipramine (1.0-15.0mg/kg) on behaviours identified in Experiment 1. The factor analyses revealed that behaviour in the oEPM can largely (77% total variance) be accounted for in terms of 3 factors: factor 1 ('depth exploration'; e.g. head-dipping on the arms), factor 2 ('cautious exploration of arms'; e.g. flatback approach), and factor 3 ('risk assessment'; stretched attend postures - SAP). Experiments 2 and 3 showed that, over the dose range used, alprazolam selectively attenuated all measures of defensiveness. Similar, though more modest, effects were seen with diazepam. Confirming the intensity of the emotional response to the oEPM (nociceptive, endocrine and behavioural), relatively few significant behavioural changes were seen in response to the anxiogenic compounds tested. Although acute fluoxetine or imipramine treatment failed to modify behaviour in the oEPM, chronic fluoxetine (but not chronic imipramine) attenuated total flat back approach and increased head dipping outside the central square. Together, the results indicate that the oEPM induces behavioural defensive responses that are sensitive to alprazolam and chronic fluoxetine.

Blockade of 5-HT(2) receptors in the periaqueductal grey matter (PAG) abolishes the anxiolytic-like effect of 5-HT(1A) receptor antagonism in the median raphe nucleus in mice.

Several lines of evidence support the involvement of serotonergic (5-HT) neurons of the median raphe nucleus (MRN) in anxiety-like behaviour. In this context, it is known that blockade of 5-HT(1A) somatodendritic autoreceptors in the midbrain raphe nuclei increases the firing rate of these neurons, disinhibiting 5-HT release in postsynaptic target areas such as amygdala, hippocampus and periaqueductal grey matter (PAG). However, while activation of 5-HT(1A) or 5-HT(2) receptors in forebrain targets such as the amygdala or hippocampus enhances anxiety-like behaviours in rodents, stimulation of both receptor subtypes in the midbrain PAG markedly reduces anxiety-like behaviour. In view of these findings, the present study investigated whether the anti-anxiety effects induced by pharmacological disinhibition of 5-HT neurons in the MRN are attenuated by the blockade of 5-HT(2) receptors within the PAG. Mice received combined intra-PAG injection with ketanserin (10 nmol/0.1 µl), a 5-HT(2) receptor antagonist, followed by intra-MRN injection of WAY-100635 (5.6 nmol/0.1 µl), a highly selective 5-HT(1A) receptor antagonist. They were then individually exposed to the elevated plus-maze (EPM), with the videotaped behavioural sessions subsequently scored for both conventional and ethological measures. The results confirmed that intra-MRN infusion of WAY100635 reduces behavioural indices of anxiety without significantly altering general activity measures, and further showed that this effect was completely blocked by intra-PAG pretreatment with an intrinsically-inactive dose of ketanserin. Together, these results suggest that 5HT(2) receptor populations located within the midbrain PAG play a significant role in the reduction of anxiety observed following disinhibition of 5-HT neurons in the MRN.

D-cycloserine enhances memory consolidation in the plus-maze retest paradigm.

Prior undrugged exposure to the elevated plus-maze (EPM) alters future behavioral strategy as well as responsivity to conventional anxiolytic agents. This EPM retest phenomenon appears to be dependent upon learning the spatial configuration of the maze on initial exposure and, in particular, the location of the relatively safe enclosed arms. As posttraining administration of the glycineB receptor partial agonist, D-cycloserine (DCS), has been shown to enhance the consolidation of many forms of memory, we have examined the effects of this compound on the EPM retest effect in male mice. The results of Experiment 1 confirmed that 5 min undrugged exposure to the EPM completed abolishes the anxiolytic efficacy of chlordiazepoxide (CDP; 15 mg/kg) on 24 hr retest. In Experiment 2, posttraining administration of DCS (7.5 and 15 mg/kg), but not CDP (15 mg/kg) or DCS (30 mg/kg), significantly and selectively increased time spent in the enclosed arms (and reciprocally decreased open arm exploration) on 24 hr retest, a finding consistent with an enhancement of consolidation. Experiment 3 used a modified EPM retest protocol to assess the effects of posttraining DCS (15 mg/kg) on behavioral responses to CDP (15 mg/kg) challenge on 24 hr retest. Using a 1-min prior exposure regimen that did not compromise the anxiolytic efficacy of CDP in control mice, the results showed that posttraining administration of DCS abolished the anxiolytic response to CDP challenge. These data strongly suggest that the EPM retest effect involves glycineB/NMDA receptor-dependent neuroplasticity. Further studies will be required to identify the neural circuitry involved.

Anxioselective profile of glycineB receptor partial agonist, D-cycloserine, in plus-maze-naïve but not plus-maze-experienced mice.

Recent successes as a pharmacological adjunct to exposure therapy has focused attention on the therapeutic potential of the glycine(B) receptor partial agonist, D-cycloserine (DCS), in certain clinical anxiety disorders. Although widely believed to reflect a facilitation of extinction learning, previous research with DCS and other glycine(B) partial agonists suggests the additional possibility of intrinsic anxiolytic activity. In the present study, ethological methods were used to profile the behavioural effects of DCS (7.5-30.0mg/kg) and the positive control chlordiazepoxide (CDP, 15 mg/kg) in mice exposed to the elevated plus-maze for the first time (plus-maze trial 1; Experiment 1) and in mice pre-exposed undrugged to the maze 24h prior to testing (plus-maze trial 2; Experiment 2). The results show that, in test-naive animals, both CDP and DCS (15 mg/kg, but not lower or higher doses) produced significant anxioselective profiles with the effects of DCS statistically weaker than those of CDP. However, as predicted by the plus-maze retest effect, CDP was without behavioural activity in test-experienced animals, while the highest dose of DCS (30 mg/kg) induced behavioural changes more consistent with mild psychomotor stimulation than anxiolysis. Present findings therefore confirm the intrinsic anxiolytic activity of DCS in untrained animals, with the observed bell-shaped dose-response function most probably indicative of varying affinities and intrinsic activities at NMDA receptor subtypes. The contrasting and comparatively limited effects of DCS in test-experienced mice suggest that prior maze exposure radically alters the extent to which NMDA receptor-related mechanisms are involved in future behavioural responses to this test environment.

5-HT2 receptor activation in the midbrain periaqueductal grey (PAG) reduces anxiety-like behaviour in mice.

It is widely acknowledged that the indoleamine neurotransmitter serotonin (5-HT) plays a dual role in the regulation of anxiety, a role that in part depends upon neuroanatomical locus of action. Thus, whereas stimulation of 5-HT 1A or 5-HT2 receptors in the limbic forebrain (amygdala, hippocampus) enhances anxiety-like responding in rodents, activation of corresponding receptor populations in the midbrain periaqueductal grey (PAG) more often than not reduce anxiety-like behaviour. The present study specifically concerns the anxiety-modulating influence of 5-HT2 receptors within the mouse PAG. Experiment 1 assessed the effects of intra-PAG infusions of the 5-HT2B/2C receptor agonist mCPP (0, 0.03, 0.1 or 0.3 nmol/0.1 microl) on the behaviour of mice exposed to the elevated plus-maze. As mCPP acts preferentially at 5-HT2B and 5-HT2C receptors, Experiment 2 investigated its effects in animals pretreated with ketanserin, a preferential 5-HT2A/2C receptor antagonist. In both cases, test sessions were videotaped and subsequently, scored for anxiety-like behaviour (e.g., percentage of open arm entries and percentage of open arm time) as well as general locomotor activity (closed arm entries). The results of Experiment 1 showed that mCPP microinfusions (0.03 and 0.1 nmol) into the PAG of mice decreased behavioural indices of anxiety without significantly altering general activity measures. In Experiment 2, the anxiolytic-like profile of intra-PAG mCPP (0.03 nmol) was substantially attenuated by intra-PAG pretreatment with an intrinsically inactive dose of the preferential 5-HT2A/2C receptor antagonist, ketanserin (10 nmol/0.1mul). Together, these data suggest that 5HT2C receptor populations within the midbrain PAG play an inhibitory role in plus-maze anxiety in mice.

Comparative effects of nonselective and subtype-selective gamma-aminobutyric acidA receptor positive modulators in the rat-conditioned emotional response test.

Benzodiazepine receptor anxiolytics show no selectivity between gamma-aminobutyric acid-A receptors containing alpha1, alpha2, alpha3 or alpha5 subunits. Pharmacological studies and data emerging from transgenic mouse models, however, predict that compounds with selective affinity and/or efficacy for gamma-aminobutyric acid-A receptor subtypes would have novel pharmacological profiles. Thus, the gamma-aminobutyric acid-A-alpha1 'affinity selective' drug zolpidem has a sedative-hypnotic profile, whereas L838,417, which has 'selective efficacy' for gamma-aminobutyric acid-A-alpha2, alpha3 and alpha5 receptors, has an anxiolytic-like profile. Here, we compare the nonselective benzodiazepine-site-positive modulators diazepam, lorazepam, midazolam, alprazolam and zopiclone with (i) gamma-aminobutyric acid-AA-alpha1 affinity selective compounds zolpidem and CL218,872 and (ii) L838,417, in the rat-conditioned emotional response test after systemic administration. Given the role of the basolateral amygdala in anxiety and the expression of alpha1, alpha2 and alpha3 subunits in this region, we also assessed the effects of bilateral infusion of L838,417 and midazolam directly into basolateral amygdala in the conditioned emotional response test. Nonselective modulators at low-moderate doses produced anxiolytic effects and sedation at higher doses. Zolpidem was inactive as an anxiolytic and engendered severe sedation, whereas CL218,872 produced an anxiolytic-like profile with minimal sedation. L838,417 produced an anxiolytic-like profile with no sedation, albeit producing behavioural disturbance at high doses. Infusion of midazolam and L838,417 into basolateral amygdala engendered anxiolytic-like effects, although both compounds were more effective after systemic injections, implicating additional brain sites in their anxiolytic-like actions after systemic administration. In conclusion, the diversity of effects of the compounds studied implicates both intrinsic efficacy and/or subtype selectivity as important determinants of anxiolytic-like effects in the rat-conditioned emotional response test.

The effect of social factors on the anxiolytic efficacy of buspirone in male rats, male mice, and men.

Earlier findings suggest that housing conditions in laboratory animals and life events in humans influence the efficacy of anxiolytic drugs. Here we report on the impact of social isolation on buspirone efficacy in male mice and rats as assessed by the elevated plus-maze. In addition, the impact of social support on buspirone efficacy was assessed in male patients. When administered 30 min before testing and irrespective of housing conditions, buspirone significantly suppressed locomotor activity both in mice (6 mg/kg) and rats (10 mg/kg) and, as such, other behavioral changes observed at this time point must be seen as behaviorally nonselective. However, these locomotor disruptive effects of buspirone were not evident in either species at longer injection-test intervals (2 and 4 h). When given 2 h prior to testing, a low (3 mg/kg) but not high (10 mg/kg) dose of buspirone increased the frequency of open arm exploration in rats (but not mice) irrespective of housing conditions. At the longest injection-test interval used (4 h), buspirone increased the duration of open arm exploration in individually housed, but not group-housed, rats. Similar, though somewhat less robust, effects were observed in male mice at this time. In a double-blind placebo-controlled study with male patients, chronic buspirone treatment (3 x 10 mg daily for 6 weeks) produced a highly significant reduction in scores on the Hamilton Rating Scale for Anxiety (HAM-A). Multiple regression analysis of social support received by patients indicated that the support of nonrelatives (but not of family or other relatives) was a strong positive predictor of buspirone efficacy. Taken together, our data support the hypothesis that social conditions affect the anxiolytic efficacy of buspirone. Results are discussed in relation to differences in the social organization of the three species investigated.

Anxiolytic-like effect of way-100635 microinfusions into the median (but not dorsal) raphe nucleus in mice exposed to the plus-maze: influence of prior test experience.

Studies in several laboratories have confirmed the anxiolytic potential of a wide range of 5-HT(1A) receptor antagonists in rats and mice, with recent evidence pointing to a postsynaptic site of action in the ventral hippocampus. It would, therefore, be predicted that blockade of 5-HT(1A) somatodendritic autoreceptors in the midbrain raphe nuclei should produce anxiogenic-like effects. To test this hypothesis, we investigated the effects of WAY-100635 microinfusions (0, 1.0 or 3.0 microg in 0.1 microl) into the dorsal (DRN) or median (MRN) raphe nuclei on behaviours displayed by male Swiss-Webster mice in the elevated plus-maze. As this test is sensitive to prior experience, the effects of intra-raphe infusions were examined both in maze-naive and maze-experienced subjects. Sessions were videotaped and subsequently scored for conventional indices of anxiety (open arm avoidance) and locomotor activity (closed arm entries), as well as a range of ethological measures (e.g. risk assessment). In maze-naive mice, intra-MRN (but not intra-DRN) infusions of WAY-100635 (3.0 microg) increased open arm exploration and reduced risk assessment. Importantly, these effects could not be attributed to a general reduction in locomotor activity. A similar, though somewhat weaker, pattern of behavioural change was observed in maze-experienced animals. This unexpected anxiolytic effect of 5-HT(1A) autoreceptor blockade in the MRN cannot be accounted for by a disinhibition of 5-HT release in forebrain targets (e.g. hippocampus and amygdala), where stimulation of postsynaptic 5-HT(1A) receptors enhances anxiety-like responses. However, as the MRN also projects to the periaqueductal gray matter (PAG), an area known to be sensitive to the anti-aversive effects of 5-HT, it is argued that present results may reflect increased 5-HT release at this crucial midbrain locus within the neural circuitry of defense.

Effects of intra-hippocampal infusion of WAY-100635 on plus-maze behavior in mice. Influence of site of injection and prior test experience.

The positive profile of systemically-administered 5-HT(1A) receptor antagonists in several rodent models of anxiolytic activity suggests an important role for postsynaptic 5-HT(1A) receptor mechanisms in anxiety. To test this hypothesis, we investigated the effects of WAY-100635 microinfusions (0, 0.1, 1.0 or 3.0 microg in 0.2 microl) into the dorsal (DH) or ventral (VH) hippocampus on behaviours displayed by male Swiss-Webster mice in the elevated plus-maze. As prior experience is known to modify pharmacological responses in this test, the effects of intra-hippocampal infusions were examined both in maze-naïve and maze-experienced subjects. Test videotapes were scored for conventional indices of anxiety (% open arm entries/time) and locomotor activity (closed arm entries), as well as a range of ethological measures (e.g. risk assessment). In maze-naïve mice, intra-VH (but not intra-DH) infusions of WAY-100635 (3.0 microg but not lower doses) increased open arm exploration and reduced risk assessment. These effects were observed in the absence of significant changes in locomotor activity. In contrast, neither intra-VH nor intra-DH infusions of WAY-100635 altered the behaviour of maze-experienced mice. These findings suggest that postsynaptic 5-HT(1A) receptors in the ventral (but not dorsal) hippocampus play a significant role both in the mediation of plus-maze anxiety in mice and in experientially-induced alterations in responses to this test.