Pierre Mormède

Stress and emotionality: a multidimensional and genetic approach.

The use of behavioural tests aiming to assess the psychological components of stress in animals has led to divergent and sometimes arbitrary interpretations of animal behaviour. This paper presents a critical evaluation of behavioural methods currently used to investigate stress and emotionality. One of its main goals is to demonstrate, through experimental evidence, that emotionality may no longer be seen as a unidimensional construct. Accordingly, following a discussion about concepts, we propose a multiple-testing approach, paralleled by factor analyses, as a tool to dissociate and study the different dimensions of emotionality. Within this multidimensional context, genetic studies (illustrated here by different rat models) are shown to be particularly useful to investigate the neurobiology of stress/emotionality. A genetic approach can be used (i) to broaden and dissect the variability of responses within and between populations and (ii) to search for the molecular bases (i.e. genes and gene products) which underlie such a variability.

A multiple-test study of anxiety-related behaviours in six inbred rat strains.

Recent studies have underlined the impact of genetic factors in anxiety profiles. In this context, we have initiated a series of experiments aiming to select, among six inbred strains of rats, a pair of strains that contrasts the most in fear-related behaviours measured in the open field, the elevated plus-maze, the black and white box and the social interaction test. Significant interstrain differences were found for all behavioural measures. A factor analysis of all variables produced three independent factors explaining 85.1% of the total variance. Factor 1 had high loadings from variables related to the approach/avoidance towards aversive stimuli (e.g., center of the open field, open arms of the plus-maze and white compartment of the black/white box). Variables related to general activity in novel environments (e.g., total locomotion in the open field and closed-arm entries in the plus-maze) loaded highly on Factor 2. Defecation and time of social interaction loaded positively on Factor 3. To verify whether elevated plus-maze variables loading on Factor 1 were associated to anxiety, the effects of single doses of diazepam and pentylenetetrazole were examined in Lewis and SHR rats, i.e., the most contrasting strains regarding Factor 1. Variables with high loadings on this factor changed in opposite ways in response to diazepam and pentylenetetrazole treatments. This study suggests, thus, that Lewis and SHR strains may constitute a useful model for studying the neurobiological mechanisms underlying the interindividual differences in baseline levels of anxiety.

Exploration of the hypothalamic–pituitary–adrenal function as a tool to evaluate animal welfare

Measuring HPA axis activity is the standard approach to the study of stress and welfare in farm animals. Although the reference technique is the use of blood plasma to measure glucocorticoid hormones (cortisol or corticosterone), several alternative methods such as the measurement of corticosteroids in saliva, urine or faeces have been developed to overcome the stress induced by blood sampling itself. In chronic stress situations, as is frequently the case in studies about farm animal welfare, hormonal secretions are usually unchanged but dynamic testing allows the demonstration of functional changes at several levels of the system, including the sensitization of the adrenal cortex to ACTH and the resistance of the axis to feedback inhibition by corticosteroids (dexamethasone suppression test). Beyond these procedural aspects, the main pitfall in the use of HPA axis activity is in the interpretation of experimental data. The large variability of the system has to be taken into consideration, since corticosteroid hormone secretion is usually pulsatile, follows diurnal and seasonal rhythms, is influenced by feed intake and environmental factors such as temperature and humidity, age and physiological state, just to cite the main sources of variation. The corresponding changes reflect the important role of glucocorticoid hormones in a number of basic physiological processes such as energy metabolism and central nervous system functioning. Furthermore, large differences have been found across species, breeds and individuals, which reflect the contribution of genetic factors and environmental influences, especially during development, in HPA axis functioning. Usually, these results will be integrated with data from behavioral observation, production and pathology records in a comprehensive approach of farm animal welfare.

Behavioural, physiological and immunological consequences of social status and aggression in chronically coexisting resident-intruder dyads of male rats.

The behavioural and physiological consequences of social status and reciprocal fighting in resident-intruder dyads of Long Evans male rats were evaluated. Before a chronic cohabitation of 10 days, residents and intruders were individually housed for one month to increase their aggressiveness. Control animals included isolates, i.e., animals kept individually housed throughout the experiment and pair-housed rats, i.e., pairs of rats housed together from their rats in the laboratory. In 19 out of 20 dyads, a clear dominance relationship developed with an advantage to the resident in 68% of the cases. Dominants showed more exploratory activity than subordinates in a open-field test at the end of the cohabitation period; subordinates groomed longer than animals from other experimental groups. Dominants had lower pain thresholds than individually and pair-housed animals. Both dominants and subordinates had higher tyrosine hydroxylase enzymatic activities in the left adrenal than isolated and pair-housed rats. Subordinates lost body weight and had higher plasma corticosteroid concentrations than animals from the other experimental groups. In addition, they had smaller thymus glands and reduced spleen lymphocyte responses to mitogenic stimulation in vitro, in comparison to dominant animals. These results show that subordination in the dyadic resident-intruder paradigm leads to a complex syndrome of behavioural and physiological changes, some of which may be modulated by the intensity of aggressive interactions.

Serotonin and stress.

Forty-five years after its discovery, brain serotonin (5-HT) is still the subject of intense research aimed at understanding its role in stress adaptation. At the presynaptic level, numerous stressors increase nerve firing and extracellular 5-HT at the level of serotonergic cell bodies or nerve terminals. Different studies have reported stressor- and region-specific changes in extracellular 5-HT, a view challenged by electrophysiological and neurochemical evidence for a nonspecific response of serotonergic neurones to stressors when activity/arousal is taken into account. In addition, early studies indicate that stress-induced elevation in 5-HT synthesis, a key counter-regulatory process allowing serotonergic homeostasis, is mediated by specific neuroendocrine mechanisms. In addition to the multiplicity of postsynaptic 5-HT receptors and their specific regulation by corticoids, specificity to stressors is also underscored when considering one receptor type such as the 5-HT1A receptor. Stress studies should consider the past experience and the genetic status of the individual as key modulators of the serotonergic responses to stress.

Anxiety- and activity-related effects of diazepam and chlordiazepoxide in the rat light/dark and dark/light tests

We have investigated, through factor analysis, anxiety- and activity-related variables in rats placed in the light/dark box. Thus, vehicle-, diazepam (DZ)-, and chlordiazepoxide (CDP)-treated rats were submitted 30 min later to 5-min light/dark or dark/light tests (initial placements in light or dark, respectively). Following this test, the animals were tested for 5 min in an automated activity monitor. Doses of DZ (0.75-3.0 mg/kg) and CDP (2.5-10.0 mg/kg) were based on preliminary evidence for 1.5 mg/kg of DZ and 5 mg/kg of CDP being anxiolytic in the elevated plus-maze. In the light/dark test, DZ increased the number of visits to and duration in the light compartment, and locomotor activity in the dark compartment; moreover, DZ decreased the latency to enter the light compartment. These effects were, however, significant for the highest dose only. Although CDP yielded similar behavioural effects, only the highest dose had a significant effect, namely, on latency to enter the light side. Conversely, none of the other variables were benzodiazepine-sensitive. Locomotion in the activity cages was decreased by DZ and CDP, an effect significant for the highest doses of benzodiazepines (dark/light test condition only). In both tests, factor analyses revealed an anxiety-related factor (to which all variables related to the visits in the light and part of the locomotion in the dark contributed), and an activity-related factor (upon which the latency to enter the dark and part of the locomotion in the dark loaded) in the light/dark test only. It is suggested that although the light/dark and dark/light tests capture an approach/avoidance dimension, DZ and CDP are more effective in the former test. Compared to the light/dark test, however, the plus-maze may be more sensitive to the anxiolytic effects of DZ and CDP.

Differential effects of social stress on central serotonergic activity and emotional reactivity in Lewis and spontaneously hypertensive rats.

Social stress by repeated defeat has been shown to be endowed with neuroendocrine and behavioural effects that render this stress model useful to identify adaptive mechanisms. Among these mechanisms, those related to central serotonergic systems (e.g., hippocampal 5-HT1A receptors, cortical 5-HT2A receptors) have been particularly underlined. Nonetheless, how (i) the neuroendocrine and behavioural effects of social stress are affected by the genetic status of the animal, and (ii) this status affects the relationships between central serotonergic systems and adaptive processes has not been studied so far. The present study has thus analysed the effects of repeated defeat (once a day for seven days) by Long-Evans resident rats upon the psychoneuroendocrine profile of Lewis rats and spontaneously hypertensive rats previously characterized for their contrasting social and anxiety-related behaviours. Repeated defeat decreased in a time-dependent manner, body weight growth and food intake in both strains, these decreases being, however, more severe and longer lasting in Lewis rats. This strain-dependent difference could not be accounted for by differences in physical contacts with the resident rats as the number of attacks and their latency throughout the stress period were similar between spontaneously hypertensive and Lewis rats. When exposed to an elevated plus-maze test of anxiety, the unstressed Lewis rats entered less the open arms than their spontaneously hypertensive counterparts, thus confirming that Lewis rats are more anxious than spontaneously hypertensive rats. This difference was amplified by stress as the latter increased anxiety-related behaviours in Lewis rats only. These strain- and stress-related differences were associated with differences in locomotor activity, this being increased in unstressed Lewis compared with spontaneously hypertensive rats; moreover, stress triggered hypolocomotion in the former but not the latter strain. Lastly, in the forced swimming test. Lewis rats spent more time immobile than spontaneously hypertensive rats with stress increasing immobility in a strain-independent manner. Beside the aforementioned metabolic changes, the activity of the hypothalamo-pituitary-adrenal axis was slightly stimulated in a strain-independent manner by the stressor, as assessed by increased corticosterone levels and adrenal weights, and decreased thymus weights. In Lewis, but not in spontaneously hypertensive rats, midbrain serotonin metabolism was increased by stress, a difference associated with an increased Bmax value of cortical [3H]ketanserin binding at 5-HT2A receptors. On the other hand, the Bmax value of hippocampal [3H]8-hydroxy-2-(di-n-propylamino)tetralin binding at 5-HT1A receptors was decreased by stress, this reduction being amplified in spontaneously hypertensive compared with Lewis rats. This study shows that the psychoneuroendocrine responses to social stress may have a genetic origin, and that the use of socially stressed Lewis and spontaneously hypertensive rats may provide an important paradigm to study adaptive processes. However, whether the aforementioned strain-dependent differences in central serotonergic systems (partly or totally) underlie the distinct profiles of emotivity measured in spontaneously hypertensive and Lewis rats, is discussed in the context of the relationships between serotonergic systems and behavioural responses to novel environments.

Behavioral, neuroendocrine and serotonergic consequences of single social defeat and repeated fluoxetine pretreatment in the Lewis rat strain.

We have analysed some behavioral, neuroendocrine and serotonergic consequences of a single (30-min) social defeat followed by 14-18 h of sensory contact with the aggressor, in Lewis rats, an inbred strain highly sensitive to chronic social stressors [Berton O. et al. (1998) Neuroscience 82, 147-159]. In addition, we have investigated how the aforementioned consequences are affected by pretreatment with the selective serotonin reuptake inhibitor, fluoxetine (7.5 mg/kg/day for 21 days). A single social defeat triggered hypophagia and body weight loss, and increased anxiety in the elevated plus-maze. It did not affect baseline plasma adrenocorticotropic hormone levels and renin activity, but decreased plasma corticosterone levels. On the other hand, the responses of the latter variables to subsequent acute forced swim stress were blunted (corticosterone) or amplified (adrenocorticotropic hormone, renin activity) by prior defeat. The density of hippocampal serotonin transporters, but not that of hippocampal serotonin-1A and cortical serotonin-2A receptors, was decreased by a single social defeat; in addition, neither tryptophan availability and serotonin synthesis/metabolism, nor serotonin-1A autoreceptor-mediated functions (inhibition of serotonin synthesis, hyperphagia) were affected. Fluoxetine pretreatment diminished social defeat-induced hypophagia, body weight loss and anxiety without affecting these variables in control animals. This pretreatment increased plasma corticosterone levels in resting and acutely stressed rats, but abolished social defeat-elicited corticosterone hyporesponsiveness to acute forced swim stress. Except for a decrease in midbrain serotonin transporter density, fluoxetine did not affect the other serotonergic indices analysed herein, i.e. serotonin-1A and serotonin-2A receptor densities, serotonin synthesis/metabolism. A single social defeat in Lewis rats produces behavioral and endocrine alterations that may model some aspects of human anxiety disorders. In this paradigm, prior fluoxetine treatment is endowed with adaptive behavioral, and possibly neuroendocrine, effects without affecting the key elements of central serotonergic systems analysed herein.

Modulation of the locomotor response to amphetamine by corticosterone

Abstract In the present experiments, we investigated the influence of chronic modifications of circulating levels of corticosterone on the locomotor response to amphetamine. Different groups of rats were adrenalectomized and implanted subcutaneously with pellets releasing different amounts of corticosterone (0–200 mg). A wide range of corticosterone concentrations was reached in order to saturate selectively either the type I (mineralocorticoid) or the type II (glucocorticoid) corticosteroid receptors. The locomotor response to d -amphetamine (1.5 mg/kg) was studied 10–14 days later. We found that adrenalectomy reduced the response to d -amphetamine by 33% and that a normal response was restored with pellets releasing physiological concentrations of corticosterone (50-mg pellets), and was potentiated in animals with pellets releasing high amounts of corticosterone mimicking chronic stress situations (200-mg pellets). The correlation between plasma corticosterone concentration, locomotor activity following d -amphetamine and thymus weight, which is a reliable indicator of glucocorticoid action, shows that the influence of the locomotor response to d -amphetamine administration is likely to be mediated via a type II receptor. Since the locomotor activating effect of peripheral administration of d -amphetamine has been shown to depend on the integrity of the dopaminergic innervation of the nucleus accumbens, the effect of d -amphetamine at different doses (0, 1, 3, 10μg/gm1) injected directly into the nucleus accumbens was studied. The results demonstrated that removing the circulating corticosterone induced a similar decrease of the locomotor activity elicited by d -amphetamine injection in the nucleus accumbens. This response was restored in animals with the 50- and 200-mg pellets. This modulatory action of corticosterone on the reactivity of the dopaminergic system to psychostimulants may represent a critical step for the interchangeability between Stressors and psychostimulants in sensitization processes.

Effects of repeated fluoxetine on anxiety-related behaviours, central serotonergic systems, and the corticotropic axis in SHR and WKY rats

In keeping with the anxiolytic property of selective serotonin reuptake inhibitors (SSRIs) in humans, we have examined in the spontaneously hypertensive rat (SHR) and the Wistar-Kyoto (WKY) rat, which display low and high anxiety, respectively, some psychoneuroendocrine effects of a repeated treatment with the SSRI fluoxetine (5 or 10 mg/kg daily, for 3 weeks). Two days after the last injection, plasma levels of fluoxetine were not detectable whereas those of its metabolite, norfluoxetine, were present to similar extents in both strains. By means of the elevated plus-maze test (29-30 h after the 13th administration of fluoxetine) and an open field test (48 h after the last injection of fluoxetine), it was observed that fluoxetine pretreatment did not yield anxiolysis; hence, some, but not all, behaviours were indicative of anxiety and hypolocomotion (as assessed through principal component analyses and acute diazepam studies). In both strains, the 10 mg/kg dose of fluoxetine decreased hypothalamus 5-HT and 5-HIAA levels, and reduced midbrain and/or hippocampus [3H]citalopram binding at 5-HT transporters, but did not affect [3H]8-hydroxy-2-(di-N-propylamino)tetralin binding at hippocampal 5-HT1A receptors. However, the fluoxetine-elicited reduction in hippocampal 5-HT transporter binding was much more important in WKY than in SHR rats, this strain-dependent effect being associated in WKY rats with a reduction in cortical [3H]ketanserin binding at 5-HT2A receptors. Lastly, in WKY rats, repeated fluoxetine administration increased adrenal weights and the plasma corticosterone response to open field exposure, but did not affect the binding capacities of hippocampal mineralocorticoid and glucocorticoid receptors. These data show that key psychoneuroendocrine responses to repeated fluoxetine administration may be strain-dependent, and that repeated fluoxetine administration does not yield anxiolysis, as assessed by two standard tests of emotivity.