Roser Nadal

Long-term neuroendocrine and behavioural effects of a single exposure to stress in adult animals

There is now considerable evidence for long-lasting sequels of stress. A single exposure to high intensity predominantly emotional stressors such as immobilisation in wooden-boards (IMO) induces long-term (days to weeks) desensitization of the hypothalamic-pituitary-adrenal (HPA) response to the same (homotypic) stressor, whereas the response to novel (heterotypic) stressors was enhanced. In addition, long-lasting changes in behaviour have been described after a single exposure to brief or more prolonged sessions of shocks, predator, predator odour, underwater stress or a combination of three stressors on 1 day. The most consistent changes are reduced entries into the open arms of the elevated plus-maze and enhanced acoustic startle response, both reflecting enhanced anxiety. However, it is unclear whether there is any relationship between the intensity of the stressors, as evaluated by the main physiological indexes of stress (e.g. HPA axis), the putative traumatic experience they represent and their long-term behavioural consequences. This is particularly critical when trying to model post-traumatic stress disorders (PTSD), which demands a great effort to validate such putative models.

Acute effects of ketamine in the holeboard, the elevated-plus maze, and the social interaction test in Wistar rats

Although noncompetitive NMDA receptor antagonists have shown an anxiolyticlike profile in several studies, such effects have not been observed consistently. Previous studies with ketamine, a noncompetitive NMDA antagonist, have employed only shock tests of anxiety based on conflict procedures. In the present experiment, the effect of an acute low dose of ketamine (7 mg/kg) was examined in adult male Wistar rats tested in three nonconflict tests: holeboard, social interaction, and elevated plus‐maze paradigms. The results showed that ketamine decreased time spent in active social interaction and the number of rearings and central activity in the social interaction test. It also decreased the number of entries into and the percentage of time spent in open arms and the total number of entries in the elevated plus‐maze. No significant effect was observed in head dipping in the holeboard test, although the number of crossings did increase. These results suggest an anxiogeniclike effect of ketamine in contrast with results previously described for noncompetitive NMDA receptor antagonists. These effects of ketamine are more similar to those described for stimulant drugs such as caffeine, cocaine, or amphetamine in anxiety tests. Depression and Anxiety 5:29–33, 1997.

7,8-dihydroxyflavone, a TrkB receptor agonist, blocks long-term spatial memory impairment caused by immobilization stress in rats.

Post‐traumatic stress disorder (PTSD) patients show cognitive deficits, but it is unclear whether these are a consequence of the pathology or a pre‐existing factor of vulnerability to PTSD. Animal models may help to demonstrate whether or not exposure to certain stressors can actually induce long‐lasting (LL; days) impairment of hippocampus‐dependent memory tasks and to characterize neurobiological mechanisms. Adult male rats were exposed to 2‐h immobilization on boards (IMO), a severe stressor, and spatial learning in the Morris water maze (MWM) was studied days later. Exposure to IMO did not modify learning or short‐term memory in the MWM when learning started 3 or 9 days after IMO, but stressed rats did show impaired long‐term memory at both times, in accordance with the severity of the stressor. New treatments to prevent PTSD symptoms are needed. Thus, considering the potential protective role of brain‐derived neurotrophic factor (BDNF) on hippocampal function, 7,8‐dihydroxyflavone (7,8‐DHF), a recently characterized agonist of the BDNF receptor TrkB, was given before or after IMO in additional experiments. Again, exposure to IMO resulted in LL deficit in long‐term memory, and such impairment was prevented by the administration of 7,8‐DHF either 2 h prior IMO or 8 h after the termination of IMO. The finding that IMO‐induced impairment of spatial memory was prevented by pharmacological potentiation of TrkB pathway with 7,8‐DHF even when the drug was given 8 h after IMO suggests that IMO‐induced impairment is likely to be a LL process that is strongly dependent on the integrity of the BDNF‐TrkB system and is susceptible to poststress therapeutic interventions. 7,8‐DHF may represent a new therapeutic approach for early treatment of subjects who have suffered traumatic experiences.

A single exposure to immobilization causes long-lasting pituitary-adrenal and behavioral sensitization to mild stressors

We have previously reported that a single exposure to immobilization (IMO) in rats causes a long-term desensitization of the hypothalamic-pituitary-adrenal (HPA) response to the same (homotypic) stressor. Since there are reports showing that a single exposure to other stressors causes sensitization of the HPA response to heterotypic stressors and increases anxiety-like behavior, we studied in the present work the long-term effects of IMO on behavioral and HPA response to mild superimposed stressors. In Experiments 1 and 2, adult male Sprague-Dawley rats were subjected to 2 h of IMO and then exposed for 5 min to the elevated plus-maze (EPM) at 1, 3 or 7 days after IMO. Blood samples were taken at 15 min after initial exposure to the EPM. Increases in anxiety-like behavior and HPA responsiveness to the EPM were found at all times post-IMO. Changes in the resting levels of HPA hormones did not explain the enhanced HPA responsiveness to the EPM (Experiment 3). In Experiments 4 and 5, we studied the effects of a single exposure to a shorter session of IMO (1 h) on behavioral and HPA responses to a brief and mild session of foot-shocks done 10 days after IMO. Neither previous IMO nor exposure to shocks in control rats modified behavior in the EPM. However, a brief session of shocks in previously IMO-exposed rats dramatically increased anxiety in the EPM. HPA and freezing responses to shocks were similar in control and previous IMO groups. Therefore, a single exposure to IMO appears to induce long-lasting HPA and behavioral sensitization to mild superimposed stressors, although the two responses are likely to be at least partially independent. Long-term effects of IMO on the susceptibility to stress-induced endocrine and emotional disturbances may be relevant to the characterization of animal models of post-traumatic stress.

Influence of reactivity to novelty and anxiety on hypothalamic-pituitary-adrenal and prolactin responses to two different novel environments in adult male rats.

Since stressor-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis is involved in some stress-related pathologies, much attention has been paid in laboratory animals to the study of the relationship between endocrine, particularly HPA, responsiveness to stressors and other individual characteristics, such as reactivity to novelty and fear/anxiety. In the present study, adult male rats were classified as high or low reactive to novelty (HR versus LR), as a function of the horizontal activity displayed during 30 min in a circular corridor, and as high or low anxiety (HA versus LA) as a function of the time spent in the open arms of the elevated plus-maze. Then, the behavioural and hormonal response to two distinct novel environments (the hole-board and the light-dark) was assessed in the same subjects, using a counterbalanced design. Plasma prolactin, ACTH and corticosterone responses to the hole-board were higher than to the light-dark, a good correlation between the two tests being found for each hormone. Whereas the hormonal response to the novel environments was not affected by anxiety, HR rats showed a consistently higher HPA response than LR rats when the criteria to classify the animals were the activity during the first 15 min in the circular corridor, but not when the activity during the second 15 min was considered. Neither trait affected prolactin response. The present results demonstrate a good within-individual consistency of the endocrine response to novel environments and support the hypothesis of a higher HPA response to stressors for HR versus LR rats. In contrast, no contribution of fear/anxiety to endocrine responsiveness was observed.

Environmental enrichment effects in social investigation in rats are gender dependent

Environmental enrichment (E) can change neuronal structure, improves learning in various tasks and increases cerebral plasticity. However, no E effects were found in a test measuring social olfactory discrimination memory and, although they have been mainly measured in males, gender differences have been reported in other tests. The aims of the present study were to evaluate gender differential effects of E in the social discrimination paradigm which also involves social olfactory discrimination and in the elevated plus-maze test (EPM) for measuring anxiety. E procedure consisted of a combination of social and physical factors; groups of 11-12 Sprague-Dawley rats were separated by sex in large cages with physical stimulus for a period of 8 weeks starting immediately after weaning. Differential gender E effects appeared in the social exploratory patterns: enriched males showed increased exploratory behaviour towards juvenile rats in comparison to control males, whereas no differences were found in females. No effects of E in social discrimination memory were observed. In the EPM, both enriched male and female rats showed less anxious behaviour than non-enriched animals.

Sex-dependent effects of maternal deprivation and adolescent cannabinoid treatment on adult rat behaviour

Early life experiences such as maternal deprivation (MD) exert long‐lasting changes in adult behaviour and reactivity to stressors. Adolescent exposure to cannabinoids is a predisposing factor in developing certain psychiatric disorders. Therefore, the combination of the two factors could exacerbate the negative consequences of each factor when evaluated at adulthood. The objective of this study was to investigate the long‐term effects of early MD [24 hours at postnatal day (PND) 9] and/or an adolescent chronic treatment with the cannabinoid agonist CP‐55,940 (0.4 mg/kg, PND 28–42) on diverse behavioural and physiological responses of adult male and female Wistar rats. We tested them in the prepulse inhibition (PPI) of the startle response and analysed their exploratory activity (holeboard) and anxiety (elevated plus maze, EPM). In addition, we evaluated their adrenocortical reactivity in response to stress and plasma leptin levels. Maternal behaviour was measured before and after deprivation. MD induced a transient increase of maternal behaviour on reuniting. In adulthood, maternally deprived males showed anxiolytic‐like behaviour (or increased risk‐taking behaviour) in the EPM. Adolescent exposure to the cannabinoid agonist induced an impairment of the PPI in females and increased adrenocortical responsiveness to the PPI test in males. Both, MD and adolescent cannabinoid exposure also induced sex‐dependent changes in plasma leptin levels and body weights. The present results indicate that early MD and adolescent cannabinoid exposure exerted distinct sex‐dependent long‐term behavioural and physiological modifications that could predispose to the development of certain neuropsychiatric disorders, though no synergistic effects were found.

The hypothalamic-pituitary-adrenal and glucose responses to daily repeated immobilisation stress in rats: Individual differences

It is accepted that there are important individual differences in the vulnerability to stress-induced pathologies, most of them associated to the hypothalamic-pituitary and sympatho-medullo-adrenal axes, the two prototypical stress-responsive systems. However, there are few studies specifically aimed at characterising individual differences in the physiological response to daily repeated stress in rats. In the present work, male rats were submitted to repeated immobilisation (IMO) stress (1 h daily for 13 days) and several samples were taken at specific days and time points. Animals only subjected to blood sampling procedure served as controls. Daily adrenocorticotropic-hormone (ACTH), corticosterone and glucose responses to immobilisation (that included the post-immobilisation period) progressively declined over the days. In addition, repeated immobilisation resulted in decreased relative thymus weight, increased relative adrenal weight, elevated corticotropin-releasing factor (CRF) mRNA levels in the hypothalamic paraventricular nucleus (PVN), and down-regulation of glucocorticoid receptor gene transcription in hippocampus CA1. However, only CRF mRNA levels in the paraventricular nucleus correlated with the ACTH (on day 1) and corticosterone responses (from day 4-13) to immobilisation. When the animals were classified in three groups on the basis of their plasma ACTH levels immediately after the first immobilisation, individual differences in the ACTH response progressively disappeared on successive exposures to the stressor, whereas those in corticosterone and glucose were more sustained. The present results suggest that there are individual differences in the physiological response to stress that tend to be reduced rather than accentuated by repeated exposure to the stressor. Nevertheless, this buffering effect of repeated stress was dependent on the particular variable studied.

Litter size affects emotionality in adult male rats

The role of natural variations in pre-weaning litter size in rodent adult emotionality and the importance of maternal care as a possible mediating factor have been frequently neglected. To address these issues, maternal behaviour of Sprague-Dawley dams differing in natural number of pups was studied for the first seven postnatal days. Later, adult behaviour of representative male offspring was studied in the elevated plus-maze, the circular corridor, the dark-light box and the forced swimming test. Three groups of offspring were selected in function of the number of littermates: L<10 group (less than 10 pups per dam), L10-15 (between 10 and 15 pups per dam) and L>15 group (more than 15 pups per dam). L<10 litters showed a reduced habituation of activity across time in a circular corridor and as compared to L>15 litters, L<10 litters showed a lower activity during the first 5 min of exposure to the circular corridor. L<10 litters had also higher signs of anxiety in the elevated plus-maze, in comparison to the other two groups. In addition, L<10 litters showed in the forced swimming test reduced struggling and more mild swimming behavior than the other two groups. These abnormalities in L<10 litters are not explained by maternal behavior since they received individually more maternal care than L>15, as assessed by total licking-grooming observed during the whole observation period divided by number of pups. Although previous data from several laboratories have demonstrated that low maternal care is associated with heightened emotionality at adulthood, the present results suggest an important contribution of spontaneous litter size to adult emotional behavior that cannot be explained by concomitant changes in maternal care.

Characterization of central and peripheral components of the hypothalamus-pituitary-adrenal axis in the inbred Roman rat strains.

Several studies performed in outbred Roman high- and low-avoidance lines (RHA and RLA, respectively) have demonstrated that the more anxious line (RLA) is characterized by a higher hypothalamic-pituitary-adrenal (HPA) response to certain stressors than the less anxious one (RHA). However, inconsistent results have also been reported. Taking advantage of the generation of an inbred colony of RLA and RHA rats (RHA-I and RLA-I, respectively), we have characterized in the two strains not only resting and stress levels of peripheral HPA hormones but also central components of the HPA axis, including CRF gene expression in extra-hypothalamic areas. Whereas resting levels of ACTH and corticosterone did not differ between the strains, a greater response to a novel environment was found in RLA-I as compared to RHA-I rats. RLA-I rats showed enhanced CRF gene expression in the paraventricular nucleus (PVN) of the hypothalamus, with normal arginin-vasopressin gene expression in both parvocellular and magnocellular regions of the PVN. This enhanced CRF gene expression is not apparently related to altered negative corticosteroid feedback as similar levels of expression of brain glucorticoid and mineralocorticoid receptors were found in the two rat strains. CRF gene expression tended to be higher in the central amygdala and it was significantly higher in the dorsal region of the bed nucleus of stria terminalis (BNST) of RLA-I rats, while no differences appeared in the ventral region of BNST. Considering the involvement of CRF and the BNST in anxiety and stress-related behavioral alterations, the present data suggest that the CRF system may be a critical neurobiological substrate underlying differences between the two rat strains.