Efthimia Kitraki

Gender-dependent alterations in corticosteroid receptor status and spatial performance following 21 days of restraint stress

The effects of 21-day exposure to restraint stress on hippocampal corticosteroid receptors and on spatial performance of male and female rats were evaluated. Stressed male animals exhibited a decrease in glucocorticoid receptor immunoreactivity in the CA1 area and the dentate gyrus. At the same time, stressed males tested on Morris water maze showed delayed learning and worse memory scores, compared with the control males. By contrast, stressed females exhibited an increase in glucocorticoid receptor immunoreactivity in CA1, similar learning ability and improved memory scores, compared with control females. In addition, stressed females showed a significant increase in mineralocorticoid receptor immunoreactivity in the CA3 area compared with controls. These data show that 21 days of restraint stress affect hippocampal corticosteroid receptors and spatial performance in a gender-specific manner. The observed changes in corticosteroid receptor levels following stress, may be causatively linked to the stress-induced alterations on spatial learning and memory.

Long-Lasting Effects of Stress on Glucocorticoid Receptor Gene Expression in the Rat Brain

Stressful stimuli are known to affect glucocorticoid receptor (GR) mRNA levels in the rat brain. The aim of this study was to examine the duration of chronic stress-induced changes in GR gene expression in the male rat hippocampus and cerebellum. By using in situ hybridization histochemistry, we detected a statistically significant down-regulation of GR mRNA both in the hippocampus and in the cerebellum of rats stressed for 8, 10 and 14 days. The same degree of down-regulation could also be detected in the above brain areas of rats stressed for 14 days and left undisturbed for 48 h or 8 days after stress. To examine the effects of subsequent stressors on the expression of down-regulated GR mRNA in the hippocampus of chronically stressed rats, we determined, by Northern blotting, GR mRNA levels in the hippocampi of rats stressed for 14 days and subsequently exposed to either short- or long-duration stressors. The down-regulated levels of GR mRNA remained practically unaffected when a subsequent new stressor was applied. Our results show that chronic stress-induced down-regulation of GR mRNA in the rat brain can be extended for periods longer than the initial/causative stimulus, irrespective of the presence of a novel stimulus.

Effects of hyperactivity of the maternal hypothalamic-pituitary-adrenal (HPA) axis during pregnancy on the development of the HPA axis and brain monoamines of the offspring

Offspring of mothers with adrenal hyperactivity during pregnancy have been reported to have changes in brain monoamines and altered emotional, reactive, sexual and maternal behavior. Since the hypothalamic‐pituitary‐adrenal (HPA) axis is known to be involved in the expression of such behaviors and is itself under monoaminergic control, we examined the development of the HPA axis and brain monoamines in pups whose mothers had adrenal hyperactivity, reflecting administration of ACTH during the last third of their pregnancy. The adrenals of the experimental animals weighed less and had aberrant morphology. The abnormal histology was more pronounced in the adrenals of the experimental females than of the males, suggesting that females were more vulnerable to the prenatal treatment. In both experimental males and females, basal plasma corticosterone levels were higher compared to the controls, while after exposure to stress, experimental animals attained lower plasma corticosterone levels than the controls. In the brain of the experimental animals, dopaminergic activity appeared to be decreased, while serotonergic activity increased. Our results indicate that the prenatal treatment affected brain development in the offspring and as a consequence programmed the developing HPA axis in such a way as to hyperfunction under basal conditions, leading to its exhaustion and its inability to react properly to stress.

Spatial Performance and Corticosteroid Receptor Status in the 21‐Day Restraint Stress Paradigm

Abstract: Twenty‐one days of restraint stress has been shown to affect hippocampal plasticity, neurogenesis, and spatial memory. Hippocampal glucocorticoid receptors (GR) and mineralocorticoid receptors (MR) are the main mediators of stress response and learning/memory processes. We studied the performance of male and female rats on a hippocampal‐dependent spatial task after 21 days of restraint in relation to the stress‐induced changes of GR and MR status in their hippocampi. Reduced GR immunostaining was detected in the dentate gyrus and CA1 area of stressed male rats. Stressed male rats performed worse than the male control rats on the Morris water maze. In contrast, unaltered (in the dentate gyrus) or increased (in CA1) GR immunoreactivity was seen in the hippocampus of stressed female rats. Stressed female rats had an improved memory score in the task compared with the female control rats. In addition, stressed female rats showed increased MR immunostaining in the CA3 area, which is known to be severely affected by stress. The observed sexually dimorphic effects of 21‐day restraint in spatial learning and memory may be associated with the sex‐dependent changes in hippocampal corticosteroid receptor status after stress.

Forced Swimming Differentially Affects Male and Female Brain Corticosteroid Receptors

Corticosteroid receptors are key mediators of the neuroendocrine response to stress. Previously, we have determined the effects of restraint stress on the regulation of corticosteroid receptor genes in the brain and pituitary of male and female rats. Significant gender- and regional-specific regulation of receptor mRNAs was observed. To further investigate the stressor specificity in the same context, we have determined glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) mRNAs following exposure to swimming stress paradigms applied alone, or in combination with restraint stress. Our data revealed stressor-specific alterations in GR or MR mRNA levels, which were more pronounced in males, the gender most affected by swimming stress. No alterations in GR or MR mRNA levels were detected in the female hippocampus and hypothalamus upon exposure to swimming paradigms, while in males the same stressors down-regulated GR mRNA in the hippocampus (chronic exposure) and up-regulated both genes in the hypothalamus (acute exposure). In the frontal cortex, acute swimming stress caused a reciprocal change in GR mRNA levels in the two sexes. The above difference is not due to circulating ovarian steroids, since ovariectomy did not change the female pattern of GR gene expression following acute stress. Our results further showed a hypothalamic-pituitary-adrenal axis facilitation to a novel superimposed stressor expressed at the level of limbic corticosteroid receptors: When chronically restrained rats of both sexes were exposed to acute swimming stress, a reduced GR/MR mRNA ratio, implying reduced feedback axis sensitivity, was detected in both the hippocampus and the hypothalamus. In conclusion, our work provides additional evidence on stressor, gender and region specificity in the regulation of brain corticosteroid receptors.

Enriched environment influences hormonal status and hippocampal brain derived neurotrophic factor in a sex dependent manner

The present study is aimed at testing the hypothesis that an enriched environment (EE) induces sex-dependent changes in stress hormone release and in markers of increased brain plasticity. The focus was on hypothalamic-pituitary-adrenocortical (HPA) axis activity, plasma levels of stress hormones, gene expression of glutamate receptor subunits and concentrations of brain-derived neurotrophic factor (BDNF) in selected brain regions. Rats exposed to EE were housed in groups of 12 in large cages with various objects, which were frequently changed, for 6 weeks. Control animals were housed four per cage under standard conditions. In females the EE-induced rise in hippocampal BDNF, a neurotrophic factor associated with increased neural plasticity, was more pronounced than in males. Similar sex-specific changes were observed in BDNF concentrations in the hypothalamus. EE also significantly attenuated oxytocin and aldosterone levels only in female but not male rats. Plasma testosterone positively correlated with hippocampal BDNF in female but not male rats housed in EE. In male rats housing in EE led to enhanced levels of testosterone and adrenocorticotropic hormone (ACTH), this was not seen in females. Hippocampal glucocorticoid but not mineralocorticoid receptor levels decreased in rats housed in EE irrespective of sex. Housing conditions failed to modify mRNA levels of glutamate receptor type 1 (Glur1) and metabotropic glutamate receptor subtype 5 (mGlur5) subunits of glutamate receptors in the forebrain. Moreover, a negative association between corticosterone and BDNF was observed in both sexes. The results demonstrate that the association between hormones and changes in brain plasticity is sex related. In particular, testosterone seems to be involved in the regulatory processes related to neuroplasticity in females.

Contribution of Sex and Cellular Context in the Regulation of Brain Corticosteroid Receptors following Restraint Stress

The two subtypes of corticosterone receptors in the rat brain play a pivotal role in the modulation of the stress response. Appropriate control of their gene expression is therefore critical for the maintenance of cellular and organism homeostasis. In this study, we investigated the contribution of gender and of the cellular environment of certain brain areas to the expression of both types of corticosteroid receptors, following restraint stress. Adult Wistar rats of both sexes were subjected to acute, chronic or to a combined chronic plus acute stress regimen, and the expression of glucocorticoid and mineralocorticoid receptors was evaluated in their hippocampus, hypothalamus, pituitary and frontal cortex, by using Northern blot analysis. Significant sex differences were observed in the first three brain areas examined as to the stress-induced expression of corticosteroid receptors. Among these, females showed a distinct mechanism of regulating glucocorticoid/mineralocorticoid receptor ratio in the hippocampus upon chronic stress, while the female hypothalamus was more prone than the male to changing corticosteroid receptor expression in response to restraint stress. In another set of experiments, we assessed the influence of ovarian steroids on stress-induced corticosteroid receptor expression in the above brain areas by analyzing ovariectomized rats exposed to short-term restraint. Our results showed that although ovarian steroids affect the stress-induced expression of receptor genes in a region-specific manner, their elimination does not appear to lead to the male pattern of expression. These findings provide further evidence for the existence of both regional and gender specificity in the regulation of brain and pituitary corticosteroid receptors following stress, and support the hypothesis of a distinct male and female neuroendocrine axis in response to stress.

Glucocorticoid receptor gene expression in the embryonic rat brain.

The early ontogenetic pattern of glucocorticoid receptor (GR) gene expression was studied in the rat brain through embryonic days (E) 12 to 17. Using a [35S]-labelled GR antisense RNA probe for in situ hybridization, we first detected GR mRNA in E13 embryos. The strongest signal was in Rathkes pouch, but the hypothalamic, and to a lesser degree the pontine and rhinencephalic neuroepithelium were also moderately labelled. Significant levels of GR mRNA were also detected in the choroid plexus and the epithelia lining the ventricles on E13. Receptor gene expression was further extended by E15 to the neuroepithelium and the differentiating field of several neuronal structure primordia, including the basal ganglia, rhinencephalon, hippocampus, pons and cerebellum. On E17, GR gene expression was in addition detected in the amygdala, subiculum and olfactory bulb and cortex. The integrity of the mRNA transcripts revealed by in situ hybridization was assessed by Northern blot analysis of total RNA from embryonic brain and pituitary. A major approximately 7-kb transcript was detected throughout embryonic development. An adult-like GR protein was shown by immunoblotting analysis to be expressed in brain and pituitary extracts already by E13. Based on our results, we postulate a receptor-mediated regulatory role for glucocorticoids in the embryonic development of the rat brain.

Maternal behavior of dams treated with ACTH during pregnancy

Experimental female rats were injected with ACTH during the last third of their pregnancy. This treatment resulted in prolongation of gestation and in abnormal development of the young. The number of resorptions, stillbirths, and congenitally malformed pups was increased and those that appeared normal had lower body weights. The experimental treatment also severely affected the ability of the dams to exhibit normal maternal behavior. Significant individual differences were noted in the sensitivity of the dams to the experimental treatment. Cross-fostering experiments revealed that experimental dams exhibited normal maternal behavior towards control foster pups, after an initial delay of 24 h. When experimental pups, born after a prolonged gestation, or delivered by caesarian section after the normal duration of gestation, were given to control mothers, normal maternal behavior was observed, but the survival rate of the young was not increased.

Impaired Neuroendocrine Response to Stress following a Short-Term Fat-Enriched Diet

Unbalanced diets and stressful situations disrupt energy homeostasis and are implicated in the development of severe pathologies. The present study investigated the effects of a 7-day diet, enriched in corn oil (20%) and proportionally lower in protein and carbohydrate, on the major regulators of energy expenditure and stress response of adult male Wistar rats exposed to acute swimming stress at the end of the dietary treatment. Food intake and body weight gain were lower in diet-fed as compared with normal-chow-fed controls. The circulating leptin levels were elevated in both nonstressed and stressed diet-fed rats, while the glucose levels were significantly increased only in the diet-fed group subjected to stress. The plasma insulin levels were not affected by the diet, but were significantly reduced in acutely stressed rats. Acute swimming increased corticosterone levels both in chow-fed and diet-fed rats. No significant effect of diet was observed on corticosterone levels. Northern blot analysis showed increased glucocorticoid receptor mRNA levels in the hypothalamus of normally fed rats subjected to stress. This increase was not observed in the diet-fed stressed group, which on the contrary showed reduced glucocorticoid receptor mRNA levels following stress. The data presented indicate that even a moderately unbalanced, fat-enriched diet can within a short time disrupt the metabolic neuroendocrine balance and the stress response, rendering the organism more vulnerable to potential stressful insults.