慢性束缚应激大鼠中枢糖皮质激素受体变化及中药复方的影响

目的:研究慢性束缚应激时大鼠脑内糖皮质激素受体的变化以及逍遥散、四君子汤、金匮肾气丸三种中药复方对其影响.方法:制作大鼠束缚应激模型,用特制束缚架连续束缚7 d与21 d,每天3 h用免疫组织化学方法结合图像分析检测中枢(海马CA1区、齿状回、大脑皮质)糖皮质激素受体的变化.结果:慢性束缚应激后,大鼠海马CA1区、大脑皮层和齿状回GR免疫反应阳性细胞平均总面积和阳性细胞数目在慢性应激的早期(7d模型组)明显增多(P＜0.05),免疫反应强度明显增强(P＜0.01).在慢性应激的后期(21 d模型组),则表现为相关脑区GR免疫反应阳性细胞平均总面积和阳性细胞数目均明显减少(P＜0.05),免疫反应强度明显减弱(P＜0.01).中药复方各组相关脑区神经元GR免疫反应阳性细胞平均总面积和阳性细胞数目较21 d模型组明显增高,免疫反应强度明显增强,三给药组之间并无明显差异,说明三给药组均能使GR含量保持于较高的状态,同时能保持GR免疫活性,其中又以逍遥散作用为明显.结论:逍遥散、四君子汤和金匮肾气丸明显逆转糖皮质激素受体下降趋势.     

Effect of Treadmill Exercise on Blood Glucose,Serum Corticosterone Levels and Glucocorticoid Receptor Immunoreactivity in the Hippocampus in Chronic Diabetic Rats

Abnormal excess of glucocorticoid is one of feature characteristics in type 2 diabetes. In the present study, we investigated the effect of treadmill exercise at chronic diabetic stages on glucocorticoid receptor (GR) immunoreactivity in the hippocampal CA1 region and dentate gyrus, which are very vulnerable to diabetes. For this study, we used Zucker diabetic fatty (ZDF) rats and Zucker lean control (ZLC) rats. Twenty-three-week-old ZLC and ZDF rats were put on the treadmill with or without running for 7 weeks and sacrificed at 30 weeks of age. Treadmill exercise significantly decreased diabetes-induced blood glucose and serum corticosteroid levels although they did not drop to control levels. In sedentary ZLC rats, GR immunoreactivity was detected in pyramidal cells of the CA1 region as well as in granule cells of the dentate gyrus. In the sedentary ZDF rats, GR immunoreactivity was significantly increased in these regions. However, treadmill exercise significantly decreased GR immunoreactivity in these regions. These results indicate that treadmill exercise in chronic diabetic rats significantly decreased GR immunoreactivity in the hippocampal CA1 region and dentate gyrus, although blood glucose and serum corticosteroid levels did not fully recover to normal state.     

Liver glucocorticoid receptor and heat shock protein 70 levels in rats exposed to different stress models

The aim of the present study was to define the stress-induced pattern of cytosolic glucocorticoid receptor (GR) and Hsp70 protein in the liver of male Wistar rats exposed to different stress models: acute (2 h/day) immobilization or cold (4 degrees C); chronic (21 days) isolation, crowding, swimming or isolation plus swimming and combined (chronic plus acute stress). Changes in plasma levels of corticosterone were studied by radioimmunoassay (RIA). The results obtained by Western immunoblotting showed that both acute stressors led to a significant decrease in cytosolic GR and Hsp70 levels. Compared to acute stress effects, only a weak decrease in the levels of GR and Hsp70 was demonstrated in chronic stress models. Chronically stressed rats, which were subsequently exposed to novel acute stressors (immobilization or cold), showed a lower extent of GR down-regulation when compared to acute stress. The exception was swimming, which partially restores this down-regulation. The observed changes in the levels of these major stress-related cellular proteins in liver cytosol lead to the conclusion that chronic stressors compromise intracellular GR down-regulation in the liver.     

Differential effects of corticosterone and dexamethasone on hippocampal neurogenesis in vitro

Prenatal stress during fetal development results in the blockade of neurogenesis in the dentate gyrus in adulthood. Present study was undertaken to investigate the dominant role of the glucocorticoid receptors in corticosterone actions on the neurogenesis of fetal hippocampal progenitor cells. For that purpose, expressions of key molecules affected by corticosterone and dexamethasone were compared during proliferation and differentiation of the hippocampal progenitor cells. Corticosterone (2 microM) significantly decreased the number of bromodeoxyuridine-labeled cells (about 50%) and caused the dendritic atrophy in microtubule-associated protein 2-labeled cells. The expressions of NeuroD, BDNF, and NR1 mRNA levels and protein levels of p-ERK and p-CREB were remarkably decreased by corticosterone in a dose-dependent manner. In contrast, dexamethasone, a glucocorticoid receptor (GR) specific agonist, had an inhibitory effect on proliferation, but not differentiation. It is concluded that corticosterone elicits its effects on neurogenesis including proliferation and differentiation whereas stimulation of the glucocorticoid receptor is sufficient to decrease only proliferation.     

Differential involvement of amygdala corticosteroid receptors in visceral hyperalgesia following acute or repeated stress

Symptoms of irritable bowel syndrome (IBS) are exacerbated by stress. Previously, we demonstrated that the stress hormone corticosterone applied directly to the amygdala induced visceral hypersensitivity through the actions of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). However, the involvement of amygdaloid GR and MR in the regulation of visceral sensitivity following psychological stress is unknown; therefore, the goal of the present study was to determine the relative importance of amygdaloid GR and MR in the regulation of visceral sensitivity in a rodent model of behavioral stress. Male F-344 rats were stereotaxically implanted with micropellets bilaterally on the dorsal margin of the amygdala containing the GR antagonist mifepristone, the MR antagonist spironolactone, or cholesterol as a control. Animals were then exposed to 1 h of water-avoidance stress (WAS) or sham stress for 1 day (acute) or 7 days (repeated). Visceral sensitivity was assessed either 1 h or 24 h after the final session of WAS and quantified as the number of contractions of the external abdominal oblique, a visceromotor response, in response to colorectal distension at pressures of 0-60 mmHg. Acute stress induced transient visceral hyperalgesia, which was absent 24 h after WAS and independent of GR and MR. Conversely, repeated WAS induced sustained visceral hyperalgesia that was abolished by specifically targeting the amygdala with GR and MR antagonists. These results demonstrate that the amygdala corticosteroid system plays an essential role in mediating the effects of repeated WAS on visceral sensitivity. Furthermore, our findings suggest that amygdaloid GR and MR may be involved in IBS symptomatology.     

The glucocorticoid agonist activities of mifepristone (RU486) and progesterone are dependent on glucocorticoid receptor levels but not on EC50 values

Mifepristone is an antagonist of the glucocorticoid receptor (GR) that also has significant agonist activity in some cell types. We examined the partial agonist activity of mifepristone in COS-7 cells transfected with increasing amounts of a glucocorticoid receptor expression vector pmGR. As pmGR levels increased, the response of the reporter, pMTVCAT to dexamethasone increased, consistent with increasing levels of receptor expression; the response to mifepristone also increased but at a higher rate, resulting in increasing mifepristone agonist and decreasing antagonist activity. In contrast, increasing pMTVCAT levels increased CAT activity induced by both dexamethasone and mifepristone, but did not change the relative agonist activity of mifepristone. We also examined the relationship between agonist activity and receptor level in a series of clones of the E8.2.A3 cell line expressing various levels of GR. Again, the relative agonist activity of mifepristone increased as GR increased. This increase was not due to changes in the dose response curves to these two ligands since their EC50 values were independent of receptor levels. These results indicate that the degree of glucocorticoid agonist activity exhibited by mifepristone is dependent on the concentration of GR in the cell. Similar results were obtained with another partial agonist of the GR, progesterone, whereas the complete antagonist ZK98.299 had no agonist activity under any condition. Taken together, these results suggest that the phenomenon of receptor concentration-dependence is a property of partial GR agonists in general.     

Immunoreactivities and Levels of Mineralocorticoid and Glucocorticoid Receptors in the Hippocampal CA1 Region and Dentate Gyrus of Adult and Aged Dogs

Corticosteroids are important factors in the maintenance of homeostasis in the brain. They are regulated via the interaction with two corticosteroid receptor systems—the mineralocorticoid (MR) and glucocorticoid receptor (GR). In the present study, we observed age-related changes in serum cortisol levels, and immunoreactivities and protein levels of MR and GR in the hippocampal CA1 region and dentate gyrus. The serum cortisol levels were significantly high (about twofold) in the aged group compared to that in the adult group. In the adult dog (2–3 years old), MR and GR immunoreactivity was detected in neurons in the pyramidal layer of the CA1 region, and in the granular and multiform layers of the dentate gyrus. In the aged dog (10–12 years old), MR immunoreactivity in the CA1 region was significantly decreased, especially, in the dentate multiform layer. In contrast, GR immunoreactivity in the aged dog was slightly decreased in the CA1 region and dentate gyrus. In the Western blot analysis, MR protein level in the aged dog was significantly lower compared to that of the adult dog; GR protein level in the aged dog was not significantly decreased. This result indicates that the reduction of MR immunoreactivity and protein level in the hippocampus of the aged dog may be associated with neural dysfunction in the aged hippocampus.     

A Single-Day Treatment with Mifepristone Is Sufficient to Normalize Chronic Glucocorticoid Induced Suppression of Hippocampal Cell Proliferation

Background

Chronic stress or prolonged administration of glucocorticoids suppresses proliferation and/or survival of newborn cells in adult rat dentate gyrus. Earlier we showed that administration of the glucocorticoid receptor antagonist mifepristone during the final 4 days of a 21 days period of corticosterone treatment fully normalized the number of newborn cells. Here we aimed to better understand how mifepristone achieves this effect and questioned whether an even shorter (single day) mifepristone treatment (instead of 4 days) also suffices to normalize neurogenesis.

Methods

We investigated various steps of the neurogenic process, using the immunohistochemical markers BrdU, doublecortin, proliferating cell nuclear antigen as well as glial fibrillary acidic protein, after 17 or 21 days of corticosterone (versus vehicle) treatment.

Results

Corticosterone primarily attenuates the proliferation of cells which subsequently develop into neurons; this is fully reversed by mifepristone. Surprisingly, the corticosteroid effects on neurogenesis can even be fully re-set by a single-day treatment with mifepristone (on day 18), despite the continued corticosterone exposure on subsequent days.

Conclusions

Our results emphasize that studies into the therapeutical efficacy of new antidepressants, especially those targeting HPA-activity or the glucocorticoid receptor, should explore the possibility to reduce treatment duration.     

Stress-induced changes of glucocorticoid receptor in rat liver.

The effect of corticosterone injection and of acute and repeated stress on rat liver cytosol glucocorticoid receptor was studied to ascertain whether corticosterone-induced glucocorticoid receptor (GR) regulation also takes place in intact animals as it does in adrenalectomized ones. Adult male rats were exposed to six different stressors (swimming, 10 mg/kg histamine i.p., 500 mU/kg vasopressin s.c., heat, immobilization and cold) acutely or three times daily for 18 days (repeated stress). Each of the stressors applied acutely provoked a pronounced increase of plasma corticosterone with subsequent induction of hepatic tyrosine aminotransferase activity. Depletion of cytosol receptor was however only noticed after swimming and histamine injection. On the other hand, sustained hypersecretion of corticosterone evoked by repeated stress significantly reduced the number of GR in rat liver cytosol without any change in Kd. It is concluded that in the presence of intact adrenal glands cytosol receptors are more resistant to corticosterone-induced depletion than in their absence. Further, repeated stress causes down-regulation of GR in the liver, most probably by sustained corticosterone secretion, yet the effect of other stress factors cannot be excluded.     

The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test

Pre-clinical and clinical studies have employed treatment with glucocorticoid receptor (GR) antagonists in an attempt to limit the deleterious behavioral and physiological effects of excess glucocorticoids. Here, we examined the effects of GR antagonists on neuroendocrine and behavioral stress responses, using two compounds: mifepristone, a GR antagonist that is also a progesterone receptor antagonist, and CORT 108297, a specific GR antagonist lacking anti-progestin activity. Given its well-documented impact on neuroendocrine and behavioral stress responses, imipramine (tricyclic antidepressant) served as a positive control. Male rats were treated for five days with mifepristone (10 mg/kg), CORT 108297 (30 mg/kg and 60 mg/kg), imipramine (10 mg/kg) or vehicle and exposed to forced swim test (FST) or restraint stress. Relative to vehicle, imipramine potently suppressed adrenocorticotropin hormone (ACTH) responses to FST and restraint exposure. Imipramine also decreased immobility in the FST, consistent with antidepressant actions. Both doses of CORT 108297 potently suppressed peak corticosterone responses to FST and restraint stress. However, only the higher dose of CORT 108297 (60 mg/kg) significantly decreased immobility in the FST. In contrast, mifepristone induced protracted secretion of corticosterone in response to both stressors, and modestly decreased immobility in the FST. Taken together, the data indicate distinct effects of each compound on neuroendocrine stress responses and also highlight dissociation between corticosterone responses and immobility in the FST. Within the context of the present study, our data suggest that CORT 108297 may be an attractive alternative for mitigating neuroendocrine and behavioral states associated with excess glucocorticoid secretion.     

Stress facilitates body weight gain in genetically predisposed rats on medium-fat diet

To assess the interaction between stress and energy homeostasis, we immobilized male Sprague-Dawley rats prone to diet-induced obesity (DIO) or diet resistance (DR) once for 20 min and then fed them either low-fat (4.5%) chow or a medium-fat (31%), high-energy (HE) diet for 9 days. Stressed, chow-fed DIO rats gained less, while stressed DIO rats on HE diet gained more body weight and had higher feed efficiency and plasma leptin levels than unstressed controls. Neither stress nor diet affected DR body weight gain. While stress-induced plasma corticosterone levels did not differ between phenotypes, DIO rats were initially more active in an open field and had higher hippocampal dentate gyrus and CA1 glucocorticoid receptor (GR) mRNA than DR rats, regardless of prior stress or diet. HE diet intake was associated with raised dentate gyrus and CA1 GR and amygdalar central nucleus (CeA) corticotropin-releasing hormone (CRH) mRNA expression, while stress was associated with reduced hypothalamic dorsomedial nucleus Ob-R mRNA and CeA CRH specifically in DIO rats fed HE diet. Thus a single stress triggers a complex interaction among weight gain phenotype, diet, and stress responsivity, which determines the body weight and adiposity of a given individual.     

Hippocampal glucocorticoid receptor expression in the tree shrew: Regulation by psychosocial conflict

Summary 1. This study was conducted to determine whether chronic psychosocial conflict alters the expression of glucocorticoid receptor (GR) mRNA in the hippocampus of male tree shrews (Tupaia belangeri).2. To generate probes for thein situ hybridization, the tree shrew GR gene was partly cloned. There was a 90% homology between the deduced amino acid sequence of the cloned tree shrew GR and that of the corresponding human GR sequence.³⁵S-Labeled riboprobes which had been transcribed from the tree shrew GR clone hybridized to pyramidal neurons in all subregions of the tree shrew hippocampal formation and to granule neurons in the dentate gyrus.3. Afterin situ hybridization, the expression of GR mRNA was semiquantitatively determined by counting silver grains over single neurons of the hippocampal formation of psychosocially stressed tree shrews and control animals. After 12 days of social conflict, the number of silver grains in the CA1 and CA3 pyramidal neurons of stressed animals was significantly lower than in controls. No statistically significant differences in mRNA expression were observed in the pyramidal neurons of the subiculum and in the granule neurons of the dentate gyrus.4. The present results suggest that psychosocial stress leads to a site-specific down-regulation of hippocampal GR via modification of mRNA expression.     

Epigenetic mechanisms in the dentate gyrus act as a molecular switch in hippocampus-associated memory formation

We make strong memories of significant events in our lives which may serve to increase our resilience and adaptation capacity to deal with future challenges. It is well established that the neurotransmitter glutamate and the ERK MAPK intracellular signaling pathway play a principal role in memory formation. In addition, stress-associated hormones like glucocorticoids released during such events are known to strengthen formation of memories. But, how do these hormones work? Do they interact with the ERK MAPK pathway or otherwise? What are the more distal, epigenomic effects? We discovered in rats and mice that confrontation with a psychological challenge (e.g. forced swimming, Morris water maze) would lead, through NMDA-ERK signaling, to MSK1 and Elk-1 activation in dentate gyrus neurons (a part of the hippocampus involved in encoding of memories) resulting in histone H3 S10-phosphorylation and K14-acetylation, H4 hyper-acetylation, gene induction and formation of memories of the event. Moreover, glucocorticoid hormones via the glucocorticoid receptor (GR) greatly facilitated the epigenomic mechanisms and cognitive performance. Therefore, we propose that formation of enduring memories of significant events requires an interaction of GRs with the NMDA/ERK/MSK1/Elk-1 signaling pathways to allow an optimal epigenomic activation pattern in dentate gyrus neurons to accommodate their altered neurophysiological function.     

The Early Stage Formation of PI3K-AMPAR GluR2 Subunit Complex Facilitates the Long Term Neuroprotection Induced by Propofol Post-Conditioning in Rats

Previously, we have shown that the phosphoinositide-3-kinase (PI3K) mediated acute (24 h) post-conditioning neuroprotection induced by propofol. We also found that propofol post-conditioning produced long term neuroprotection and inhibited the internalization of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR2 subunit up to 28 days post middle cerebral artery occlusion (MCAO). However, the relationship between PI3K with AMPA receptor GluR2 subunit trafficking in propofol post-conditioning has never been explored. Here we showed that propofol post-conditioning promoted the binding of PI3K to the C-terminal of AMPA receptor GluR2 subunit and formed a complex within 1 day after transient MCAO. Interestingly, the enhanced activity of PI3K was observed in the hippocampus of post-conditioning rats at day 1 post ischemia, whereas the decrease of AMPA receptor GluR2 subunit internalization was found up to 28 days in the same group. Administration of PI3K selective antagonist wortmannin inhibited the improvement of spatial learning memory and the increase of neurogenesis in the dentate gyrus up to 28 days post ischemia. It also reversed the inhibition of AMPA receptor GluR2 internalization induced by propofol post-conditioning. Together, our data indicated the critical role of PI3K in regulating the long term neuroprotection induced by propofol post-conditioning. Moreover, this role was established by first day activation of PI3K and formation of PI3K-AMPA receptor GluR2 complex, thus stabilized the structure of postsnaptic AMPA receptor and inhibited the internalization of GluR2 subunit during the early stage of propofol post-conditioning.     

Localization and regulation of glucocorticoid and mineralocorticoid receptor messenger RNAs in the hippocampal formation of the rat

Messenger RNAs coding for glucocorticoid (GR) and mineralocorticoid (MR) receptor proteins were localized to discrete subfields of the hippocampal formation by in situ hybridization histochemistry, using cRNA probes of approximately equivalent specific activity. Both GR and MR mRNAs were present in all subfields examined; GR mRNA was of greatest abundance in CA1, while MR mRNA was most densely labeled in CA3. In all subfields examined, MR mRNA was considerably more abundant than GR mRNA. Removal of circulating glucocorticoids by adrenalectomy precipitated an up-regulation of GR mRNA in subfields CA1-2 and the dentate gyrus, which was reversed by dexamethasone replacement. High doses of dexamethasone significantly down-regulated GR mRNA in CA3. In contrast, adrenalectomy produced significant up-regulation of MR mRNA only in subfield CA1-2. The data indicate that steroid receptor mRNAs are differentially distributed in hippocampus, and that sensitivity to steroids occurs within defined structural domains of the hippocampal formation.