Differential Regulation of Hippocampal Glucocorticoid Receptors mRNA and Fast Feedback: Relevance to Post-Traumatic Stress Disorder

Hippocampal glucocorticoid receptors (GR and MR) play an important role in glucocorticoid negative feedback. Abnormalities in negative feedback are found in depression and in post-traumatic stress disorder (PTSD), suggesting that GR and MR might be involved in the pathophysiology of these disorders. Enhanced negative feedback, the PTSD-specific neuroendocrine abnormality, can be induced in animals using a single prolonged stress (SPS) paradigm (a number of different stressors in one prolonged session, ‘no stress’ interval and a testing session one week later). In the current study, we examined hippocampal GR and MR mRNA distribution in the same animals that exhibited altered negative feedback following the SPS. Seven groups of adult Sprague-Dawley male rats (seven animals each) were used in two studies, comparing unstressed controls to acutely stressed animals (SPS: 24 h group), SPS animals (seven and 14 days), and SPS+chronic stress animals. GR and MR mRNA distribution across hippocampal subfields was studied using in-situ hybridization with ³⁵S-labelled cRNA probes. Acute stress produced down-regulation of GR and MR mRNA across all hippocampal subfields. Seven days later (SPS-7 group), there was a differential recovery, with GR mRNA reaching higher than the prestress levels, and MR mRNA remaining down-regulated. The same differential regulation was present in the 14-day group. Chronically stressed animals that exhibited normal fast feedback also had normalization in their GR and MR mRNA levels. The MR/GR ratio was decreased only in animals that had enhanced fast feedback. These findings suggest that the increase in GR, in hippocampus is involved in the fast feedback hypersensitivity observed in the SPS animals, and might also underlie enhanced dexamethasone sensitivity found in PTSD. Since differential activation of GR and MR can modulate memory, behavioural responsivity, anxiety and fear, change in MR/GR ratio might also explain other PTSD-related phenomena.     

Synthesis of isotopically labelled versions of adenosine agonist GR79236

Versions of adenosine receptor agonist GR79236 (1 ), labelled either with carbon-14 (9 ) at C-8 of the purine ring or with tritium (15 ) in the cyclopentyl ring, were prepared in overall yields of 64% and 25% respectively. A mass labelled [M+4] version (24 ) containing carbon-13, nitrogen-15, and deuterium was also prepared in 3% yield. Copyright © 2000 John Wiley & Sons, Ltd.     

滋阴清热方对MRL／1pr狼疮鼠皮质醇及糖皮质激素受体mRNA表达的影响

目的 观察MRL／1pr狼疮鼠皮质醇及糖皮质激素爱体（GR）mRNA表达的变化及滋阴清热方对其水平的影响,探讨中药减轻激素不良反应的机理。方法40只MRL／1pr狼疮模型鼠随机分为4组,即模型组、中药组、西药组、中西药组。分别给予生理盐水、滋阴清热方、强的松悬液、滋阴清热复方＋强的松悬液灌胃,均每日1次,每次0．5mL,连续用药10周。另选用昆明小鼠10只作为正常对照组。应用放射免疫法测定MRL／1pr狼疮鼠糖皮质醇水平,反转录酶一聚合酶链反应法（RT—PCR）检测GRmRNA,观察各组皮质醇、GRmRNA表达的变化。结果模型组血清皮质醇水平与正常组比较,P〉0．05,而GRocmRNA表达明显低于正常组（P〈0．05）。西药组GRαmRNA、GRBmRNA下调水平高于中药组和中西药组（P〈0．01或P〈0．05）。结论滋阴清热方可上调MRL／1pr狼疮鼠GRmRNA表达水平,从而增强糖皮质激素的临床疗效,减少激素不良反应。     

急性缺氧后早期豚鼠耳蜗糖皮质激素受体表达的变化

目的观察缺氧后早期豚鼠耳蜗糖皮质激素受体（glucocorticoid receptor,GR）的变化特点。方法利用低氧舱使动物缺氧,应用免疫组化SP法结合图像分析技术检测缺氧后豚鼠耳蜗GR的表达。结果缺氧后GR在豚鼠耳蜗各回均有不同程度的染色,主要阳性部位是螺旋韧带、血管纹、螺旋器和螺旋神经节。与正常组相比缺氧后早期GR在耳蜗各阳性部位的表达均明显减弱,其中在螺旋韧带和血管纹的表达减弱最明显,有统计学意义（P〈0．01）。结论缺氧可使GR在豚鼠耳蜗中的表达减弱。缺氧对耳蜗的损伤可能和GR表达的减弱有关。     

Identification of an amino acid residue important for binding of methiothepin and sumatriptan to the human 5-HT(1B) receptor.

Site-directed mutagenesis of the human 5-HT_1B receptor was performed to investigate the role of the amino acid residues cysteine 326 and tryptophan 327 in transmembrane region VI and aspartic acid 352 in transmembrane region VII in ligand binding. Binding studies were performed with the antagonist radioligand [³H]GR125743 on mutant and wild-type receptors stably expressed in Chinese hamster ovary cells (CHO)-K1 cells. Substitution of tryptophan 327 by alanine resulted in decreased affinities of all ligands tested. The most prominent changes in affinity were observed for the antagonist methiothepin and the antimigraine drug sumatriptan, which were reduced approximately 300- and 60-fold, respectively. Nevertheless, the affinity of 5-HT remained the same. Replacement of the aspartic acid 352 by alanine reduced high-affinity binding of 5-HT. Substitution of cysteine 326 by alanine had minor effects on ligand binding. Some of these results agree with the results from mutagenesis studies of the corresponding amino acids in other receptors. However, some notable differences also emerge showing that functional roles of individual amino acid residues must be tested experimentally in each receptor subtype.     

Combined treatment with exogenous estradiol and progesterone increases the incidence of breast cancer in TA2 mice without ovaries

TA2 mice have a high incidence of spontaneous breast cancer without chemical stimulus. There are two proposed explanations for this phenomenon: one is gravidity and the frequency of pregnancies, and the other is related to the presence of the mouse mammary tumor virus (MMTV). MMTV is hormonally regulated and indirectly promotes tumor formation by leading to the activation of Wnt oncogenes through insertional mutagenesis. In order to clarify the relationship between estrogen, progesterone, MMTV, Wnt oncogenes and breast cancer, ovaries from virgin female TA2 mice were removed and the mice were treated with exogenous estradiol and progesterone in different patterns. This study found that the combination of exogenous estradiol and progesterone induced breast cancer formation in TA2 mice without ovaries. MMTV-LTR mRNA exhibited the highest expression in tumor tissue from the combination treatment group (CT). Mammary tissue from mice in the CT group had the highest Wnt1, Wnt5a, Wnt5b and Wnt10b mRNA expression levels. These results indicate that estradiol and progesterone act in a synergistic manner to upregulate MMTV, which subsequently induces breast cancer in TA2 mice. Various members of the Wnt gene family may play specific roles in different stages of carcinogenesis in TA2 mice.     

Chronic blockade of glucocorticoid receptors by RU486 enhances lipopolysaccharide-induced depressive-like behaviour and cytokine production in rats

Although accumulating evidence supports a role for cytokines in the pathophysiology of depression, the cytokine hypothesis of depression is debatable. It has been suggested that neuroendocrine and immune systems acting in concert may have roles in the development and the maintenance of the disease. Glucocorticoid receptor (GR) is the key element which exerts both anti-inflammatory and cytokine-inhibiting effects. Whether functional changes of GR are involved in the pathophysiology of cytokine-induced depression remains elusive. In the present study, the effects of both acute and chronic GR blockade on depressive-like behaviour and cytokine production induced by lipopolysaccharides (LPS), cytokine inducer, were investigated in rats. Acute or chronic blockade of GR was achieved by a single administration or repeated administrations, respectively, of the GR antagonist RU486 (RU). Behavioural measurements, including saccharin preference, locomotor activity, and immobility time, were assessed. The serum levels of proinflammatory cytokines (TNFα, IL-1β, and IFNγ) were determined by ELISA. The results showed that LPS induced significant but transient depressive-like behaviour. Repeated, but not single, administration of RU significantly enhanced and prolonged LPS-induced depressive-like behaviour and an increase in the serum production of TNFα and IFNγ. These results indicate that the effective blockade of GR enhanced the depressive-like behaviour induced by cytokines. Findings from this study suggest that GR dysfunction may be an important contributing factor to the development of cytokine-related depression. These findings add to the growing evidence of mechanisms by which cytokines influence depression.     

Unpredictable chronic stress model in zebrafish (Danio rerio): behavioral and physiological responses

Zebrafish (Danio rerio) have emerged as a promising model organism to study development, toxicology, pharmacology, and neuroscience, among other areas. Despite the increasing number of studies using zebrafish, behavioral studies with this species are still elementary when compared to rodents. The aim of this study was to develop a model of unpredictable chronic stress (UCS) in zebrafish. We evaluated the effects of UCS protocol during 7 or 14 days on behavioral and physiological parameters. The effects of stress were evaluated in relation to anxiety and exploratory behavior, memory, expression of corticotrophin-releasing factor (CRF) and glucocorticoid receptor (GR), and cortisol levels. As expected, UCS protocol increased the anxiety levels, impaired cognitive function, and increased CRF while decreased GR expression. Moreover, zebrafish submitted to 7 or 14 days of UCS protocol presented increased cortisol levels. The protocol developed here is a complementary model for studying the neurobiology and the effects of chronic stress in behavioral and physiological parameters. In addition, this protocol is less time consuming than standard rodent models commonly used to study chronic stress. These results confirm UCS in zebrafish as an adequate model to preclinical studies of stress, although further studies are warranted to determine its predictive validity.