Characterization of the vulnerability to repeated stress in Fischer 344 rats: possible involvement of microRNA-mediated down-regulation of the glucocorticoid receptor.

In the present study, we established and characterized an animal model of vulnerability to repeated stress. We found that control Sprague-Dawley (SD) rats showed a gradual decrease in the HPA axis response following 14 days of repeated restraint stress, whereas Fischer 344 (F344) rats did not show such HPA axis habituation. Similar habituation was observed in the expression of c-fos mRNA, corticotropin-releasing hormone hnRNA, and phospho-CREB and phospho-ERK proteins in the hypothalamic paraventricular nucleus (PVN) of SD rats, but not in the F344 rats. In addition, repeatedly restrained F344 rats exhibited decreased cell proliferation in the dentate gyrus of the hippocampus and increased anxiety-related behaviours, while repeatedly restrained SD rats exhibited a selective enhancement of hippocampal cell proliferation in the ventral area. Moreover, we found a lower expression of glucocorticoid receptor (GR) protein, but not mRNA, in the PVN of F344 rats compared to SD rats. We also identified that microRNA (miR)-18a inhibited translation of GR mRNA in cultured neuronal cells and that increased expression of miR-18a in the PVN was observed in F344 rats compared with SD rats. These strain differences in GR protein levels were not found in the hippocampus and prefrontal cortex, and the expression of miR-18a was much lower in these brain regions than in the PVN. Our results suggest that F344 rats could be a useful animal model for studying vulnerability to repeated stress, and that miR-18a-mediated down-regulation of GR translation may be an important factor to be considered in susceptibility to stress-related disorders.

Reduced expression of glyoxalase-1 mRNA in mood disorder patients.

Glyoxalase-1 (Glo1) is an antioxidant enzyme which detoxifies alpha-ketoaldehydes to prevent the accumulation of pro-oxidant compounds, such as methylglyoxal, in all cell types. Glo1 has been suggested to be involved in anxiety disorders, autism, and Alzheimer's disease. Mood disorders have a high rate of comorbidity with anxiety disorders although, to date, little is known of the involvement of Glo1 in the pathophysiology of these conditions. In the present study, we examined the expression levels of Glo1 mRNA in peripheral white blood cells of mood disorder patients to understand the role of Glo1 in mood disorders. Quantitative real-time polymerase chain reaction experiments revealed that reduced expression of Glo1 mRNA was observed in major depressive and bipolar disorder patients in a current depressive state, as compared with healthy control subjects. In contrast, the expression of Glo1 mRNA in major depressive and bipolar patients, in a remissive state, showed no significant alteration when compared with healthy control subjects. These results suggest that the aberrant expression of Glo1 might be involved in the pathophysiology of mood disorders.

The effect of placebo administration on the first-night effect in healthy young volunteers.

The first-night effect is a well-known phenomenon that is considered to result from a subject's lack of adaptation to the unfamiliar environment of a sleep laboratory and to the technical equipment used for polysomnography. The effect has been explored as a laboratory model for transient insomnia. The main characteristics of this effect are short total sleep time (TST) and rapid eye movement (REM) sleep, a lower sleep efficiency index, and longer REM sleep latency. Previous studies have reported that personality traits (such as trait anxiety) are a potential cause of the first-night effect and that the placebo effect is closely related to the anxiety levels of the subjects. To the best of our knowledge, there are no reports regarding the effects of a placebo on first-night sleep. This omission can be explained by the fact that the polysomnographic recordings obtained during the first night of a study are generally excluded from the analysis in order to avoid the inclusion of the first-night effect. In the present study, 8 male university students were subjected to polysomnographic examinations during drug-free, placebo-administration, and benzodiazepine-administration conditions in order to clarify the placebo effect on sleep during consecutive nights, particularly on the first night. The recordings for each condition were conducted for 4 consecutive nights. A placebo or 5 mg nitrazepam was administered at 2230 h using a double-blind crossover design, while no drug was administered during the drug-free condition. There was a 10-day interval between the examination of each condition. Polysomnographic recording was started at 2300 h and continued until the natural awakening of the subjects on the next morning. Subsequently, the subjects were requested to fill in a rating scale that is used to evaluate the subjective perception of sleep. An increase in stage-2 sleep associated with the first-night effect was observed on the first night during the drug-free and placebo-administration conditions. However, REM sleep reduction associated with the first-night effect was detected on the first night during the drug-free condition; this decrease in REM sleep was counteracted by the placebo during the placebo-administration condition. The nitrazepam, but not the placebo, decreased both slow-wave sleep (SWS) and REM sleep. The values for the tendency to fall asleep, feeling refreshed upon awakening in the morning, and the tension upon awakening in the morning were improved to a greater extent by the placebo and nitrazepam administrations than when no drug was administered. These results demonstrate the possibility that placebo administration may have a hypnotic/anxiolytic effect and may improve transient insomnia without causing SWS and REM sleep reductions.

Expression and functional study of estrogen receptor-related receptors in human prostatic cells and tissues.

Estrogen receptor-related receptors (ERRs; alpha, beta, gamma) are orphan nuclear receptors and constitutively active without binding to estrogen. Like estrogen receptors (ERs), ERRs bind to estrogen receptor elements and estrogen receptor element-related repeats. Growing evidence suggests that ERRs can cross-talk with ERs in different cell types via competition for DNA sites and coactivators. We hypothesize that ERRs might play regulatory roles in normal and neoplastic prostatic cells by sharing similar ER-mediated pathways or acting independently. In this study, we investigated mRNA and protein expression patterns of three ERR members in normal human prostate epithelial cells, established cell lines, cancer xenografts, and prostatic tissues. Additionally, effects of transient transfection of ERRs on prostatic cell proliferation and ER expression were also examined. RT-PCR showed that ERRalpha and ERRgamma transcripts were detected in most cell lines and xenografts, whereas ERRbeta was detected in normal epithelial cells and few immortalized cell lines but not in most cancer lines. Similar results were demonstrated in clinical prostatic specimens. Western blottings and immunohistochemistry confirmed similar expression patterns that ERR proteins were detected as nuclear proteins in epithelial cells, whereas their expressions became reduced or undetected in neoplastic prostatic cells. Transient transfection confirmed that ERRs were expressed in prostatic cells as nuclear proteins and transcriptionally active in the absence of estradiol. Transfection results showed that overexpression of ERRs inhibited cell proliferation and repressed ERalpha transcription in PC-3 cells. Our study shows that ERRs, which are coexpressed with ERs in prostatic cells, could regulate cell growth and modulate ER-mediated pathways via interference on ERalpha transcription in prostatic cells.

Flavone and isoflavone phytoestrogens are agonists of estrogen-related receptors.

While estrogen-related receptors (ERRalpha, ERRbeta, and ERRgamma) share a high amino acid sequence homology with estrogen receptors (ERs), estrogens are not ligands of ERRs. Structure-function studies from this and other laboratories have revealed that ERRs have small ligand-binding pockets and have provided evidence to show that these receptors can activate gene transcription in a constitutive manner. To address the question as to whether there is any agonist for ERRs, our laboratory recently performed virtual ligand screening on ERRalpha that predicted flavone and isoflavone phytoestrogens to be ligands of this receptor. Our mammalian cell transfection and mammalian two-hybrid experiments revealed that three isoflavones (genistein, daidzein, and biochanin A) and one flavone (6,3',4'-trihydroxyflavone) behaved as agonists of ERRs. These phytoestrogens induced the activity of ERRalpha at concentrations that are comparable to those for the activation of ERalpha and ERbeta. In this study, we also used the results of ERRalpha ligand-binding site mutant, F232A, to verify our ERRalpha hypothetical computer model. Our recent ERR research has determined for the first time that flavone and isoflavone phytoestrogens are agonists of ERRs. In addition, our studies have demonstrated that an approach that combines structure-based virtual screening and receptor functional assays can identify novel ligands of orphan nuclear receptors.

A qualitative and quantitative analysis of rhythmic activities during a mental task and sleep spindles.

The frequency, configuration, and distribution of sleep spindles are similar to some of the rhythmic activities seen during task performance. In the present study, the relationship between rhythmic activities during sleep and arithmetic addition was investigated in male university students with (n = 10) and without (n = 10) frontal midline theta activity (Fmtheta). Electroencephalograms (EEGs) during addition in both groups were compared at frontal and central areas on three consecutive days. Polysomnograms were recorded at the same regions on four consecutive nights for each group. The amount of theta rhythm during a mental task (Fmtheta) and in nocturnal sleep at Fz and Cz electrodes was greater for the Fmtheta group than for the non-Fmtheta group, while the amount of beta rhythm at both sites was smaller in the Fmtheta group than in the non-Fmtheta group. There were no differences between the groups in the amount of alpha rhythm at either site. The frequency of alpha rhythm at Fz and Cz in both situations was slower for the Fmtheta group than for the non-Fmtheta group, but there were no differences in the frequency of theta and beta rhythms between the groups at either site. These results suggest that rhythmic activities during a mental task and in sleep may correlate with each other.

The relationship between rhythmic activities during a mental task and sleep spindles: a correlative analysis.

In a previous study, we suggested that the characteristics of theta, alpha, and beta rhythms during a mental task were similar to those during sleep. Building upon the previous data, correlations between rhythmic activities during a mental task and during sleep were investigated in the present study. Patterns of correlation and no correlation between rhythmic activities during the mental task were similar to those during sleep for subjects with and without frontal midline theta (Fmtheta) activity. In the Fmtheta group, there were no correlations between rhythmic activities in the two situations, while in the non-Fmtheta subjects, theta and alpha rhythms showed a positive correlation with one another, and theta and beta rhythms correlated negatively during sleep. In both groups, there were many correlations between rhythmic activities during the mental task and those in Sleep Stage 2, while there were few correlations between rhythmic activities during the mental task and those in other sleep stages. These results suggest that the mechanism generating rhythmic activities during the appearance of rhythmic activities induced by a mental task may be closely related to those of rhythmic activities during sleep, and that the membrane potentials in reticular thalamic (RE) neurons during the appearance of rhythmic activities induced by a mental task may be nearly equivalent to that in Sleep Stage 2, and that the correlation pattern between the rhythmic activities in each group may be well explained by the appearance pattern of each rhythm in the previous report.

Altered gene expression of histone deacetylases in mood disorder patients.

Chromatin remodeling such as changes in histone acetylation has been suggested to play an important role in the pathophysiology and treatment of mood disorders. In the present study, we investigated whether the expression of histone deacetylase (HDAC) genes are altered in mood disorder patients. We used quantitative real-time PCR to measure the mRNA levels of 11 HDACs (HDAC1-11) in peripheral white blood cells of major depressive disorder (MDD) and bipolar disorder (BPD) patients during depressive and remissive episodes and in the first-degree relatives of BPD patients. In addition, we investigated the effect of antidepressants and mood stabilizers on the mRNA levels of HDACs using mice. In MDD, the expression of HDAC2 and -5 mRNA was increased in a depressive state, but not in a remissive state, compared to controls. In BPD, the expression of HDAC4 mRNA was increased only in a depressive state, and the expression of HDAC6 and -8 was decreased in both depressive and remissive states compared to controls, whereas the first-degree relatives did not show any significant alteration in expression levels. Animal study showed that the expression of HDAC2 and -5 or HDAC4, -6 and -8 mRNAs in the mouse leukocytes were not affected by chronic treatment with antidepressants or mood stabilizers. Our data suggest that aberrant transcriptional regulation caused by the altered expression of HDACs is associated with the pathophysiology of mood disorders.