Identification of synaptic activity-dependent genes by exposure of cultured cortical neurons to tetrodotoxin followed by its withdrawal

Activity-dependent gene expression is one of the key mechanisms of synaptic plasticity that form the basis of higher order functions such as learning and memory. In the present study, we surveyed for activity-dependent genes by analyzing gene expression changes accompanying reversible inhibition of synaptic activity by tetrodotoxin (TTX) using two types of DNA microarrays; our focused oligo DNA microarray "Synaptoarray" and the commercially available high-density array. Cerebral cortical cells from E18 rat embryos were cultured for 14 days to ensure synaptogenesis, then treated with 1 muM TTX for 48 hr without detectable effect on cell viability. Synaptic density estimated by the amount of Synapsin I and Synaptotagmin I was decreased 21-24% by TTX treatment, but recovered to the control level 48 hr after TTX withdrawal. Comparison of gene expression profiles by competitive hybridization of fluorescently labeled cRNA from TTX-treated and control cells showed an overall downregulation of the genes on the Synaptoarray by TTX-treatment with different recovery rates after TTX withdrawal. With 16 representative genes, microarray data were validated by real-time PCR analysis. Genes most severely downregulated by TTX and upregulated above the control level at 5 hr after TTX withdrawal were munc13-1 (involved in docking and priming of synaptic vesicles) and Shank2 (involved in the postsynaptic scaffold). In addition, comprehensive screening at 5 hr after TTX withdrawal using high density arrays resulted in additional identification of Rgs2, a regulator of trimeric G-protein signaling, as an activity-dependent gene. These three genes are thus likely to be key factors in the regulation of synaptic plasticity. (c) 2007 Wiley-Liss, Inc.     

Review of mental‐health‐related stigma in Japan

The aim of this study is to understand the nature and characteristics of mental‐health‐related stigma among Japanese people. We searched relevant studies in English or Japanese published since 2001 using MEDLINE and PsycINFO, and found 19 studies that examined mental‐health‐related stigma in Japan. Regarding knowledge about mental illness, reviewed studies showed that in the Japanese general population, few people think that people can recover from mental disorders. Psychosocial factors, including weakness of personality, are often considered the cause of mental illness, rather than biological factors. In addition, the majority of the general public in Japan keep a greater social distance from individuals with mental illness, especially in close personal relationships. Schizophrenia is more stigmatized than depression, and its severity increases the stigmatizing attitude toward mental illness. The literature also showed an association between more direct social contact between health professionals and individuals with mental illness and less stigmatization by these professionals. Less stigmatization by mental health professionals may be associated with accumulation of clinical experience and daily contact with people who have mental illness. Stigmatizing attitudes in Japan are stronger than in Taiwan or Australia, possibly due to institutionalism, lack of national campaigns to tackle stigma, and/or society's valuing of conformity in Japan. Although educational programs appear to be effective in reducing mental‐health‐related stigma, future programs in Japan need to address problems regarding institutionalism and offer direct social contact with people with mental illness.     

Epidemiological studies on patients with a persistent vegetative state

Persistent vegetative state after severe brain damage was studied epidemiologically in 110 cases from 1973 to 1976. The causes of brain damage were varied. More than one-third of the cases were due to trauma, and about one-fifth were the result of vascular accidents. Three year observation revealed that 65% of the patients died during this period. Mean survival time for dead patients was 38 months. Reactivity, clinical signs, EEG findings, methods of management, and results of various trials of treatment were investigated in connection with the patient's prognosis.     

Protective effects of idebenone and α-tocopherol on β-amyloid-(1–42)-induced learning and memory deficits in rats: implication of oxidative stress in β-amyloid-induced neurotoxicity in vivo

Amyloid β-peptide (Aβ), the major constituent of the senile plaques in the brains of patients with Alzheimer's disease, is cytotoxic to neurons and has a central role in the pathogenesis of the disease. Previous studies have suggested that oxidative stress is involved in the mechanisms of Aβ-induced neurotoxicity in vitro. In the present study, we examined whether oxidative stress contributes to learning and memory deficits caused by continuous intracerebroventricular infusion of Aβ-(1–42). In the Aβ-(1–42)-infused rats, spontaneous alternation behaviour in a Y-maze and spatial memory in a water maze task were significantly impaired, as compared with Aβ-(40–1)-infused control rats. The retention of passive avoidance learning was also significantly impaired by treatment with Aβ-(1–42). Potent antioxidants idebenone and α-tocopherol prevented the behavioural deficits in Y-maze and water maze, but not passive avoidance, tasks in Aβ-(1–42)-infused rats when they were repeatedly administered by mouth once a day from 3 days before the start of Aβ infusion to the end of behavioural experiments. Lipid peroxide levels in the hippocampus and cerebral cortex of Aβ-(1–42)-infused rats did not differ from those in control animals, and neither idebenone nor α-tocopherol affected the lipid peroxide levels. These results suggest that treatment with antioxidants such as idebenone and α-tocopherol prevents learning and memory deficits caused by Aβ.     

Heterogeneity and potentiation of AMPA type of glutamate receptors in rat cultured microglia

alpha-amino-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptor in rat cultured microglia were analyzed precisely using flop- and flip-preferring allosteric modulators of AMPA receptors, 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro-phenoxyacetamide (PEPA) and cyclothiazide (CTZ), respectively. Glutamate (Glu)- or kainite (KA)-induced currents were completely inhibited by a specific blocker of AMPA receptor, LY300164, indicating that functional Glu-receptors in cultured microglia are mostly AMPA receptor but not KA receptor in many cells. Glu- and KA-induced currents were potentiated by PEPA and CTZ in a concentration-dependent manner. The ratio of the potentiation by PEPA to the potentiation by cyclothiazide varied with cells between 0.1 and 0.9, suggesting cell-to-cell heterogeneity of AMPA receptor subunits expressed in microglia. Quantitative RT-PCR revealed that GluR1-3 mainly occurred in the flip forms, which agreed with the stronger potentiation of receptor currents by CTZ vs. PEPA. Finally, the potentiation of microglial AMPA receptors by PEPA and CTZ inhibited the Glu-induced release of tumor necrosis factor-alpha (TNF-alpha) unpredictably. The increase in TNF-alpha release by Glu or KA required extracellular Na+ and Ca2+ ions but not mitogen-activated protein kinase (MAPK), suggesting the effects of PEPA and CTZ were not due to the inhibition of MAPK. These results suggest that potentiation of microglial AMPA receptors serves as a negative feedback mechanism for the regulation of TNF-alpha release and may contribute to the ameliorating effects of allosteric modulators of AMPA receptors.     

Atrazine-induced aromatase expression is SF-1 dependent: implications for endocrine disruption in wildlife and reproductive cancers in humans

BACKGROUND: Atrazine is a potent endocrine disruptor that increases aromatase expression in some human cancer cell lines. The mechanism involves the inhibition of phosphodiesterase and subsequent elevation of cAMP. METHODS: We compared steroidogenic factor 1 (SF-1) expression in atrazine responsive and non-responsive cell lines and transfected SF-1 into nonresponsive cell lines to assess SF-1's role in atrazine-induced aromatase. We used a luciferase reporter driven by the SF-1-dependent aromatase promoter (ArPII) to examine activation of this promoter by atrazine and the related simazine. We mutated the SF-1 binding site to confirm the role of SF-1. We also examined effects of 55 other chemicals. Finally, we examined the ability of atrazine and simazine to bind to SF-1 and enhance SF-1 binding to ArPII. RESULTS: Atrazine-responsive adrenal carcinoma cells (H295R) expressed 54 times more SF-1 than nonresponsive ovarian granulosa KGN cells. Exogenous SF-1 conveyed atrazine-responsiveness to otherwise nonresponsive KGN and NIH/3T3 cells. Atrazine induced binding of SF-1 to chromatin and mutation of the SF-1 binding site in ArPII eliminated SF-1 binding and atrazine-responsiveness in H295R cells. Out of 55 chemicals examined, only atrazine, simazine, and benzopyrene induced luciferase via ArPII. Atrazine bound directly to SF-1, showing that atrazine is a ligand for this "orphan" receptor. CONCLUSION: The current findings are consistent with atrazine's endocrine-disrupting effects in fish, amphibians, and reptiles; the induction of mammary and prostate cancer in laboratory rodents; and correlations between atrazine and similar reproductive cancers in humans. This study highlights the importance of atrazine as a risk factor in endocrine disruption in wildlife and reproductive cancers in laboratory rodents and humans.     

Model for paraaortic lymph node metastasis produced by orthotopic implantation of ovarian carcinoma cells in athymic nude mice

Lymph node metastasis through the lymphatic vessels is a critical step in determining the outcome of ovarian cancer patients, and prognosis should be improved by preventing lymph node metastasis. However, experimental models for lymph node metastasis of ovarian carcinoma are not available. We developed an orthotopic transplantation model to study this process in nude mice using the human ovarian carcinoma cell lines, KF and MH. Highly metastatic sublines (KF-LN3 and MH-LN3) were selected in vivo in nude mice by repeated orthotopic transplantation, lymph node metastasis formation and culturing the tumour cells in vitro. Because this model seems to correspond to the advanced clinical stage of ovarian carcinomas, it should be useful in understanding the molecular biology of ovarian carcinomas and in the development of therapeutic modalities against lymph node metastasis.     

A case of advanced gastric cancer with simultaneous multiple bone metastases and double occurrence of disseminated intravascular coagulation successfully controlled with combined chemotherapy

A 41-year-old man was found to have advanced gastric cancer with simultaneous multiple bone metastases when pyloric stenosis was being diagnosed in our hospital. We performed gastrojejunostomy from the lower third of the stomach to the upper third of the duodenum to relieve the obstruction. However, at 8 days after surgery, disseminated intra-vascular coagulation (DIC) occurred. Therefore, the patient was administered combined chemotherapy with TS-1 plus low-dose cisplatin in addition to anti-DIC therapy. TS-1 (150 mg/day) and cisplatin (10 mg/body intravenously over the course of 30 minutes) were administered on days 1 to 5, 8 to 12, and 15 to 19 (weekday-on/weekend-off schedule). There was remarkable response to this chemotherapy, and the patient was shifted from inpatient to outpatient treatment. The treatment course was repeated for 4 cycles until remission was observed. Because of hematologic relapse due to DIC at 6 months after the first treatment, he was readmitted for administration of combined chemotherapy. Fortunately, DIC once again responded to the same chemotherapy regimen. In this pathologic condition, combined chemotherapy is unavoidable when DIC occurs with cancer. Accordingly, it is necessary that an effective combined chemotherapy with mild bone marrow suppression be chosen. A companion drug should be chosen in consideration of delayed homo-toxicity and of the possibility of relapse into DIC in the drug withdrawal period. In addition, it is indispensable that careful consideration be given to the most favorable dose and regimen.