Molecular mechanism of long-term effects of neurotransmitters

作为研究脑内生物活性物质如何通过即刻早期基因引起长期效应的研究的一部分，研究了类固醇激素对培养的大鼠边缘神经元存活率的影响。先前证实，在培养的边缘神经元中，糖皮质激素以剂量依赖的方式增加红藻氨酸诱导的细胞毒性，而雌激素在低于10℃时可提高培养神经元的存活率。为了阐明雌激素的这种作用，我们用c-fos原位杂交技术对大鼠边缘系统进行了全面的雌激素干预。结果，我们证实了雌激素诱导c-fos激活，而糖皮质激素不能。已知糖皮质激素可抑制AP-1位点。这些结果表明，雌激素和糖皮质激素相互调节即刻早期基因。雌激素的这种作用可能与我们的实验数据有关，即雌激素有时会导致培养的海马神经元内钙离子的长期升高。我们的假设是，即刻早期基因的下一个延迟基因可能是肝细胞生长因子(HGF)基因，因为HGF也与神经组织损伤后的修复有关，而糖皮质激素抑制HGF mRNA的激活。用Northern印迹法检测红藻氨酸对大鼠海马区HGF和c-Met基因表达的影响。海人酸致伤后6~48h可诱导HGF和c-met基因的表达。类固醇激素对红藻氨酸诱导的HGF mRNA的影响正在进行中。为了更准确地证实雌激素对神经元的影响，我们使用了雌激素受体基因转染的神经母细胞瘤细胞系。通过在该细胞系中添加雌激素，神经母细胞瘤细胞分化为成熟的神经元。我们正在使用这种被雌激素分化的细胞系来实验雌激素对神经元的影响。

As a part of the study on how biologically active substances in the brain cause long-term effects through immediate early genes, effects of steroid hormones on survival rate of cultured rat limbic neurons were investigated.It was previously confirmed that in cultured limbic neurons glucocorticoid increased kainic acid-induced cytotoxicity dose-dependently, while estrogen increased survival rate of cultured neurons at concentrations lower than 10^<-6>M.To elucidate this effect of estrogen, in situ hybridization of c-fos was performed on the rat limbic system after general administration of estrogen.As the results, we confirmed that estrogen induced c-fos activation, while glucocorticoid administration did not. It has been known that glucocorticoid inhibits AP-1 site. These results suggested that estrogen and glucocorticoid reciprocally regulated immediate early genes. Such effect of estrogen is probably related with our experimental data that estrogen sometimes causes long-term elevation of Ca^<2+> within cultured hippocampal neurons.Our hypothesis is that the next targent gene to the immediate early gene may be hepatocyte growth factor (HGF) gene since HGF is related with repairing also nervous tissue after injury and glucocorticoid inhibits HGF mRNA activation. Northern blotting analysis of HGF and c-met mRNA was performed on the rat hippocampus after general administration of kainic acid. HGF and c-met mRNA were induced 6 to 48 hours after kainic acid injection. Effect of steroid hormones on this kainic acid-induced HGF mRNA was under progress.To confirm the effect of estrogen on neurons more precisely, neuroblastoma cell line into which estrogen receptor gene was transfected was used. By adding estrogen to this cell line, the neuroblastoma cells differentiated into matured neurons. We are experimenting the effect of estrogen on neurons using this cell line differentiated by estrogen.