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时早期基因产生长期影响的一部分，我们研究了类固醇激素对培养大鼠边缘神经元存活率的影响。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了kainic酸诱导的细胞毒性，而雌激素在浓度低于10^<-6>M时提高了培养神经元的存活率。为了阐明雌激素的这种作用，我们在给药后对大鼠边缘系统进行了c-fos的原位杂交。结果，我们证实雌激素诱导c-fos激活，而糖皮质激素没有。已知糖皮质激素抑制AP-1位点。这些结果表明雌激素和糖皮质激素相互调节直接早期基因。雌激素的这种作用可能与我们的实验数据有关，雌激素有时会导致培养海马神经元内Ca^<2+>的长期升高。我们的假设是，下一个直接早期基因的靶基因可能是肝细胞生长因子（HGF）基因，因为HGF也与损伤后神经组织的修复有关，糖皮质激素抑制HGF mRNA的激活。用Northern blotting法对大鼠海马组织进行HGF和c-met mRNA的分析。kainic酸注射后6 ~ 48 h诱导HGF和c-met mRNA的表达。类固醇激素对kainic酸诱导的HGF mRNA的影响尚在研究中。为了更准确地证实雌激素对神经元的影响，我们使用转染雌激素受体基因的神经母细胞瘤细胞系。通过向该细胞系添加雌激素，神经母细胞瘤细胞分化为成熟的神经元。我们正在用这种由雌激素分化的细胞系试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.