Studies on neurotoxic mechanisms by excitotoxins

本研究涉及大脑中基因转录的调节，以评估特定的l -谷氨酸嗜离子受体亚型可能参与兴奋毒素对神经元毒性的机制。转录因子是对特定核心核苷酸序列具有高亲和力的核蛋白，可调节RNA聚合酶II的活性，RNA聚合酶II负责细胞核中基因组DNA形成mRNA。全身给药n-甲基- d -天冬氨酸（NMDA）导致小鼠海马中转录因子激活蛋白-1 （AP1）的DNA结合活性选择性和剧烈增强。在低温恒温器的帮助下冷冻冠状切片，然后在双眼显微镜下用干冰上的塑料毛细管冲孔出所需的区域。在CA1和CA3锥体细胞中没有发现这种增强现象，只在齿状颗粒细胞中可见。齿状回的增强是短暂的，在给药后2小时达到峰值，4小时后下降，这种增强对NMDA通道阻滞剂的拮抗作用很敏感。免疫组化分析显示，NMDA诱导齿状回c-Jun和c-Fos蛋白表达，但CA1和CA3亚区不表达。相比之下，kainic酸（KA）在CA1和CA3锥体层以及齿状颗粒层诱导AP1 DNA结合的剧烈和长时间增强。给药KA而非NMDA导致AP1在邻近区域的结合明显增强，但不包括锥体层和颗粒层。KA诱导CA1和CA3锥体层严重神经元死亡，但不影响齿状颗粒神经元。这些结果表明，调节特定蛋白质的从头合成可能是与兴奋毒素诱导的神经元细胞死亡相关的机制的基础。

The present study deals with modulation of gene transcription in the brain, in order to evaluate possible involvement of particular ionotropic receptor subtypes for L-glutamic acid in mechanisms underlying neuronal toxicity by excitotoxins. Transcription factors are nuclear proteins with high affinity for a particular core nucleotide sequence to modulate the activity of RNA polymerase II that is responsible for formation of mRNA from genomic DNA in the nucleus. The systemic administration of N-methyl-D-aspartic acid (NMDA) led to selective and drastic potentiation of DNA binding activity of the transcription factor activator protein-1 (AP1) in murine hippocampus. Frozen coronal sections were made with the aid of a cryostat, followed by punching out of the desired regions by a plastic capillary on dry ice under a binocular microscope. The potentiation was only seen in the dentate granule cells, but not in the CA1 and CA3 pyramidal cells. The potentiation in the dentate gyrus was transient with a peak at 2 h after administration and a decline within 4 h later, which occurred in a manner sensitive to antagonism by an NMDA channel blocker. Immunohistochemical analysis revealed that NMDA induced expression of both c-Jun and c-Fos proteins in the dentate gyrus, but not in the CA1 and CA3 subfields. By contrast, kainic acid (KA) induced drastic and prolonged potentiation of AP1 DNA binding in the CA1 and CA3 pyramidal layers in addition to dentate granule layers. The administration of KA but not NMDA led to marked potentiation of AP1 binding in areas neighboring but excluding pyramidal and granule layers. KA induced severe neuronal death in the CA1 and CA3 pyramidal layers without affecting dentate granular neurons. These results suggest that modulation of de novo synthesis of particular proteins may underlie mechanisms associated with neuronal cell death induced by excitotoxins.