Single molecule analysis for nuclear DNAof the neuron in excitotoxicity

1.三磷酸肌醇途径作为海马神经元兴奋性毒性死亡信号的研究我们试图确定海马神经元兴奋性毒性过程中快速核变化的机制。我们观察到：（1）谷氨酸或N-甲基-D-天冬氨酸（NMDA）使细胞质和核质内三磷酸肌醇（IP_3）浓度升高，核质内Ca^2+浓度升高（2）IP_3可引起透化神经元[Ca ^<2 +>]_n升高和细胞核改变; IP_3介导的Ca^<2+>释放的阻断剂显著抑制谷氨酸或NMDA引起的核变化。谷氨酸激活与IP_3通路相关的NMDA和代谢型受体，使[Ca^<2+>]_n持续高于生理水平，并在兴奋性毒性中迅速损伤核DNA。 关于我们 在兴奋性毒性中迅速诱导DNA断裂我们试图在微分干涉显微镜下直接分析从少数发生核变化的神经元中提取的DNA。在培养的大鼠海马神经元中，随着细胞核的变化，用微量吸管吸取3-5个神经元。用蛋白酶K和苯酚提取它们的DNA，用核酸染料染色，并在全内反射荧光（TIRF）显微镜下观察，这使我们能够观察用荧光染料染色的单分子。虽然从经历核粒化的神经元中提取的DNA在未涂覆的玻璃上显示出布朗运动，但它们在带正电荷的聚-D-赖氨酸涂覆的玻璃上显示出各种类型的碎片。三维共聚焦成像还显示，DNA原位断裂的神经元与核颗粒。先进的DNA光学成像技术显示，谷氨酸在兴奋性毒性的早期阶段诱导了DNA片段化。少

1.Inositol trisphosphate pathway as an excitotoxic death signal to the nucleus in hippocampal neuronsWe sought to determine the mechanism underlying the rapid nuclear change during excitotoxicity in hippocampal neurons. We observed: 1) glutamate or N-methyl-D-aspartate (NMDA) increased cytoplasmic and nucleoplasmic inositol trisphosphate (IP_3) concentrations detected with green fluorescent protein, and increased nucleoplasmic Ca^<2+> concentration ([Ca^<2+>]_n) that was consistently associated with the nuclear changes, 2) IP_3 induced an increase in [Ca^<2+>]_n and the nuclear changes in permeabilized neurons, and 3) a blocker of IP_3-mediated Ca^<2+> release significantly inhibited the nuclear changes induced by glutamate or by NMDA. Glutamate activates NMDA and metabotropic receptors both linked to the IP_3 pathway, increases [Ca^<2+>]_n persisting above the physiological level, and rapidly damages the nuclear DNA in excitotoxicity.2.Advanced optical imaging reveals that glutamate r … More apidly induces DNA fragmentation in excitotoxicityWe attempted to directly analyze the DNA extracted from a few neurons undergoing the nuclear change under the differential interference contrast microscope. In cultured rat hippocampal neurons, following the nuclear changes, 3-5 neurons were aspirated with micropipette. Their DNAs were extracted with proteinase K and phenol, stained with a nucleic acid dye, and observed under the total internal reflection fluorescence (TIRF) microscope, which allows us to observe the single molecules stained with fluorescent dye. While DNAs extracted from the neurons undergoing the nuclear granulation showed Brownian motion on the non-coated glass, they showed various types of fragmentation on the poly-D-lysine coated glass, which was positively charged. Three dimensional confocal imaging also revealed that DNA in situ showed fragmentation in the neuron with nuclear granulation. The advanced optical imaging for nuclear DNA reveals that glutamate induced DNA fragmentation in the early phase of excitotoxicity. Less