Single molecule analysis for nuclear DNAof the neuron in excitotoxicity
| Project/Area Number |
14571306
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Cerebral neurosurgery
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| Research Institution | Hamamatsu University School of Medicine |
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Principal Investigator |
YAMAMOTO Seiji Hamamatsu University School of Medicine, Photon Medical Research Center, Associate Professor, 光量子医学研究センター, 助教授 (60144094)
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| Co-Investigator(Kenkyū-buntansha) |
TERAKAWA Susumu Hamamatsu University School of Medicine, Photon Medical Research Center, Associate Professor, 光量子医学研究センター, 教授 (50014246)
SAKURAI Takashi Hamamatsu University School of Medicine, Photon Medical Research Center, Research Associate, 光量子医学研究センター, 助手 (50283362)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 2003: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 2002: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | excitotoxicity / DNA fragmentation / inositol trisphosphate / nuclear calcium signal / total internal reflection fluorescence microscope / single molecule imagine / ミトコンドリア障害 |
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| Research Abstract |
1.Inositol trisphosphate pathway as an excitotoxic death signal to the nucleus in hippocampal neurons
We 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 excitotoxicity
We 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
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Report
(3 results)
Research Products
(14 results)
