| Budget Amount *help |
¥2,500,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥300,000)
Fiscal Year 2007: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2006: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
We investigated hippocampal neurons to determine whether alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) treatment alone induced comparable changes to those induced by glutamate in cortical neurons.
A nuclear granular structure and cellular swelling appeared after AMPA treatment in a dose-dependent manner under the video-enhanced contrast-differential interference contrast (VEC-DIC) microscope. The AMPA receptor antagonist,4-(8-methyl-9H-1,3-dioxolo [4,5-h] [2,3] benzodiazepin-5-yl)-benzenamine hydrochlorid (GYKI-52466), blocked the inducement by continuous exposre to100μM AMPA for1h. By contrast, NMDA receptor blocker, MK-801, did not block them. To detect the types of cell death, necrosis or apoptosis, AMPA-treated hippocampal neurons were stained with propidium iodide (PI) and fluorescein-4-isothiocyanate (FITC) conjugated annexin V (annexin V-FITC). Continuous exposure to AMPA for 1h induced necrosis in a dose-dependent manner. GYKI-52466inhibited the necrosis, but
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MK-801 did not. DNA fragmentation was assessed using comet assay. The treatment with AMPA (10μM-1mM) for 1h significantly increased the percentage of severer DNA damage. AMPA induced rapid Ca^<2+> increases in the nucleus before the nuclear granulation appeared. AMPA also rapidly induced IP_3production, since green fluorescent protein-tagged pleckstrin homology domain of PLC-δ1(GFP-PHD) was translocated from the plasmamembrane to the cytoplasm in response to increased concentration of IP3. However, it is necessary to investigate for the involvement of nuclear Ca^<2+> increase leading to nuclear granulation and DNA fragmentation. These results indicated after increase of nuclear Ca^<2+>, and led to DNA fragmentation as an early stage of necrosis without involvement of NMDA receptors. Three kinds of GluR1 shRNA plasmids were used to investigate whether they suppressed AMPA-mediated excitotoxicity. Hippocampal neurons expressed GluR1 shRNAs did not suppress cellular swelling and nuclear granulation after AMPA application, however, it must be investigated whether GluR1 suppression is not involved in AMPA-dependent excitotoxicity in detail. Less
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