Histochemical and physiological study of delayd neuronal death

迟发性神经元死亡的组织化学和生理学研究

• 批准号: 08671608
• 负责人: OGURO Keiji
• 金额: $ 0.45万
• 依托单位: Jichi Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08671608/
• 关键词: cerebral ischemi; gluramate recepto; Ca^<2+>-ATPase; protein kinase C; slice; hippocampus; mongolian gerbi; Ca^+-ATPase; Ca^<2+>-ATPase

1.Making plasmamembrane Ca^<2+>-ATPase antibody, immunohistochemistryUsing Ca^<2+>-ATPase type II antibody we made a immunohistochemical mapping of that enzyme in normal rat and gerbil brain. In hippocampus, the distribution of the enzyme is similar to that one which we have detected enzymehistochemically. That is, the enzyme is diffusely located on the plasma membrane of pyramidal neurons, axon, dendrite. There is no differences in distributional density between CA1, CA3 and dentate gyrus.2.Physiological study of gerbil hippocampal slicesWe studied N-methyl-D-aspartate (NMDA) receptor-mediated synaptic potentials in CA1 pyramidal neurons using hippocampal slices of the gerbils after transient forebrain ischemia. In the presence of 6-cyanc 7-nitroquinoxaline-2,3-dione (CNQX) and bicucullin, stimulation on Schaffer collateral/commissural fibers induced field excitatory postsynaptic potentials (fEPSP) activated by NMDA receptors. We found that in many slices after ischemia, low frequency … More stimulation (0.1-10Hz) to input fibers caused repeated depression and potentiation of the NMDA-mediated fEPSP.The cyclic changes in fEPSP amplitude were dependent on stimulus frequency, ranging from 0.08 to 2.5 cycle/min. The cyclic changes were blocked by application of 1 bis (o-aminophenoxy) ethane-N,N,N', N'-tetraacety1, tetraacetoxymethy1 ester (BAPTA-AM), a membrane permeable Ca^<2+> chelator, but they were little affected by application of vera-pamil or by reducing Ca^<2+>in bathing solution. Intracellular recordings showed periodic depolarizations of membrane potential synchroniz with depression of EPSP.The cyclic phenomenon was significantly attenuated by application of 1-(5-soquinolinylsulfony1)-2-methylpiperazine (H-7) and K252a, protein kinase C (PKC) antagonist.These results suggest that stimulus dependent NMDA-receptor activation, medi-ated by PKC,takes place the postischemic CA1 neurons and the cyclic change may reflect abnormal intracellular Ca^<2+> signaling process towards neuronal degeneration re-sulted in periodic membrane depolarization.3.Potential mapping of the gerbil hippocampus stimulated on the contralateral commisural fibersWe made potential mapping of postischemic gerbil hippocampus by recording EPSP induced by contralateral commisural fiber stimulation. We revealed that CA1 pyramidal neurons are in the hyper excitatory state in the early stage (2-8h) following ischemic insult and LTP is significantly increased in that period compared with the non-ischemic group. This is the first report of the abnormal physiological conditions in the early postischemic period in vivo. Less

1.制作质膜Ca~(2+)&Gt；-ATPase抗体，免疫组织化学：用Ca~(2+)&Gt；-ATPase II型抗体对正常大鼠和沙土鼠脑内该酶进行免疫组织化学定位。在海马区，该酶的分布与我们用酶组织化学方法检测到的相似。也就是说，该酶弥漫地分布在锥体神经元、轴突、树突的质膜上。沙土鼠海马片的生理学研究利用沙土鼠短暂性前脑缺血后的海马片，研究了N-甲基-D-天冬氨酸(NMDA)受体介导的CA1区锥体神经元突触电位。在6-环-7-硝基-2，3-二酮(CNQX)和荷包牡丹碱存在下，刺激Schaffer侧支/连合纤维可诱发由NMDA受体激活的场兴奋性突触后电位(FEPSP)。我们发现，在缺血后的许多脑片中，低频…更多的刺激(0.1~10 Hz)引起NMDA介导的fEPSP的反复抑制和增强，fEPSP幅度的周期性变化依赖于刺激频率，范围为0.08~2.5个周期/分钟。1-二(邻氨基苯氧基)乙烷-N，N，N‘，N’-四乙酸四乙酯(BAPTA-AM)是一种膜透性的钙离子螯合剂，可阻断这种周期性变化，但维拉帕米的应用或降低浴液中的钙离子对其影响不大。细胞内记录显示，膜电位的周期性去极化与EPSP的抑制同步。应用1-(5-异喹啉磺基)-2-甲基哌嗪(H-7)和蛋白激酶C(PKC)拮抗剂K252a可显著减轻细胞周期现象。这些结果表明，在PKC的介导下，刺激依赖的NMDA受体激活发生在缺血后的CA1神经元，这种周期性变化可能反映了细胞内钙离子的异常。3.沙土鼠对侧交感神经纤维的电位标测我们通过记录对侧交感神经纤维刺激诱发的EPSP，对沙土鼠脑缺血后的海马区进行电位标测。结果表明，脑缺血早期(2~8h)CA1区锥体神经元处于高兴奋性状态，LTP较非缺血组明显升高。这是在体脑缺血后早期出现异常生理状态的首次报道。较少

项目成果

Tsubokawa H,Oguro K,et al: "Intracellular inositol 1,3,4,5-tetrakisphosphate enhances the calcium current in hippocampal CA1 neurons of the gerbil after ischemia." J Physiol.(London). 497. 67-78 (1996)

Tsubokawa H、Oguro K 等人：“细胞内肌醇 1,3,4,5-四磷酸可增强沙鼠海马 CA1 神经元缺血后的钙电流。”

Ogura k, Masuzawa T, Kawai N et al.: "Cyclic changes in NMDA receptor activation in hippocampal CAl neurons after ischemia" Neurosience Research. 29. 273-281 (1997)

Ogura k、Masuzawa T、Kawai N 等人：“缺血后海马 CA1 神经元中 NMDA 受体激活的循环变化”神经科学研究。

Oguro K,et al.: "Histochemical study of Ca^<2+>-ATPase activity in ischemic CA1 pyramidal neurons in the gerbil hippocampus." Acta Neurropathol Berl. 90. 448-453 (1995)

Oguro K,et al.：“沙鼠海马缺血性 CA1 锥体神经元 Ca^2-ATP 酶活性的组织化学研究。”