Histochemical and physiological study of delayd neuronal death

1.利用Ca^<2+>-ATPase II型抗体对正常大鼠和沙鼠脑内该酶进行免疫组化定位。在海马体中，酶的分布与我们用酶组织化学方法检测到的相似。即该酶弥散分布于锥体神经元、轴突、树突的质膜上。CA1、CA3与齿状回的分布密度无明显差异。沙鼠海马切片的生理研究我们利用沙鼠短暂性前脑缺血后的海马切片研究了n -甲基- d -天冬氨酸（NMDA）受体介导的CA1锥体神经元突触电位。在6-氰基- 7-硝基喹啉-2,3-二酮（CNQX）和二库林存在下，刺激Schaffer侧支/交联纤维可诱导NMDA受体激活的场兴奋性突触后电位（fEPSP）。我们发现，在缺血后的许多切片中，输入纤维的低频…More刺激（0.1-10Hz）引起nmda介导的fEPSP的反复抑制和增强。fEPSP振幅的循环变化与刺激频率有关，范围为0.08 ~ 2.5 cycle/min。应用1双（邻氨基酚氧基）乙烷-N，N，N‘， N’-四乙酰1，四乙酰氧基甲基酯（BAPTA-AM）（一种膜透性Ca^<2+>螯合剂）可阻断循环变化，但应用vera-pamil或在沐浴液中还原Ca^<2+>对循环变化影响不大。细胞内记录显示膜电位周期性去极化与EPSP抑制同步。应用1-(5-soquinolinylsulfony1)-2- methyl哌嗪（H-7）和PKC拮抗剂K252a可显著减弱这种循环现象。这些结果表明，刺激依赖的nmda受体激活，由PKC介导，发生在缺血后的CA1神经元中，这种循环变化可能反映了细胞内异常的Ca^<2+>信号传导过程导致神经元变性，从而导致周期性的膜去极化。对侧互神经纤维刺激沙鼠海马电位作图通过记录对侧互神经纤维刺激诱导的EPSP，对沙鼠海马电位作图。结果表明，CA1锥体神经元在缺血损伤后早期（2-8h）处于高兴奋状态，LTP明显高于非缺血组。这是第一次在体内报道缺血后早期的异常生理状况。少

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