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Elucidation of molecular mechanism of synaptic exocytosis and its modification mechanism in mammalian hippocampal autapse.

阐明哺乳动物海马突触胞吐作用的分子机制及其修饰机制。

基本信息

批准号：
09680819
负责人：
YAMAGUCHI Kazuhiko
金额：
$ 1.86万
依托单位：
Kyorin University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

Mechanism underlying synaptic exocytosis has not been revealed yet, though many kinds of genes of proteins relating to exocytosis have been cloned and these amino acid sequences have been deduced. To investigate the physiological role of syntaxin 1A, a key protein for synaptic exocytosis, antibody against syntaxin 1A was applied into cell soma of the cultured rat hippocampal neuron through a whole cell patch-pipette in the present project. First, we analyzed effects of anti-syntaxin 1A antibody on the autaptic transmission of hippocampal neuron. Intracellular application of the antibody enhanced the autaptic transmission, that is, antibody against syntaxin lA enhanced the amplitude of the autaptic EPSC, while it did not change the asynchronous EPSC amplitude distribution. Quantal size of asynchronous EPSC was not affected by antibody. This result indicated that the antibody did not enhanced sensitivity of glutamate receptors located on the postsynaptic membrane, but it enhanced neurotransmitter release from the presynaptic terminal. This result suggested a suppressive role for syntaxin 1A in exocytosis at the mammalian central synapse. Second, to elucidate the modulatory mechanism of the synaptic exocytosis, the dependence of EPSC amplitude and paired pulse modification (ppm) on extracellular Ca^<2+> concentration was analyzed using the autapse of the cultured rat dentate neuron. Increase in cAMP concentration by forskolin caused enhancement of synaptic exocytosis Without changing ppm, suggesting principal reason of the enhancement of the synaptic exocytosis by forskolin from dentate neuron was increase in the size of docked vesicle pool (readily releasable pool) or number of release sites. This mechanism would play an important role in long-term potentiation of synaptic exocytosis at CA3 of mammalian hippocampus.

尽管已经克隆了多种与胞吐作用相关的蛋白质基因并推导了这些氨基酸序列，但突触胞吐作用的机制尚未被揭示。为了研究突触胞吐作用的关键蛋白突触融合蛋白 1A 的生理作用，在本项目中，通过全细胞贴片移液器将抗突触融合蛋白 1A 的抗体通过全细胞贴片移液管施加到培养的大鼠海马神经元的细胞体中。首先，我们分析了抗突触蛋白1A抗体对海马神经元自动传递的影响。细胞内应用该抗体增强了自体传递，即抗突触蛋白1A的抗体增强了自体EPSC的幅度，而它并没有改变异步EPSC的幅度分布。异步EPSC的量子大小不受抗体影响。该结果表明该抗体没有增强位于突触后膜上的谷氨酸受体的敏感性，但它增强了突触前末端的神经递质释放。这一结果表明突触融合蛋白 1A 在哺乳动物中央突触的胞吐作用中具有抑制作用。其次，为了阐明突触胞吐作用的调节机制，使用培养的大鼠齿状神经元的自动分析来分析EPSC幅度和配对脉冲修饰(ppm)对细胞外Ca 2+ 浓度的依赖性。毛喉素增加 cAMP 浓度导致突触胞吐作用增强在不改变 ppm 的情况下，表明毛喉素增强齿状神经元突触胞吐作用的主要原因是停泊囊泡池（易于释放池）的大小或释放位点数量的增加。该机制在哺乳动物海马CA3突触胞吐作用的长期增强中发挥重要作用。