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

虽然已经克隆了多种与突触胞吐相关的蛋白质基因，并推导出了这些氨基酸序列，但突触胞吐作用的机制还没有被揭示。为了研究突触胞吐的关键蛋白--合成素1A的生理作用，本课题通过全细胞贴片移液管将抗合成素1A的抗体应用于培养的大鼠海马神经细胞胞体。首先，我们分析了抗合成素1A抗体对海马神经元自体传递的影响。细胞内应用该抗体增强了自体EPSC的传递，即抗Synaxin 1a抗体增强了自体EPSC的幅度，但不改变自体EPSC的非同步幅度分布。异步化EPSC的量子大小不受抗体的影响。结果表明，该抗体不能增强突触后膜上谷氨酸受体的敏感性，但能促进突触前终末的神经递质释放。这一结果提示，在哺乳动物中央突触的胞吐过程中，Synaxin 1A起着抑制作用。其次，为了阐明突触胞吐的调控机制，利用培养的大鼠齿状回神经元，分析了细胞外钙离子浓度对EPSC幅值和双脉冲调制(Ppm)的影响。增加cAMP浓度可使突触胞吐增加，但不改变ppm，提示齿状神经元突触胞吐增加的主要原因是对接的囊泡池(易释放池)的大小或释放部位的数量增加。这一机制可能在哺乳动物海马区CA3区突触胞吐作用的长期增强中发挥重要作用。

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.