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

虽然已经克隆了许多与胞吐作用相关的蛋白质基因，并推导出了这些蛋白质的氨基酸序列，但胞吐作用的机制尚未完全阐明。为探讨突触外排的关键蛋白syntaxin 1A的生理作用，本研究采用全细胞膜片吸管法将syntaxin 1A抗体导入体外培养的大鼠海马神经元索马胞体。首先，我们分析了抗syntaxin 1A抗体对海马神经元自发性传递的影响。细胞内应用抗syntaxin 1A抗体可增强自发性传递，即抗syntaxin 1A抗体可增强自发性EPSC的振幅，而不改变非同步性EPSC的振幅分布。抗体不影响异步EPSC的量子大小。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梢的神经递质释放。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。第二，为了阐明突触胞吐的调节机制，利用培养的大鼠齿状回神经元的自显影，分析了EPSC振幅和成对脉冲修正（ppm）对细胞外Ca^2+浓度的依赖性。Forskolin增加cAMP浓度引起突触胞吐作用增强，但不改变ppm，提示Forskolin增强齿状核神经元突触胞吐作用的主要原因是增加了停靠囊泡池（易释放池）的大小或释放位点的数量。这一机制可能在哺乳动物海马CA3区突触胞吐的长时程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.