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海马神经元突触囊泡释放特征及可释放囊泡含量定量分析

徐淑君, 王萍, 夏迪

宁波大学医学院，宁波 315211； 中国科学院上海生命科学研究院神经所神经科学国家重点实验室，上海 200031

摘要

中枢神经系统突触前的神经末梢只有少量的突触囊泡存在，突触囊泡数目的多少和融合模式将影响突触传递的效率。对突触囊泡数目的多少和释放模式的研究依赖于有效的研究方法。在本研究中，与膜亲和力不同的荧光染料用于标记体外培养的海马神经元的功能性突触囊泡。通过场电位和高钾刺激，动态观察荧光强度的变化，结果显示在第一轮刺激中，与膜亲和力低的染料FM2-10 脱色的比例(93.0%±5.9 %)显著大于与膜亲和力高的染料FM1-43 (57.9%±3.5%)。但是，第二和第三轮刺激中FM1-43 脱色的比例分别为(24.0±2.3)%，(8.6±1.5)%，显著大于FM2-10 的脱色比例[(1.4±3.8)%，(2.3±1.6)%]。这个结果提示快速内吞模式不仅存在于囊泡的第一次释放，同时还存在于囊泡回收后的再次释放。另一方面，高频刺激和高渗蔗糖溶液这两种方法同时用于检测体外混合培养13~14 天的抑制性神经元的可释放囊泡池(readily releasable pool, RRP)的大小。结果显示，用高渗蔗糖溶液估计的RRP 的大小[(200±23.0) pC]显著大于用高频刺激估计的RRP 的大小[(51.1±10.5) pC]。分析其 可能的原因是用双patch 的方法分析的是两个神经元之间的联系，而在混合培养的系统中，一个神经元有可能受多个神经元的 支配，用高渗溶液刺激则使所有的突触前RRP 都释放，所以用这种方法计算的RRP 值要大的多。因此为了排除混合培养的 神经元中突触联系的多少对RRP 值的影响，用高频刺激的方法来估计RRP 值的大小更合理。

关键词： 内吞; 突触囊泡; 全细胞记录; 培养细胞; 海马

分类号：R338.1+3

Quantitative analysis of synaptic vesicle release and readily releasable pool size in hippocampal neurons

XU Shu-Jun, WANG Ping, XIA Di

Medical School, Ningbo University, Ningbo 315211, China； Institute of Neuroscience and Key Laboratory of Neurobiology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

Abstract

In central nervous system only a limited number of vesicles exist in the presynaptic terminals. The size and fusion modes of the vesicles were particularly important because of their potential impact on neuronal communications. Efficient methods were needed to analyze the recycling kinetics of synaptic vesicle and the size of readily releasable pool (RRP). In this study, fluorescent dyes with different affinity for membranes (FM1-43 with high affinity and FM2-10 with low affinity) were used to stain the functional synaptic vesicles of cultured hippocampal neurons and the kinetics of vesicle recycling was measured. The results showed that the destaining proportion was larger for FM2-10 than that for FM1-43 during the first trial, while it was greater for FM1-43 than FM2-10 during the second and third trials (first round, 93.0%±5.9% versus 57.9%±3.5% for FM2-10 and FM1-43, respectively, P<0.0001; second round, 1.4%±3.8% versus 24.0%±2.3%, P<0.0001; third round, 2.3%±1.6% versus 8.6%±1.5%, P=0.005). The results indicated that rapid endocytosis existed not only in the first round but also occurred when the vesicles were reused. Moreover, Both high-frequency stimuli and hypertonic sucrose stimuli were used to estimate the RRP sizes in the mix cultured hippocampal inhibitory neurons at 13- 14 days in vitro (DIV). We found that the RRP size estimated by hypertonic sucrose stimuli [(200±23.0) pC] was much larger than that estimated by high-frequency stimuli [(51.1±10.5) pC]. One possible reason for the discrepancies in RRP estimates is that in mix cultured conditions, one neuron may receive inputs from several neurons and hypertonic sucrose stimuli will cause RRP of all those neurons release, while using dual patch recording, only the connection between two neurons was analyzed. Thus, to exclude out the impacts of inputs numbers on RRP sizes, it is more reasonable to use high-frequency stimuli to estimate the RRP size in mix cultured neurons.

Key words: endocytosis; synaptic vesicles; whole cell recording; cultured cell; hippocampus

收稿日期：2009-08-07　　录用日期：2009-10-16

通讯作者：徐淑君　　E-mail: xushujun@nbu.edu.cn

引用本文：

徐淑君, 王萍, 夏迪. 海马神经元突触囊泡释放特征及可释放囊泡含量定量分析[J]. 生理学报 2009; 61 (6): 505-510.

XU Shu-Jun, WANG Ping, XIA Di. Quantitative analysis of synaptic vesicle release and readily releasable pool size in hippocampal neurons. Acta Physiol Sin 2009; 61 (6): 505-510 (in Chinese with English abstract).