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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。
Only a small number of synaptic vesicles exist in the presynaptic nerve endings of the central nervous system. The number of synaptic vesicles and the mode of fusion will affect the efficiency of synaptic transmission. The study of the number of synaptic vesicles and the mode of release relies on effective research methods. In the present study, fluorescent dyes with different membrane affinity were used to label functional synaptic vesicles in hippocampal neurons cultured in vitro. The change of fluorescence intensity was observed dynamically by field and potassium stimulation. The results showed that in the first stimulation, the ratio of decolorization to FM2-10 with low affinity to membrane (93.0% ± 5.9%) was significantly higher than that with high membrane affinity Dye FM1-43 (57.9% ± 3.5%). However, the decolorization rates of FM1-43 in the second and third rounds of stimulation were (24.0 ± 2.3)% and (8.6 ± 1.5)%, respectively, which were significantly higher than those of FM2-10 [(1.4 ± 3.8)% and ± 1.6)%]. This result suggests that the rapid endocytic mode exists not only in the first release of vesicles but also in the release of vesicles after release. On the other hand, both high-frequency stimulation and hypertonic sucrose solutions were simultaneously used to detect the size of the readily releasable pool (RRP) of inhibitory neurons cultured in vitro for 13 to 14 days. The results showed that the size of RRP estimated with hypertonic sucrose solution [(200 ± 23.0) pC] was significantly greater than that estimated with high frequency stimulation [(51.1 ± 10.5) pC]. The possible reason for this analysis is that the two-patch method is used to analyze the relationship between two neurons. In a mixed-culture system, one neuron may be dominated by multiple neurons. When stimulated with hypertonic solution, So that all the pre-synaptic RRP are released, so using this method to calculate the RRP value much larger. Therefore, in order to rule out the influence of synaptic connections on RRP in mixed cultured neurons, it is more reasonable to estimate the RRP value by high-frequency stimulation.