Xu Shu-Jun, Wang Ping, Xia Di
Medical School, Ningbo University, Ningbo 315211, China.
Sheng Li Xue Bao. 2009 Dec 25;61(6):505-10.
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.
在中枢神经系统中，突触前终末仅存在有限数量的囊泡。囊泡的大小和融合模式尤为重要，因为它们可能会对神经元通讯产生影响。需要高效的方法来分析突触囊泡的循环动力学以及可快速释放池（RRP）的大小。在本研究中，使用对膜具有不同亲和力的荧光染料（高亲和力的FM1-43和低亲和力的FM2-10）对培养的海马神经元的功能性突触囊泡进行染色，并测量囊泡循环的动力学。结果显示，在第一次试验期间，FM2-10的脱色比例大于FM1-43，而在第二次和第三次试验期间，FM1-43的脱色比例大于FM2-10（第一轮，FM2-10和FM1-43分别为93.0%±5.9%和57.9%±3.5%，P<0.0001；第二轮，1.4%±3.8%和24.0%±2.3%，P<0.0001；第三轮，2.3%±1.6%和8.6%±1.5%，P=0.005）。结果表明，快速内吞作用不仅存在于第一轮，而且在囊泡重复使用时也会发生。此外，还使用高频刺激和高渗蔗糖刺激来估计体外培养13-至14天（DIV）的混合培养海马抑制性神经元中的RRP大小。我们发现，高渗蔗糖刺激估计的RRP大小[（200±23.0）pC]远大于高频刺激估计的大小[（51.1±10.5）pC]。RRP估计值存在差异的一个可能原因是，在混合培养条件下，一个神经元可能从多个神经元接收输入，高渗蔗糖刺激会导致所有这些神经元的RRP释放，而使用双膜片钳记录时，仅分析两个神经元之间的连接。因此，为了排除输入数量对RRP大小的影响，在混合培养的神经元中使用高频刺激来估计RRP大小更为合理。
