Legendre P, Korn H
Departement des Biotechnologies, Institut Pasteur, Paris, France.
Eur J Neurosci. 1994 Oct 1;6(10):1544-57. doi: 10.1111/j.1460-9568.1994.tb00545.x.
To extend our study of the inhibitory synaptic network we have developed an isolated whole-brain preparation of the 52-h-old zebrafish (Brachydanio rerio) in which the structural and functional integrity of the brain is preserved. We report the characterization of quantal inhibitory events and the correlation of their properties with those of the underlying activated channels. During whole-cell recordings of the Mauthner cells, applications of 10(-6) M tetrodotoxin greatly reduced the frequency and amplitude of the spontaneously occurring synaptic events, which were dominated by Cl--dependent inhibitory postsynaptic currents (IPSCs). Lowering Ca2+ and adding Mg2+ to tetrodotoxin-containing solutions resulted in a further decrease in amplitude of the recorded synaptic currents, the remaining ones being considered as miniature IPSCs (mIPSCs). Applications of 0.5-1 microM strychnine in the presence of tetrodotoxin eliminated > 90% of the inhibitory currents in the preparation. The amplitude histograms of these mIPSCs exhibited two initial equally spaced peaks, followed by a skewed distribution for higher values. The first two components were well fitted by the sum of two Gaussian curves, giving a mean quantal amplitude of 35.7 pA (at a holding potential of -50 mV) and a coefficient of variation of 0.25 for the first peak. Outside-out recordings showed at least two classes of glycine receptor channels, one having multiple conductance levels with a main state of 81-86 pS and another displaying only one opening level of 41-43 pS. These two mean conductance states had similar mean open times, of 0.6-1 and 4.5-6 ms respectively. In addition, three mean closed times were observed for the 41-43 pS level. The shortest group (0.6-1 ms) was considered as representing gaps within bursts. Burst analysis revealed three mean burst durations, of 0.6, 4 and 35 ms. Comparisons of the amplitude of the first class of mIPSCs and of the open channel conductances indicated that one quantum opens 14-22 channels. Moreover, the correspondence between the mean decay time of mIPSCs and the mean open time or medium burst duration (4-5 ms) suggests that glycine-activated channels open only once in response to a single exocytosis. The pre- and postsynaptic origins of mIPSCs amplitude fluctuations are discussed in the context of multivesicular release versus the hypothesis of postsynaptic receptor saturation.
为了扩展我们对抑制性突触网络的研究，我们开发了一种52小时大的斑马鱼（Brachydanio rerio）全脑分离标本，其中大脑的结构和功能完整性得以保留。我们报告了量子抑制性事件的特征及其特性与潜在激活通道特性的相关性。在对Mauthner细胞进行全细胞记录期间，应用10^(-6) M河豚毒素可大大降低自发发生的突触事件的频率和幅度，这些事件主要由氯离子依赖性抑制性突触后电流（IPSC）主导。降低钙离子浓度并向含河豚毒素的溶液中添加镁离子会导致记录的突触电流幅度进一步降低，剩余的电流被视为微小IPSC（mIPSC）。在河豚毒素存在的情况下应用0.5 - 1 microM士的宁可消除标本中> 90%的抑制性电流。这些mIPSC的幅度直方图显示出两个初始等间距的峰值，随后是较高值的偏态分布。前两个成分可以很好地用两条高斯曲线的总和拟合，在保持电位为 -50 mV时，第一个峰值的平均量子幅度为35.7 pA，变异系数为0.25。外侧向外记录显示至少有两类甘氨酸受体通道，一类具有多个电导水平，主要状态为81 - 86 pS，另一类仅显示一个41 - 43 pS的开放水平。这两个平均电导状态具有相似的平均开放时间，分别为0.6 - 1 ms和4.5 - 6 ms。此外，在41 - 43 pS水平观察到三个平均关闭时间。最短的一组（0.6 - 1 ms）被认为代表爆发内的间隙。爆发分析揭示了三个平均爆发持续时间，分别为0.6、4和35 ms。对第一类mIPSC的幅度和开放通道电导的比较表明，一个量子打开14 - 22个通道。此外，mIPSC的平均衰减时间与平均开放时间或中等爆发持续时间（4 - 5 ms）之间的对应关系表明，甘氨酸激活的通道在单次胞吐作用下仅打开一次。我们在多泡释放与突触后受体饱和假说的背景下讨论了mIPSC幅度波动的突触前和突触后起源。
