非稳态条件下皮层锥体神经元单钙通道电流的波动分析
Fluctuation analysis in nonstationary conditions: single Ca channel current in cortical pyramidal neurons
摘要：
波动分析是一种方法，它允许测量离子通道的单通道电流，即使它太小而不能直接用膜片钳技术解决。这是电压门控Ca 2+通道（VGCC）的情况。它们存在于所有哺乳动物中枢神经元中，控制突触前递质的释放、突触后信号传导和突触整合。然而，在生理浓度的细胞外Ca 2+中，它们的单通道电流的幅度很小，并且没有很好地确定。但是，测量这个数量是必不可少的估计功能VGCC的膜和通道相关的Ca 2+信号结构域的大小，并了解Ca 2+信号的随机性。在这里，我们记录了VGCC电流在有核补丁从第5层锥体神经元在大鼠新皮层，在生理外部Ca 2+（1-2 mM）。由于VGCC电流的快速下降，传统波动分析所需的电流响应的整体平均被证明是不切实际的。因此，我们开发了一种更强大的方法，使用平均电流拟合的个人电流响应和带通滤波。此外，电压斜坡刺激被证明是有用的。通过对仿真数据的分析，验证了该方法的准确性.在1 mM的外部Ca 2+浓度和-20 mV的膜电位下，我们发现平均单通道电流幅度约为0.04 pA，在2 mM外部Ca 2+时增加至0.065 pA，波动的弛豫时间常数在0.2- 0.12pA的范围内。0.8毫秒。结果是相关的了解树突状细胞的Ca 2+尖峰在新皮层第5层锥体神经元的随机特性。与报告的方法，单通道电流幅度的本地VGCC可以准确地解决，尽管条件不稳定的rundown。
英文摘要：
Fluctuation analysis is a method which allows measurement of the single channel current of ion channels even when it is too small to be resolved directly with the patch clamp technique. This is the case for voltage-gated Ca2+ channels (VGCCs). They are present in all mammalian central neurons, controlling presynaptic release of transmitter, postsynaptic signaling and synaptic integration. The amplitudes of their single channel currents in a physiological concentration of extracellular Ca2+, however, are small and not well determined. But measurement of this quantity is essential for estimating numbers of functional VGCCs in the membrane and the size of channel-associated Ca2+ signaling domains, and for understanding the stochastic nature of Ca2+ signaling. Here, we recorded the VGCC current in nucleated patches from layer 5 pyramidal neurons in rat neocortex, in physiological external Ca2+ (1-2 mM). The ensemble-averaging of current responses required for conventional fluctuation analysis proved impractical because of the rapid rundown of VGCC currents. We therefore developed a more robust method, using mean current fitting of individual current responses and band-pass filtering. Furthermore, voltage ramp stimulation proved useful. We validated the accuracy of the method by analyzing simulated data. At an external Ca2+ concentration of 1 mM, and a membrane potential of -20 mV, we found that the average single channel current amplitude was about 0.04 pA, increasing to 0.065 pA at 2 mM external Ca2+, and 0.12 pA at 5 mM. The relaxation time constant of the fluctuations was in the range 0.2-0.8 ms. The results are relevant to understanding the stochastic properties of dendritic Ca2+ spikes in neocortical layer 5 pyramidal neurons. With the reported method, single channel current amplitude of native VGCCs can be resolved accurately despite conditions of unstable rundown.
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