Centre de Recherche Université Laval Robert-Giffard, Université Laval Québec, QC, Canada.
Front Cell Neurosci. 2011 Oct 10;5:20. doi: 10.3389/fncel.2011.00020. eCollection 2011.
Cell to cell communication in the central nervous system is encoded into transient and local membrane potential changes (ΔVm). Deciphering the rules that govern synaptic transmission and plasticity entails to be able to perform V(m) recordings throughout the entire neuronal arborization. Classical electrophysiology is, in most cases, not able to do so within small and fragile neuronal subcompartments. Thus, optical techniques based on the use of fluorescent voltage-sensitive dyes (VSDs) have been developed. However, reporting spontaneous or small ΔV(m) from neuronal ramifications has been challenging, in part due to the limited sensitivity and phototoxicity of VSD-based optical measurements. Here we demonstrate the use of water soluble VSD, ANNINE-6plus, with laser-scanning microscopy to optically record ΔV(m) in cultured neurons. We show that the sensitivity (>10% of fluorescence change for 100 mV depolarization) and time response (sub millisecond) of the dye allows the robust detection of action potentials (APs) even without averaging, allowing the measurement of spontaneous neuronal firing patterns. In addition, we show that back-propagating APs can be recorded, along distinct dendritic sites and within dendritic spines. Importantly, our approach does not induce any detectable phototoxic effect on cultured neurons. This optophysiological approach provides a simple, minimally invasive, and versatile optical method to measure electrical activity in cultured neurons with high temporal (ms) resolution and high spatial (μm) resolution.
细胞间通讯在中枢神经系统中被编码为短暂和局部的膜电位变化 (ΔVm)。要破译支配突触传递和可塑性的规则，就必须能够在整个神经元分支中进行 V(m) 记录。在大多数情况下，经典的电生理学无法在小而脆弱的神经元亚区中做到这一点。因此，已经开发了基于使用荧光电压敏感染料 (VSD) 的光学技术。然而，由于基于 VSD 的光学测量的有限灵敏度和光毒性，报告神经元分支中的自发或小 ΔV(m) 一直具有挑战性。在这里，我们展示了使用水溶性 VSD，ANNINE-6plus，与激光扫描显微镜结合，用于在培养神经元中光学记录 ΔV(m)。我们表明，该染料的灵敏度（对于 100 mV 去极化，荧光变化超过 10%）和时间响应（亚毫秒级）允许即使没有平均也可以稳健地检测动作电位 (AP)，从而可以测量自发神经元放电模式。此外，我们表明可以记录反向传播的 APs，沿着不同的树突部位和树突棘内。重要的是，我们的方法不会对培养的神经元产生任何可检测的光毒性作用。这种光电生理方法提供了一种简单、微创且多功能的光学方法，可用于以高时间（ms）分辨率和高空间（μm）分辨率测量培养神经元中的电活动。
