Submillisecond optical reporting of membrane potential in situ using a neuronal tracer dye.
Submillisecond optical reporting of membrane potential in situ using a neuronal tracer dye.
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DOI：
10.1523/jneurosci.1240-09.2009
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发表时间：
2009-07-22
期刊：
影响因子：
--
通讯作者：
DiGregorio DA
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文献类型：
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作者：
Bradley J;Luo R;Otis TS;DiGregorio DA
神经科学的一个主要目标是开发易于使用、光毒性有限、并达到检测单个神经元单个动作电位所需的速度和灵敏度的膜电位光学记录器。在这里，我们提出了一种新颖的、双分量光学方法来实现这些目标。利用Förster共振能量转移(FRET)技术，将神经元示踪染料DiO与膜分配对电压敏感的二苯甲胺(DPA)结合，获得与膜电位变化相关的光信号。在HEK293细胞中，DIO/DPA在衍射极限激光光斑照射下，每100 mV(τ̣~0.1ms)获得56%的去极化诱导荧光变化，而在神经元培养和脑片中，动作电位(AP)每100 mV产生大于25%的ΔF/F。DIO/DPA提供的高灵敏度使得能够在单次试验中从体细胞、轴突或树突膜隔间检测到阈值下活动和高频AP。意识到DPA可以抑制兴奋性，我们分析了原代培养神经元和脑片中单个AP的幅度和持续时间、猝发特性和自发放电，发现它们在高达2µM的DPA下没有被检测到的改变，而在5µM的DPA下仅有轻微的扰动。这些发现证实了一种简单、非侵入性的方法，该方法依赖于神经元示踪染料来监测电信号流，并为研究完整神经元回路中的信号提供了独特的灵活性。
A major goal in neuroscience is the development of optical reporters of membrane potential that are easy to use, have limited phototoxicity, and achieve the speed and sensitivity necessary for detection of individual action potentials in single neurons. Here we present a novel, two-component optical approach that attains these goals. By combining DiO, a fluorescent neuronal tracer dye, with dipicrylamine (DPA), a molecule whose membrane partitioning is voltage-sensitive, optical signals related to changes in membrane potential based on Förster resonance energy transfer (FRET) are reported. Using DiO/DPA in HEK 293 cells with diffraction-limited laser spot illumination, depolarization-induced fluorescence changes of 56 % per 100 mV (τ̣ ~ 0.1 ms) were obtained, while in neuronal cultures and brain slices, action potentials (APs) generated a ΔF/F per 100 mV of greater than 25%. The high sensitivity provided by DiO/DPA enabled the detection of subthreshold activity and high frequency APs in single trials from somatic, axonal, or dendritic membrane compartments. Recognizing that DPA can depress excitability, we assayed the amplitude and duration of single APs, burst properties, and spontaneous firing in neurons of primary cultures and brain slices and found that they are undetectably altered by up to 2 µM DPA and only slightly perturbed by 5 µM DPA. These findings substantiate a simple, non-invasive method that relies on a neuronal tracer dye for monitoring electrical signal flow, and offers unique flexibility for the study of signaling within intact neuronal circuits.