Neuronal Networks in Health and Disease Laboratory, Institute of Experimental Epileptology and Cognition Research, Life and Brain Center, University of Bonn Medical Center, 53127 Bonn, Germany.
Department of Neurology, The David Geffen School of Medicine at UCLA, Los Angeles, CA 90095.
Proc Natl Acad Sci U S A. 2021 Feb 9;118(6). doi: 10.1073/pnas.2020235118.
Voltage sensing with genetically expressed optical probes is highly desirable for large-scale recordings of neuronal activity and detection of localized voltage signals in single neurons. Most genetically encodable voltage indicators (GEVI) have drawbacks including slow response, low fluorescence, or excessive bleaching. Here we present a dark quencher GEVI approach (dqGEVI) using a Förster resonance energy transfer pair between a fluorophore glycosylphosphatidylinositol-enhanced green fluorescent protein (GPI-eGFP) on the outer surface of the neuronal membrane and an azo-benzene dye quencher (D3) that rapidly moves in the membrane driven by voltage. In contrast to previous probes, the sensor has a single photon bleaching time constant of ∼40 min, has a high temporal resolution and fidelity for detecting action potential firing at 100 Hz, resolves membrane de- and hyperpolarizations of a few millivolts, and has negligible effects on passive membrane properties or synaptic events. The dqGEVI approach should be a valuable tool for optical recordings of subcellular or population membrane potential changes in nerve cells.
利用基因表达的光学探针进行电压感应对于大规模记录神经元活动和检测单个神经元中的局部电压信号非常理想。大多数可基因编码的电压指示剂（GEVI）都存在缺点，包括响应速度慢、荧光强度低或过度漂白。在这里，我们提出了一种暗猝灭 GEVI 方法（dqGEVI），该方法利用细胞膜外表面的荧光素糖基磷脂酰肌醇增强型绿色荧光蛋白（GPI-eGFP）和在电压驱动下快速在膜内移动的偶氮苯染料猝灭剂（D3）之间的Förster 共振能量转移对。与以前的探针相比，该传感器的单光子漂白时间常数约为 40 分钟，具有高时间分辨率和保真度，可用于检测 100 Hz 时的动作电位触发，分辨率为几毫伏的膜去极化和超极化，并且对被动膜特性或突触事件几乎没有影响。dqGEVI 方法应该是用于记录神经细胞亚细胞或群体膜电位变化的光学记录的有价值的工具。
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