1] Applied Physics Program, School of Engineering and Applied Science, Harvard University, Cambridge, Massachusetts, USA. [2].
1] Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada. [2].
Nat Methods. 2014 Aug;11(8):825-33. doi: 10.1038/nmeth.3000. Epub 2014 Jun 22.
All-optical electrophysiology-spatially resolved simultaneous optical perturbation and measurement of membrane voltage-would open new vistas in neuroscience research. We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and QuasAr2, which show improved brightness and voltage sensitivity, have microsecond response times and produce no photocurrent. We engineered a channelrhodopsin actuator, CheRiff, which shows high light sensitivity and rapid kinetics and is spectrally orthogonal to the QuasArs. A coexpression vector, Optopatch, enabled cross-talk-free genetically targeted all-optical electrophysiology. In cultured rat neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials (APs) in dendritic spines, synaptic transmission, subcellular microsecond-timescale details of AP propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell-derived neurons. In rat brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without the use of conventional electrodes.
全光学电生理学——空间分辨的同时光学扰动和细胞膜电压测量——将为神经科学研究开辟新的视野。我们开发了两种基于古菌视紫红质的电压指示剂，QuasAr1 和 QuasAr2，它们具有更高的亮度和电压灵敏度，响应时间为微秒级，并且不产生光电流。我们设计了一种通道视紫红质激活剂 CheRiff，它具有高光敏性和快速动力学特性，并且与 QuasArs 光谱正交。一个共表达载体 Optopatch，实现了无串扰的基因靶向全光学电生理学。在培养的大鼠神经元中，我们将 Optopatch 与图案化的光学激发相结合，探测树突棘中的逆行动作电位 (AP)、突触传递、AP 传播的亚秒级细节以及网络中许多神经元的同时发射。Optopatch 测量揭示了人干细胞源性神经元中内在兴奋性的自稳态调节。在大鼠脑片中，Optopatch 以高信噪比诱导和报告动作电位和亚阈事件。Optopatch 平台可实现高通量、空间分辨的电生理学，而无需使用传统电极。
