Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.
UCL Queen Square Institute of Neurology, University College London, London, UK.
Nat Methods. 2019 Aug;16(8):763-770. doi: 10.1038/s41592-019-0471-2. Epub 2019 Jul 15.
Current techniques for monitoring GABA (γ-aminobutyric acid), the primary inhibitory neurotransmitter in vertebrates, cannot follow transients in intact neural circuits. To develop a GABA sensor, we applied the design principles used to create the fluorescent glutamate receptor iGluSnFR. We used a protein derived from a previously unsequenced Pseudomonas fluorescens strain and performed structure-guided mutagenesis and library screening to obtain intensity-based GABA sensing fluorescence reporter (iGABASnFR) variants. iGABASnFR is genetically encoded, detects GABA release evoked by electric stimulation of afferent fibers in acute brain slices and produces readily detectable fluorescence increases in vivo in mice and zebrafish. We applied iGABASnFR to track mitochondrial GABA content and its modulation by an anticonvulsant, swimming-evoked, GABA-mediated transmission in zebrafish cerebellum, GABA release events during interictal spikes and seizures in awake mice, and found that GABA-mediated tone decreases during isoflurane anesthesia.
目前用于监测 GABA（γ-氨基丁酸）的技术，脊椎动物中的主要抑制性神经递质，无法跟踪完整神经回路中的瞬变。为了开发 GABA 传感器，我们应用了用于创建荧光谷氨酸受体 iGluSnFR 的设计原则。我们使用了一种源自先前未测序的荧光假单胞菌菌株的蛋白质，并进行了结构导向的突变和文库筛选，以获得基于强度的 GABA 感应荧光报告（iGABASnFR）变体。iGABASnFR 是基因编码的，可检测急性脑切片中传入纤维电刺激引起的 GABA 释放，并在体内产生易于检测的荧光增加，在小鼠和斑马鱼中均如此。我们应用 iGABASnFR 来追踪线粒体 GABA 含量及其在抗惊厥药、游泳诱发的、斑马鱼小脑 GABA 介导的传递中的调制，在清醒小鼠的发作间期棘波和发作期间的 GABA 释放事件，发现异氟烷麻醉期间 GABA 介导的音调降低。
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