학술논문
A genetically encoded fluorescent sensor for in vivo imaging of GABA
Document Type
Report
Author
Source
Nature Methods. August 2019, Vol. 16 Issue 8, 763
Subject
Language
English
ISSN
1548-7091
Abstract
Author(s): Jonathan S. Marvin [sup.1] , Yoshiteru Shimoda [sup.2] , Vincent Magloire [sup.2] , Marco Leite [sup.2] , Takashi Kawashima [sup.1] , Thomas P. Jensen [sup.2] , Ilya Kolb [sup.1] [...]
Current techniques for monitoring GABA ([gamma]-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. The genetically encoded GABA sensor iGABASnFR allows visualizing GABA signaling in vivo. Its application is demonstrated in mouse slices, in the awake mouse and in behaving zebrafish.
Current techniques for monitoring GABA ([gamma]-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. The genetically encoded GABA sensor iGABASnFR allows visualizing GABA signaling in vivo. Its application is demonstrated in mouse slices, in the awake mouse and in behaving zebrafish.