Abstract
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.
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Acknowledgements
We would like to thank Catherine S. Nicholson-Guthrie (Indiana University) for the gift of the Pseudomonas fluorescens strain CNG89; D. Stern, S. Picard, A. Lemire and D. Kao for helping sequence the genome of CNG89; D.Walpita for rat neuronal culture; J. Macklin and R. Patel for collecting two-photon spectra; and A. Abdelfattah and C.-L. Hsu for assistance with brain slice recordings. Y.S., M.L. and D.M.K. are supported by the Medical Research Council (grant no. MR/L01095X/1) and Wellcome Trust (grant nos. 095580/Z/11/Z and 212285/Z/18/Z). V.M. is supported by Epilepsy Research UK (grant no. P1702). T.P.J. and D.A.R. are supported by the Wellcome Trust Principal Fellowship (no. 212251/Z/18/Z) and European Research Council Advanced Grant (no. 323113). M.A. is supported by Simons Collaboration on the Global Brain Research Awards 325171 and 542943SPI. This work was supported by the Howard Hughes Medical Institute.
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J.S.M. and L.L.L. were responsible for protein engineering and experimental design and led the project. T.P.J. and D.A.R. performed hippocampal slice imaging. I.K. was responsible for somatosensory slice electrophysiology. K.P. and O.N. performed visual cortex volume imaging. Y.S., V.M., M.L. and D.M.K. produced the mouse epilepsy model. E.L.K. and N.J.L performed mitochondrial experiments. T.K. and M.B.A. designed and performed the zebrafish experiments.
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Supplementary Video 1
Light-sheet imaging of zebrafish cerebellum during fictive swimming. Companion video to Fig. 5. Fluorescence in the region indicated by the arrow changes during bouts of swimming.
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Marvin, J.S., Shimoda, Y., Magloire, V. et al. A genetically encoded fluorescent sensor for in vivo imaging of GABA. Nat Methods 16, 763–770 (2019). https://doi.org/10.1038/s41592-019-0471-2
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DOI: https://doi.org/10.1038/s41592-019-0471-2
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