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A genetically encoded fluorescent sensor for in vivo imaging of GABA

Nature Methods volume 16, pages 763–770 (2019)Cite this article

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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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Fig. 1: Sensor design and characterization in neuronal culture.
Fig. 2: Recording of stimulus-evoked extracellular GABA transients using iGABASnFR transfected acute brain slices.
Fig. 3: iGABASnFR response during interictal spiking.
Fig. 4: GABA sensor behavior during polyspikes and seizures.
Fig. 5: GABA response measured with iGABASnFR.F102Y.Y137L fluorescence in a fictive model of swimming in zebrafish larvae.

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Data availability

All data from this study are available upon request. All constructs have been deposited at Addgene (112159–112180). Sequences have been deposited in GenBank(MH392466, MH392467 and MH392468). Protein structure has been uploaded to the Protein Data Bank (6DGV). AAV virus is available from Addgene.

Code availability

All analysis code used in this study is available upon request.

References

  1. Fritschy, J.-M. & Panzanelli, P. GABAA receptors and plasticity of inhibitory neurotransmission in the central nervous system. Eur. J. Neurosci. 39, 1845–1865 (2014).

    Article  Google Scholar 

  2. Padgett, C. L. & Slesinger, P. A. GABAB receptor coupling to G-proteins and ion channels. Adv. Pharm 58, 123–147 (2010).

    Article  CAS  Google Scholar 

  3. Sodickson, D. L. & Bean, B. P. GABAB receptor-activated inwardly rectifying potassium current in dissociated hippocampal CA3 neurons. J. Neurosci. 16, 6374–6385 (1996).

    Article  CAS  Google Scholar 

  4. Macdonald, R. L. & Olsen, R. W. GABAA receptor channels. Annu. Rev. Neurosci. 17, 569–602 (1994).

    Article  CAS  Google Scholar 

  5. van der Zeyden, M., Oldenziel, W. H., Rea, K., Cremers, T. I. & Westerink, B. H. Microdialysis of GABA and glutamate: analysis, interpretation and comparison with microsensors. Pharmacol. Biochem. Behav. 90, 135–147 (2008).

    Article  Google Scholar 

  6. Dana, H. et al. Sensitive red protein calcium indicators for imaging neural activity. eLife 5, 413 (2016).

    Article  Google Scholar 

  7. Chen, T.-W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).

    Article  CAS  Google Scholar 

  8. Dana, H. et al. High-performance GFP-based calcium indicators for imaging activity in neuronal populations and microcompartments. Nat. Methods https://doi.org/10.1038/s41592-019-0435-6 (2019).

    Article  CAS  Google Scholar 

  9. Marvin, J. S. et al. Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR. Nat. Methods 15, 936–939 (2018).

    Article  CAS  Google Scholar 

  10. Masharina, A., Reymond, L., Maurel, D., Umezawa, K. & Johnsson, K. A fluorescent sensor for GABA and synthetic GABAB receptor ligands. J. Am. Chem. Soc. 134, 19026–19034 (2012).

    Article  CAS  Google Scholar 

  11. Grimley, J. S. et al. Visualization of synaptic inhibition with an optogenetic sensor developed by cell-free protein engineering automation. J. Neurosci. 33, 16297–16309 (2013).

    Article  CAS  Google Scholar 

  12. Jensen, T. P., Zheng, K., Tyurikova, O., Reynolds, J. P. & Rusakov, D. A. Monitoring single-synapse glutamate release and presynaptic calcium concentration in organised brain tissue. Cell Calcium 64, 102–108 (2017).

    Article  CAS  Google Scholar 

  13. Jensen, T. P. et al. Multiplex imaging relates quantal glutamate release to presynaptic Ca2+ homeostasis at multiple synapses in situ. Nat. Commun. 10, 1414–1414 (2019).

    Article  Google Scholar 

  14. Marvin, J. S., Schreiter, E. R., Echevarría, I. M. & Looger, L. L. A genetically encoded, high-signal-to-noise maltose sensor. Proteins 79, 3025–3036 (2011).

    Article  CAS  Google Scholar 

  15. Geng, Y. et al. Structure and functional interaction of the extracellular domain of human GABA(B) receptor GBR2. Nat. Neurosci. 15, 970–978 (2012).

    Article  CAS  Google Scholar 

  16. Planamente, S. et al. A conserved mechanism of GABA binding and antagonism is revealed by structure-function analysis of the periplasmic binding protein Atu2422 in Agrobacterium tumefaciens. J. Biol. Chem. 285, 30294–30303 (2010).

    Article  CAS  Google Scholar 

  17. Planamente, S. et al. Structural basis for selective GABA binding in bacterial pathogens. Mol. Microbiol. 86, 1085–1099 (2012).

    Article  CAS  Google Scholar 

  18. Guthrie, G. D. & Nicholson-Guthrie, C. S. Gamma-Aminobutyric acid uptake by a bacterial system with neurotransmitter binding characteristics. Proc. Natl Acad. Sci. USA 86, 7378–7381 (1989).

    Article  CAS  Google Scholar 

  19. Marvin, J. S. et al. The rational design of allosteric interactions in a monomeric protein and its applications to the construction of biosensors. Proc. Natl Acad. Sci. USA 94, 4366–4371 (1997).

    Article  CAS  Google Scholar 

  20. Grimm, J. B. et al. A general method to fine-tune fluorophores for live-cell and in vivo imaging. Nat. Methods 14, 987–994 (2017).

    Article  CAS  Google Scholar 

  21. Marvin, J. S. & Hellinga, H. W. Manipulation of ligand binding affinity by exploitation of conformational coupling. Nat. Struct. Biol. 8, 795–798 (2001).

    Article  CAS  Google Scholar 

  22. Engel, D. et al. Plasticity of rat central inhibitory synapses through GABA metabolism. J. Physiol. 535, 473–482 (2001).

    Article  CAS  Google Scholar 

  23. Brunton, L., Chabner, B. & Knollman, B. Goodman and Gilman’s The Pharmacological Basis of Therapeutics 12th edn (McGraw Hill Professional, 2011).

  24. Putluri, N. et al. Metabolomic profiling reveals a role for androgen in activating amino acid metabolism and methylation in prostate cancer cells. PLoS ONE 6, e21417 (2011).

    Article  CAS  Google Scholar 

  25. Benson, D. L., Watkins, F. H., Steward, O. & Banker, G. Characterization of GABAergic neurons in hippocampal cell cultures. J. Neurocytol. 23, 279–295 (1994).

    Article  CAS  Google Scholar 

  26. Dodge, F. A. & Rahamimoff, R. Co-operative action of calcium ions in transmitter release at the neuromuscular junction. J. Physiol. 193, 419–432 (1967).

    Article  CAS  Google Scholar 

  27. Adesnik, H., Bruns, W., Taniguchi, H., Huang, Z. J. & Scanziani, M. A neural circuit for spatial summation in visual cortex. Nature 490, 226–231 (2012).

    Article  CAS  Google Scholar 

  28. Zhang, S. et al. Selective attention. Long-range and local circuits for top-down modulation of visual cortex processing. Science 345, 660–665 (2014).

    Article  CAS  Google Scholar 

  29. Nelson, L. E. et al. The sedative component of anesthesia is mediated by GABA(A) receptors in an endogenous sleep pathway. Nat. Neurosci. 5, 979–984 (2002).

    Article  CAS  Google Scholar 

  30. Westphalen, R. I. & Hemmings, H. C. Selective depression by general anesthetics of glutamate versus GABA release from isolated cortical nerve terminals. J. Pharmacol. Exp. Ther. 304, 1188–1196 (2003).

    Article  CAS  Google Scholar 

  31. Farrant, M. & Nusser, Z. Variations on an inhibitory theme: phasic and tonic activation of GABA(A) receptors. Nat. Rev. Neurosci. 6, 215–229 (2005).

    Article  CAS  Google Scholar 

  32. Cohen, I., Navarro, V., Clemenceau, S., Baulac, M. & Miles, R. On the origin of interictal activity in human temporal lobe epilepsy in vitro. Science 298, 1418–1421 (2002).

    Article  CAS  Google Scholar 

  33. Trevelyan, A. J., Sussillo, D. & Yuste, R. Feedforward inhibition contributes to the control of epileptiform propagation speed. J. Neurosci. 27, 3383–3387 (2007).

    Article  CAS  Google Scholar 

  34. Schevon, C. A. et al. Evidence of an inhibitory restraint of seizure activity in humans. Nat. Commun. 3, 1060 (2012).

    Article  Google Scholar 

  35. Magloire, V., Mercier, M. S., Kullmann, D. M. & Pavlov, I. GABAergic interneurons in seizures: investigating causality with optogenetics. Neuroscientist https://doi.org/10.1177/1073858418805002 (2018).

  36. Kätzel, D., Nicholson, E., Schorge, S., Walker, M. C. & Kullmann, D. M. Chemical-genetic attenuation of focal neocortical seizures. Nat. Commun. 5, 3847 (2014).

    Article  Google Scholar 

  37. Ziemann, A. E. et al. Seizure termination by acidosis depends on ASIC1a. Nat. Neurosci. 11, 816–822 (2008).

    Article  CAS  Google Scholar 

  38. Raimondo, J. V. et al. Tight coupling of astrocyte pH dynamics to epileptiform activity revealed by genetically encoded pH sensors. J. Neurosci. 36, 7002–7013 (2016).

    Article  CAS  Google Scholar 

  39. Ahrens, M. B. et al. Brain-wide neuronal dynamics during motor adaptation in zebrafish. Nature 485, 471–477 (2012).

    Article  CAS  Google Scholar 

  40. Scalise, K., Shimizu, T., Hibi, M. & Sawtell, N. B. Responses of cerebellar Purkinje cells during fictive optomotor behavior in larval zebrafish. J. Neurophysiol. 116, 2067–2080 (2016).

    Article  Google Scholar 

  41. Tanabe, K. et al. Atypical protein kinase C regulates primary dendrite specification of cerebellar Purkinje cells by localizing Golgi apparatus. J. Neurosci. 30, 16983–16992 (2010).

    Article  CAS  Google Scholar 

  42. Wu, J. et al. Genetically encoded glutamate indicators with altered color and topology. ACS Chem. Biol. 13, 1832–1837 (2018).

    Article  CAS  Google Scholar 

  43. Somogyi, P. & Klausberger, T. Defined types of cortical interneurone structure space and spike timing in the hippocampus. J. Physiol. 562, 9–26 (2005).

    Article  CAS  Google Scholar 

  44. Ohno-Shosaku, T. et al. Presynaptic cannabinoid sensitivity is a major determinant of depolarization-induced retrograde suppression at hippocampal synapses. J. Neurosci. 22, 3864–3872 (2002).

    Article  CAS  Google Scholar 

  45. Jo, S. et al. GABA from reactive astrocytes impairs memory in mouse models of Alzheimer’s disease. Nat. Med 20, 886–896 (2014).

    Article  CAS  Google Scholar 

  46. Emir, U. E., Tuite, P. J. & Öz, G. Elevated pontine and putamenal GABA levels in mild-moderate Parkinson disease detected by 7 Tesla proton MRS. PLoS ONE 7, e30918 (2012).

    Article  CAS  Google Scholar 

  47. Raymond, L. A. et al. Pathophysiology of Huntington’s disease: time-dependent alterations in synaptic and receptor function. Neuroscience 198, 252–273 (2011).

    Article  CAS  Google Scholar 

  48. Simpson, M. D., Slater, P., Deakin, J. F., Royston, M. C. & Skan, W. J. Reduced GABA uptake sites in the temporal lobe in schizophrenia. Neurosci. Lett. 107, 211–215 (1989).

    Article  CAS  Google Scholar 

  49. Chao, H.-T. et al. Dysfunction in GABA signalling mediates autism-like stereotypies and Rett syndrome phenotypes. Nature 468, 263–269 (2010).

    Article  CAS  Google Scholar 

  50. Kunkel, T. A., Bebenek, K. & McClary, J. Efficient site-directed mutagenesis using uracil-containing DNA. Meth. Enzym. 204, 125–139 (1991).

    Article  CAS  Google Scholar 

  51. Studier, F. W. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005).

    Article  CAS  Google Scholar 

  52. Rama, S. Shift and mean algorithm for functional imaging with high spatio-temporal resolution. Front. Cell. Neurosci. 9, 446 (2015).

    Article  Google Scholar 

  53. Vladimirov, N. et al. Light-sheet functional imaging in fictively behaving zebrafish. Nat. Methods 11, 883–884 (2014).

    Article  CAS  Google Scholar 

  54. White, R. M. et al. Transparent adult zebrafish as a tool for in vivo transplantation analysis. Cell Stem Cell 2, 183–189 (2008).

    Article  CAS  Google Scholar 

  55. Kawakami, K. et al. A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish. Dev. Cell 7, 133–144 (2004).

    Article  CAS  Google Scholar 

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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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Authors and Affiliations

  1. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA

    Jonathan S. Marvin, Takashi Kawashima, Ilya Kolb, Ondrej Novak, Kaspar Podgorski, Misha B. Ahrens & Loren L. Looger

  2. UCL Queen Square Institute of Neurology, University College London, London, UK

    Yoshiteru Shimoda, Vincent Magloire, Marco Leite, Thomas P. Jensen, Dmitri A. Rusakov & Dimitri M. Kullmann

  3. Louisiana State University Health Sciences Center, Shreveport, LA, USA

    Erika L. Knott & Nancy J. Leidenheimer

  4. Second Medical Faculty, Charles University, Prague, Czech Republic

    Ondrej Novak

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Contributions

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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Correspondence to Loren L. Looger.

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The authors declare no competing interests.

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Peer review information: Nina Vogt was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Supplementary Figures 1–22

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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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