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Monitoring hippocampal glycine with the computationally designed optical sensor GlyFS


Fluorescent sensors are an essential part of the experimental toolbox of the life sciences, where they are used ubiquitously to visualize intra- and extracellular signaling. In the brain, optical neurotransmitter sensors can shed light on temporal and spatial aspects of signal transmission by directly observing, for instance, neurotransmitter release and spread. Here we report the development and application of the first optical sensor for the amino acid glycine, which is both an inhibitory neurotransmitter and a co-agonist of the N-methyl-d-aspartate receptors (NMDARs) involved in synaptic plasticity. Computational design of a glycine-specific binding protein allowed us to produce the optical glycine FRET sensor (GlyFS), which can be used with single and two-photon excitation fluorescence microscopy. We took advantage of this newly developed sensor to test predictions about the uneven spatial distribution of glycine in extracellular space and to demonstrate that extracellular glycine levels are controlled by plasticity-inducing stimuli.

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Fig. 1: Design of the optical glycine FRET sensor (GlyFS).
Fig. 2: Characterization of GlyFS using two-photon excitation (2PE) fluorescence microscopy (λ2PE = 800 nm).
Fig. 3: Measuring extracellular glycine levels using GlyFS in hippocampal tissue.
Fig. 4: Extracellular glycine levels reported by GlyFS are lower at dendritic spines.
Fig. 5: GlyFS identifies activity patterns that control extracellular glycine levels.


  1. 1.

    Okumoto, S. et al. Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors. Proc. Natl. Acad. Sci. USA 102, 8740–8745 (2005).

    Article  CAS  Google Scholar 

  2. 2.

    Namiki, S., Sakamoto, H., Iinuma, S., Iino, M. & Hirose, K. Optical glutamate sensor for spatiotemporal analysis of synaptic transmission. Eur. J. Neurosci. 25, 2249–2259 (2007).

    Article  Google Scholar 

  3. 3.

    Marvin, J. S. et al. An optimized fluorescent probe for visualizing glutamate neurotransmission. Nat. Methods 10, 162–170 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

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

    Article  CAS  Google Scholar 

  5. 5.

    Betz, H. Glycine receptors: heterogeneous and widespread in the mammalian brain. Trends Neurosci. 14, 458–461 (1991).

    Article  CAS  Google Scholar 

  6. 6.

    Xu, T.-L. & Gong, N. Glycine and glycine receptor signaling in hippocampal neurons: diversity, function and regulation. Prog. Neurobiol. 91, 349–361 (2010).

    Article  CAS  Google Scholar 

  7. 7.

    Johnson, J. W. & Ascher, P. Glycine potentiates the NMDA response in cultured mouse brain neurons. Nature 325, 529–531 (1987).

    Article  CAS  Google Scholar 

  8. 8.

    Schell, M. J. The N-methyl d-aspartate receptor glycine site and d-serine metabolism: an evolutionary perspective. Philos. Trans. R. Soc. Lond. B 359, 943–964 (2004).

    Article  CAS  Google Scholar 

  9. 9.

    Citri, A. & Malenka, R. C. Synaptic plasticity: multiple forms, functions, and mechanisms. Neuropsychopharmacology 33, 18–41 (2008).

    Article  Google Scholar 

  10. 10.

    Nabavi, S. et al. Engineering a memory with LTD and LTP. Nature 511, 348–352 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Nong, Y. et al. Glycine binding primes NMDA receptor internalization. Nature 422, 302–307 (2003).

    Article  CAS  Google Scholar 

  12. 12.

    Ferreira, J. S. et al. Co-agonists differentially tune GluN2B-NMDA receptor trafficking at hippocampal synapses. Elife 6, e25492 (2017).

    Article  Google Scholar 

  13. 13.

    Henneberger, C., Papouin, T., Oliet, S. H. R. & Rusakov, D. A. Long-term potentiation depends on release of d-serine from astrocytes. Nature 463, 232–236 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Papouin, T. et al. Synaptic and extrasynaptic NMDA receptors are gated by different endogenous coagonists. Cell 150, 633–646 (2012).

    Article  CAS  Google Scholar 

  15. 15.

    Le Bail, M. et al. Identity of the NMDA receptor coagonist is synapse specific and developmentally regulated in the hippocampus. Proc. Natl. Acad. Sci. USA 112, E204–E213 (2015).

    Article  CAS  Google Scholar 

  16. 16.

    Chen, R.-Q. et al. Role of glycine receptors in glycine-induced LTD in hippocampal CA1 pyramidal neurons. Neuropsychopharmacology 36, 1948–1958 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Winkelmann, A. et al. Changes in neural network homeostasis trigger neuropsychiatric symptoms. J. Clin. Invest. 124, 696–711 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Hashimoto, A., Oka, T. & Nishikawa, T. Extracellular concentration of endogenous free d-serine in the rat brain as revealed by in vivo microdialysis. Neuroscience 66, 635–643 (1995).

    Article  CAS  Google Scholar 

  19. 19.

    Berntsson, R. P.-A., Smits, S. H. J., Schmitt, L., Slotboom, D.-J. & Poolman, B. A structural classification of substrate-binding proteins. FEBS Lett. 584, 2606–2617 (2010).

    Article  CAS  Google Scholar 

  20. 20.

    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  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Van Durme, J. et al. A graphical interface for the FoldX forcefield. Bioinformatics 27, 1711–1712 (2011).

    Article  CAS  Google Scholar 

  22. 22.

    Morris, G. M. et al. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J. Comput. Chem. 30, 2785–2791 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Bajar, B. T., Wang, E. S., Zhang, S., Lin, M. Z. & Chu, J. A guide to fluorescent protein FRET pairs. Sensors (Basel) 16, 1488 (2016).

    Google Scholar 

  24. 24.

    Whitfield, J. H. et al. Construction of a robust and sensitive arginine biosensor through ancestral protein reconstruction. Protein Sci. 24, 1412–1422 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Deuschle, K. et al. Construction and optimization of a family of genetically encoded metabolite sensors by semirational protein engineering. Protein Sci. 14, 2304–2314 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Fritz, R. D. et al. A versatile toolkit to produce sensitive FRET biosensors to visualize signaling in time and space. Sci. Signal. 6, rs12 (2013).

    Article  CAS  Google Scholar 

  27. 27.

    Chen, X., Zaro, J. L. & Shen, W.-C. Fusion protein linkers: property, design and functionality. Adv. Drug Deliv. Rev. 65, 1357–1369 (2013).

    Article  CAS  Google Scholar 

  28. 28.

    Piston, D. W. & Kremers, G.-J. Fluorescent protein FRET: the good, the bad and the ugly. Trends Biochem. Sci. 32, 407–414 (2007).

    Article  CAS  Google Scholar 

  29. 29.

    Hamberger, A. & Nyström, B. Extra- and intracellular amino acids in the hippocampus during development of hepatic encephalopathy. Neurochem. Res. 9, 1181–1192 (1984).

    Article  CAS  Google Scholar 

  30. 30.

    Clifton, B. E. & Jackson, C. J. Ancestral protein reconstruction yields insights into adaptive evolution of binding specificity in solute-binding proteins. Cell Chem. Biol. 23, 236–245 (2016).

    Article  CAS  Google Scholar 

  31. 31.

    Okubo, Y. et al. Imaging extrasynaptic glutamate dynamics in the brain. Proc. Natl. Acad. Sci. USA 107, 6526–6531 (2010).

    Article  Google Scholar 

  32. 32.

    Ermolyuk, Y. S. et al. Independent regulation of basal neurotransmitter release efficacy by variable Ca2+influx and bouton size at small central synapses. PLoS Biol. 10, e1001396 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Maravall, M., Mainen, Z. F., Sabatini, B. L. & Svoboda, K. Estimating intracellular calcium concentrations and buffering without wavelength ratioing. Biophys. J. 78, 2655–2667 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Roux, M. J. & Supplisson, S. Neuronal and glial glycine transporters have different stoichiometries. Neuron 25, 373–383 (2000).

    Article  CAS  Google Scholar 

  35. 35.

    Tinberg, C. E. & Khare, S. D. Computational Protein Design. 363–373 (Humana Press, New York, NY, 2017).

    Google Scholar 

  36. 36.

    Tannous, B. A. et al. Metabolic biotinylation of cell surface receptors for in vivo imaging. Nat. Methods 3, 391–396 (2006).

    Article  CAS  Google Scholar 

  37. 37.

    Panatier, A. et al. Glia-derived d-serine controls NMDA receptor activity and synaptic memory. Cell 125, 775–784 (2006).

    Article  CAS  Google Scholar 

  38. 38.

    Horio, M. et al. Levels of d-serine in the brain and peripheral organs of serine racemase (Srr) knock-out mice. Neurochem. Int. 59, 853–859 (2011).

    Article  CAS  Google Scholar 

  39. 39.

    Matsui, T. et al. Functional comparison of d-serine and glycine in rodents: the effect on cloned NMDA receptors and the extracellular concentration. J. Neurochem. 65, 454–458 (1995).

    Article  CAS  Google Scholar 

  40. 40.

    Tønnesen, J., Inavalli, V. V. G. K. & Nägerl, U. V. Super-resolution imaging of the extracellular space in living brain tissue. Cell 172, 1108–1121.e15 (2018).

    Article  CAS  Google Scholar 

  41. 41.

    Rusakov, D. A. & Kullmann, D. M. Extrasynaptic glutamate diffusion in the hippocampus: ultrastructural constraints, uptake, and receptor activation. J. Neurosci. 18, 3158–3170 (1998).

    Article  CAS  Google Scholar 

  42. 42.

    Bethge, P., Chéreau, R., Avignone, E., Marsicano, G. & Nägerl, U. V. Two-photon excitation STED microscopy in two colors in acute brain slices. Biophys. J. 104, 778–785 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Cubelos, B., Giménez, C. & Zafra, F. Localization of the GLYT1 glycine transporter at glutamatergic synapses in the rat brain. Cereb. Cortex 15, 448–459 (2005).

    Article  Google Scholar 

  44. 44.

    Bergeron, R., Meyer, T. M., Coyle, J. T. & Greene, R. W. Modulation of N-methyl-d-aspartate receptor function by glycine transport. Proc. Natl Acad. Sci. USA 95, 15730–15734 (1998).

    Article  CAS  Google Scholar 

  45. 45.

    Martina, M. et al. Glycine transporter type 1 blockade changes NMDA receptor-mediated responses and LTP in hippocampal CA1 pyramidal cells by altering extracellular glycine levels. J. Physiol. (Lond.) 557, 489–500 (2004).

    Article  CAS  Google Scholar 

  46. 46.

    Danglot, L., Rostaing, P., Triller, A. & Bessis, A. Morphologically identified glycinergic synapses in the hippocampus. Mol. Cell. Neurosci. 27, 394–403 (2004).

    Article  CAS  Google Scholar 

  47. 47.

    Langer, J. & Rose, C. R. Synaptically induced sodium signals in hippocampal astrocytes in situ. J. Physiol. (Lond.) 587, 5859–5877 (2009).

    Article  CAS  Google Scholar 

  48. 48.

    Chen, P. E. et al. Modulation of glycine potency in rat recombinant NMDA receptors containing chimeric NR2A/2D subunits expressed in Xenopus laevis oocytes. J. Physiol. (Lond.) 586, 227–245 (2008).

    Article  CAS  Google Scholar 

  49. 49.

    Le Meur, K., Galante, M., Angulo, M. C. & Audinat, E. Tonic activation of NMDA receptors by ambient glutamate of non-synaptic origin in the rat hippocampus. J. Physiol. (Lond.) 580, 373–383 (2007).

    Article  CAS  Google Scholar 

  50. 50.

    Zhang, L.-H., Gong, N., Fei, D., Xu, L. & Xu, T.-L. Glycine uptake regulates hippocampal network activity via glycine receptor-mediated tonic inhibition. Neuropsychopharmacology 33, 701–711 (2008).

    Article  CAS  Google Scholar 

  51. 51.

    Woitecki, A. M. H. et al. Identification of synaptotagmin 10 as effector of NPAS4-mediated protection from excitotoxic neurodegeneration. J. Neurosci. 36, 2561–2570 (2016).

    Article  CAS  Google Scholar 

  52. 52.

    Anders, S. et al. Spatial properties of astrocyte gap junction coupling in the rat hippocampus. Philos. Trans. R. Soc. Lond. B 369, 20130600 (2014).

    Article  CAS  Google Scholar 

  53. 53.

    Ortinski, P. I. et al. Selective induction of astrocytic gliosis generates deficits in neuronal inhibition. Nat. Neurosci. 13, 584–591 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Minge, D. et al. Heparan sulfates support pyramidal cell excitability, synaptic plasticity, and context discrimination. Cereb. Cortex 27, 903–918 (2017).

    PubMed  PubMed Central  Google Scholar 

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We thank Dr. O'Mara (Australian National University) for helpful discussions. Research was funded by the Human Frontiers Science Program Young Investigator Award (HFSP to H.J., C.H., and C.J.J.; grant number: RGY0084/2012), German Academic Exchange Service (DAAD-Go8) Travel Fellowship (to C.H. and C.J.J.), NRW-Rückkehrerprogramm (to C.H.), the European Union (ITN EU-GliaPhD) and German Research Foundation (DFG, SFB1089 B03, SPP1757 HE6949/1 and HE6949/3, to C.H.).

Author information




W.H.Z., J.A.M., V.V., J.H.W. and C.J.J. designed, produced and analyzed the sensor. M.K.H., J.H.W., A.B.W., W.H.Z., D.M., B.B. and C.H. performed and analyzed all experiments using two-photon excitation and electrophysiology in acute brain slices. M.K.H., J.H.W., I.S.-R., P.E.G., H.J., S.S. and A.B.W. performed studies on GlyFS expressed by cells. C.H., C.J.J. and H.J. designed the study. C.H., C.J.J. and W.H.Z. wrote the initial manuscript, to which all authors subsequently contributed.

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Correspondence to Colin J. Jackson or Christian Henneberger.

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Zhang, W.H., Herde, M.K., Mitchell, J.A. et al. Monitoring hippocampal glycine with the computationally designed optical sensor GlyFS. Nat Chem Biol 14, 861–869 (2018).

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