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An optimized fluorescent probe for visualizing glutamate neurotransmission

Abstract

We describe an intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) with signal-to-noise ratio and kinetics appropriate for in vivo imaging. We engineered iGluSnFR in vitro to maximize its fluorescence change, and we validated its utility for visualizing glutamate release by neurons and astrocytes in increasingly intact neurological systems. In hippocampal culture, iGluSnFR detected single field stimulus–evoked glutamate release events. In pyramidal neurons in acute brain slices, glutamate uncaging at single spines showed that iGluSnFR responds robustly and specifically to glutamate in situ, and responses correlate with voltage changes. In mouse retina, iGluSnFR-expressing neurons showed intact light-evoked excitatory currents, and the sensor revealed tonic glutamate signaling in response to light stimuli. In worms, glutamate signals preceded and predicted postsynaptic calcium transients. In zebrafish, iGluSnFR revealed spatial organization of direction-selective synaptic activity in the optic tectum. Finally, in mouse forelimb motor cortex, iGluSnFR expression in layer V pyramidal neurons revealed task-dependent single-spine activity during running.

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Figure 1: Sensor development and in vitro characterization.
Figure 2: Characterization of iGluSnFR in neuron-astrocyte coculture.
Figure 3: Two-photon glutamate uncaging–evoked iGluSnFR signals in acute hippocampal slices.
Figure 4: Imaging glutamate in mouse retina in vitro.
Figure 5: Glutamatergic input into C. elegans AVA neurons and resulting somatic [Ca2+] signal.
Figure 6: In vivo imaging of awake behavior and motor task–associated glutamate transients in mouse primary motor cortex.

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References

  1. 1

    Kullmann, D.M. & Asztely, F. Extrasynaptic glutamate spillover in the hippocampus: evidence and implications. Trends Neurosci. 21, 8–14 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

    Haydon, P.G. GLIA: listening and talking to the synapse. Nat. Rev. Neurosci. 2, 185–193 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

    Nedergaard, M., Ransom, B. & Goldman, S.A. New roles for astrocytes: redefining the functional architecture of the brain. Trends Neurosci. 26, 523–530 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Choi, D.W. Excitotoxic cell death. J. Neurobiol. 23, 1261–1276 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Parsons, C.G., Danysz, W. & Quack, G. Glutamate in CNS disorders as a target for drug development: an update. Drug News Perspect. 11, 523–569 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Benveniste, H., Drejer, J., Schousboe, A. & Diemer, N.H. Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J. Neurochem. 43, 1369–1374 (1984).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Innocenti, B., Parpura, V. & Haydon, P.G. Imaging extracellular waves of glutamate during calcium signaling in cultured astrocytes. J. Neurosci. 20, 1800–1808 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Hu, Y., Mitchell, K.M., Albahadily, F.N., Michaelis, E.K. & Wilson, G.S. Direct measurement of glutamate release in the brain using a dual enzyme-based electrochemical sensor. Brain Res. 659, 117–125 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Kerr, J.N. & Denk, W. Imaging in vivo: watching the brain in action. Nat. Rev. Neurosci. 9, 195–205 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    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  PubMed  PubMed Central  Google Scholar 

  11. 11

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

    Article  PubMed  PubMed Central  Google Scholar 

  12. 12

    Okumoto, S. Imaging approach for monitoring cellular metabolites and ions using genetically encoded biosensors. Curr. Opin. Biotechnol. 21, 45–54 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Tian, L. et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat. Methods 6, 875–881 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 14

    Frommer, W.B., Davidson, M.W. & Campbell, R.E. Genetically encoded biosensors based on engineered fluorescent proteins. Chem. Soc. Rev. 38, 2833–2841 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    de Lorimier, R.M. et al. Construction of a fluorescent biosensor family. Protein Sci. 11, 2655–2675 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    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 

  17. 17

    Hires, S.A., Zhu, Y. & Tsien, R.Y. Optical measurement of synaptic glutamate spillover and reuptake by linker optimized glutamate-sensitive fluorescent reporters. Proc. Natl. Acad. Sci. USA 105, 4411–4416 (2008).

    Article  CAS  Google Scholar 

  18. 18

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Alicea, I. et al. Structure of the Escherichia coli phosphonate binding protein PhnD and rationally optimized phosphonate biosensors. J. Mol. Biol. 414, 356–369 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Hu, Y. et al. Crystal structure of a glutamate/aspartate binding protein complexed with a glutamate molecule: structural basis of ligand specificity at atomic resolution. J. Mol. Biol. 382, 99–111 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Nadler, J.V. Aspartate release and signalling in the hippocampus. Neurochem. Res. 36, 668–676 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Clements, J.D. Transmitter timecourse in the synaptic cleft: its role in central synaptic function. Trends Neurosci. 19, 163–171 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Losonczy, A. & Magee, J.C. Integrative properties of radial oblique dendrites in hippocampal CA1 pyramidal neurons. Neuron 50, 291–307 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    Miller, R.F. Cell communication mechanisms in the vertebrate retina the proctor lecture. Invest. Ophthalmol. Vis. Sci. 49, 5184–5198 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  25. 25

    Margolis, D.J. & Detwiler, P.B. Different mechanisms generate maintained activity in ON and OFF retinal ganglion cells. J. Neurosci. 27, 5994–6005 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Shimamoto, K. et al. dl-threo-β-benzyloxyaspartate, a potent blocker of excitatory amino acid transporters. Mol. Pharmacol. 53, 195–201 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Berntson, A. & Taylor, W.R. Response characteristics and receptive field widths of on-bipolar cells in the mouse retina. J. Physiol. (Lond.) 524, 879–889 (2000).

    Article  CAS  Google Scholar 

  28. 28

    Schwartz, G.W. et al. The spatial structure of a nonlinear receptive field. Nat. Neurosci. 15, 1572–1580 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Chalfie, M. et al. The neural circuit for touch sensitivity in Caenorhabditis elegans. J. Neurosci. 5, 956–964 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    White, J.G., Southgate, E., Thomson, J.N. & Brenner, S. The structure of the nervous system of the nematode Caenorhabditis elegans. Phil. Trans. R. Soc. Lond. B 314, 1–340 (1986).

    Article  CAS  Google Scholar 

  31. 31

    Mellem, J.E., Brockie, P.J., Zheng, Y., Madsen, D.M. & Maricg, A.V. Decoding of polymodal sensory stimuli by postsynaptic glutamate receptors in C. elegans. Neuron 36, 933–944 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Brockie, P.J., Madsen, D.M., Zheng, Y., Mellem, J. & Maricg, A.V. Differential expression of glutamate receptor subunits in the nervous system of Caenorhabditis elegans and their regulation by the homeodomain protein UNC-42. J. Neurosci. 21, 1510–1522 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Husson, S.J. et al. Optogenetic analysis of a nociceptor neuron and network reveals ion channels acting downstream of primary sensors. Curr. Biol. 22, 743–752 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Akerboom, J. et al. Optimization of a GCaMP calcium indicator for neural activity imaging. J. Neurosci. 32, 13819–13840 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Yang, G., Pan, F. & Gan, W.B. Stably maintained dendritic spines are associated with lifelong memories. Nature 462, 920–924 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Albrecht, J. et al. Extracellular concentrations of taurine, glutamate, and aspartate in the cerebral cortex of rats at the asymptomatic stage of thioacetamide-induced hepatic failure: modulation by ketamine anesthesia. Neurochem. Res. 25, 1497–1502 (2000).

    Article  CAS  Google Scholar 

  37. 37

    Forde, B.G. & Lea, P.J. Glutamate in plants: metabolism, regulation, and signalling. J. Exp. Bot. 58, 2339–2358 (2007).

    Article  CAS  Google Scholar 

  38. 38

    Sano, C. History of glutamate production. Am. J. Clin. Nutr. 90, 728S–732S (2009).

    Article  CAS  Google Scholar 

  39. 39

    Nguyen, Q.T. et al. An in vivo biosensor for neurotransmitter release and in situ receptor activity. Nat. Neurosci. 13, 127–132 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Singer, J.H. & Diamond, J.S. Vesicle depletion and synaptic depression at a mammalian ribbon synapse. J. Neurophysiol. 95, 3191–3198 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. 41

    Moussawi, K., Riegel, A., Nair, S. & Kalivas, P.W. Extracellular glutamate: functional compartments operate in different concentration ranges. Front. Syst. Neurosci. 5, 94 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    Crowe, J. et al. 6xHis-Ni-NTA chromatography as a superior technique in recombinant protein expression/purification. Methods Mol. Biol. 31, 371–387 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Harnett, M.T., Makara, J.K., Spruston, N., Kath, W.L. & Magee, J.C. Synaptic amplification by dendritic spines enhances input cooperativity. Nature 491, 599–602 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Magee, J.C. Dendritic hyperpolarization-activated currents modify the integrative properties of hippocampal CA1 pyramidal neurons. J. Neurosci. 18, 7613–7624 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    Ji, N., Magee, J.C. & Betzig, E. High-speed, low-photodamage nonlinear imaging using passive pulse splitters. Nat. Methods 5, 197–202 (2008).

    Article  CAS  Google Scholar 

  47. 47

    Borghuis, B.G. et al. Imaging light responses of targeted neuron populations in the rodent retina. J. Neurosci. 31, 2855–2867 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    Pologruto, T.A., Sabatini, B.L. & Svoboda, K. ScanImage: flexible software for operating laser scanning microscopes. Biomed. Eng. Online 2, 13 (2003).

    Article  PubMed  PubMed Central  Google Scholar 

  49. 49

    Wang, Y.V., Weick, M. & Demb, J.B. Spectral and temporal sensitivity of cone-mediated responses in mouse retinal ganglion cells. J. Neurosci. 31, 7670–7681 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Chalasani, S.H. et al. Dissecting a circuit for olfactory behaviour in Caenorhabditis elegans. Nature 450, 63–70 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Lister, J.A., Robertson, C.P., Lepage, T., Johnson, S.L. & Raible, D.W. nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate. Development 126, 3757–3767 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52

    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  PubMed  PubMed Central  Google Scholar 

  53. 53

    Westerfield, M . The Zebrafish Book 2nd edn. (University of Oregon Press, Eugene, Oregon, USA, 1994).

  54. 54

    Asakawa, K. & Kawakami, K. Targeted gene expression by the Gal4-UAS system in zebrafish. Dev. Growth Differ. 50, 391–399 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. 55

    Akitake, C.M., Macurak, M., Halpern, M.E. & Goll, M.G. Transgenerational analysis of transcriptional silencing in zebrafish. Dev. Biol. 352, 191–201 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. 56

    Tennant, K.A. et al. The organization of the forelimb representation of the C57BL/6 mouse motor cortex as defined by intracortical microstimulation and cytoarchitecture. Cereb. Cortex 21, 865–876 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  57. 57

    Yang, G., Pan, F., Parkhurst, C.N., Grutzendler, J. & Gan, W.B. Thinned-skull cranial window technique for long-term imaging of the cortex in live mice. Nat. Protoc. 5, 201–208 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank J. Macklin for two-photon spectrophotometry and two-photon lifetime measurements of purified proteins; H. White and S. Winfried for tissue culture; B. Shields and A. Hu for mouse brain dissection and neuronal culture; Molecular Biology and Media Prep Shared Resources for DNA preparation and sequencing and for media preparation; M. Ramirez and K. Ritola for virus production; D. Kim and the GECI Project for advice and use of the neuronal culture rig; and K. Svoboda, J. Magee and A. Hantman for helpful conversations. All affiliations are HHMI Janelia Farm. HHMI supported this work.

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J.S.M. and L.L.L. conceived of the project. J.S.M. designed iGluSnFR and performed in vitro and cultured cell characterization; L.T. characterized cultured neurons and astrocytes; M.T.H. performed glutamate uncaging experiments; B.G.B. and J.B.D. characterized retina; A.G. and C.I.B. characterized worms; S.L.R. and M.B.O. characterized zebrafish; J.C. and W.-B.G. characterized mouse motor cortex; J.A. and E.R.S. provided RCaMP1e; T.-W.C. assisted with data analysis; S.A.H. and L.L.L. provided global perspective and planning of experiments across species.

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Correspondence to Loren L Looger.

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B.G.B. owns Borghuis Instruments, which manufactures and sells the specialized syringe that was used for intraocular virus injections in this study.

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Marvin, J., Borghuis, B., Tian, L. et al. An optimized fluorescent probe for visualizing glutamate neurotransmission. Nat Methods 10, 162–170 (2013). https://doi.org/10.1038/nmeth.2333

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