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ABSTRACT The strength of an excitatory synapse depends on its ability to release glutamate and on the density of postsynaptic receptors. Genetically encoded glutamate indicators (GEGIs)

allow eavesdropping on synaptic transmission at the level of cleft glutamate to investigate properties of the release machinery in detail. Based on the sensor iGluSnFR, we recently developed

accelerated versions of GEGIs that allow investigation of synaptic release during 100-Hz trains. Here, we describe the detailed procedures for design and characterization of fast iGluSnFR

variants in vitro, transfection of pyramidal cells in organotypic hippocampal cultures, and imaging of evoked glutamate transients with two-photon laser-scanning microscopy. As the released

glutamate spreads from a point source—the fusing vesicle—it is possible to localize the vesicle fusion site with a precision exceeding the optical resolution of the microscope. By using a

spiral scan path, the temporal resolution can be increased to 1 kHz to capture the peak amplitude of fast iGluSnFR transients. The typical time frame for these experiments is 30 min per

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VIEWED BY OTHERS GLUTAMATE INDICATORS WITH IMPROVED ACTIVATION KINETICS AND LOCALIZATION FOR IMAGING SYNAPTIC TRANSMISSION Article Open access 04 May 2023 ALL-OPTICAL REPORTING OF INHIBITORY

RECEPTOR DRIVING FORCE IN THE NERVOUS SYSTEM Article Open access 16 October 2024 DETERMINANTS OF SYNAPSE DIVERSITY REVEALED BY SUPER-RESOLUTION QUANTAL TRANSMISSION AND ACTIVE ZONE IMAGING

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Download references ACKNOWLEDGEMENTS We thank I. Ohmert and S. Graf for the preparation of organotypic cultures and excellent technical assistance. This study was supported by the German

Research Foundation through Research Unit FOR 2419 P4 (T.G.O.) and P7 (J.S.W.), Priority Programs SPP 1665 (T.G.O.) and SPP 1926 (J.S.W.), Collaborative Research Center grant SFB 936 B7

(T.G.O.), and BBSRC grants BB/M02556X/1 (K.T.) and BB/S003894 (K.T.). We thank R. Y. Tsien (University of California, San Diego) for providing pCI syn tdimer2. AUTHOR INFORMATION Author

notes * J. Simon Wiegert Present address: Research Group Synaptic Wiring and Information Processing, Center for Molecular Neurobiology Hamburg, Hamburg, Germany * Nordine Helassa Present

address: Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, UK * Silke Kerruth Present address: Department of

Biophysical Chemistry, J. Heyrovský Institute of Physical Chemistry, Prague, Czech Republic AUTHORS AND AFFILIATIONS * Institute for Synaptic Physiology, Center for Molecular Neurobiology

Hamburg, Hamburg, Germany Céline D. Dürst, J. Simon Wiegert, Christian Schulze & Thomas G. Oertner * Molecular and Clinical Sciences Research Institute, St George’s, University of

London, London, UK Nordine Helassa, Silke Kerruth, Catherine Coates & Katalin Török * School of Biosciences, University of Kent, Canterbury, UK Michael A. Geeves Authors * Céline D.

Dürst View author publications You can also search for this author inPubMed Google Scholar * J. Simon Wiegert View author publications You can also search for this author inPubMed Google

Scholar * Nordine Helassa View author publications You can also search for this author inPubMed Google Scholar * Silke Kerruth View author publications You can also search for this author

inPubMed Google Scholar * Catherine Coates View author publications You can also search for this author inPubMed Google Scholar * Christian Schulze View author publications You can also

search for this author inPubMed Google Scholar * Michael A. Geeves View author publications You can also search for this author inPubMed Google Scholar * Katalin Török View author

publications You can also search for this author inPubMed Google Scholar * Thomas G. Oertner View author publications You can also search for this author inPubMed Google Scholar

CONTRIBUTIONS C.D.D., J.S.W., K.T., and T.G.O. designed the experiments and prepared the manuscript. C.D.D. performed synaptic imaging experiments. N.H., S.K., C.C., and M.G. created and

characterized novel iGluSnFR variants, C.S. wrote software to acquire and analyze GEGI data. CORRESPONDING AUTHOR Correspondence to Thomas G. Oertner. ETHICS DECLARATIONS COMPETING INTERESTS

The authors declare no competing interests. ADDITIONAL INFORMATION JOURNAL PEER REVIEW INFORMATION: _Nature Protocols_ thanks Edwin R. Chapman, Yulong Li, Jason Vevea and other anonymous

reviewer(s) for their contribution to the peer review of this work. PUBLISHER’S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional

affiliations. RELATED LINKS KEY REFERENCE USING THIS PROTOCOL Helassa, N. et al. _Proc. Natl. Acad. Sci. USA_ 115, 5594–5599 (2018): https://www.pnas.org/content/115/21/5594 INTEGRATED

SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURE 1 BLEACHING OF IGLUSNFR FLUORESCENCE DURING A 100-TRIAL SINGLE-BOUTON EXPERIMENT DOES NOT AFFECT GLUTAMATE-INDUCED RESPONSES. (A) Raw traces

(~100 trials) of iGluSnFR signals measured in a single presynaptic terminal in response to single APs elicited every 10 s. Note downward slope of baseline in every trial due to bleaching of

iGluSnFR, and slow decrease of F0 over the time course of the experiment (17 min). Partial recovery between trials is likely due to lateral diffusion of unbleached iGluSnFR molecules in the

axonal membrane. (B) Decrease in iGluSnFR resting fluorescence (F0) over the time course of the experiment (17 min). (C) Peak amplitude of individual trials expressed as relative change in

fluorescence (ΔF/F0) is stable over the time course of the experiment. (D) Peak amplitude (ΔF/F0) is independent of F0. SUPPLEMENTARY INFORMATION SUPPLEMENTARY TEXT AND FIGURES Supplementary

Figure 1 REPORTING SUMMARY RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Dürst, C.D., Wiegert, J.S., Helassa, N. _et al._ High-speed imaging of

glutamate release with genetically encoded sensors. _Nat Protoc_ 14, 1401–1424 (2019). https://doi.org/10.1038/s41596-019-0143-9 Download citation * Received: 08 October 2018 * Accepted: 22

January 2019 * Published: 15 April 2019 * Issue Date: May 2019 * DOI: https://doi.org/10.1038/s41596-019-0143-9 SHARE THIS ARTICLE Anyone you share the following link with will be able to

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