Protocol | Published:

High-speed imaging of glutamate release with genetically encoded sensors

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 synapse.

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Data availability

The data that support the findings of this study are available from the corresponding author on request.

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.

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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

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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

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.

Competing interests

The authors declare no competing interests.

Correspondence to Thomas G. Oertner.

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.

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Fig. 1: Overview of the protocol workflow for the development of glutamate sensors and two-photon imaging of glutamate transients in individual synapses.
Fig. 2: Characterization of GEGIs.
Fig. 3: iGluSnFR expression in CA3 pyramidal cells in an organotypic slice culture of rat hippocampus.
Fig. 4: Localization of fluorescence transients in low and high [Ca2+]o.
Fig. 5: Signal extraction of GEGI transients from a single Schaffer collateral bouton in CA1.
Fig. 6: Release statistics of neighboring boutons on the same axon.
Fig. 7: Resolving high-frequency transmission with ultrafast GEGI, iGluu.
Supplementary Figure 1: Bleaching of iGluSnFR fluorescence during a 100-trial single-bouton experiment does not affect glutamate-induced responses.

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