Effects of fluorescent glutamate indicators on neurotransmitter diffusion and uptake

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Abstract

Genetically encoded fluorescent glutamate indicators (iGluSnFRs) enable neurotransmitter release and diffusion to be visualized in intact tissue. Synaptic iGluSnFR signal time courses vary widely depending on experimental conditions, often lasting 10-100 times longer than the extracellular lifetime of synaptically released glutamate estimated with uptake measurements. iGluSnFR signals typically also decay much more slowly than the unbinding kinetics of the indicator. To resolve these discrepancies, here we have modeled synaptic glutamate diffusion, uptake and iGluSnFR activation to identify factors influencing iGluSnFR signal waveforms. Simulations suggested that iGluSnFR competes with transporters to bind synaptically released glutamate, delaying glutamate uptake. Accordingly, synaptic transporter currents recorded from iGluSnFR-expressing astrocytes in mouse cortex were slower than those in control astrocytes. Simulations also suggested that iGluSnFR reduces free glutamate levels in extrasynaptic spaces, likely limiting extrasynaptic receptor activation. iGluSnFR and lower-affinity variants nonetheless provide linear indications of vesicle release, underscoring their value for optical quantal analysis.

Data availability

MATLAB code used to perform the simulations in this study are included with the manuscript and supporting files. The IgorPro experiment file containing all of the simulation data is available athttps://nih.box.com/s/ttnppg1kzc4ur9d4ahmfpl2j0m7rvfsm.Source data files for Figure 4 are available at https://tufts.app.box.com/s/ptkpd4wig9njz2y9egocgenu3utkehae.

Article and author information

Author details

Moritz Armbruster

Department of Neuroscience, Tufts University School of Medicine, Boston, United States

Competing interests
The authors declare that no competing interests exist.
Chris G Dulla

Neuroscience, Tufts University School of Medicine, Boston, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-6560-6535
Jeffrey S Diamond

NINDS, NIH, Bethesda, United States

For correspondence
diamondj@ninds.nih.gov

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-1770-2629

Funding

National Institute of Neurological Disorders and Stroke (NS003039)

Jeffrey S Diamond

National Institute of Neurological Disorders and Stroke (NS113499)

Chris G Dulla

National Institute of Neurological Disorders and Stroke (NS104478)

Chris G Dulla

National Institute of Neurological Disorders and Stroke (NS100796)

Chris G Dulla

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: All animal protocols were approved by the Tufts Institutional Animal Care and Use Committee (protocol #B2019-48).

Copyright

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.