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Counting the Number of Glutamate Molecules in Single Synaptic Vesicles

preprint
submitted on 30.08.2019 and posted on 03.09.2019 by Yuanmo Wang, Hoda fathali, devesh mishra, Thomas Olsson, Jacqueline Keighron, karolina skibicka, Ann-Sofie Cans

Analytical tools for direct quantitative measurements of glutamate, the principal excitatory neurotransmitter in brain, are lacking. Here, we introduce a new enzyme-based amperometric sensor technique for direct counting of the number of glutamate molecules stored inside single synaptic vesicles. An ultra-fast enzyme-based glutamate sensor is placed into a solution of isolated synaptic vesicles, which stochastically rupture at the sensor surface in a potential dependent manner by applying a constant negative potential. High-speed (10 kHz) amperometry is used to record sub-millisecond current spikes, which represent glutamate release from single vesicles that burst open. Glutamate quantification is achieved by a calibration curve that is based on measurements of glutamate release from vesicles pre-filled with various concentrations of glutamate. Our measurements show that a single synaptic vesicle encapsulates about 8000 glutamate molecules, which is comparable to the measured exocytotic quantal glutamate release in the nucleus accumbens of mouse brain tissue. Hence, this new methodology introduces the means to quantify ultra-small amounts of glutamate and to study synaptic vesicle physiology, pathogenesis and drug treatments for neuronal disorders where glutamate is involved.

Funding

The Swedish Research Council, The Swedish Brain Foundation, Ragnar Söderberg Foundation, The Novo Nordisk Foundation, The Wallenberg Center for Molecular and Translational Medicine at the University of Gothenburg, Ernst and Fru Rådman Colliander Stiftelse, Wilhelm and Martina Lundgren Stiftelse, Magnus Bergvall Stiftelse

History

Email Address of Submitting Author

cans@chalmers.se

Institution

Chalmers University of Technology

Country

Sweden

ORCID For Submitting Author

0000-0002-3059-2399

Declaration of Conflict of Interest

Authors declare no conflict of interest.

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in Journal of the American Chemical Society

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