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Counting the Number of Glutamate Molecules in Single Synaptic Vesicles

Journal article
Authors Y. M. Wang
H. Fathali
Devesh Mishra
T. Olsson
J. D. Keighron
Karolina P Skibicka
A. S. Cans
Published in Journal of the American Chemical Society
Volume 141
Issue 44
Pages 17507-17511
ISSN 0002-7863
Publication year 2019
Published at Institute of Neuroscience and Physiology, Department of Physiology
Wallenberg Centre for Molecular and Translational Medicine
Pages 17507-17511
Language en
Links dx.doi.org/10.1021/jacs.9b09414
Keywords release, transmitter, mechanisms, adsorption, kinetics, Chemistry
Subject categories Chemical Sciences

Abstract

Analytical tools for quantitative measurements of glutamate, the principal excitatory neurotransmitter in the brain, are lacking. Here, we introduce a new enzyme-based amperometric sensor technique for the counting of glutamate molecules stored inside single synaptic vesicles. In this method, 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 at a constant negative potential. The continuous amperometric signals are sampled at high speed (10 kHz) to record sub-millisecond 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 glutamate concentrations. Our measurements show that an isolated single synaptic vesicle encapsulates about 8000 glutamate molecules and is comparable to the measured exocytotic quantal glutamate release in amperometric glutamate sensing 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.

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