Abstract
Microdialysis coupled to HPLC is the preferred method for quantification of glutamate (Glu) concentrations, both in normal and pathological conditions. However, HPLC is a time consuming technique that suffers from poor temporal resolution. Here we describe an alternative method to measure glutamate concentrations in small-volume dialysis samples by quantifying hydrogen peroxide released by glutamate oxidase using the Amplex Red method. This system permits continuous automatic sample collection and the detection of a fluorescent reaction product, resorufin, which provides a measure of the glutamate concentration. Quantification can be carried out in small microdialysis samples to allow a temporal resolution of 60 s. Both in vitro and in vivo tests showed that this method was reproducible and reliable, detecting Glu along a linear scale. To validate the proposed method, extracellular Glu concentrations in the rat brain were measured and correlated with electrophysiological activity prior, during and after seizure induction with 4-aminopyridine. This method may be adapted to monitor other biologically active compounds, including acetylcholine and glucose, as well as other compounds that generate hydrogen peroxide as a reaction product and may be used as an alternative to other neurochemical methods.
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Acknowledgments
We wish to thank Alejandro Castellanos-Hernández for his technical assistance in evaluating EEG activity. This work was funded primarily by personal funds (Alberto Morales-Villagrán) and by CONSEJO NACIONAL DE CIENCIA Y TECNOLOGIA (CONACYT-CB grants: 105807 and 106179).
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Morales-Villagrán, A., Beltrán-Ramírez, R., López-Pérez, S.J. et al. A Capillary Fraction Collector Coupled to a Fluorescence Reader: A Novel Device to Continuously Quantify Glutamate During Microdialysis. Neurochem Res 37, 1457–1464 (2012). https://doi.org/10.1007/s11064-012-0736-y
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DOI: https://doi.org/10.1007/s11064-012-0736-y