A Capillary Fraction Collector Coupled to a Fluorescence Reader: A Novel Device to Continuously Quantify Glutamate During Microdialysis

• DOI: 10.1007/s11064-012-0736-y
• Publication Date: 2012-03-07T06:23:04Z
• Authors: Alberto Morales-V...; Raúl Beltrán-Ramírez; Silvia J. López-P...; Verónica Palomera...; Laura Medina-Ceja
• Keywords: Microdialysis; Neurochemical; Dialysis tubing

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.