title: A capillary fraction collector coupled to a fluorescence reader: a novel device to continuously quantify glutamate during microdialysis.
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.
authors.affiliation: Departamento de Biología Celular y Molecular, CUCBA, Universidad de Guadalajara, Guadalajara, Jalisco, Mexico. amorales@cucba.udg.mx
authors.forename: Alberto
authors.initials: A
authors.lastname: Morales-Villagrán
authors.forename: Raúl
authors.initials: R
authors.lastname: Beltrán-Ramírez
authors.forename: Silvia J
authors.initials: SJ
authors.lastname: López-Pérez
authors.forename: Verónica
authors.initials: V
authors.lastname: Palomera-Ávalos
authors.forename: Laura
authors.initials: L
authors.lastname: Medina-Ceja
mesh_terms: D000818:Animals; D001522:Behavior, Animal; D004569:Electroencephalography; D005453:Fluorescence; D018698:Glutamic Acid / Q000032:analysis*; D008297:Male; D017551:Microdialysis; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results
chemical_list: D018698:Glutamic Acid