Endogenous Glu and GABA analysis
Glu and GABA in the dialysate were measured by HPLC coupled with
fluorometric detection as previously described
(Mela et al. , 2012;
Paolone et al. , 2015). Thirty
microliters of o-phthaldialdehyde/mercaptoethanol reagent were added to
30 μl aliquots of sample, and 50 μl of the mixture were automatically
injected (Triathlon autosampler; Spark Holland, Emmen, Netherlands) onto
a 5-C18 Hypersil ODS analytical column (3 mm inner diameter, 10 cm
length; Thermo-Fisher, USA) perfused at a flow rate of 0.48 ml
min-1 (Jasco PU-2089 Plus quaternary pump; Jusco,
Tokyo, Japan) with a mobile phase containing 0.1 M sodium acetate, 10%
methanol and 2.2% tetrahydrofuran (pH 6.5). Glu and GABA were detected
by means of a fluorescence spectrophotometer FP-2020 plus (Jasco, Tokyo,
Japan) with the excitation and the emission wavelengths set at 370 and
450 nm respectively. Under these conditions, the limits of detection for
Glu and GABA were ~1 nM and 0.5 nM, and their retention
times ~3.5 min and ~18.0 min,
respectively. Chromatogram analysis was made by a blinded experimenter
using a dedicated ChromNav software (Jasco, Tokyo, Japan).