Simultaneous determination of total and extracellular concentrations of the amino acid neurotransmitters in cat visual cortex by microbore liquid chromatography and electrochemical detection

Abstract

To investigate the influence of a partial sensory deprivation on the total and extracellular concentration of the amino acid neurotransmitters in cat visual cortex, two microbore HPLC methods were developed for the simultaneous determination of aspartate, glutamate, glycine, taurine and γ-aminobutyric acid in cat brain extracts or microdialysis samples. For the determination of the total neurotransmitter concentrations in the visual cortex, the brains were quickly frozen and 200-μm cryostat sections were made. From these sections tissue samples of 2×2 mm 2 containing the six cortical layers were dissected out of the central and peripheral parts of area 17. After homogenisation and centrifugation, the supernatants were used for quantitative amino acid analysis using an o-phthalaldehyde– tert.-butylthiol pre-column derivatisation HPLC gradient elution method on a microbore column (100×1 mm I.D.; C 8) and single electrochemical detection. Microdialysis samples from area 17 were obtained every 15 min using 2-mm probes perfused with synthetic cerebrospinal fluid at a flow-rate of 1 μl/min. After o-phthalaldehyde– tert.-butylthiol derivatisation they were analysed on a microbore column by isocratic elution and dual electrochemical detection. The instrumentation and the different separation parameters were optimised and standard curve, recovery, analytical precision and detection limits for each neurotransmitter were determined.