SIMULTANEOUS DETERMINATION OF TOTAL AND EXTRACELLULAR CONCENTRATIONS OF THE AMINO-ACID NEUROTRANSMITTERS IN CAT VISUAL-CORTEX BY MICROBORE LIQUID-CHROMATOGRAPHY AND ELECTROCHEMICAL DETECTION

To investigate the influence of a partial sensory deprivation on the t otal and extracellular concentration of the amino acid neurotransmitte rs in cat visual cortex, two microbore HPLC methods were developed for the simultaneous determination of aspartate, glutamate, glycine, taur ine and gamma-aminobutyric acid in cat brain extracts or microdialysis samples. For the determination of the total neurotransmitter concentr ations in the visual cortex, the brains were quickly frozen and 200-mu m cryostat sections were made. From these sections tissue samples of 2X2 mm(2) containing the six cortical layers were dissected out of the central and peripheral parts of area 17. After homogenisation and cen trifugation, the supernatants were used for quantitative amino acid an alysis using an o-phthaldehyde-tert.-butylthiol pre-column derivatisat ion HPLC gradient elution method on a microbore column (100x1 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 sy nthetic cerebrospinal fluid at a flow-rate of 1 mu l/min. After o-phth alaldehyde-tert.-butylthiol derivatisation they were analysed on a mic robore column by isocratic elution and dual electrochemical detection. The instrumentation and the different separation parameters were opti mised and standard curve, recovery, analytical precision and detection limits for each neurotransmitter were determined. (C) 1998 Elsevier S cience B.V.