Measurement of GABA and glutamate in vivo levels with high sensitivity and frequency

In the present protocol, we demonstrate a high-performance liquid chromatography (HPLC) system that enables detection of very low amounts of γ-aminobutyric acid (GABA) (0.03 pmol) and glutamate (0.8 pmol). The HPLC system consists of two pumps, an electrochemical detector, a high-pressure six-way switching valve, a guard column, a microbore column, and a column oven. A microdialysis probe was implanted in the right parietal cortex in rats. Dialysates were collected every 5 min and were split into two equal aliquots for separate analysis of GABA and glutamate. After derivatization with o-phthalaldehyde (OPA), samples were isocratically separated and purified by the guard column. To make the peak of GABA or glutamate appear in an opportune place in a chromatogram, a six-way switching valve was used to control the eluate containing GABA or glutamate to be led to the microbore column and electrochemical detector. By the use of this system, decrease in extracellular concentration of GABA, which precedes the appearance of electrical discharge initiated by hyperbaric oxygen (HBO₂) exposure, was detected by microdialysis at the time resolution of 5 min.