The measurement of the release of endogenous GABA from rat brain slices by liquid chromatography with electrochemical detection.
A method for the determination of GABA by derivatization with 2,4,6-trinitrobenzenesulphonic acid and subsequent separation and quantitation by HPLC with electrochemical detection was characterized with respect to specificity, reproducibility and sensitivity. No other amino acid occurring in significant amounts in the brain was found to interfere; however, adequate separation of the derivatives of GABA and tryptophan must be carefully checked in each experiment. The sensitivity of the method is essentially determined by baseline noise, which mainly depends on the quality of the HPLC pump; under our conditions, it was about 2 ng/ml analyte. The coefficients of variation determined at two different concentrations relevant for the subsequent experiments were well below 10%. The method proved useful for the assessment of endogenous release of GABA from superfused rat cortical slices by electrical stimulation, which, in contrast to the basal release, was found to be completely calcium-dependent at stimulation frequencies of 5 and 12 Hz, under our conditions. Both stimulated and basal release of GABA was enhanced 4-5-fold by the inhibitor of GABA uptake, SK&F 89976 (10 microM).
Waldmeier
P C
PC
Research Department, Ciba-Geigy Ltd., Basel, Switzerland.
Wicki
P
P
Feldtrauer
J J
JJ
Baumann
P A
PA
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000378:metabolism* / Q000502:physiology; D002118:Calcium / Q000494:pharmacology / Q000502:physiology; D002851:Chromatography, High Pressure Liquid; D004558:Electric Stimulation; D004563:Electrochemistry; D066298:In Vitro Techniques; D008297:Male; D009557:Nipecotic Acids / Q000494:pharmacology; D010477:Perfusion; D051381:Rats; D011919:Rats, Inbred Strains; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D000596:Amino Acids; D009557:Nipecotic Acids; D005680:gamma-Aminobutyric Acid; C045598:N-(4,4-diphenyl-3-butenyl)nipecotic acid; D002118:Calcium