HPLC conditions are critical for the detection of GABA by microdialysis.
In microdialysis studies, neither exocytotic release of gamma-aminobutyric acid (GABA), nor the presence of GABA type B (GABA(B)) autoreceptors, have been clearly established. It was investigated whether the chromatographic separation of GABA may have contributed to discrepancies in the literature. After extending the profile of the HPLC chromatogram to a retention time of 60 min, it was observed that various unknown compounds of biological origin co-eluted near the GABA peak. The retention time of GABA appeared to be extremely sensitive to pH; even at a retention time of around 60 min there was only a small pH window (5.26 +/- 0.01) where GABA was consistently well separated from co-eluting compounds. GABA determined by the improved assay was sensitive to tetrodotoxin (TTX), calcium depletion and the GABA(B) autoreceptor agonist baclofen. The present results illustrate that if the proper analytical conditions are applied, extracellular GABA can be sampled and quantified by microdialysis in free-moving animals. However, when the time-curves are considered, there is a striking delay of about 15-30 min before the effects of TTX, calcium depletion or baclofen are observed, as compared to the reported response of neurotransmitters such as dopamine (less than 5 min). It is assumed that the glial cells serve as a buffer between the GABA synapse and the microdialysis probes. It is proposed that microdialysis samples measure synaptic GABA indirectly, through glial cells surrounding the synapses.
Department of Biomonitoring and Sensoring, University Centre of Pharmacy, Groningen, The Netherlands. krea@farm.rug.nl
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Rea
T I F H
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Cremers
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Westerink
D000779:Anesthetics, Local / Q000494:pharmacology; D000818:Animals; D001418:Baclofen / Q000031:analogs & derivatives / Q000494:pharmacology; D001596:Benzylamines / Q000494:pharmacology; D001921:Brain / Q000033:anatomy & histology / Q000187:drug effects / Q000378:metabolism*; D001923:Brain Chemistry / Q000502:physiology*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D005110:Extracellular Space / Q000187:drug effects; D018756:GABA Antagonists / Q000494:pharmacology; D008297:Male; D017551:Microdialysis / Q000379:methods*; D009557:Nipecotic Acids / Q000494:pharmacology; D010721:Phosphinic Acids / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013779:Tetrodotoxin / Q000494:pharmacology; D013997:Time Factors; D005680:gamma-Aminobutyric Acid / Q000032:analysis* / Q000494:pharmacology
D000779:Anesthetics, Local; D001596:Benzylamines; D018756:GABA Antagonists; D009557:Nipecotic Acids; D010721:Phosphinic Acids; C052329:phaclofen; C083008:CGP 52432; C030278:nipecotic acid; D013779:Tetrodotoxin; D005680:gamma-Aminobutyric Acid; D001418:Baclofen; D011188:Potassium