Measurement of GABAA receptor function in rat cultured cerebellar granule cells by the Cytosensor microphysiometer.
1. gamma-Aminobutyric acid (GABA), acting via the GABAA receptor, increased the extracellular acidification rate of rat primary cultured cerebellar granule cells, measured by the Cytosensor microphysiometer. 2. The optimal conditions for the measurement of GABAA receptor function in cerebellar granule cells by microphysiometry were: cells seeded at 9-12 x 10(5) cells/transwell cup and maintained in vitro for 8 days, GABA stimulation performed at 25 degrees C, with a stimulation time of 33 s. 3. GABA stimulated a concentration-dependent increase in the extracellular acidification rate with an EC50 of 2.0 +/- 0.2 microM (mean +/- s.e.mean, n = 7 experiments) and maximal increase (Emax) over basal response of 15.4 +/- 1.2%. 4. The sub-maximal GABA-stimulated increase in acidification rate could be potentiated by the 1,4-benzodiazepine, flunitrazepam (100 nM). The 10 nM GABA response showed the maximal benzodiazepine facilitation (GABA alone, 1.4 microV s-1, GABA + flunitrazepam, 3.8 microV s-1, mean increment over basal, n = 7). 5. The GABA-stimulated increase in acidification rate was inhibited by the GABAA antagonist, bicuculline (100 microM) (90% inhibition at 1 mM GABA). 6. The results of this study show that activation of GABAA receptors in rat cerebellar granule cells caused an increase in the extracellular acidification rate; an effect which was potentiated by benzodiazepines and inhibited by a GABAA receptor antagonist. This paper defines the conditions and confirms the feasibility of using microphysiometry to investigate GABAA receptor function in primary cultured CNS neurones. The microphysiometer provides a rapid and sensitive technique to investigate the regulation of the GABAA receptor in populations of neurones.
Brown
M J
MJ
Division of Neuroscience, School of Biological Sciences, University of Manchester.
Wood
M D
MD
Coldwell
M C
MC
Bristow
D R
DR
D000818:Animals; D001569:Benzodiazepines / Q000494:pharmacology; D001640:Bicuculline / Q000494:pharmacology; D002452:Cell Count / Q000187:drug effects; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005445:Flunitrazepam / Q000494:pharmacology; D018756:GABA Antagonists / Q000494:pharmacology*; D018757:GABA Modulators / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D051381:Rats; D017207:Rats, Sprague-Dawley; D011963:Receptors, GABA-A / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology*
D018756:GABA Antagonists; D018757:GABA Modulators; D011963:Receptors, GABA-A; D001569:Benzodiazepines; D005680:gamma-Aminobutyric Acid; D005445:Flunitrazepam; D001640:Bicuculline