Measurement of GABAA receptor function in rat cultured cerebellar granule cells by the Cytosensor microphysiometer

1. γ-Aminobutyric acid (GABA), acting via the GABA_A 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 GABA_A receptor function in cerebellar granule cells by microphysiometry were: cells seeded at 9–12×10⁵ cells/transwell cup and maintained in vitro for 8 days, GABA stimulation performed at 25°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 μM (mean±s.e.mean, n=7 experiments) and maximal increase (E_max) 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 μV s⁻¹, GABA+flunitrazepam, 3.8 μV s⁻¹, mean increment over basal, n=7).
5. The GABA-stimulated increase in acidification rate was inhibited by the GABA_A antagonist, bicuculline (100 μM) (90% inhibition at 1 mM GABA).
6. The results of this study show that activation of GABA_A 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 GABA_A receptor antagonist. This paper defines the conditions and confirms the feasibility of using microphysiometry to investigate GABA_A receptor function in primary cultured CNS neurones. The microphysiometer provides a rapid and sensitive technique to investigate the regulation of the GABA_A receptor in populations of neurones.