Modulation of cerebellar GABAA receptors by Benzodiazepine receptor inverse agonists

Pringle, Ashley Ker (1994) Modulation of cerebellar GABAA receptors by Benzodiazepine receptor inverse agonists. University of Southampton, Doctoral Thesis.

Abstract

Single unit extracellular recordings were made from Purkinje cells of the in vitro cerebellar slice preparation. Application of 0.05-1.0mM γ-aminobutyric acid (GABA) produced a concentration-dependent reduction in the spontaneous firing rate of these cells. GABA responses could be bidirectionally modulated by benzodiazepine (BDZ) receptor ligands, being potentiated by the agonists RU32007 and attenuated by the inverse agonist R019-4603. These effects of the BDZs could be blocked by the BDZ antagonist flumazenil. The BDZ ligands produced simulatneous changes in the firing rate of the cells, RU32007 reducing whilst Ro19-4603 increasing the rate. These changes were attributed to modulation of tonically released GABA. Responses to 0.1mM GABA were also attenuated by the imidazopyrimidine inverse agonist RU34347. Associated with this effect was a decrease in the firing rate of the cells. Application of RU34347 for short time periods (40-60secs) produced an inhibitory response similar to that observed following GABA application. This response was partially blocked by both flumazenil and bicuculline (a GABA antagonist).

A study of stimulus-evoked inhibition was also made by placing a bipolar stimulating electrode into the molecular layer of the cerebellum. Following stimulation, a period of inhibition occurred, results being recorded in the form of peri-stimulus time histograms (PSTH). Ro19-4603 (10pM-1nM) produced a dose-dependent decrease in the period of inhibition. RU34347 (10nM-10fM) produced an increase in the duration of the inhibition although the concentration-response relationship was biphasic. Between 10nM and 10pM a greater effect was observed as the concentration was decreased, the peak effect being being seen at 10pM and this could be antagonised by 1μM flumazenil. As the concentration was reduced further, a reduction in the magnitude of effect was observed, the minimum effective concentration being 100fM. It was also found that the increase in the duration of inhibition induced by 10nM RU34347 was only fully antagonised by 10μM flumazenil, a much higher concentration than would be predicted from the relative affinities of the two compounds at classical BDZ receptors.

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