Biphasic actions of the benzodiazepine receptor inverse agonist RU34347 in the rat cerebellar slice.

We have characterised the biphasic response of the benzodiazepine receptor inverse agonist RU34347 in the rat cerebellar slice preparation using extracellular electrophysiological recordings from Purkinje cells. RU34347 (10 fM-10 nM) produced an increase in GABA(A)-mediated inhibition at between 10 fM and 10 nM, a response normally associated with benzodiazepine agonists. This response was biphasic, being dose dependent between 10 fM and the peak effect at 10 pM, and inversely related to concentration between 100 pM and 10 nM. Associated with this increase in inhibition was a decrease in firing rate, also showing a biphasic concentration-response relationship. The agonist-like response was composed of two elements, an initial increase occurring after 5 min followed by a slow decline over the next 20 min. This second, declining, phase was more evident at higher concentrations of RU34347. The peak effect seen at 10 pM was fully antagonised by 1 microM flumazenil, a benzodiazepine receptor antagonist. This concentration of flumazenil also antagonised the decrease in firing rate induced by 10 pM RU34347. The response to 10 nM RU34347 was further characterised since the largest second phase decline was demonstrated at this concentration. 10 nM flumazenil fully antagonised the second, declining, phase of the response, but not the magnitude of the initial increase. A partial antagonism of the peak effect was seen with 1 microM flumazenil, and a full antagonism at 10 microM flumazenil. This effect was similar to that observed with Ro15-4513 in a previous study. Therefore, we investigated the binding of RU34347 to diazepam-insensitive benzodiazepine receptors. [3H]Ro15-4513 was displaced by Ro15-4513, flumazenil and Ro19-4603, but not by RU34347. Therefore, although the electrophysiological data correlate well with that previously reported for Ro15-4513, RU34347 does not displace Ro15-4513 binding at the diazepam-insensitive benzodiazepine receptor. Therefore, either both ligands bind to complimentary sites on the same receptor complex, or both induce a similar physiological response through an action on different receptors.