Flumazenil-insensitive benzodiazepine effects in vitro and in vivo

OBJECTIVE To identify benzodi⁃azepine(BZD)effects that are insensitive to the classical BZD binding site antagonist,flumazenil.Whether the flumazenil-insensitive BZD effects have selectivity on different GABAA receptor sub⁃types was also investigated.METHODS The high-concentration effects of BZDs and their sensitivity to flumazenil were determined on recombi⁃nant synaptic(α1β2γ2,α2β2γ2,α5β2γ2)and extra-synaptic(α4β2δ)GABAA receptors using the voltage-clamp electrophysiology technique.The in vivo evaluation of flumazenil-insensitive BZD effects was conducted in mice loss of right reflex(LORR)test.RESULTS Diazepam induced a biphasic potentiation for theα1β2γ2,α2β2γ2 andα5β2γ2 receptor channels,but did not affect theα4β2δreceptor.In contrast to the nanomolar com⁃ponent of potentiation,the second potentiation elicited by micromolar diazepam was insensitive to flumazenil.Midazolam,clonazepam,and loraz⁃epam at 200μmol·L-1 exhibited similar flumaze⁃nil-insensitive effects onα1β2γ2,α2β2γ2 andα5β2γ2 receptors,whereas the potentiation induced by 200μmol·L-1 zolpidem or triazolam was abol⁃ished by flumazenil.Consistent with the in vitro results,flumazenil antagonized the zolpidem(50 mg·kg-1)-induced LORR,but not those induced by 50 mg·kg-1 diazepam or 100 mg·kg-1 midazolam.CONCLUSION The existence of non-classical BZD binding sites on certain GABAA receptor subtypes and the flumazenil-insensitive effects depend on the chemical structures of the allosteric modulators.