Flumazenil-insensitive benzodiazepine binding sites in GABAA receptors contribute to benzodiazepine-induced immobility in zebrafish larvae

Abstract

AimsBenzodiazepines (BZDs) produce various pharmacological actions by binding to and allosterically regulating GABAA receptors. Several in vitro studies have demonstrated diazepam, the prototypic BZD, produces a high-dose action that cannot be countered with the classical BZD-binding site antagonist flumazenil. Here, we investigate the existence and behavioral relevance of non-classical BZD binding sites in zebrafish larvae. Main methodsZebrafish larvae were treated with a series of BZDs alone or combined with flumazenil, bicuculline (a non-selective GABAA receptor antagonist), or RO 15–4513 (a general BZD antagonist and a proposed antagonist interacting with α+/β− interfaces in α4/6/β3δ receptors), and their locomotor activities and behavioral phenotypes were recorded. Key findingsDiazepam-induced hypolocomotion (sedation-like state) at low doses (10 and 20 mg L−1) was effectively antagonized by flumazenil or bicuculline, while diazepam-induced immobility (anesthesia-like state) at higher dose (30 mg L−1) was prevented by bicuculline (3 mg L−1) but not flumazenil, even at doses up to 150 mg L−1. Ro 15–4513 also failed to efficiently antagonize diazepam-induced immobility. Immobility induced by high dose of another 1,4-BZD, clonazepam, was also resistant to flumazenil. SignificanceThese results provide direct in vivo evidence for non-classical BZD-binding sites, which may be located at the second transmembrane domain of GABAA receptors and contribute to BZD-induced anesthesia.