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

Aims: Benzodiazepines (BZDs) produce various pharmacological actions by binding to and allosterically regulating GABA(A) 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 methods: Zebrafish larvae were treated with a series of BZDs alone or combined with flumazenil, bicuculline (a non-selective GABA(A) receptor antagonist), or RO 15-4513 (a general BZD antagonist and a proposed antagonist interacting with alpha+/beta- interfaces in alpha(4/6)/beta(3)delta receptors), and their locomotor activities and behavioral phenotypes were recorded. Key findings: Diazepam-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. Significance: These results provide direct in vivo evidence for non-classical BZD-binding sites, which may be located at the second transmembrane domain of GABA(A) receptors and contribute to BZD-induced anesthesia.