Flumazenil-insensitive benzodiazepine binding sites in GABA_A 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 α+/β- interfaces in α_4/6/β₃δ 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.

KEYWORDS: {{ getKeywords(articleDetailText.words) }}