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

AIMS

Benzodiazepines (BZDs) produce various pharmacological actions by binding to and allosterically regulating GABA 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 receptor antagonist), or RO 15-4513 (a general BZD antagonist and a proposed antagonist interacting with α+/β- interfaces in α/βδ 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) was effectively antagonized by flumazenil or bicuculline, while diazepam-induced immobility (anesthesia-like state) at higher dose (30 mg L) was prevented by bicuculline (3 mg L) but not flumazenil, even at doses up to 150 mg L. 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 receptors and contribute to BZD-induced anesthesia.