• Yanqing Cao, Hui Yan, Gang Yu, and Ruibin Su.
• State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Key Laboratory of Neuropsychopharmacology, Beijing Institute of Pharmacology and Toxicology, Beijing, China.
• Life Sci. 2019 Dec 15; 239: 117033.

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.Copyright © 2019 Elsevier Inc. All rights reserved.

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