Requirement of α₅-GABA_A receptors for the development of tolerance to the sedative action of diazepam in mice

Despite its pharmacological relevance, the mechanism of the development of tolerance to the action of benzodiazepines is essentially unknown. The acute sedative action of diazepam is mediated via α₁-GABA_A receptors. Therefore, we tested whether chronic activation of these receptors by diazepam is sufficient to induce tolerance to its sedative action. Knock-in mice, in which the α₁-, α₂-, α₃-, or α₅-GABA_A receptors had been rendered insensitive to diazepam by histidine-arginine point mutation, were chronically treated with diazepam (8 d; 15 mg · kg^-1 · d^-1) and tested for motor activity. Wild-type, α ₂(H101R), and α₃(H126R) mice showed a robust diminution of the motor-depressant drug action. In contrast, α₅(H105R) mice failed to display any sedative tolerance. α₁(H101R) mice showed no alteration of motor activity with chronic diazepam treatment. Autoradiography with [³H]flumazenil revealed no change in benzodiazepine binding sites. However, a decrease in α₅-subunit radioligand binding was detected selectively in the dentate gyrus with specific ligands. This alteration was observed only in diazepam-tolerant animals, indicating that the manifestation of tolerance to the sedative action of diazepam is associated with a downregulation of α₅-GABA_A receptors in the dentate gyrus. Thus, the chronic activation of α₅-GABA_A receptors is crucial for the normal development of sedative tolerance to diazepam, which manifests itself in conjunction with α₁-GABA_A receptors.