ABSTRACT
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 alpha1-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 thealpha1-,alpha2-,alpha3-, oralpha(5)-GABA(A) receptors had been rendered insensitive to diazepam by histidine-arginine point mutation, were chronically treated with diazepam (8 d; 15 mg x kg(-1) x d(-1)) and tested for motor activity. Wild-type, alpha2(H101R), and alpha3(H126R) mice showed a robust diminution of the motor-depressant drug action. In contrast, alpha5(H105R) mice failed to display any sedative tolerance. alpha1(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 alpha5-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 alpha5-GABA(A) receptors in the dentate gyrus. Thus, the chronic activation of alpha(5)-GABA(A) receptors is crucial for the normal development of sedative tolerance to diazepam, which manifests itself in conjunction with alpha1-GABA(A) receptors.
Subject(s)
Brain Chemistry/drug effects , Diazepam/pharmacology , Hypnotics and Sedatives/pharmacology , Receptors, GABA-A/physiology , Alkynes , Allosteric Regulation/drug effects , Amino Acid Substitution , Animals , Benzodiazepines/pharmacokinetics , Binding Sites , Corpus Striatum/chemistry , Crosses, Genetic , Dentate Gyrus/chemistry , Down-Regulation , Drug Tolerance/physiology , Female , Hippocampus/chemistry , Imidazoles/pharmacokinetics , Mice , Motor Activity/drug effects , Motor Cortex/chemistry , Nucleus Accumbens/chemistry , Point Mutation , Protein Subunits , Radioligand Assay , Receptors, GABA-A/chemistry , Receptors, GABA-A/deficiency , Receptors, GABA-A/drug effects , Receptors, GABA-A/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/physiologyABSTRACT
Classical benzodiazepines such as diazepam are widely used tranquillisers and hypnotics in various neuropsychiatric diseases including alcohol-related disorders. One of the major drawbacks of benzodiazepine therapy, however, is an exacerbation of the sedative and hypnotic effects associated with alcohol intake, even at low doses. Even though the gamma-aminobutyric acid (GABA)A receptor complex is a common target for the actions of both classes of drugs, the molecular mechanisms underlying the enhanced pharmacological properties of the combined use of benzodiazepines and alcohol remain to be identified. The present experiments aimed at clarifying which of the GABAA receptor subtypes mediate the augmented hypnotic-like and sedative effects of combined diazepam and alcohol using the righting reflex and motor activity assays, respectively, in histidine-to-arginine point mutated mice that possess diazepam-insensitive alpha1-, alpha2-, alpha3- or alpha5-GABAA receptors. The combination of diazepam (2 or 3 mg/kg) and ethanol (3 g/kg) induced loss of righting reflex with a significantly dose-dependent increase of the latency to its full recovery in wild-type, alpha1(H101R), alpha3(H126R) and alpha5(H105R) but not in alpha2(H101R) mice. A combined treatment with diazepam (1 mg/kg) and ethanol (2.5 g/kg) precipitated motor inhibition similarly in wild-type and alpha2(H101R) mice. Responsiveness of the alpha2(H101R) mice to ethanol alone was similar to that of wild-type mice. These results demonstrate that induction of loss of righting reflex by combined diazepam and alcohol is closely dependent on the activation of the alpha2-GABAA receptors by the benzodiazepine whereas precipitation of sedation involves GABAA receptors other than the alpha2-GABAA receptors.
