Abstract

Chronic administration of benzodiazepine agonists produces behavioral tolerance. For induction of tolerance, the use-dependent down-regulation of γ-aminobutyric acid_A(GABA_A)/benzodiazepine receptors is a potential cellular mechanism. We previously identified GABA_A receptors on clathrin-coated vesicles from rat brain, suggesting that surface receptors can be internalized via endocytosis. To examine a role for coated vesicles in GABA_A receptor down-regulation in vivo, fractions were obtained from mouse brain microsomes through density centrifugation and treatment with 0.1% Triton X-100. This coated vesicle preparation was enriched in clathrin subunits and clathrin light-chain kinase and had twice the level of [³H]flunitrazepam binding as did vesicles not exposed to Triton. Adult mice were treated with lorazepam (2 mg/kg/day) for 7 days via osmotic minipump, achieving a serum level of 103 ± 8.9 ng/ml. The level of flunitrazepam bound to coated vesicles was increased by 83 ± 13% in the lorazepam-treated mice compared with vehicle-treated controls. TheB_max value for [³H]flunitrazepam binding to synaptic membranes from lorazepam-treated animals was 33 ± 4% lower than that of controls. The amount of GABA_A receptor alpha-1 subunits, as quantified by Western blotting, followed a similar pattern. Relative to controls, immunoreactivity for alpha-1 subunits in coated vesicles from lorazepam-treated mice was increased by 60.0 ± 10.3%, whereas that in synaptic membranes declined by 12 ± 6%. These results indicate that lorazepam-dependent GABA_A receptor sequestration occurs in mouse brain. Furthermore, it is suggested that this sequestration may play a role in GABA_A receptor down-regulation in vivo.