Selective labelling of diazepam-insensitive GABA A receptors in vivo using [ 3H]Ro 15-4513

Abstract

1 Classical benzodiazepines (BZs), such as diazepam, bind to GABA A receptors containing α1, α2, α3 or α5 subunits that are therefore described as diazepam-sensitive (DS) receptors. However, the corresponding binding site of GABA A receptors containing either an α4 or α6 subunit do not bind the classical BZs and are therefore diazepam-insensitive (DIS) receptors; a difference attributable to a single amino acid (histidine in α1, α2, α3 and α5 subunits and arginine in α4 and α6). 2 Unlike classical BZs, the imidazobenzodiazepines Ro 15-4513 and bretazenil bind to both DS and DIS populations of GABA A receptors. In the present study, an in vivo assay was developed using lorazepam to fully occupy DS receptors such that [ 3H]Ro 15-4513 was then only able to bind to DIS receptors. 3 When dosed i.v., [ 3H]Ro 15-4513 rapidly entered and was cleared from the brain, with approximately 70% of brain radioactivity being membrane-bound. Essentially all membrane binding to DS + DIS receptors could be displaced by unlabelled Ro 15-4513 or bretazenil, with respective ID 50 values of 0.35 and 1.2 mg kg -1. 4 A dose of 30 mg kg -1 lorazepam was used to block all DS receptors in a [ 3H]Ro 15-1788 in vivo binding assay. When predosed in a [ 3H]Ro 15-4513 binding assay, lorazepam blocked [ 3H]Ro 15-4513 binding to DS receptors, with the remaining binding to DIS receptors accounting for 5 and 23% of the total (DS plus DIS) receptors in the forebrain and cerebellum, respectively. 5 The in vivo binding of [ 3H]Ro 15-4513 to DIS receptors in the presence of lorazepam was confirmed using α1H101R knock-in mice, in which α1-containing GABA A receptors are rendered diazepam insensitive by mutation of the histidine that confers diazepam sensitivity to arginine. In these mice, and in the presence of lorazepam, there was an increase of in vivo [ 3H]Ro 15-4513 binding in the forebrain and cerebellum from 4 and 15% to 36 and 59% of the total (i.e. DS plus DIS) [ 3H]Ro 15-4513 binding observed in the absence of lorazepam. © 2005 Nature Publishing Group All rights reserved.