TARGETING OF THE BENZODIAZEPINE BINDING SITE

DESCRIPTION: The overall goal of the proposed research is to define the role of the benzodiazepine (BZ) binding site in the GABA/A receptors of mice during neuronal development, synaptic function and certain behaviors. Gene targeting by Cre-mediated recombination on engineered lox/P sites will be used. The BZ binding site will be disrupted in vivo either by conditional gene knockout of the GABA/A recently gamma/2 subunit or engineering a specific point mutation in the GABA/A receptor alpha/1 subunit. In both mouse lines the total number of GABA/A receptors is expected to be comparable to the number found in the wild type animal, but the affinity for BZ ligands is expected to be drastically reduced. The specific aims are as follows: 1. To remove the high affinity BZ binding site of GABA/A receptors in specific brain regions in mice by targeted deletion of an essential exon in the beta/2 subunit. The spatial and temporal control of the knockout of this subunit in mice brain will be performed by intercrossing with another mice that is transgene expressing Cre recombinase in specific brain regions. 2. To disrupt the high affinity BZ binding site type I in GABA/A receptors by targeted mutations of a specific amino acid in the alpha/1 subunit. The targeted amino acid is the resides His 100 which when mutated to Arg yields receptors which are essentially insensitive to BZ agonists, despite the normal gating by GABA. 3. To investigate the impact of lack of BZ modulation in both mouse lines for CNS function, in particular, regarding neuronal development with synaptic transmission. The analyses will involved electrophysiological, radio-ligand and immunocytochemical techniques. 4. To investigate, in both mouse lines, the impact of lack of a BZ modulation on the behavioral effects of several BZ ligands (each with a different intrinsic activity) and of other substances such as ethanol, volatile anesthetics and barbiturates.