Background: Gamma-aminobutyric acid type A receptors (GABAA-Rs) are the major inhibitory receptors in the mammalian brain and are modulated by a number of sedative/hypnotic drugs including benzodiazepines and anesthetics. The significance of specific GABAA-Rs subunits with respect to behavior and in vivo drug responses is incompletely understood. The gamma2 subunit is highly expressed throughout the brain. Global gamma2 knockout mice are insensitive to the hypnotic effects of diazepam and die perinatally. Heterozygous gamma2 global knockout mice are viable and have increased anxiety-like behaviors. To further investigate the role of the gamma2 subunit in behavior and whole animal drug action, we used gene targeting to create a novel mouse line with attenuated gamma2 expression, i.e., gamma2 knockdown mice.
Results: Knockdown mice were created by inserting a neomycin resistance cassette into intron 8 of the gamma2 gene. Knockdown mice, on average, showed a 65% reduction of gamma2 subunit mRNA compared to controls; however gamma2 gene expression was highly variable in these mice, ranging from 10-95% of normal. Immunohistochemical studies demonstrated that gamma2 protein levels were also variably reduced. Pharmacological studies using autoradiography on frozen brain sections demonstrated that binding of the benzodiazepine site ligand Ro15-4513 was decreased in mutant mice compared to controls. Behaviorally, knockdown mice displayed enhanced anxiety-like behaviors on the elevated plus maze and forced novelty exploration tests. Surprisingly, mutant mice had an unaltered response to hypnotic doses of the benzodiazepine site ligands diazepam, midazolam and zolpidem as well as ethanol and pentobarbital. Lastly, we demonstrated that the gamma2 knockdown mouse line can be used to create gamma2 global knockout mice by crossing to a general deleter cre-expressing mouse line.
Conclusion: We conclude that: 1) insertion of a neomycin resistance gene into intron 8 of the gamma2 gene variably reduced the amount of gamma2, and that 2) attenuated expression of gamma2 increased anxiety-like behaviors but did not lead to differences in the hypnotic response to benzodiazepine site ligands. This suggests that reduced synaptic inhibition can lead to a phenotype of increased anxiety-like behavior. In contrast, normal drug effects can be maintained despite a dramatic reduction in GABAA-R targets.