GABAA receptor γ2 subunit knockdown mice have enhanced anxiety-like behaviour but unaltered hypnotic response to benzodiazepines -: art. no. 30

Background: Gamma-aminobutyric acid type A receptors (GABA(A)-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 GABA(A)-Rs subunits with respect to behavior and in vivo drug responses is incompletely understood. The gamma 2 subunit is highly expressed throughout the brain. Global gamma 2 knockout mice are insensitive to the hypnotic effects of diazepam and die perinatally. Heterozygous gamma 2 global knockout mice are viable and have increased anxiety-like behaviors. To further investigate the role of the gamma 2 subunit in behavior and whole animal drug action, we used gene targeting to create a novel mouse line with attenuated gamma 2 expression, i.e., gamma 2 knockdown mice. Results: Knockdown mice were created by inserting a neomycin resistance cassette into intron 8 of the gamma 2 gene. Knockdown mice, on average, showed a 65% reduction of gamma 2 subunit mRNA compared to controls; however gamma 2 gene expression was highly variable in these mice, ranging from 10-95% of normal. Immunohistochemical studies demonstrated that gamma 2 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 g2 knockdown mouse line can be used to create gamma 2 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 gamma 2 gene variably reduced the amount of gamma 2, and that 2) attenuated expression of gamma 2 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 GABA(A)-R targets.