GABAA receptor gamma 2 subunit knockdown mice have enhanced anxiety-like behavior but unaltered hypnotic response to benzodiazepines

Abstract

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.

Figure 1

Gene targeting of the γ2 subunit of the GABA_A-R and development of γ2 knockdown mice. (A) Targeting strategy used to produce γ2 knockdown mice. Relevant region of endogenous gene (+), targeting vector, correctly targeted knockdown γ2 (F), and cre-recombined knockout alleles (f) are shown. Relative locations of relevant restriction sites, exons (numbered orange boxes), loxP sites (blue triangles), plasmid backbone (wavy line), and positive (NEO) and negative (PGK-TK) selection cassettes are shown. (B) Southern Blot analysis of BglII digested tail DNAs. Probe A hybridizes to a 9.2 kb BglII fragment from the wild type endogenous allele and a 3.4 kb BglII fragment from the targeted allele.

Figure 2

Northern blot analysis. (A) Sample northern blot analysis of wild type (+/+) and homozygous knockdown (F/F) adult whole brain total RNA hybrizided with a γ2 cDNA or a human β-actin probe as a loading control. (B) Densitometric analysis of northern blots. The ratio of band density between γ2 and β-actin hybridization for wild type (n = 23) and homozygous knockdown (n = 36) mice was plotted. On each individual blot, the ratio of wild type band densities was averaged and normalized to 100. All samples on each blot were then compared to the average of wild type band densities on that blot. A total of 4 different blots were included in this analysis. The horizontal bar in each column represents the mean for that genotype.

Figure 3

Immunohistochemical distribution of γ2 subunit of the GABA_A-R in sections from individual (A) wild type and (B) homozygous knockdown mice. Note the high level of abundance of γ2 immunoreactivity in olfactory bulb (OB), cerebral cortex (CC), hippocampus (HP), substantia nigra (SN), and cerebellum (CB) of wild type samples. The amount of γ2 is variably reduced in many brain regions of the knockdown samples.

Figure 4

Behavioral characterization of knockdown mice. (A-C) Elevated plus maze. Knockdown mice (A) entered open arms less often and (B) for less time, however, (C) total entries into arms did not differ from wild type mice. (D) Knockdown mice were also less active in the forced exploration test. The bars are means ± SEM. *p < .01, **p < .001.

Figure 5

Hypnotic sensitivity. No significant differences in the hypnotic effects of ethanol, pentobarbital, zolpidem, midazolam, or diazepam were observed. The bars are means ± SEM.