A schizophrenia-related sensorimotor deficit links alpha 3-containing GABAA receptors to a dopamine hyperfunction

Abstract

Overactivity of the dopaminergic system in the brain is considered to be a contributing factor to the development and symptomatology of schizophrenia. Therefore, the GABAergic control of dopamine functions was assessed by disrupting the gene encoding the alpha3 subunit of the GABA(A) receptor. alpha3 knockout (alpha3KO) mice exhibited neither an obvious developmental defect nor apparent morphological brain abnormalities, and there was no evidence for compensatory up-regulation of other major GABA(A)-receptor subunits. Anxiety-related behavior in the elevated-plus-maze test was undisturbed, and the anxiolytic-like effect of diazepam, which is mediated by alpha2-containing GABA(A) receptors, was preserved. As a result of the loss of alpha3 GABA(A) receptors, the GABA-induced whole-cell current recorded from midbrain dopamine neurons was significantly reduced. Spontaneous locomotor activity was slightly elevated in alpha3KO mice. Most notably, prepulse inhibition of the acoustic startle reflex was markedly attenuated in the alpha3KO mice, pointing to a deficit in sensorimotor information processing. This deficit was completely normalized by treatment with the antipsychotic D2-receptor antagonist haloperidol. The amphetamine-induced hyperlocomotion was not altered in alpha3KO mice compared with WT mice. These results suggest that the absence of alpha3-subunit-containing GABA(A) receptors induces a hyperdopaminergic phenotype, including a severe deficit in sensorimotor gating, a common feature among psychiatric conditions, including schizophrenia. Hence, agonists acting at alpha3-containing GABA(A) receptors may constitute an avenue for an effective treatment of sensorimotor-gating deficits in various psychiatric conditions.

Fig. 1.

Targeting of the GABA_A-receptor α3 subunit (Gabra3) gene and molecular analysis. (A) Scheme of targeting strategy. Thin lines represent genomic sequences not included in the targeting vector. Thick lines represent genomic sequences included in the targeting vector. Dotted lines show pKS vector sequences. The neomycin-resistance marker (neo) is flanked by two FRT sites and the exon 4 by two loxP sites. The targeting vector is linearized with SpeI within the region of homology and fully integrated into the genome (targeted allele 1). Subsequent expression of the Flp transgene in mice eliminates the neomycin-resistance marker (targeted allele 2). (B) Southern blot analysis using 5′-flanking, 3′-flanking, and neo probes. ES1, ES cell line RW4; ES2, ES cell line AB2; clone 10, Gabra3-targeted clone. Mice 510, 513, 516, 517, 526, 529, and 530 were offspring of a mouse harboring both the targeted allele 1 and the hACTB::Flp transgene. Mouse 517 has lost the neomycin-resistance cassette (targeted allele 2). (C and D) Confirmation of the knockout at the mRNA and protein levels. mRNA prepared from brain without cerebellum and cortex was used for RT-PCR analysis (C). Western blotting was performed by using GABA_A-receptor subunit-specific antibodies (D). (E) Immunohistochemical analysis of GABA_A-receptor subunits with subtype-specific polyclonal antibodies recognizing the α1, α2, and α3 subunits.

Fig. 2.

GABA currents and locomotor activity in α3KO mice. (A) GABA-induced currents of midbrain dopamine neurons. Bath application of GABA elicited significantly smaller whole-cell currents in midbrain dopamine neurons from α3KO animals than from WT littermates (calibration bars, 25 pA, 6 sec). *, P < 0.05. (B) Locomotor activity in the open field was indexed by distance moved per 5-min bin. A slight elevation was revealed in the α3KO mice, regardless of gender, and this effect was most consistently seen in the first half of the test; the effect then subsided before reemerging toward the end of the test session. *, significant difference between α3KO and WT mice at the corresponding bins.

Fig. 3.

Elevated-plus-maze test. (A) A difference in locomotor activity (distance moved in the entire maze) was also detected in the elevated-plus-maze test (*, P < 0.05). (B) Anxiety-related behavior in untreated mice, as exemplified by the illustrated measure of percent time spent in the open arms, remained comparable between the two genotypes. (C) Both α3KO mice and the WT controls responded to diazepam, which promoted exploration of the open arms of the elevated-plus-maze test. *, significant difference from the saline (sal) control condition in the respective genotype.

Fig. 4.

PPI of the acoustic startle response. (A) PPI was indexed by percent inhibition, defined as the percent reduction in reactivity in prepulse-plus-pulse trials relative to pulse-alone trials. Increasing prepulse intensity led to increased magnitude of PPI. PPI was disrupted in α3KO mice, specifically in the middle two prepulse intensities (*, P < 0.05). (B) The PPI deficiency in α3KO mice was restored to WT levels by haloperidol. The drug significantly elevated percent PPI in the mutant across all levels of prepulse, but was effective in the WT mice only at the lowest prepulse intensity (*, P < 0.05). Comparisons between α3KO and WT mice in the saline condition again revealed a significant deficiency of PPI (*, P < 0.05) in the α3KO group at all except the lowest prepulse intensity. (C) Analysis restricted to either the α3KO or the WT mice indicated that a significant drug effect emerged only in the mutant (*, P < 0.05) but not in the WT mice.

Fig. 5.

The locomotor response to systemic amphetamine was assessed over 120 min. Amphetamine (2.5 mg/kg i.p.) led to a pronounced increase in activity. The response to the drug was not altered by the mutation. A marginal increase in locomotor activity was again observed.