Defective motor behavior and neural gene expression in RIIbeta-protein kinase A mutant mice

Abstract

Motor behavior is modulated by dopamine-responsive neurons in the striatum, where dopaminergic signaling uses G-protein-coupled pathways, including those that result in the activation of cAMP-dependent protein kinase (PKA). The RIIbeta isoform of PKA is highly enriched in the striatum, and targeted disruption of the RIIbeta gene in mice leads to a dramatic reduction in total PKA activity in this region. Although the mutant mice show typical locomotor responses after acute administration of dopaminergic drugs, they display abnormalities in two experience-dependent locomotor behaviors: training on the rotarod task and locomotor sensitization to amphetamine. In addition, amphetamine induction of fos is absent, and the basal expression of dynorphin mRNA is reduced in the striatum. These results demonstrate that motor learning and the regulation of neuronal gene expression require RIIbeta PKA, whereas the acute locomotor effects of dopaminergic drugs are relatively unaffected by this PKA deficiency.

Fig. 1.

Targeted disruption of RIIβ. A, Genomic locus, targeting vector, and predicted structure of targeted locus. The targeting vector replaces the coding region of exon 1 of the RIIβ gene with a neomycin resistance cassette (neo). Restriction enzyme sites shown include the following: A,AatII; E, EcoRI;H, HindIII; R,RsrI. The probe fragment used to identify disrupted alleles in ES cells and mice is shown. B, Genomic Southern blot of tail DNA from offspring of a cross of heterozygotes. DNA was digested with HindIII and probed with the fragment shown in A. The wild-type allele yields a 4.7 kb band, and the disrupted allele yields a 3.0 kb band. Genotypes are indicated above each lane: wild type (+/+), heterozygous (+/−), and homozygous mutant (−/−). C, Northern blot of brain total RNA (10 μg) from wild-type (+/+) and homozygous mutant (−/−) mice, probed with a 350 bp riboprobe specific for RIIβ. The migration of RIIβ mRNA is indicated. D, In situhybridization of wild-type (WT) and homozygous mutant (RIIβ ko) brain slices, using the RIIβ-specific riboprobe. Expression is high in the cortex and striatum and is low in the globus pallidus.

Fig. 2.

RIIβ is the major PKA isoform in the striatum.A, Western blot of several regions of wild-type mouse brain probed with RIIβ-specific antiserum. Lanes (fromleft to right), cblm, cerebellum; bstm, brainstem; hpth, hypothalamus; hppc, hippocampus; cllc, colliculi; mdbr, midbrain; strm, striatum; nctx, neocortex. B, Western blot analysis of PKA subunit isoform levels in striatum, using homogenates from three wild-type (+/+) and three RIIβ knock-out (−/−) mice. Blots were probed with antibodies to the indicated PKA subunits. C, Kinase assay with homogenates of the indicated brain regions from wild-type (+/+) and mutant (−/−) mice. Phosphorylation of the PKA substrate Kemptide was assayed in the presence (Total) or absence (Basal) of 5 μm cAMP. Error bars represent SEM. D, PKA activation curves with striatal homogenates from wild-type (wt) and RIIβ^−/− (mutant) mice. Half-maximal activation (dotted lines) was achieved at ∼4 μm cIMP in wild-type mice and 1 μm cIMP in mutants. cIMP was used instead of cAMP because of its lower affinity for PKA R subunits (see Materials and Methods).

Fig. 3.

Impaired performance of RIIβ knock-out mice on the rotarod task. A, RIIβ^−/−(open squares; n = 11) and wild-type control mice (closed squares; n = 13) were tested for their ability to stay on the accelerating rotarod. Ten trials were conducted on the first day and four on the second day (error bars represent SEM). ANOVA for repeated measures revealed a significant effect of trials 1–10 on day 1 in both wild-type (F_(9,108) = 7.62; p < 0.001) and RIIβ knock-out mice (F_(9,90) = 5.11; p < 0.001) as well as a significant effect of genotype (F_(1,22) = 14.07,p < 0.002 on day 1;F_(1,22) = 10.24, p < 0.005 on day 2). B, When tested at a lower rate of acceleration, RIIβ^−/− (open squares;n = 18) mice were impaired significantly, as compared with wild-type control mice (closed squares;n = 17; F_(1,33) = 60.76,p < 0.001 on day 1;F_(1,33) = 26.90, p < 0.001 on day 2).

Fig. 4.

RIIβ mutants have no obvious cerebellar or locomotor behavioral defects. A, Hind paw footprints were recorded, and representative prints of a wild-type (+/+) and RIIβ mutant (−/−) mouse are shown. B, Mean stride length and mean range in stride length in the paw print assay were similar in both wild-type (+/+; n = 10) and RIIβ mutant (−/−; n = 9) mice. C, Locomotor activity in response to a novel environment was recorded in an open-field arena, and similar responses were observed in wild-type (+/+; n = 10) and RIIβ mutant (−/−;n = 9) mice. Error bars represent SEM in all panels.

Fig. 5.

Response of RIIβ mutants to dopaminergic agents.A, Acute locomotor responses. Wild-type (solid bars) and RIIβ^−/− (stippled bars) mice were treated with saline (n = 5 WT and 5 KO), a 10 mg/kg dose of d-amphetamine (amph; n = 5 WT and 6 KO), or a 20 mg/kg dose of cocaine (n = 6 WT and 9 KO). Locomotor activity was determined in an open field by recording photobeam breaks. Error bars represent SEM. B, Enhanced sensitization to low-dose amphetamine. Wild-type (solid bars) and RIIβ^−/− (stippled bars) mice were treated with saline (n = 5 WT and 5 KO), a 2.5 mg/kg dose of d-amphetamine (n = 5 WT and 6 KO), or a 5 mg/kg dose ofd-amphetamine (n = 6 WT and 6 KO) for 5 d, and locomotion was determined. Locomotor responses to three other doses were determined also (data not shown). When saline treatment was compared with 1.0, 2.5, 5.0, and 10 mg/kg doses of amphetamine, repeated measures ANOVA revealed a significant effect of genotype (F_(1,42) = 41.07; p < 0.001). C, D1 agonist-induced grooming behavior. Wild-type (solid bars) and RIIβ^−/−(stippled bars) mice were treated with saline (n = 8 WT and 8 KO) or a 5.0 mg/kg dose of SKF81297 (n = 18 WT and 18 KO). Each mouse was observed for grooming and other activities; the percentage of time spent grooming is reported as mean ± SEM. Grooming increased significantly with SKF81297 in both genotypes (p < 0.02), but mutants responded somewhat less than wild types (p = 0.05). D, Acute locomotor responses to D1 and D2 agonists. Wild-type (solid bars) and RIIβ^−/− (stippled bars) mice were treated with saline (n = 8 WT and 11 KO), an 8.0 mg/kg dose of SKF38393 (n = 5 WT and 7 KO), or a 2.5 mg/kg dose of quinpirole (quin; n = 5 WT and 5 KO), and locomotor responses were determined.

Fig. 6.

RIIβ knock-out mice lack the induction ofc-fos mRNA in the dorsomedial striatum by amphetamine.A, In situ hybridization forc-fos mRNA is shown as dark-field photomicrographs. Representative images from dorsomedial striatum demonstrate thatc-fos mRNA expression is induced in wild-type mice (WT) 1 hr after intraperitoneal injection of 10 mg/kg d-amphetamine (bottom panels) but is not induced in RIIβ^−/− mutants (RIIβ KO). Images from saline-treated animals of each genotype are shown also (top panels). Scale bar, 50 μm.B, Densitometry measurements from several forebrain regions of wild-type (solid bars) or RIIβ^−/− (stippled bars) mice treated with saline (−) or 10 mg/kg d-amphetamine (+). RIIβ^−/− mice do not induce c-fos in the dorsomedial (DM Str) region, as compared with wild-type mice (p < 0.001). Other regions shown are dorsolateral striatum (DL Str) and cingulate cortex (Cing Cx). Error bars represent SEM (n = 3–6 mice for each condition).

Fig. 7.

Dynorphin mRNA is reduced in RIIβ knock-out mice. A, Dynorphin mRNA is expressed in a mainly normal pattern in RIIβ^−/− mice (RIIβ KO) but at reduced levels in the dorsolateral and dorsomedial regions. Wild-type (WT) brain is shown for comparison.Ovals indicate the regions sampled for the densitometric analysis shown in B. B, Quantitative densitometry of three regions of the striatum from wild-type (solid bars; n = 7) and RIIβ^−/− mice (stippled bars;n = 7) shows that no significant difference is observed in the ventrolateral striatum, but reductions are seen in the dorsolateral (p < 0.001) and dorsomedial (p < 0.002) regions. Error bars represent SEM. DL, Dorsolateral; DM, dorsomedial;Str, striatum; VL, ventrolateral.