Mice lacking dopamine D2 and D3 receptors have spatial working memory deficits

Abstract

Mice deficient for dopamine D(2) and D(3) receptors exhibit blunted c-fos responses to D(1) agonist stimulation. Stereologic cell counting revealed decreased numbers of medial prefrontal cortex neurons that express Fos immunoreactivity in all layers, particularly in the prelimbic and anterior cingulate subregions. Pretreatment of these mutants with a single, low dose of methamphetamine (METH) led to a sustained increase in the number of neurons that express Fos immunoreactivity in response to a D(1) agonist challenge, which was most significant in prelimbic and anterior cingulate subregions. The increased c-fos responses reached wild-type-like levels in METH-pretreated D(2) mutants but remained submaximal in METH-pretreated D(3) mutants. Additional studies tested the performance of wild type and mutants in a delayed alternation test, a cognitive task critically dependent on optimal activation of prefrontal cortical D(1) receptors by synaptically released dopamine. Both D(2) and D(3) mutants exhibited deficits in their spatial working memory, with increasing impairments at increasing delays. Whereas METH pretreatment rescued the spatial working memory of D(2) mutants, it had no effect on D(3) mutants. These data suggest that the sustained improvement of spatial working memory in METH-pretreated D(2) mutants is attributable to D(1) receptor-mediated mechanisms.

Fig. 1.

D₁ agonist-stimulated forebrain c-fos mRNA expression. Northern blot of c-fos mRNA extracted from wild-type and D₁, D₂, and D₃knock-out mice 60 min after injection of two full D₁agonists SKF82958 (1 mg/kg) or SKF81297 (2 mg/kg). Eachlane contains 10 μg of total RNA extracted from tissues pooled from two animals per genotype. A, SKF82958-stimulated c-fos mRNA expression in wild-type (WT) and D₁ −/− mice. Thelane marked D1* shows basal levels of c-fos mRNA detected in D₁ mutants, and thelane marked D1 shows c-fosmRNA detected in these mutants after SKF82958 injection. Note the absence of c-fos mRNA induction in SKF-treated D₁ knock-out mice. B, Like SKF82958 (for a quantitative comparison, see Schmauss, 2000), the full D₁ agonist SKF81297 also elicits blunted c-fos mRNA responses in D₂ −/− (lanes marked D2) and D₃−/− mice (lanes marked D3) that can be reversed by METH pretreatment (5 mg/kg). In this experiment, METH was administered 8 d before SKF81297 injection.

Fig. 2.

SKF82958-stimulated c-fos mRNA expression in drug-naive and METH-pretreated D₂/D₃ double mutants. In situ hybridization of c-fos mRNA expressed 60 min after SKF82958 (1 mg/kg) administration to drug-naive (saline-pretreated) wild type (WT) and drug-naive and METH-pretreated homozygous D₂/D₃double mutants (DM). METH (5 mg/kg) was administered 1 week before SKF administration. Sections were hybridized to a ³⁵S-labeled antisense riboprobe comprising 540 nucleotides of the mouse c-fos mRNA. Images on film were digitized using the MCID image analysis system and colorized uniformly to highlight c-fos signal intensities. Note the blunted SKF-stimulated c-fos mRNA expression in drug-naive double mutants that is completely reversed by METH pretreatment.M, Motor cortices; Str, striatum. Scale bar, 1 mm.

Fig. 3.

SKF82958-induced expression of Fos immunoreactivity in the PFC of wild type. Top, Schematic illustration of the regional extent of the mouse PFC in a cross section taken 5.5 mm rostral to the interaural line (Hof et al., 2000) and its cytoarchitecture visualized in a photomicrograph of an NeuN-immunolabeled section. Bottom, Expression of Fos immunoreactivity detected 60 min after saline or SKF82958 administration to wild type. In animals treated with saline, only a few nuclei express Fos immunoreactivity. SKF82958 induces a robust nuclear expression of Fos immunoreactivity in both the deep and superficial layers of the PFC. There is no obvious difference in the expression of SKF-stimulated Fos immunoreactivity between drug-naive and METH-pretreated wild type. M, Motor cortices; Str, striatum. Scale bar, 0.5 mm.

Fig. 4.

SKF82958-stimulated expression of Fos immunoreactivity in the prelimbic subregion of the PFC of D₂ and D₃ single mutants. Top, Detection of Fos immunoreactivity expressed 60 min after SKF82958 administration to drug-naive wild type (WT) and D₂ (D₂ −/−) and D₃ (D₃ −/−) mutants. Saline-treated mice of all genotypes expressed comparably low levels of Fos immunoreactivity (data not shown). In response to equivalent doses of SKF, drug-naive D₂ and D₃ mutants express blunted levels of Fos immunoreactivity throughout the prelimbic cortex.Bottom, Detection of Fos immunoreactivity (brown immunoperoxidase labeling) in neuronal nuclei (blue thionin counterstain) of the PFC of drug-naive wild-type and D₂ and D₃ mutants visualized at 100× magnification. Larger neurons were identified by nuclear size, and smaller neurons typically have only one or two thionin-labeled nuclear densities, a feature distinct from astrocytes. Scale bar, 0.25 mm.

Fig. 5.

SKF82958-stimulated expression of Fos immunoreactivity in the prelimbic subregion of the PFC of drug-naive and METH-pretreated D₂ and D₃ single mutants. The blunted c-fos responses of drug-naive D₂(D₂ −/−) and D₃(D₃ −/−) mutants are reversed when mutants were treated with METH (5 mg/kg) 1 week before an SKF82958 challenge. Scale bar, 0.25 mm.

Fig. 6.

Stereological comparison of the numbers of Fos-immunoreactive (Fos-ir) neurons in the PFC of wild type and D₂ and D₃ single mutants. Mean ± SEM percentages of neurons expressing SKF-induced Fos immunoreactivity (calculated as percentage of the total number of neurons) in the superficial (II/III) and deep (V/VI) layers of the IL, PL, and AC cortices of saline- or METH-pretreated mice. In D₂ and D₃ singlemutants, the numbers of neurons expressing Fos immunoreactivity are reduced in all subregions of the PFC compared with wild type (WT) (reductions rank, AC > PL > IL). c-fos responses to SKF82958 are increased by METH pretreatment of the mutants. This increase is larger in D₂ mutants but remains submaximal (relative to wild type) in all three subregions of the PFC of D₃mutants.

Fig. 7.

T-Maze performance of wild type and D₂and D₃ single mutants at four retention times. The mean ± SEM percentage of correct arm entries was determined from tests using eight animals per genotype. Each delay period was tested twice, and the means of both days were statistically compared (ANOVA) between wild type and mutants. A, Comparison of wild type (WT) and D₃ −/− mutants trained in parallel. B, Comparison of second group of wild type trained in parallel with D₂ −/− mutants. Delay periods (holding box retention times) are indicated on theabscissa. The dashed line across thebars at 50% correct arm entries indicates chance performance.

Fig. 8.

Comparison of the T-maze performance of drug-naive and METH-pretreated wild type and D₂ and D₃mutants at three retention times. A, Comparison of drug-naive and METH-pretreated wild type tested in parallel with drug-naive and METH-pretreated D₃ mutants.B, Comparison of drug-naive and METH-pretreated wild type tested in parallel with drug-naive and METH-pretreated D₂ mutants. Delay periods are indicated on theabscissa, and the dashed line across thebars at 50% correct arm entries indicates chance performance. Each delay period was tested twice, and the means of both days were statistically compared (ANOVA) between genotypes and treatment groups. Statistical differences are indicated ontop of the corresponding bars. In both series of experiments and for all three delay periods, no statistical differences were found between drug-naive and METH-pretreated wild type (wild type–D₃ series, p = 0.09,p = 0.32, and p = 0.22 at 15, 20, and 30 sec delay, respectively; wild type–D₂ series,p = 0.09, p = 0.10, andp = 0.32 at 15, 20, and 30 sec delay, respectively).

Fig. 9.

SKF82958-stimulated c-fos mRNA expression detected 1–7 d after METH pretreatment. Northern blot of frontal cortical c-fos mRNA expressed 60 min after injection of SKF82958 (1 mg/kg) in drug-naive (lanes marked SKF) and METH-pretreated D₂ (D2 −/−) and D₃ (D3 −/−) mice. Lanesmarked 1, 3, 5, and7 illustrate SKF-stimulated c-fos mRNA levels detected 1, 3, 5, and 7 d, respectively, after METH (5 mg/kg) pretreatment.