D2-like dopamine receptors mediate the response to amphetamine in a mouse model of ADHD

Abstract

The mechanisms underlying the effects of psychostimulants in attention deficit hyperactivity disorder (ADHD) are not well understood, but indirect evidence implicates D2 dopamine receptors. Here we dissect the components of dopaminergic neurotransmission in the hyperactive mouse mutant coloboma to identify pre- and postsynaptic elements essential for the effects of amphetamine in these mice. Amphetamine treatment reduced locomotor activity in coloboma mice, but induced a robust increase in dopamine overflow suggesting that abnormal regulation of dopamine efflux does not account for the behavioral effect. However, the D2-like dopamine receptor antagonists haloperidol and raclopride, but not the D1-like dopamine receptor antagonist SCH23390, blocked the amphetamine-induced reduction in locomotor activity in coloboma mice, providing direct evidence that D2-like dopamine receptors mediate the effect of amphetamine in these mice. With the precedent established that it is possible to directly antagonize this response, this strategy should prove useful for identifying novel therapeutics in ADHD.

Figure 1

Effect of the D2-like dopamine receptor antagonist haloperidol on amphetamine-mediated locomotor activity. Coloboma (A) and control mice (B) were treated with saline or 4 mg/kg amphetamine and challenged with haloperidol. Compared to vehicle treatment, amphetamine significantly increased locomotor activity in control mice (***p < 0.001, paired Student’s t test) but significantly reduced locomotor activity in coloboma mice (*p<0.05, paired Student’s t test). Two-factor ANOVA with repeated measures revealed a significant effect of genotype (F_1,14 = 17.77, p < 0.001) and dose of haloperidol (F_3,42 = 4.00, p < 0.05) on amphetamine-mediated locomotor activity. Post hoc analyses using paired Student’s t tests demonstrated a significant increase in amphetamine-mediated locomotor activity after treatment with 0.05 mg/kg haloperidol in both control and coloboma mice (# p < 0.05). Treatment with haloperidol alone produced a significant genotype × dose interaction effect (two-factor ANOVA with repeated measures; F_2,28 = 3.90, p < 0.05). Post hoc analyses (paired Student’s t tests) demonstrated a significant reduction in the locomotor activity of both control and coloboma mice after treatment with 0.3 mg/kg haloperidol alone compared to vehicle (*p < 0.05; **p < 0.01). Data are presented as beam breaks accumulated in 1 hr following drug treatment and are expressed as mean ± SEM (n = 8/genotype/dose).

Figure 2

Effect of the D2/D3 dopamine receptor antagonist raclopride on amphetamine-mediated locomotor activity. Coloboma (A) and control mice (B) were treated with saline or 4 mg/kg amphetamine and challenged with raclopride. Compared to vehicle treatment, amphetamine significantly increased locomotor activity in control mice (***p < 0.001, paired Student’s t test) but significantly reduced locomotor activity in coloboma mice (*p<0.05, paired Student’s t test). Two-factor ANOVA with repeated measures revealed a significant genotype × dose interaction effect (F_3,42 = 3.28, p < 0.05) on amphetamine-mediated locomotor activity. Post hoc analyses using paired Student’s t tests demonstrated a significant increase in amphetamine-mediated locomotor activity after treatment with 0.3 mg/kg raclopride in coloboma mice (# p < 0.05), but a trend (p = 0.06) for a reduction in the motor activity of normal mice at the same dose. Treatment with raclopride alone produced a significant genotype × dose interaction effect (two-factor ANOVA with repeated measures; F_2,28 = 5.34, p < 0.05). Compared to vehicle treatment (paired Student’s t tests), all doses of raclopride significantly reduced locomotor activity in control mice (*p < 0.05; **p < 0.01); 0.3 and 1 mg/kg significantly reduced the locomotor activity of coloboma mice with a trend toward reduction with 0.1 mg/kg (p = 0.06). Data are presented as beam breaks accumulated in 1 hr following drug treatment and are expressed as mean ± SEM (n = 8/genotype/dose).

Figure 3

Effect of the D1/D5 dopamine receptor antagonist SCH23390 on amphetamine-mediated locomotor activity. Coloboma (A) and control mice (B) were treated with saline or 4 mg/kg amphetamine and challenged with SCH23390. Compared to vehicle treatment, amphetamine significantly increased locomotor activity in control mice (**p < 0.01, paired Student’s t test) but significantly reduced locomotor activity in coloboma mice (*p < 0.05, paired Student’s t test). Two-factor ANOVA with repeated measures revealed a significant effect of genotype (F_1,12 = 25.22, p < 0.001) and dose of SCH23390 (F_3,36 = 18.41, p < 0.0001) on amphetamine-mediated locomotor activity. 0.2 mg/kg SCH23390 resulted in a significant reduction in amphetamine-mediated behavior in both control and coloboma mice (##p < 0.01). Two-factor ANOVA with repeated measures for treatment with SCH23390 alone revealed significant main effects of genotype (F_1,12 = 10.09, p < 0.01) and dose (F_2,24 = 4.45, p < 0.05). Compared to vehicle treatment (paired Student’s t tests), 0.2 mg/kg SCH23390 significantly reduced locomotor activity in control and coloboma mice (**p < 0.01; ***p < 0.001); 0.05 mg/kg SCH23390 significantly reduced the locomotor activity of coloboma mice (*p < 0.05). Data represent total beam breaks in 1 hr after drug administration and are expressed as mean ± SEM (n=7/genotype/dose).

Figure 4

Effect of D1- and D2-like dopamine receptor antagonists on apomorphine-mediated locomotor activity. Coloboma (A) and control mice (B) were treated with saline or 1 mg/kg apomorphine and challenged with SCH23390 or haloperidol. Two-factor ANOVA with repeated measures revealed a significant genotype × treatment interaction effect (F_3,42 = 4.95, p < 0.005). Post hoc analyses demonstrated that, compared to vehicle treatment, apomorphine significantly reduced locomotor activity in coloboma mice, but increased locomotor activity in control mice (*p < 0.05, paired Student’s t test). A significant increase in apomorphine-mediated locomotor activity was observed after treatment with 0.05 mg/kg haloperidol in coloboma mice (# p < 0.05). Data represent total beam breaks in 1 hr after drug administration and are expressed as mean ± SEM (n=8/genotype/dose).

Figure 5

Basal extracellular dopamine concentrations in striatum. A, Striatal dopamine concentrations were measured by no net flux microdialysis in alert freely moving coloboma (n = 8) and control mice (n = 9). Values are expressed as mean ± SEM. * p < 0.05, Student’s t test. B. The extraction fraction, an indirect measure of dopamine reuptake, was determined by calculating the slope of the linear regression analysis of the perfused concentration of dopamine (C_in) versus the perfused dopamine concentration minus the dialysate dopamine concentration (C_in − C_out). Values are expressed as mean ± SEM. No significant difference was observed between the extraction fractions from control and coloboma mice.

Figure 6

Amphetamine-induced dopamine efflux in striatum. A–D, Mice (n = 6–10/genotype/dose) were injected with 2 mg/kg (A), 4 mg/kg (B), 8 mg/kg (C) amphetamine or 4 mg/kg amphetamine plus 0.05 mg/kg haloperidol (D) and microdialysis samples collected in 20 min intervals. Basal values (‘B’) were calculated by averaging the dopamine concentrations from the 4 samples prior to amphetamine injection. Data are expressed as means ± SEM. E, The absolute amphetamine-induced increase in extracellular dopamine efflux in experiments A–D was determined by calculating the area under the curve (A.U.C.) after subtracting basal extracellular dopamine concentrations. Two-factor ANOVA revealed main effects of genotype (F_1,53 = 7.58, p < 0.01) and treatment (F_3,53 = 6.85, p < 0.001).

Figure 7

D2 dopamine receptor mediated responses. A, Control and coloboma mice (n = 11–12/genotype/dose) were injected with the D2-like dopamine receptor antagonist spiperone. Catalepsy was assessed every 20 min for 2 hr. Data represent average catalepsy time from the 6 test intervals and are expressed as mean ± SEM. Two-factor ANOVA with repeated measures revealed main effects of genotype (F_1,18 = 5.28, p < 0.05) and dose (F_5,90 = 17.37, p < 0.0001). B, Control and coloboma mice (n = 8/genotype/dose) were challenged with the D2/D3 dopamine receptor-selective agonist quinpirole and locomotor activity was assessed. Data are presented as percent of vehicle treatment to normalize for gross differences in baseline locomotor activity and are expressed as mean ± SEM. Two-factor ANOVA with repeated measures revealed main effects of genotype (F_1,14 = 6.24, p < 0.05) and dose (F_4,56 = 4.18, p < 0.01). C, Quinpirole-mediated inhibition of 0.1 μM forskolin-stimulated adenylate cyclase activity. Data are expressed as a percent of forskolin-induced adenylate cyclase activity without agonist. Data represent mean ± SEM (n = 8/genotype). Two-factor ANOVA with repeated measures revealed main effects of genotype (F_1,14 = 5.65, p < 0.05) and dose (F_2,28 = 4.73, p < 0.05).