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

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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.

Introduction

Attention deficit hyperactivity disorder (ADHD) is characterized by inattention, impulsivity and hyperactivity. A familiar feature of ADHD is the response to psychostimulants such as methylphenidate (Ritalin) and d-amphetamine (Adderall). Both compounds are indirect agonists that increase extracellular monoamine concentrations (Ferris et al., 1972, Heikkila et al., 1975). In ADHD patients, low doses of stimulants produce beneficial behavioral effects by reducing excess motor activity and enhancing concentration. Although the efficacy of psychostimulants in ADHD was recognized nearly 70 years ago (Bradley, 1937), the biological mechanisms are not understood.

Many theories, including several that focus on dopaminergic transmission, have emerged to explain the effects of psychostimulants. The rate dependency hypothesis suggests that the effect of psychostimulants in hyperactive children is a phenomenon driven by behavioral state (Glick and Milloy, 1973, Sahakian and Robbins, 1977). The locomotor response to psychostimulants is an inverted U-shaped function: low doses increase locomotor activity and high doses induce focused behaviors (stereotypy) that compete with and consequently reduce locomotor activity. Drug-naive ADHD patients are at the highest point on this inverted U-shaped curve. Accordingly, the psychostimulant-induced reduction in motor activity is ascribed to an increase in stereotyped behavior. Others propose that low doses of stimulants reduce synaptic catecholamine concentrations or the amplitude of impulse-induced dopamine release (McCracken, 1991, Seeman and Madras, 1998, Solanto, 1998). Low doses of direct-acting dopamine agonists preferentially interact with presynaptic receptors to inhibit dopamine release and therefore reduce locomotor activity (Skirboll et al., 1979). By extension, it is proposed that low doses of the indirect agonists methylphenidate and amphetamine reduce locomotor activity in ADHD patients through a similar mechanism of action. Others suggest that psychostimulant therapy compensates for insufficient dopamine transmission in the frontal cortex while reducing nigrostriatal overactivity (Castellanos, 1997). Despite these and other well-developed hypotheses, there is little empirical evidence to explain the therapeutic mechanism of psychostimulants in ADHD.

Animal models of ADHD provide an opportunity to explore pathogenic and therapeutic mechanisms. The coloboma mouse mutant is one such model. These mice exhibit hyperactivity, inattention and delayed developmental milestones (Hess et al., 1992, Heyser et al., 1995, Bruno et al., 2007) plus abnormal monoaminergic regulation (Raber et al., 1997, Jones and Hess, 2003). These defects are attributable, at least in part, to a reduction in the expression of SNAP-25 (Hess et al., 1996), a protein essential for neurotransmitter exocytosis (Söllner et al., 1993). The phenotype results from a hemizygous deletion mutation that includes the Snap25 gene (Hess et al., 1992); several independent research groups have determined that there is also a genetic association between the SNAP25 gene and ADHD in humans (Barr et al., 2000, Brophy et al., 2002, Mill et al., 2002, Kustanovich et al., 2003, Brookes et al., 2006). Indeed, a recent meta-analysis also supports this association (Faraone et al., 2005). Similar to its effects on ADHD patients, amphetamine reduces locomotor activity in coloboma mice (Hess et al., 1996). Here we dissect the components of dopaminergic neurotransmission in coloboma mice to identify elements essential for the effect of amphetamine.

Section snippets

Mice

Coloboma (Cm/+) mice were bred and housed in group cages at the Johns Hopkins University vivarium. In all experiments, coloboma mice and wild type control littermates (4–9 months of age) were age- and sex-matched, although there is no significant difference in the locomotor activity between males and females. Experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the National Institutes of Health.

Drugs and receptor nomenclature

Drugs were purchased from Sigma (St.

D2-like dopamine receptor antagonism blocks the amphetamine-induced reduction in locomotor activity

Consistent with previous results (Hess et al., 1996), 4 mg/kg amphetamine significantly reduced the locomotor hyperactivity exhibited by coloboma mice (p < 0.05; Fig. 1A). Stereotypic behavior was not observed at this dose of amphetamine (data not shown), suggesting that the reduction in locomotor activity in coloboma mice was not attributable to an increase in competing focused repetitive behaviors. To determine if the response to amphetamine is dependent on a specific dopamine receptor subtype,

Discussion

The indirect and direct dopamine agonists amphetamine and apomorphine reduced the hyperactivity exhibited by coloboma mice. This reduction in activity was not accompanied by an increase in stereotyped behavior, eliminating rate dependency as an explanation for the effect. An increase in basal extracellular dopamine was observed in coloboma mice, which may, in part, account for the hyperactivity inasmuch as increases in dopaminergic tone also occur in other models of hyperactivity (Giros et al.,

Acknowledgments

We thank Drs. Michelle D. Jones and Irwin Lucki for consultation on the microdialysis experiments, Dr. Kristy J. Bruno for helpful discussion and Catherine Weisz for technical support. Supported by U.S. Public Health Service Grant R01 NS34845.

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