The α2C-adrenergic receptor mediates hyperactivity of coloboma mice, a model of attention deficit hyperactivity disorder
Introduction
Attention deficit hyperactivity disorder (ADHD) is characterized by hyperactivity, inattention, and impulsivity and is a common pediatric neuropsychiatric disorder (Olfson, 1992, Faraone et al., 2003, CDC, 2005). The stimulants amphetamine and methylphenidate, which are indirect agonists that increase extracellular monoamine concentrations by inhibiting reuptake and/or promoting release, are the primary treatments for ADHD (Robison et al., 1999). Amphetamine benefits some patients, methylphenidate benefits others, and still others respond to both or neither medication (Winsberg et al., 1974, Arnold et al., 1978). It is not understood how stimulants ameliorate symptoms of some patients or why subpopulations of patients respond differently to stimulants. A better understanding of the mechanisms underlying ADHD would resolve these questions.
The efficacy of stimulants suggests that dopaminergic and/or noradrenergic dysregulation contribute to the expression of ADHD. In support of this assertion, genetic association studies implicate molecules that regulate both dopaminergic and noradrenergic neurotransmission, including receptors and transporters (Cook et al., 1995, Comings et al., 1996, Comings et al., 1999, Comings et al., 2000, Daly et al., 1999, Faraone et al., 2001, Holmes et al., 2002, Maher et al., 2002, Grady et al., 2003, Roman et al., 2003, Kustanovich et al., 2004). Further, in several different studies assessing catecholamine utilization, both the dopamine metabolite homovanillic acid (HVA) and the norepinephrine metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG) are correlated with the behavioral signs of ADHD in children. For example, ADHD patients display a positive correlation between severity of hyperactivity and HVA concentration in cerebrospinal fluid (Castellanos et al., 1994, Castellanos et al., 1996), and between scores on the Test of Variables of Attention (a standardized reaction time test that measures sustained attention and impulsivity) and norepinephrine metabolites in urine (Llorente et al., 2006). Diversity in psychostimulant response, genetic polymorphisms, and neurochemical abnormalities suggests that ADHD is not attributable to any single pathophysiologic mechanism. Consequently, it is unlikely that a universal treatment will ameliorate symptoms of all ADHD patients. In the absence of specific therapeutic targets, the treatment strategy progresses from stimulants to nonstimulant drugs that increase synaptic norepinephrine (reuptake inhibitors), to drugs that are thought to decrease noradrenergic transmission (clonidine, guanfacine, or propranolol—alone or in combination with stimulants) (Silver, 1999, Pliszka, 2003).
Coloboma mice may be a useful tool for developing rational therapeutic strategies for ADHD. These mice exhibit spontaneous hyperactivity caused by a semidominant deletion mutation that includes the Snap25 gene. This mutation results in a 50% reduction in the expression of the protein SNAP-25 (Hess et al., 1992). SNAP-25 concentrates in presynaptic terminals and is expressed in neurons throughout the brain, with the highest levels of expression found in the neocortex, hippocampus, anterior thalamic nuclei, substantia nigra, and cerebellar granule cells (Oyler et al., 1989). SNAP-25 is associated with the plasma membrane of axon terminals and has been identified as a component of the machinery essential for docking synaptic vesicles at the presynaptic membrane in readiness for Ca2+-triggered neurotransmitter exocytosis (Sollner et al., 1993a, Sollner et al., 1993b). Important for this work, several independent research groups identify an 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). Also similar to ADHD patients, amphetamine ameliorates hyperactivity of coloboma mice (Hess et al., 1996), suggesting a relationship between abnormal catecholamine regulation and hyperactivity in these mice. Indeed, brain norepinephrine concentrations are increased in coloboma mice compared to control littermates, and depletion of norepinephrine ameliorates hyperactivity of coloboma mice (Jones et al., 2001, Jones and Hess, 2003), suggesting that dysregulation of norepinephrine contributes to the coloboma mouse phenotype. To determine the adrenergic receptor subtypes that regulate locomotor hyperactivity in coloboma mice, we systematically tested the effects of noradrenergic compounds on locomotor activity of coloboma mice, beginning with nonselective drugs and progressing to increasingly selective compounds.
Section snippets
Mice
Coloboma (Cm/+) mice and control (+/+) C3H/HeSnJ mice (Jackson Laboratories, Bar Harbor, Maine) were bred and housed in group cages (2–4 mice/cage) with corncob bedding at Johns Hopkins University vivarium (lights on at 7 a.m. and off at 9 p.m.). Mutants and controls were age- and sex-matched male and female mice; most mutant and control pairs were littermates. Water and standard laboratory rodent food (2018SX; Harlan Teklad, Madison, WI) were available ad libitum throughout all experiments.
Results
Consistent with previous results (Hess et al., 1992), coloboma mice were significantly more active than control littermates (Fig. 1A; Student's t test, t(62) = 8.70, P < 0.0001). In addition to overt hyperactivity, coloboma mice also exhibit head bobbing, which may substantively contribute to total beam breaks. Therefore, a more detailed analysis was performed to distinguish fine movements–defined as repetitive breaks of a single beam, which would include behaviors such as head bobbing–from
Discussion
The major obstacle to successfully treating ADHD without the use of stimulants is that the most beneficial noradrenergic targets of pharmacotherapy are unknown. Here we used coloboma mice to isolate potential targets. The results presented here suggest that β-adrenergic receptors do not regulate the locomotor hyperactivity of coloboma mice. Instead, our systematic investigation of the locomotor response of coloboma mice to adrenergic drugs implicated α2-adrenergic receptors–specifically, α2C
Acknowledgments
Supported by United States Public Health Service Grant R01 NS34845 (EJH) and Kirshstein-National Research Service Award Individual Fellowship F31 NS42489 (KJB).
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