A recently developed animal model that may be relevant to ADHD is the DAT knockout mouse, which shows about a 300-fold decrease in the rate of clearance of extracellular dopamine42 due to the lack of the gene that encodes DAT-1. These knockout mice also show evidence of behavioral abnormalities similar to those observed in ADHD, although there remain several important issues regarding dopamine autoreceptor downregulation, serotonergic tone, and activation of trace amine receptors in these animals that need to be addressed with regard to relevance to ADHD.43
DAT knockout mice demonstrate a behavioral phenotype that, on the surface, appears to mirror symptomatology associated with ADHD.43 For example, hyperactivity was one of the earliest observations in these mice, which was subsequently revealed to be particularly sensitive to a novel environment, where locomotor activity was determined to be 12-fold higher in the knockouts compared with wild-type controls. Further, while locomotor habituation to the environment (decreased activity following prolonged exposure to the environment) was evident in wild types, DAT knockout mice remained hyperactive even after 4h. In addition, repetitive exposure of the knockout animals to the activity environment only resulted in augmentation of the locomotor response. Lack of habituation to novel stimuli was also observed in a version of the novel object test as well as a modified Y-maze. Taken together, these data suggest that the DAT knockouts are not only hyperactive, but are less able to adapt to novel stimuli and may exhibit reward-like behavior. These data are also in agreement with siRNA knockdown44 or pharmacological inhibition of DAT45 in mice, which also produce pronounced hyperactivity.
Impaired cognitive function is also evident in DAT knockout mice. In a spatial working memory test using a radial arm maze, knockout mice were essentially unable to acquire the test conducted over 21 sessions. Knockout mice also had significantly higher preservation errors compared to wild-type mice and these errors remained elevated for the duration of the study, suggesting that the knockouts had difficulty suppressing inappropriate responses.43
Psychostimulants such as methylphenidate, amphetamine, and cocaine (all at relatively high doses) paradoxically robustly attenuated hyperactivity in DAT knockout animals in a novel environment.43 Interestingly, these 'calming-like' effects of the stimulants were delayed, but long-lasting, particularly for methylphenidate (up to 4h). Further, the effects of methylphenidate were dose-dependent in the DAT knockouts, but methylphenidate dosing over the same range in wild-type controls produced the more usual enhancement of activity, following an inverted U-shaped dose response. While these data were exciting, subsequent work described in the same article provided some puzzling information: extracellular dopamine concentrations in the striatum of DAT knockouts, measured by in vivo microdialysis in freely moving mice after the administration of methylphenidate, did not change, in contrast to wildtype controls, which were significantly elevated. The most likely reason for this is that dopamine levels in the DAT knockouts are already highly elevated and were probably already at ceiling. This posed a dilemma: if dopaminergic stimulants can act to decrease hyperactivity dramatically in a novel environment in DAT knockout mice, and dopamine levels are not affected, what is the mechanism of action of psychostimulants? Further experiments with the selective norepinephrine transporter (NET) inhibitor nisoxetine demonstrated no effect. However, the serotonin transporter (SERT) inhibitor (selective serotonin reuptake inhibitor (SSRI)), fluoxetine, as well as the nonselective serotonin receptor agonist quipazine and 5-HT precursor substrates, 5-hydroxytryptophan and L-tryptophan, also dramatically decreased hyperactivity in the DAT knockout mice.43 Striatal dopamine levels were not affected by these various 5-HT treatments, however. Additional experiments with fluoxetine and the dopamine receptor agonist apomorphine in dopamine-depleted DAT knockout mice suggested that the serotonergic effects on decreasing hyperactivity were mediated downstream of dopaminergic neurotransmission, with later findings indicating that limbic brain regions were of especial importance. The relevance of these findings to ADHD is currently unclear, as SSRIs are not effective in treating ADHD and one of the side effects of these drugs in the clinic is motor stimulation.46 Similarly, the norepinephrine reuptake (NET) inhibitor, atomoxetine (Strattera), is somewhat effective in treating ADHD, while nisoxetine was without effect on hyperactivity in the DAT knockout mice.43 Despite these limitations, DAT knockouts may be useful in elucidating the nondopaminergic neural mechanisms underlying ADHD.
Disruption of the DAT gene in mice significantly increases extracellular concentrations of dopamine and leads to a reduction in mRNA levels for the DRD1- and DRD2-like receptors in the striatum,47-49 findings that run somewhat counter to those found in ADHD patients. In clinical ADHD, however, patients with the DAT1 polymorphism show mutations within the 3' untranslated portion of the mRNA, i.e., outside the protein-coding region. Thus, it remains unclear how this might affect dopamine reuptake in ADHD compared to the complete lack of DAT function in DAT1 knockout mice. Further, there is evidence to suggest that the DAT1 is actually overexpressed in ADHD patients.
According to a single photon emission computed tomography (SPECT) study, the DAT is elevated by approximately 70% in adults with ADHD.50 Other SPECT studies have also demonstrated that the efficacy of the stimulant methylphenidate is attributed to an approximate 50% block of DAT: maximal clinical efficacy is also observed at a time (approximately 60min after oral dosing) when 50% DAT blockade is achieved.51 In contrast, evidence exists for dopamine hypofunctional prefrontal cortex in both DAT knockouts and in ADHD, which may be responsible for mediating the attentional/cognitive impairments observed in both cases. Thus, while the behavioral abnormalities observed in DAT knockouts is intriguing, the direct relevance of DAT knockout to ADHD remains to be determined.
6.05.4.2.3 Synaptosome-associated protein of 25kDa (SNAP-25, coloboma mutant)
The coloboma mutation arose from neutron irradiation mutagenesis studies, producing a deletion on chromosome 2 that disrupted coding of four known genes for the proteins phospholipase b1 and b4, jagged 1, and SNAP-25.52'53 The mutation is homozygous lethal. Adult heterozygote mice are viable, although a distinctive ocular dysmorphology leading to 'sunken' eyes in some mice limits behavioral testing.54
SNAP-25 mutants are hyperactive when spontaneous locomotor activity is assessed in novel environments, reaching two- to fourfold above basal activity (Figure 2) of control littermates.55 However, this hyperactivity is very variable and is hypothesized to reflect a loss of control of activity rather than a simple increase in basal activity. This can be confounded by head-bobbing and other stereotypies such as repeated jumping against the wall on one side of the activity arena (Figure 2). The hyperactivity is attenuated by administration of relatively high doses of amphetamine (4mgkg _ 1), without producing further stereotypies.55 Unfortunately, given the ocular dysmorphology evident in many SNAP-25 mutants, the effects of SNAP-25 deletion on attention/cognitive function in vivo is not known. However, SNAP-25 constitutes an integral protein for synaptic vesicle fusion and neurotransmitter release studies suggest that impaired neurotransmitter (and perhaps neuropeptide) release is regionally specific.56 SNAP-25 has also been shown to be important for hippocampal-based memory consolidation in several studies in rats utilizing long-term potentiation54,57,58; intracerebroventricular antisense oligonucleotide also induced deficits in several cognitive tests such as long-term inhibitory avoidance and fear conditioning,59 although these tests may hold little relevance for cognitive deficits observed in ADHD.
The psychostimulant amphetamine attenuated hyperactivity in coloboma mice, while it increased activity, as expected, in littermate controls.55 Thus, by reversing the dopamine plasma membrane pump, increased dopamine is released in regions such as the dorsal striatum where it is low due to impaired vesicular release caused by deficient SNAP-25. In contrast, methylphenidate does not reverse hyperactivity, but instead increased activity in both controls and SNAP-25 mutants.55 Little other pharmacological work has been performed and thus, the predictive validity of SNAP-25 mutants is unclear.
Behavioral changes in the coloboma mouse are associated with a mutation of the gene encoding the important synaptosome-associated protein, SNAP-25. Polymorphism analysis of the gene encoding SNAP-25 in humans identified biased transmission of the haplotypes of the alleles of two polymorphisms, implicating SNAP-25 in the etiology of ADHD.16,60,61 However, neurobiological deficits associated with mutation of SNAP-25 in mice, such as decreased neuronal number in neocortex, hippocampal dysfunction, and ocular dysmorphology, are not consistent with ADHD in the clinic.
6.05.4.2.4 6-Hydroxydopamine (6-OHDA) lesions
Exposure of rat pups to 6-OHDA, usually via intracerebroventricular or intracisternal injection, selectively lesions dopamine projections to the frontal cortex, resulting in an age-dependent increase in spontaneous locomotor activity.27 While rats lesioned in this manner also show evidence of cognitive impairment, these are also age-dependent. Impulsivity is not present in 6-OHDA-lesioned rats and, since impulsivity and cognitive impairment are evident in adults with ADHD, these rats may be a useful model regarding elucidation of mechanisms that cause hyperactivity in ADHD.
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