The current study provides the first evidence that oral dosing of therapeutically relevant levels of MPH improves PFC cognitive function in rats. This same dose regimen has been found to decrease locomotor activity in rats, reinforcing the idea that rats can be used as an appropriate animal model for examining medications used to treat ADHD patients.
Performance of the spatial delayed alternation task in a T maze is very relevant to many aspects of ADHD. Optimal performance of this task requires spatial working memory (remembering which side was most recently entered), response inhibition (inhibiting the tendency to return to the location where the animal was last rewarded) and the ability to sustain attention and suppress the distractions of being put into the start box. Thus, the delayed alternation task assesses many of the PFC operations that are known to be problematic in ADHD.
The current study found that moderate doses of MPH produced a highly significant improvement in delayed alternation performance. This improvement likely reflects enhanced PFC cognitive performance, as there were no changes in response time characteristic of motor or motivational changes. Indeed, given that MPH reduces eating, it is unlikely that simple changes in motivation for food reward could account for the improvement in performance.
It is also noteworthy that higher doses of MPH impaired delayed alternation performance in a large subset of animals. This impairment manifested in a perseverative profile of errors in which the rats continued to choose the same incorrect arm of the maze. Perseveration is also seen with 1) PFC lesions [23–26], 2) infusion of a high dose of a DA D1 or NE α1 agonist into the PFC [27, 28], or 3) stress exposure, which causes high levels of NE and DA release in PFC . Future studies will be needed to determine whether higher doses of MPH impair delayed alternation performance due to excessive catecholamine release in PFC, and if so, which receptor(s) underlie these impairing actions.
Comparison to cognitive effects of MPH in humans
The MPH profile observed in rats in the current study is very similar to that in seen in humans. Oral administration of clinical doses of MPH have been found to improve spatial working memory, response inhibition, set-shifting and other PFC cognitive functions in both "normal" college students  and in children and adults with ADHD [31–35]. Imaging studies have shown more efficient dorsolateral PFC activity (BOLD) following MPH doses that improve spatial working memory, consistent with improved PFC cognitive function . Interestingly, in adults with ADHD, childhood ratings of ADHD (both self-reported and informant ratings) correlated with response to methylphenidate on the spatial working memory task . Thus, studies of spatial working memory performance are likely very relevant to the therapeutic effects of ADHD medications.
In the current study, higher doses of MPH impaired spatial working memory performance in a large number of animals. These findings are consistent with the original Lyon-Robbins analysis of stimulant actions in rodents that found increasingly perseverative responses with increasing dose of stimulant administration . It is noteworthy that these "higher" doses are still much lower than those used in most other rodent studies, accentuating the fact that previous research in animals has often focused on MPH doses that are too high. Similar to our findings in rats, clinicians are often concerned that higher doses of MPH can induce perseverative thinking in patients e.g. [37, 38]. For example, higher doses of MPH (e.g. 1.0 mg/kg) can increase perseverative errors on the Wisconsin Card Sorting Task when the test is novel [39, 40]. Perseverative errors were not increased, and indeed were reduced by MPH, when the Wisconsin Card Sorting Task was given repeatedly ; however, this condition minimizes the need for flexible thinking, as the switching rule is "discovered" only during the first experience of the task. Douglas et al. also found no evidence of perseveration on other tasks, such as the Trails B, and concluded that doses below 0.9 mg/kg produced dose-related improvements in cognitive flexibility. As repeated daily doses can add together, they cautioned that doses above 0.6 mg/kg were not recommended. Thus, under optimal dosage conditions, MPH appears to improve flexible thinking in patients, but higher doses may produce a perseverative profile similar to that seen in rodents.
Receptor mechanisms underlying PFC cognitive enhancing effects of MPH
The identification of an MPH dose regimen that improves PFC cognitive performance in rats provides the opportunity to examine the neural mechanisms underlying MPH therapeutic actions. The current study began by examining the role of NE α2 and DA D1 receptors, given the importance of these receptors to PFC cognitive function. The study found that both the α2 antagonist, idazoxan, and the D1 antagonist, SCH23390, reversed the cognitive-enhancing effects of MPH. Care was taken to use antagonist doses that did not impair performance on their own; thus, additive effects of drug treatment cannot account for the normalization of response. Rather, the data are consistent with MPH improving performance by increasing the availability of NE and DA, which in turn stimulate α2 and D1 receptors. It is interesting that either idazoxan or SCH23390 was fully effective in reversing the MPH response. These data suggest that there may be beneficial interactions between these receptors, an area that has received little investigation.
Future studies will be needed to determine whether the enhancing effects of MPH occur in the PFC and/or elsewhere in the brain. Low, systemic doses of MPH and amphetamine are known to increase both NE and DA levels in the rat PFC while having more subtle effects in striatal regions (, and C.W. Berridge, personal communication). There are relatively low levels of DA transporters in the PFC ; thus the increase in both DA and NE levels likely occurs through blockade of NE transporters, which are thought to transport both NE and DA in the PFC .
The efficacy of idazoxan and SCH23390 in reversing the MPH response in the current study is not unexpected, given the importance of α2 and D1 receptor actions to PFC cognitive function. Catecholamine depletion in PFC produces working memory deficits as severe as ablation of the tissue itself , and D1 receptor blockade similarly weakens working memory regulation of behavior [44, 45]. In the current study, D1 blockade did not appear as completely effective as α2 receptor blockade, although both antagonists weakened the improvement such that it was statistically insignificant from vehicle. The potentially weaker effects with SCH23390 may be due to the lower dose used in some animals, and the difficulties in dealing with an inverted U dose response, where either too little or too much D1 receptor stimulation can impair performance. Under these conditions it is difficult to identify the correct dose of antagonist to perfectly normalize behavior. Individual variability in response to SCH23390 may arise from differences in endogenous D1 receptor stimulation under basal conditions. For example, D1 agonists have been shown to enhance attentional control in rats when infused into the PFC, but only in animals that were performing relatively poorly under basal conditions . D1 receptors also play a key role in striatal function, and it is possible that these actions outside the PFC also contributed to the enhancing effects of MPH on the delayed alternation task.
With some notable exceptions [47, 48], much of the ADHD field has focused on DA mechanisms in ADHD . In turn, there has been intensive focus on the DA actions of MPH, with some researchers even referring to MPH as a selective DA transporter blocker. The current data, in addition to recent biochemical studies [11, 18, 50] caution that the NE actions of stimulants such as MPH are just as important as the DA effects. This point is accentuated by the findings that one can recreate the symptoms of ADHD- increased locomotor activity, poor impulse control and weakened working memory/distractibility- by blocking α2 adrenoceptors with yohimbine infusions in the monkey PFC [51–53], respectively. Yohimbine also reduces the delay-related activity of PFC neurons, the cellular measure of working memory and response inhibition . Conversely, the α2 agonist, guanfacine, has been shown to strengthen working memory [55, 56], reduce distractibility , improve response inhibition [58–60], and increase regional cerebral blood flow in monkey PFC . Most recently, guanfacine has been shown to reduce locomotor hyperactivity and improve attentional control in the spontaneously hypertensive rat, a rodent model of ADHD (T. Sagvolden, personal communication). Experiments are in progress to determine whether MPH loses efficacy in mice with a functional knockout of the α2A adrenoceptor. The current results with idazoxan indicate that at least some of the beneficial effects of MPH arise from NE stimulation of α2 adrenoceptors.
Relevance to medications used to treat ADHD
Many of the medications used to treat ADHD increase endogenous NE stimulation of α2 adrenoceptors or mimic NE by directly stimulating these receptors. For example, like MPH, atomoxetine (Strattera) and amphetamine (Adderall) increase NE as well as DA in the PFC of rats [19, 42]. NE reuptake blockers have been shown to be very efficacious in treating ADHD symptoms, although their cardiac side effects have limited clinical utility in children [47, 62]. Future studies will need to examine whether low, oral doses of amphetamine and atomoxetine, like MPH, can improve delayed alternation performance in rats. Guanfacine mimics NE at α2 adrenoceptors, and is now in common use for the treatment of ADHD, especially in patients with tic disorders or drug abuse liability who cannot take stimulant medications . Guanfacine has been shown to improve spatial working memory performance in mice , rats , monkeys [55, 66] and humans . Thus, there is an excellent correspondance between drug effects in the laboratory and clinical efficacy in ADHD. The identification of an appropriate MPH dose regimen for use in rats should help in the development of safer and more effective medications for the treatment of ADHD.