Methylphenidate improves prefrontal cortical cognitive function through α2 adrenoceptor and dopamine D1 receptor actions: Relevance to therapeutic effects in Attention Deficit Hyperactivity Disorder

The effects of an acute, oral dose of MPH were examined over the dose range found to produce drug plasma levels in rats similar to clinical use in ADHD (0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg, oral administration 30 min before testing). MPH produced an inverted U dose response curve whereby the middle doses (1.0, 1.5, 2.0 mg/kg) generally improved performance, while higher doses (2.0, 3.0 mg/kg) often impaired performance. Representative dose/response curves are shown in Figure 1A. There were individual differences in MPH dose sensitivity that may result from differences in MPH absorption from the gastrointestinal tract, and/or variations in endogenous catecholamine levels in PFC circuits. For all animals, a dose was found between 1.0–2.0 mg/kg that significantly improved delayed alternation performance (Figure 1B; vehicle vs. MPH p = 0.002, df = 7). No change in response time was noted (mean ± SEM vehicle: 191.2 ± 39.1 sec; mean ± SEM MPH: 179.3 ± 30.3 sec; p > 0.7, df = 7). These enhancing doses were used in subsequent experiments to examine the receptor actions contributing to MPH therapeutic actions.

Six of the eight rats tested showed impairment in delayed alternation performance as the dose was raised (1.5–3 mg/kg, especially following 2–3 mg/kg). The number of perseverative errors significantly increased at these higher doses, as measured by the greatest number of consecutive entries into a single arm (Figure 1C; p < 0.05, df = 5). A perseverative response pattern is consistent with PFC dysfunction. No consistent change in response time was observed following higher, impairing doses of MPH, although some animals were faster and some slower than usual (mean ± SEM vehicle: 201.4 ± 46.2 sec; mean ± SEM MPH: 194.2 ± 107.2 sec; range vehicle: 91–398 sec; range MPH: 50–675 sec). No stereotyped behaviors, common at much higher doses, were observed in these animals. Thus, cognitive choices, but not behavior per se, showed a perseverative profile.

The α2 adrenoceptor antagonist, idazoxan, was co-administered with MPH to test whether MPH enhances performance by facilitating endogenous NE stimulation of α2 adrenoceptors (n = 5). The optimal dose of MPH was selected for each animal; a dose of idazoxan was selected (0.1 mg/kg) that had no effects on its own. As shown in Figure 2, idazoxan significantly reversed the enhancing effects of MPH. Two way analysis of variance with repeated measures (2-ANOVA-R) showed a significant effect of MPH (F(1,4) = 18.45, p = 0.01), a trend toward significant effect of idazoxan (F(1,4) = 6.63, p = 0.06), and a significant interaction between the two drugs (F(1,4) = 27.2, p = 0.006). User defined contrasts showed that MPH+vehicle significantly improved performance compared to vehicle+vehicle (F(1,4) = 53.3, p = 0.002), while idazoxan+vehicle was not significantly different than vehicle+vehicle (F(1,4) = 0.02 p = 0.90). Most importantly, animals performed significantly lower on the delayed alternation task following MPH+idazoxan treatment than when they were administered MPH+vehicle (F(1,4) = 43.4, p = 0.0028), and were not significantly different in their performance from days in which they were administered vehicle+vehicle (F(1,4) = 0.005 p = 0.95). These results are consistent with α2 adrenoceptor actions contributing to the enhancing effects of oral MPH.

The DA D1 receptor antagonist, SCH23390, was co-administered with MPH to test whether MPH enhances performance by facilitating endogenous DA stimulation of D1 receptors (n = 7). A dose of 0.1 mg/kg SCH23390 was used, unless this dose produced impairment on its own. In these cases, the dose of SCH23390 was lowered to 0.01 mg/kg (n = 3). SCH23390 significantly reduced the enhancing effects of MPH on delayed alternation performance (Figure 3). 2-ANOVA-R showed a significant effect of MPH, a significant effect of SCH23390, and a significant interaction between the two drugs (effect of MPH: F(1,6) = 11.59, p = 0.014; effect of SCH23390: F(1,6) = 9.3, p = 0.023; interaction between MPH and SCH23390: F(1,6) = 9.3, p = 0.023). User defined contrasts showed that MPH+vehicle significantly improved performance compared to vehicle+vehicle (F(1,6) = 61.45, p = 0.0002), while SCH23390+vehicle was not significantly different than vehicle+vehicle (F(1,6) = 0.0, p = 1.0). Animals performed significantly worse on the delayed alternation task following MPH+SCH23390 treatment than when they were administered MPH+vehicle (F(1,6) = 15.0, p = 0.008). Although performance remained a bit above vehicle levels of response, performance following MPH+SCH23390 treatment was not significantly different than following vehicle+vehicle (F(1,6) = 0.84, p = 0.4). These results are consistent with D1 receptor actions contributing to the enhancing effects of oral MPH.

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 [30] 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 [36]. 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 [35]. 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 [37]. 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 [41]; 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.

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 ([19], and C.W. Berridge, personal communication). There are relatively low levels of DA transporters in the PFC [42]; 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 [42].

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 [43], 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 [46]. 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 [49]. 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 [54]. Conversely, the α2 agonist, guanfacine, has been shown to strengthen working memory [55, 56], reduce distractibility [57], improve response inhibition [58‐60], and increase regional cerebral blood flow in monkey PFC [61]. 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.

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 [63]. Guanfacine has been shown to improve spatial working memory performance in mice [64], rats [65], monkeys [55, 66] and humans [67]. 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.

Rats were habituated to a T-maze (dimensions, 90 × 65 cm) until they were readily eating chocolate chips placed at the end of each arm and were acclimated to handling. After habituation, rats were trained on the delayed alternation task. On the first trial, animals were rewarded for entering either arm. Thereafter, for a total of 10 trials per session, rats were rewarded only if they entered the maze arm that was not previously chosen. Between trials the choice point was wiped with alcohol to remove any olfactory clues. The delay between trials started at "0" sec (i.e. about 1.5 sec, minimum possible for delayed alternation) and was subsequently raised in 5 sec intervals as needed to maintain performance at about 70% correct. Animals were scored for accuracy (arm chosen) and response time for each trial.

The experimenter testing the animal was unaware of drug treatment conditions. Given the need for oral administration of drug, rats were habituated to eating a small piece of cracker. All animals had learned to ingest the cracker rapidly and completely prior to the initiation of drug testing.

MPH was acquired from the National Institute of Drug Abuse. Doses were based on those identified by Kuzcenski and Segal [11]. MPH was dissolved in tap water and injected onto a small piece of Saltine cracker that was fed to the rat 30 min before cognitive testing. The doses examined were: 0 (water only), 0.5, 1.0, 1.5, 2.0, and 3.0 mg/kg). For example, the 1.0 mg/kg dose was made by dissolving 1 mg MPH in1 ml water and injecting a volume equivalent to the weight of the rat, e.g. a 450 g rat would receive 0.45 ml injected onto the cracker. Animals rapidly ate the cracker once habituated to the procedure. Doses were administered in random order with the exception that no animal began with the 3.0 mg/kg dosage.

Idazoxan was purchased from Sigma (St. Louis, MO) and administereed at a dose of 0.1 mg/kg. Idazoxan was dissolved in saline, and like MPH, was injected into the cracker for oral administration.

SCH23390 also was purchased from Sigma and dissolved in saline. SCH23390 was initially administered in a dose of 0.1 mg/kg. Animals who were impaired by this dose were subsequently administered 0.01 mg/kg SCH23390 so that additive effects could not account for MPH reversal.

The dependent variables were percent correct on the delayed alternation task (accuracy), response time, and greatest number of consecutive entries into an incorrect arm (perseveration score). Statistical comparisons utilized within subjects designs; simple comparisons utilized paired (dependent) T tests. The effects of idazoxan or SCH23390 on the MPH response were analyzed with a two-way analysis of variance with repeated measures with factors of 1) MPH and 2) antagonist, and user defined contrasts to test pairwise comparisons.