Dopamine D₁ and D₂ Receptor Family Contributions to Modafinil-Induced Wakefulness

Modafinil (2-[(diphenylmethyl) sulfinyl] acetamide; United States brand name Provigil) is a wake-promoting agent first prescribed in France to treat narcolepsy-associated somnolence in the 1990s. Modafinil is currently prescribed in the United States for narcolepsy-associated somnolence, shift-work sleep disorder, and obstructive sleep apnea syndrome (Minzenberg and Carter, 2008), and it is being investigated for treating cognitive dysfunction in schizophrenia, depression, and attention deficit hyperactivity disorder (Minzenberg and Carter, 2008). Modafinil is considered safe, because subjects taking modafinil do not experience amphetamine-like withdrawal symptoms during discontinuation. Side effects of modafinil have been observed, however, including anxiety (4%), depression (4%), dyskinesia (2%), and psychosis [1 case (Wu et al., 2008)]. Understanding the precise mechanism(s) of action for modafinil may enable the development of more selective compounds having fewer side effects.

Studies performed in vivo and in vitro have suggested several possible mechanisms for the action of modafinil, including inhibiting dopamine and norepinephrine transporters and increasing dopamine, serotonin, glutamate, and histamine release (Minzenberg and Carter, 2008). While it was suggested that effective doses of modafinil are insufficient to inhibit the dopamine transporter (DAT) and hence do not act via this mechanism (Mignot et al., 1994), a recent positron emission tomography study in monkeys indicated 50% binding of modafinil to the DAT at lower plasma concentrations than are induced by therapeutic doses (Madras et al., 2006). Moreover, mice lacking the DAT do not exhibit modafinil-induced wakefulness (Wisor et al., 2001). DAT knock-out (KO) mice exhibit altered dopamine D₁ and D₂ receptors, however, as well as other compensatory mechanisms such as norepinephrine abnormalities. Thus, the mechanism of modafinil-induced wakefulness requires further investigation.

Qu et al. (2008) combined pharmacological and genetic techniques to investigate the contribution of D₁ and D₂ receptors to modafinil-induced wakefulness. The authors used male D₂ receptor KO mice, as well as the D₁- and D₂-family antagonists SCH23390 and raclopride, respectively, to investigate possible dopaminergic mechanisms for modafinil-induced wakefulness. Mice were equipped with EEG and electromyogram electrodes in the cortex and both trapezius muscles respectively for polysomnographic recordings. Sleep–wakefulness states were recorded for 48 h, comparing baseline and experimental days. Polygraphic recordings were scored by 10 s epochs as wakefulness, rapid-eye-movement (REM), and non-REM (NREM) sleep according to published criteria.

The authors first demonstrated a long-lasting, dose-dependent increase in wakefulness after modafinil administration [Qu et al. (2008), their Fig. 1A–D (http://www.jneurosci.org/cgi/content/full/28/34/8462/F1)], in accordance with other publications. Mice exhibited increased awake time, as well as reduced NREM and REM sleep [Qu et al. (2008), their Fig. 2A (http://www.jneurosci.org/cgi/content/full/28/34/8462/F2)]. Both SCH23390 and raclopride fully blocked low-dose modafinil-induced increases in wakefulness [Qu et al. (2008), their Fig. 1E,I (http://www.jneurosci.org/cgi/content/full/28/34/8462/F1)] and attenuated the effects of modafinil at higher doses [Qu et al. (2008), their Fig. 1F–H,J–L (http://www.jneurosci.org/cgi/content/full/28/34/8462/F1)]. While the use of low doses of SCH23390 and raclopride increased the likelihood of specifically targeting D₁ and D₂ receptor families, respectively, neither drug is wholly selective within these families. Thus, the effects observed could also reflect antagonizing D₅ or D_3/4 receptors, respectively. To address this confound, the authors investigated wakefulness effects of modafinil in D₂ receptor KO mice. While low doses of modafinil increased wakefulness in wild-type mice [Qu et al. (2008), their Fig. 6A (http://www.jneurosci.org/cgi/content/full/28/34/8462/F6)], this effect was absent in KO mice [Qu et al. (2008), their Fig. 6C (http://www.jneurosci.org/cgi/content/full/28/34/8462/F6)]. Thus, higher doses of modafinil were required to induce wakefulness in D₂ KO mice [Qu et al. (2008), their Fig. 6C (http://www.jneurosci.org/cgi/content/full/28/34/8462/F6)]. These data support the authors' conclusions from initial studies that the D₂ receptor contributes to modafinil-induced wakefulness. Also consistent with their initial studies, the authors observed that the D₁ receptor antagonist SCH23390 attenuated modafinil-induced wakefulness in wild-type mice [Qu et al. (2008), their Fig. 6B (http://www.jneurosci.org/cgi/content/full/28/34/8462/F6)]. Modafinil-induced wakefulness was completely antagonized, however, by SCH23390 in D₂ receptor KO mice [Qu et al. (2008), their Fig. 6D (http://www.jneurosci.org/cgi/content/full/28/34/8462/F6)]. The authors conclude that these data support a D₂ and D₁ receptor interaction for modafinil-induced wakefulness. Thus, the authors contribute to evidence for D_1/2 receptor involvement in sleep/wake profiles.

While this combined genetic and pharmacological approach provides striking evidence for D₂ receptor and D₁ family involvement in modafinil-induced increases in wakefulness, further evidence may be required. For example, the authors' interpretation of SCH23390- and raclopride-induced attenuation of modafinil-induced increase in wakefulness may be premature. Although the pharmacological evidence was compelling, the authors did not demonstrate whether the SCH23390 or raclopride reduction in modafinil-induced wakefulness was an additive or competitive effect, as they did not examine the effects of these drugs alone on wakefulness. Previous reports suggest that SCH23390 reduces wakefulness in rats by increasing REM and NREM sleep (Ongini et al., 1993), while the D₂ antagonist increases sleep time in mice (Cohen et al., 1997), opposite to the effects of modafinil in mice reported by Qu et al. (2008). Thus, it cannot be concluded that the opposing effects of SCH23390 and raclopride on modafinil-induced wakefulness are not a result of competing orthogonal systems.

In addition, the authors argued that modafinil acts directly at D₁ and D₂ receptors, despite reporting no binding data to support this theory. Alternatively, modafinil may increase extracellular dopamine via DAT inhibition (Madras et al., 2006), with SCH23390 or raclopride blocking its effects on wakefulness by competitively blocking the availability of dopamine to activate these receptors. [¹¹C]raclopride binding is reduced in sleep-deprived humans, putatively via competition with increased endogenous extracellular dopamine after sleep deprivation (Volkow et al., 2008). Thus, mediation of modafinil-induced wakefulness via a D₁/D₂ receptor as opposed to DAT requires further evidence.

The interpretation of D₁-selective SCH23390 effects on modafinil-induced wakefulness must also be qualified. As discussed above, SCH23390 is not selective for the D₁ receptor, since it also binds to the D₅ receptor in mice and has behavioral effects in D1 KO mice (Centonze et al., 2003) at doses used in the study by Qu et al. (2008). Thus, a role for the D₅ receptor cannot be discounted. The reduced effects of modafinil on D₂ receptor KO mice provide evidence that the observed raclopride effects were likely to have been mediated by the D₂ receptor. Thus, greater evidence for a D₁ receptor involvement for modafinil-induced increases in wakefulness could be generated by assessing the effects of modafinil in D₁ KO mice, in combination with raclopride. These data would provide evidence similar to that generated by D₂ KO mice and strengthen the argument for D₁ receptor selectivity in the effects of modafinil. These findings would be particularly important given the interaction of D₅ receptors with ADHD, their localization on cholinergic neurons that are important for attentional performance, and the putative attention-enhancing effects of modafinil (Minzenberg and Carter, 2008). It would also be informative to administer raclopride in efficacious (high)-dose modafinil-pretreated D₂ KO mice, to further demonstrate D₂ selectivity of raclopride.

Modafinil does not induce wakefulness in mice lacking the DAT (Wisor et al., 2001). Qu et al. (2008) caution against the interpretation that the DAT is therefore required for modafinil-induced wakefulness because DAT KO mice exhibit abnormal D₁ and D₂ receptor regulation, which may mediate the reduced efficacy of modafinil in these mice. Moreover, Qu et al. (2008) indicate that the nomifensine- and modafinil-evoked current–voltage relationships differ on rat brain slices (Korotkova et al., 2007), suggesting a different mechanism of action than DAT selectivity. Nomifensine is, however, threefold to fourfold more selective to the norepinephrine transporter than DAT (Tatsumi et al., 1997), while modafinil binds threefold to fourfold more selectively to the DAT at clinically relevant doses (Madras et al., 2006). GBR12909 is also more selective to the DAT, exhibits a similar behavioral profile to modafinil, and was also ineffective in DAT KO mice (Wisor et al., 2001), suggesting that modafinil-induced DAT inhibition remains a viable hypothesis for the effects of modafinil. Moreover, the reduced effectiveness of modafinil in D₂ KO mice observed in the study by Qu et al. (2008), could be interpreted as a result of the 50% reduction in functional DAT levels in these mice (Dickinson et al., 1999).

The pharmacological studies by Qu et al. (2008) provide support for D₁ and D₂ receptor family involvement in wakefulness in mice. Moreover, the authors' combined genetic and pharmacological study provides evidence that the D₂ receptor is important in the arousal effects of modafinil. The authors' approach to investigating the mechanism of modafinil-induced wakefulness is an effective way to selectively examine receptor contributions to this effect. This approach could therefore be used to investigate the mechanism of modafinil-induced effects in other areas, such as locomotor activity or cognitive performance as these behaviors may be mediated by distinct mechanisms (Minzenberg and Carter, 2008). Increased knowledge of the mechanisms of the effects of modafinil may allow increasing selectivity for these effects, or the generation of drugs with reduced side effects. Furthermore, Qu et al. (2008) provide thoughtful and incisive interpretations of genetic and pharmacological manipulations when using this combined genetic and pharmacological approach. Care must therefore be taken when interpreting the need for both the D₁ and D₂ receptors in mediating the modafinil-induced increases in wakefulness. D₅ receptor contributions to modafinil-induced wakefulness cannot as yet be discounted, nor can the contribution of the DAT. However, the study by Qu et al. (2008) provides a platform from which the contributions of these receptors can be further investigated.