In conclusion we have found

In conclusion, we have found that Egr1 can play an inhibitory role on DBH promoter-driven transcription. This inhibition requires a newly identified Egr1 response ISRIB synthesis at the −227/−224 region of DBH promoter. This inhibition appears to result from Egr1 directly bound to the DBH promoter. We speculate that Egr1 may reduce DBH gene transcription in vivo, thereby leading to altered DA/NE ratio. Further studies should determine the functional role of Egr1 in conjunction with other transcription factors in the regulation of DBH gene expression in catecholaminergic neuronal systems.
Experimental procedures
Acknowledgments
Introduction It has been estimated that 50–70 million Americans suffer from chronic disorders of sleep and wakefulness that adversely affect health and longevity, making sleep disorders second only to pain in the number of patients seeking medical attention (Colten and Altevogt, 2006). Accordingly, the development of medications to treat sleep disorders is a high priority. One such drug is modafinil, which is approved for use in the treatment of the excessive daytime sleepiness associated with narcolepsy and other sleep disorders. Modafinil has a behavioral phenotype distinct from that of more traditional stimulants such as amphetamine or cocaine in that it does not produce rebound hypersomnolence and has limited dependence liability (Ballon and Feifel, 2006, Edgar and Seidel, 1997, Minzenberg and Carter, 2008). The mechanism of action(s) underlying the wake-promoting effects of modafinil remain to be elucidated (Minzenberg and Carter, 2008). Early research from the 1990s implicated the catecholamines, NE and DA, in the mechanism of action of modafinil, but the animal data are confusing, with the role that each neurotransmitter plays yet to be clarified. Of more than 100 neurotransmitter and enzyme targets, the only consistent finding in vitro is a low affinity (Ki=2–7 µM) inhibition of the DA transporter (DAT) (Mignot et al., 1994). While DAT knockout mice show decreased responsiveness to modafinil (Wisor et al., 2001), other data indicated that DA receptor antagonists were ineffective in blocking modafinil-induced arousal (Duteil et al., 1990, Lin et al., 1992, Simon et al., 1995). The compounds that consistently block the effects of modafinil are α1-adrenergic receptor (α1AR) antagonists (Duteil et al., 1990, Hermant et al., 1991, Lin et al., 1992), while α1bAR knockout mice show an attenuated response to the drug (Stone et al., 2002). However, modafinil does not bind α1ARs (Mignot et al., 1994). Modafinil is also a weak inhibitor of the norepinephrine transporter (NET) in vitro and in vivo (IC50 ∼36 μM); (Madras et al., 2006). However, the in vivo assessment of this interaction was limited, as data from only one animal was available for the most effective modafinil dose (Madras et al., 2006). While many neurotransmitters are involved in producing and maintaining arousal states, these transporters remain the only known biochemical targets of modafinil. Thus, the focus of these experiments was on the catecholaminergic systems and their interaction. A novel tool for studying the relative roles of NE and DA in behavioral phenotypes are dopamine β-hydroxylase knockout (Dbh −/−) mice. These mice completely lack NE and epinephrine (EPI), but show a compensatory increase in high-affinity state DA receptors and are hypersensitive to the behavioral effects of both direct (e.g. quinpirole) and indirect (e.g. amphetamine, cocaine) DA agonists (Schank et al., 2006, Weinshenker et al., 2002b). Previous studies examining arousal behaviorally or by electroencephalogram (EEG) have revealed that exploratory activity in a novel environment, latency to sleep following handling or exposure to environmental stimuli, and wake bout duration were attenuated in Dbh −/− mice, suggesting that NE is important for maintaining vigilance (Hunsley and Palmiter, 2003, Hunsley and Palmiter, 2004). Thus, Dbh −/− mice were used to explore the role of catecholamines in modafinil-induced arousal.