Narcoleptic Dobermans were also used for a series of pharmacological experiments to dissect modes of action of wake-promoting compounds. Amphetamine-like CNS stimulants currently used clinically for the management of sleepiness in narcolepsy enhance monoaminergic transmission presynaptically.
Figure 4. Effects of various DA and NE uptake inhibitors and amphetamine-like stimulants on the EEG arousal of narcoleptic dogs and correlation between in vivo EEG arousal effects and in vitro DAT binding affinities. The effects of compounds on daytime sleepiness was studied using 4 hrs daytime polygraphic recordings (10:00-14:00) in 4-5 narcoleptic animals. Two doses were studied for each compound. All DA uptake inhibitors and CNS stimulants dose-dependently increased EEG arousal and reduced slow wave sleep (SWS) when compared to vehicle treatment. In contrast, nisoxetine and desipramine, two potent NE uptake inhibitors had no significant effect on EEG arousal when doses which completely suppressed REM sleep were injected. Compounds with both adrenergic and dopaminergic effects (nomifensine, mazindol, D-amphetamine) were active on both EEG arousal (left panel) and REM sleep. The effects of the two doses studied for each stimulant was used to construct a rough dose-response curve (left panel). The drug dose which increased the time spent in wakefulness by 40% more than the baseline (vehicle session) was then estimated for each compound. The order of potency of the compounds obtained was: mazindol > (amphetamine) > nomifensine > GBR 12,909 > amineptine> (modafinil) > bupropion. In vitro DA transporter (DAT) binding was performed using [3H]-WIN 35,428 onto canine caudate membranes and demonstrated that the affinity of these DA uptake inhibitors varied widely between 6.5 nM and 3.3 mM. In addition, it was also found that both amphetamine and modafinil have a low, but significant affinity (same range as amineptine) for the DAT. A significant correlation between in vivo and in vitro effects was observed for all 5 DA uptake inhibitors and modafinil (right panel).
However, these compounds also lack pharmacological specificity. In order to study the mode of action of these wake-promoting compounds on daytime sleepiness, the stimulant properties of several dopaminergic and adrenergic uptake inhibitors were quantified and compared to the effects of amphetamine and modafinil using 4-hour daytime polygraphic recordings.62 In spite of their lack of effect on cataplexy, all dopaminergic uptake inhibitors induced significant EEG arousal (Fig. 4). In contrast, nisoxetine and desipramine, two potent adrenergic uptake inhibitors, had little effect on EEG arousal but significantly suppressed REM sleep. Furthermore, the in vivo potency of DA uptake inhibitors on EEG arousal correlates well with their in vitro affinity to the DA transporter (DAT), but not to the norepinephrine transporter (NET). These results are
Effect on Increase in EEG arousal Log (ED+40 [Mmol I.V.])
Effect on Increase in EEG arousal Log (ED+40 [Mmol I.V.])
consistent with the hypothesis that presynaptic modulation of dopaminergic transmission is a key property mediating the EEG arousal effects of these compounds. Interestingly, we also found that modafinil binds to the DAT site with low affinity,62,63 similar to the affinity range for amineptine (a DA uptake inhibitor which also enhances EEG arousal in our model). Thus, the DAT binding property may also contribute to the stimulant properties of modafinil. We recently demonstrated that the wake-promoting effect of modafinil and amphetamine were completely absent in mice lacking DAT (i.e., DAT KO mice).64 These results clearly indicate that DAT is required for the mediation of the wake-promoting effect of modafinil (and amphetamine), but other pharmacological properties may also involve in wake-promoting effects of modafinil.65,66
Amphetamine (which showed a potent EEG arousal property) has however, a relatively low DAT binding affinity,67 suggesting that other mechanisms, such as monoamine release (exchange diffusion through the DAT) by amphetamine, are also involved in the mechanism of EEG arousal. Amphetamine is also reported to block the vascular monoamine transporter 2 (VMAT 2) and to induce reverse-transport of monoamines into the synaptic area.68 To further assess the net effects of amphetamine on monoaminergic neurotransmission, we measured DA and NE efflux together with wake-promoting effects of amphetamine-analogs and isomers in the canine narcolepsy model.69 Polygraphic recordings demonstrated that d-amphetamine was about twice as potent as l-amphetamine, and was 6-times more potent than l-methamphetamine in increasing wakefulness, while d-amphetamine and l-amphetamine were equipotent in reducing REM sleep and cataplexy, and l-methamphetamine was about half as potent as l- and d-amphetamine.69 By measurements of extracellular levels of DA and NE, we found that d-amphetamine was more potent in increasing DA efflux than l-amphetamine, and l-methamphetamine had little effect. In contrast, there was no significant difference in the potencies of theses 3 derivatives on NE efflux. Thus, the potencies of amphetamine isomers/analogs on wakefulness correlated well with DA, but not NE efflux in the brain of narcoleptic dogs, and further exemplifies the importance of the DA system for the pharmacological control of EEG arousal.69
However, the involvement of the dopaminergic systems in the regulation of the sleep/wake process that has not been given much attention, mostly due to early electrophysiological findings demonstrating that dopaminergic neurons in the ventral tegmental area (VTA) and substantia nigra (SN) do not change their activity significantly during the sleep cycle,38 in contrast to noradrenergic cells of the locus coeruleus (LC) or serotonergic cells of the raphe which increase firing in wake versus sleep. Although firing patterns of DA neurons during slow wave sleep are different from those during wake or REM sleep, DA release in the LC and amygdala measured with microdialysis experiments failed to demonstrate the state dependent change.70 These experimental results led most investigators to believe that adrenergic tone was more important than dopaminergic transmission for the control of EEG arousal.
The dopaminergic system may thus be not so important for the normal sleep wake cycle regulation, but may play a key role for forced wakefulness by motivation and/or by stimulants, and the hypocretin-dopaminergic interaction may also involved in these alerting mechanisms. In clinical conditions, a disturbance of this mechanism may be more troublesome, since it may induce intolerable sleepiness at the situation required the high level of vigilance.
An involvement of dopaminergic system in intolerable sleepiness is also noted in some pathological conditions, such as Parkinson's disease. Frequent sleep attacks and associations with accidents are reported by patients with Parkinson's disease treated with DA D2/D3 agonists,71,72 the classes of compounds that induce drowsy states and cataplexy in the canine model of narcolepsy.48,49
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