Sleep and arousal are known to be regulated by both homeostatic and circadian processes, but the underlying molecular mechanisms are not well understood. It has been reported that the Drosophila rest/activity cycle has features in common with the mammalian sleep/wake cycle, and it is expected that use of the fly genetic model will facilitate a molecular understanding of sleep and arousal. This study reports the phenotypic characterization of a Drosophila rest/activity mutant known as fumin (fmn). fmn mutants have abnormally high levels of activity and reduced rest (sleep); genetic mapping, molecular analyses, and phenotypic rescue experiments demonstrate that these phenotypes result from mutation of the Drosophila Dopamine transporter gene. Consistent with the rest phenotype, fmn mutants show enhanced sensitivity to mechanical stimuli and a prolonged arousal once active, indicating a decreased arousal threshold. Strikingly, fmn mutants do not show significant rebound in response to rest deprivation as is typical for wild-type flies, nor do they show decreased life span. These results provide direct evidence that dopaminergic signaling has a critical function in the regulation of insect arousal (Kume, 2005).

Although fmn flies exhibit such rest/activity phenotypes, there is apparently no effect of the mutation on development or longevity. This contrasts markedly with the results observed for mutations of two other genes that have been implicated in the regulation of Drosophila rest: cyc and Shaker (Sh). Mutations in cyc or Sh reduce life span, relative to genetic background controls, although complete life-span curves were not reported for Sh and there appears to be only a small effect on longevity for the Sh¹⁰² allele. The different effects of these mutations on longevity may reflect the relatively selective effect of fmn on arousal. Alternatively, the effects of Sh and cyc mutations on life span might reflect requirements for these genes in developmental or physiological processes other than rest. Cyc protein is a broadly expressed basic helix-loop-helix transcription factor, whereas Sh is a voltage-activated potassium channel with a broad localization in the nervous system. In contrast, DAT deficits only affect dopaminergic neuromodulation and therefore might have a less general impact on development and physiological processes (Kume, 2005).

Fmn flies carry a mutation in the Drosophila dopamine transporter gene, indicating that alterations of dopamine signaling are responsible for the observed phenotypes. dDAT functions in the dopaminergic pathway, as shown by (1) dDAT protein has significant sequence similarity to comparable mammalian and invertebrate transporters, (2) dDAT gene expression is restricted to dopaminergic neurons (as expected for a presynaptic transporter), (3) this transporter has a substrate specificity paralleling that of the mammalian DATs, with dopamine and tyramine being the preferred substrates, and (4) the dDAT transporter mediates uptake of dopamine in cell-based assays and responds to dopamine when expressed in Xenopus oocytes (Kume, 2005).

Dopamine is cleared from the synaptic cleft via presynaptic DAT, and DAT mutant mice exhibit altered presynaptic autoreceptor function, dopamine clearance, and biosynthetic rate in addition to behavioral alterations including spontaneous hyperlocomotion and hyperactivity. These phenotypes are presumably caused by the elevated persistence of released dopamine in these mice. Similarly, it seems likely that increased dopamine signaling in fmn is responsible for the observed hyperactivity and shortening of the rest phase, regarded as sleep in Drosophila (Kume, 2005).

Previous studies in Drosophila implicate biogenic amines in the modulation of activity. In larval Drosophila, 5-HT, OA, TA, and DA regulate locomotion or the sensory-motor circuitry on which such behavior depends. In adult flies, evidence suggests that DA and 5-HT function to regulate locomotor activity and flight, respectively. The study by Lima (2005) is particularly informative in that it demonstrates state-dependent effects of dopamine neuronal stimulation in behaving flies. In flies with low basal activity, the response to targeted (and transient) stimulation of dopamine neurons is an increased probability of locomotor bouts, whereas a similar stimulation of flies showing high basal activity leads to an inhibition. These results are most consistent with a biphasic role for modulation of locomotor activity by released dopamine, with the highest levels of dopamine release leading to locomotor inhibition. This is in agreement with studies that have examined the responses of flies to the psycho-stimulant cocaine, an inhibitor of aminergic transporters. Cocaine stimulation of flies results in transient stereotypies and hyperactivity that are strikingly similar to those seen after cocaine exposure to vertebrate animals. However, the most severely affected flies become akinesic, consistent with a biphasic effect of high extracellular dopamine (Kume, 2005).

Based on the decreased arousal threshold and the prolonged responses of fmn flies to mechanical stimulation, it is suggested that this mutant is characterized by an arousal state with enhanced alertness associated with the expected increase in extracellular dopamine. The absence of significant rest rebound in fmn supports this conclusion, along with the finding that activity level during each arousal period is normal. This is the first direct evidence that altered arousal threshold and decreased rebound can result from perturbations of dopaminergic signaling. Previous results have indirectly implicated DA in arousal. Those studies examined animals with lesions of dopaminergic neuronal populations, changes in firing rates of dopaminergic neurons as a function of sleep states and arousal, or the actions of wake-promoting drugs, some of which modulate DAT or DA receptor activity. The analysis of fmn shows directly that a selective lesion of DAT, presumably with accompanying increased DA levels, results in an alteration of arousal threshold (Kume, 2005).

It is of interest that mouse DAT mutants, like fly fmn, have abnormal sleep, although arousal sensitivity has not been explicitly examined. DAT mutant mice show enhanced spontaneous locomotor hyperactivity that is greatly enhanced by the stimulating effects of novel environment. More importantly, mouse DAT mutants have significantly increased wake bout duration and moderately increased activity levels during the latter half of the active (night) phase of the diurnal cycle. The extension of the active phase in these mice mimics the phenotype of fmn flies that have a lengthened active period and shortened rest phase during the diurnal cycle. DAT knock-out mice also exhibit altered responses to wake-promoting drugs such as GBR12909, modafinil, and caffeine. Such mice show increased sensitivity to caffeine and decreased sensitivity to GBR12909 and modafinil, suggesting that the latter two drugs act on DAT to promote wakefulness. Modafinil has wake-promoting properties in Drosophila, and it will be of interest to determine whether modafinil action is altered in fmn as it is in DAT mutant mice. A role for DAT and dopaminergic signaling in regulating wakefulness in flies and mice provides additional evidence for a similarity between mammalian sleep and insect rest (Kume, 2005).

Dopamine is probably also important for the regulation of arousal in humans. Parkinson's disease patients, who have reduced dopamine levels, often complain of sleepiness. When treated with L-3,4-dihydroxyphenylalanine (L-DOPA), which increases dopamine levels, they recover from sleepiness, but with excessive L-DOPA, they exhibit insomnia. Moreover, patients and animals with narcolepsy, which show increased wakefulness and excessive sleepiness, show a compensational increase in brain D₂-like receptors, suggesting a reduced level of dopamine (Kume, 2005).

Although fmn mutants have higher basal levels of activity, they nonetheless exhibit diurnal and circadian rhythms in locomotor activity. However, rhythmicity is less evident compared with that observed in wild-type flies, presumably because there is a higher baseline of activity at all times of day and therefore a corresponding decrease in the amplitude of rhythmicity (Kume, 2005).

Part of the evidence that fmn is a mutation in dDAT comes from transgenic rescue using pan-neurally driven expression of dDAT. It is notable that this rescue is only partial. One obvious explanation for this partial rescue is that expression of ELAV-GAL4 in the dopamine neurons, the sole site of dDAT localization, is weak. However, even stronger dopamine neuron expression of dDAT, under the control of TH-GAL4, yields even weaker rescue. This counterintuitive result may indicate that the precise level of dDAT in dopamine neurons is critical for normal dopamine homeostasis. It is possible that homeostatic mechanisms potentially overcompensate for high DAT levels and reduced synaptic DA by hyperactivating postsynaptic receptors, as has been seen after inhibition of dopamine and serotonin neurons. Thus, dDAT may be a component of a precisely regulated dopamine homeostatic mechanism that controls arousal threshold and overall activity levels (Kume, 2005).

This study of a Drosophila DAT mutant indicates another striking parallel between Drosophila and vertebrates with regard to the functions of biogenic amine systems. It demonstrates that the regulated reuptake of dopamine by DAT is important for setting arousal threshold. Equally important, the identification of a mutation in the pharmacologically important dopamine transporter opens new avenues for use of this genetically tractable model in pharmacological and behavioral studies (Kume, 2005).

A fully functional dopamine transporter (DAT) mutant (dmDATx7) has been generated with all cysteines removed except the two cysteines in extracellular loop 2 (EL2). Random mutagenesis at either or both EL2 cysteines did not produce any functional transporter mutants, suggesting that the two cysteines cannot be replaced by any other amino acids. The cysteine-specific reagent MTSEA-biotin labeled dmDATx7 only after a DTT treatment which reduces the disulfide bond. Since there are no other cysteines in dmDATx7, the MTSEA-biotin labeling must be on the EL2 cysteines made available by the DTT treatment. This result provides the first direct evidence that the EL2 cysteines form a disulfide bond. Interestingly, the DTT treatment had little effect on transport activity suggesting that the disulfide bond is not necessary for the uptake function of DAT. These results and previous results are consistent with the notion that the disulfide bond between EL2 cysteines is required for DAT biosynthesis and/or its delivery to the cell surface (Chen, 2007).