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. Here, we report the phenotypic characterization of a Drosophila rest/activity mutant known as fumin ( fmn). We show that 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 mutantsdonotshowsignificantreboundinresponsetorestdeprivationasistypicalforwild-typeflies,nordotheyshowdecreasedlife span. These results provide direct evidence that dopaminergic signaling has a critical function in the regulation of insect arousal.

Dopamine (DA) is the only catecholaminergic neurotransmitter in the fruit fly Drosophila melanogaster. Dopaminergic neurons have been identified in the larval and adult central nervous system (CNS) in Drosophila and other insects, but no specific genetic tool was available to study their development, function, and degeneration in vivo. In Drosophila as in vertebrates, the rate-limiting step in DA biosynthesis is catalyzed by the enzyme tyrosine hydroxylase (TH). The Drosophila TH gene (DTH) is specifically expressed in all dopaminergic cells and the corresponding mutant, pale (ple), is embryonic lethal. We have performed ple rescue experiments with modified DTH transgenes. Our results indicate that partially redundant regulatory elements located in DTH introns are required for proper expression of this gene in the CNS. Based on this study, we generated a GAL4 driver transgene, TH-GAL4, containing regulatory sequences from the DTH 5' flanking and downstream coding regions. TH-GAL4 specifically expresses in dopaminergic cells in embryos, larval CNS, and adult brain when introduced into the Drosophila genome. As a first application of this driver, we observed that in vivo inhibition of DA release induces a striking hyperexcitability behavior in adult flies. We propose that TH-GAL4 will be useful for studies of the role of DA in behavior and disease models in Drosophila.

Dopaminergic neurons are thought to drive learning by signaling changes in the expectations of salient events, such as rewards or punishments. Olfactory conditioning in Drosophila requires direct dopamine action on intrinsic mushroom body neurons, the likely storage sites of olfactory memories. Neither the cellular sources of the conditioning dopamine nor its precise postsynaptic targets are known. By optically controlling genetically circumscribed subsets of dopaminergic neurons in the behaving fly, we have mapped the origin of aversive reinforcement signals to the PPL1 cluster of 12 dopaminergic cells. PPL1 projections target restricted domains in the vertical lobes and heel of the mushroom body. Artificially evoked activity in a small number of identifiable cells thus suffices for programming behaviorally meaningful memories. The delineation of core reinforcement circuitry is an essential first step in dissecting the neural mechanisms that compute and represent valuations, store associations, and guide actions.

Hearing in Drosophila depends on the transduction of antennal vibration into receptor potentials by ciliated sensory neurons in Johnston's organ, the antennal chordotonal organ. We previously found that a Drosophila protein in the vanilloid receptor subfamily (TRPV) channel subunit, Nanchung (NAN), is localized to the chordotonal cilia and required to generate sound-evoked potentials (Kim et al., 2003). Here, we show that the only other Drosophila TRPV protein is mutated in the behavioral mutant inactive (iav). The IAV protein forms a hypotonically activated channel when expressed in cultured cells; in flies, it is specifically expressed in the chordotonal neurons, localized to their cilia and required for hearing. IAV and NAN are each undetectable in cilia of mutants lacking the other protein, indicating that they both contribute to a heteromultimeric transduction channel in vivo. A functional green fluorescence protein-IAV fusion protein shows that the channel is restricted to the proximal cilium, constraining models for channel activation.

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