Dopamine is a regulator of arousal in the fruit fly

Abstract

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 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.

Figure 1.

fmn mutants exhibit elevated total daily activity levels, reduced rest, and a normal activity index. a, Double plots of locomotor activity data for three representative w and w; fmn flies in LD and DD conditions (3 d in LD and 4 d in DD). Data are plotted as number of activity counts (beam crossings) per 5 min of time. b, Population activity averages in LD and DD (n = 10 for w and fmn). c, Total daily activity plots for w and fmn flies. d, Average total daily rest for w and fmn populations. e, Activity indices for w and fmn flies. Error bars indicate the SEM; ^* indicates statistically significant differences between w and fmn flies (Student's t test; p < 0.001). In c-e, gray histograms represent the wild type; black histograms represent fmn.

Figure 2.

fmn flies have normal life span. Life-span curves are shown for fmn and control flies. Each curve represents ∼200 flies. See Materials and Methods for details of the longevity experiments.

Figure 3.

fmn is a dDAT mutation. a, Summary of the mapping of fmn by meiotic recombination. The map of the dDAT region is illustrated, showing locations of the dominant genetic markers and P-element insertions used for recombinational mapping of fmn. b, Northern blot analysis of dDAT expression. The blot was hybridized with the complete dDAT ORF (left), a 3′ partial ORF (right), or rp49 sequences (positive control). A 3′-deleted, truncated dDAT mRNA is expressed in fmn mutants. See Materials and Methods for details. c, Structures of dDAT genome and cDNA of wild type and fmn. In fmn, exon 6-intron 6 splicing is affected, presumably because of the heterologous DNA insertion into intron 6. This aberrant splicing results in the termination of dDAT protein at residue 343 in fmn. d, e, Rescue of fmn by neuronal dDAT expression, using elav-Gal4 as a driver. Quantitative activity and rest analysis was performed for w and fmn mutants using data collected in DD. The presence or absence of ELAV-GAL4 and the UAS-dDAT responder transgenes are indicated by + or -. The results indicate a partial rescue of the fmn phenotype by transgenic dDAT expression. Differences from nontransgenic and both single transgenic flies (n = 10) are statistically significant using Student's t test (^*p < 0.05).

Figure 4.

fmn mutants show enhanced responses and prolonged response times to mechanical stimuli. a, Responses to mild, moderate, and strong stimuli. fmn mutants showed a larger response than wild-type control flies to both mild and moderate strength stimuli (n = 16 for both genotypes) b, c, Histograms of the distributions of response times in w and fmn flies. Frequency (y-axis) corresponds to the number of stimulated responses of the indicated durations out of the total number of responses. d, Average response times for both genotypes (from strong stimulation). fmn mutants show longer bouts of activity after stimulation. Error bars indicate the SEM. ^* indicates a statistically significant difference between w and fmn flies (Wilcoxon rank-sum test; p < 0.001).

Figure 5.

fmn mutants have attenuated rest rebound after rest deprivation. The plots for w (a) and fmn (b) show changes in the amount of rest during a 24 h period of time. Rest % is the fraction of 5 min intervals in each 2 h time period with no activity. Rest deprivation was initiated at Zeitgeber time 16, 18, or 20, representing 6, 4, or 2 h of deprivation. Control populations in these experiments were flies of the same genotype that were not disturbed during the 24 h period. For wild-type flies in a, the differences between treated and control flies were significant (two-way ANOVA; p < 0.05) for all three periods of deprivation (2, 4, or 6 h), both during and after deprivation at CT 0 (^*). For fmn flies, differences between treated and control flies were significant during deprivation (at CT 16, 18, and 20), but there was no significant rebound observed after deprivation. Error bars indicate the SEM (n = 15 for all time points). This figure shows the results of one of two experiments that yielded almost identical results.