Dopamine Is a Regulator of Arousal in the Fruit Fly

Abstract

Introduction

In mammalian species, sleep and wakefulness represent two alternative physiological states, usually defined by the use of electroencephalographic methods. Both circadian and homeostatic processes are known to regulate the cycling between these two states (Borbely, 1982), but the underlying molecular mechanisms and the interactions between the two processes are not well defined. Evidence suggests that neuromodulators, including dopamine (DA), serotonin (5-HT), histamine, norepinephrine, and orexin, as well as small molecules such as adenosine, may function in the regulation of the mammalian sleep/wake cycle (Boutrel and Koob, 2004; Siegel, 2004). In addition, the molecular site of action of wake-promoting substances such as DA receptor agonists (Isaac and Berridge, 2003) and DA uptake inhibitors (Nishino et al., 1998), which act on the dopamine transporter and lead to increased locomotor activity (Wisor et al., 2001), indicate that dopaminergic signaling may be important for the regulation of sleep.

A large amount of evidence indicates that neuromodulators such as 5-HT, octopamine (OA), tyramine (TA), and DA are also important for the regulation of activity in invertebrates including Drosophila (Yellman et al., 1997; Cooper and Neckameyer, 1999; Marder and Bucher, 2001; Pendleton et al., 2002; Banerjee et al., 2004; Dasari and Cooper, 2004; Saraswati et al., 2004; Lima and Miesenbock, 2005). Although these compounds have a well documented role in promoting activity in insects, they have not been extensively investigated in the context of the rest/activity cycle, which has been used as a model for the mammalian sleep/wake cycle by several groups of investigators (Hendricks et al., 2000; Shaw et al., 2000; Ho and Sehgal, 2005). Sleep-like rest states have been described in at least two insects: the cockroach and the fruit fly Drosophila melanogaster (Tobler, 1983; Hendricks et al., 2000; Shaw et al., 2000). In Drosophila, periods of rest, during which flies remain immobile for a few minutes to hours, are behaviorally similar to mammalian sleep. They are (1) consolidated in a circadian manner, (2) regulated by homeostatic mechanisms, and (3) accompanied by a decreased responsiveness to outside stimuli. Additional similarities between mammalian sleep and fly rest behaviors include age-dependent changes in sleep or rest states, pharmacological effects of sleep (rest)-inducing or suppressing drugs such as anti-histaminics and caffeine, and changes in defined molecular markers of sleep (rest)/wake states (Andretic and Shaw, 2005; Ho and Sehgal, 2005). Similar to mammals, it has been suggested that rest may be essential for fly survival, with an effect on life span particularly evident with the cycle circadian mutant (cyc⁰¹) (Shaw et al., 2002; Hendricks et al., 2003a).

It seems unlikely that mammalian sleep and Drosophila rest have precisely the same physiological significance. Nonetheless, the use of genetic models is expected to facilitate general insights about the molecular pathways regulating sleep and arousal to the waking state. At present, the similarity of insect rest to mammalian sleep is based primarily on behavioral and pharmacological criteria. In this report, we describe a Drosophila hyperactive mutant called fumin (fmn; meaning sleepless in Japanese) and demonstrate that the behavioral phenotype is caused by a genetic lesion in the dopamine transporter (DAT) gene (Porzgen et al., 2001). The elimination of DAT activity generates a dramatic increase in the length of the active (waking) phase and a corresponding reduction of the inactive phase (sleep or rest), resulting in a nearly sleepless phenotype. The analysis of fmn suggests a role for dopamine in the modulation of insect arousal and highlights a similarity between insects and mammals regarding the molecular basis of arousal.

Materials and Methods

Fly stocks, crosses, and mutant identification. Flies were reared on a conventional corn meal, yeast, glucose agar medium at 25°C as described previously (Newby et al., 1991). Fly stocks used for the recombinational mapping were obtained from the Bloomington Stock Center (Bloomington, IN); the w¹¹¹⁸ stock used for a backcross is from the M. Saitoe laboratory (Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan) (Tamura et al., 2003), and all other stocks have been maintained in our laboratory for several years. The fmn mutant was discovered when we isogenized the second chromosome of a y w; tim⁰¹ fly stock for other purposes. One of several separate lines showed exceptionally high locomotor activity compared with the others. fmn was then identified as a second chromosome recessive mutation, which was segregated from tim⁰¹ by recombination, then identified as a dDAT mutation as described. The fmn mutation was backcrossed with w flies for seven generations to recombinationally separate it from other lesions.

pUAS-dDAT was generated by subcloning of PCR-amplified cDNA containing the dDAT open reading frame (ORF) into the pUAST vector and verified by sequencing. Transgenic flies were generated using standard microinjection procedures. Mutant (fmn) flies transgenic for ELAV-GAL4 (BL-458; P^{{w+mW.hs = GawB}} elav^C155) and UAS-dDAT were generated by crossing w ELAV-GAL4; fmn to w; UAS-dDAT/TM3a, Sb; fmn flies.

Locomotor activity and rest analysis. Activity was monitored by recording infrared beam crossings of individual flies housed in glass tubes (length, 6.5 cm; inside diameter, 3 mm). Events were scored at 1 min intervals using a Trikinetics (Waltham, MA) Drosophila activity monitoring system (Morales et al., 2002). Unless otherwise specified, 2- to 3-d-old male flies were used for behavioral analysis, and data were collected continuously for 3 d under either a 12 h light/dark (LD) cycle or continuous dark (DD) conditions. The full 3 d of data were used for quantitative analysis. Data were processed with a Microsoft (Redmond, WA) Excel-based program as described previously (Hendricks et al., 2003a). Five minutes with no activity was defined as a rest bout. The activity index was calculated by dividing total daily activity counts by the length of the active period, which is the total number of minutes during which a fly exhibits at least one activity event. Quantitative analyses were performed with at least 15 male flies (2-5 d after emergence), and the averages with SEMs are displayed. All experiments were replicated at least three times.

Recombinational mapping of fmn. w; fmn flies were mated to flies carrying a dominant mutation on the second chromosome (BL-1401; wg^Sp-1 Bl¹ L^rm Bc¹ Pu² Pin^B/SM5), and the female progeny were backcrossed with w; fmn males. The resulting F2 flies with various combinations of dominant phenotypic markers were assayed for activity and used to determine recombination frequencies. About one-half of the flies with either the L (Lobe; cytogenetic locus 51A2-3) or Bc (Black cell; 54F6) marker, but not both, showed the fmn behavioral phenotype, indicating that fmn maps approximately halfway between L and Bc. Subsequently, flies with both the L and fmn phenotypes were crossed with strains carrying a single P-element insertion at 54B10-13 (BL-11175), 53E6-8 (BL-10931), or 53C9-10 (BL-12056). Female offspring were backcrossed with fmn males. The resulting F2 flies with both the L and the P-element markers were again assayed for the fmn phenotype by monitoring locomotor activity. With BL-11175, BL-10931, and BL-12056, 11 of 47 (23%), 7 of 41 (17%), and 1 of 39 (3%) recombinant offspring were phenotypically fmn, indicating fmn maps to the left of and very close to 53C9-10.

Northern blot analysis. Based on the published sequence of dDAT (GenBank accession number, NM 079039), the whole ORF (1896 bp) was PCR amplified from control fly head RNA, cloned into a plasmid vector, and verified by restriction mapping and sequencing. This plasmid was used as template to make both whole ORF and 3′ partial digoxigenin-RNA (DIG-RNA) probes using the SP6 in vitro transcription system (Roche Products, Welwyn Garden City, UK). For the 3′ partial probe, RsaI-digested plasmid, which yields a 533 nucleotide probe, was used. For the positive control, a PCR-amplified DIG-DNA probe of rp49 (Vaslet et al., 1980) was used. Total RNAs were prepared from heads of control and fmn flies, using Trizol (Sigma, St. Louis, MO). Poly(A)+ RNA was prepared using an mRNA purification kit (Oligotex-dT; TaKaRa, Tokyo, Japan). Then, 0.7 μg samples of polyA⁺ RNA were fractionated through 1% agarose, transferred to a nylon membrane, hybridized with the indicated probes, and detected by chemiluminescence.

fmn dDAT gene analysis. PolyA⁺ RNA was prepared from fmn fly heads, and the 3′ end of dDAT cDNA was PCR amplified, cloned, and sequenced using the GeneRacer kit (Invitrogen, San Diego, CA). Briefly, first-strand cDNA was reverse transcribed using a polydT primer with two general primer sites. Nested PCR was performed first with 5′-ATC CTT CAC CAA CTC GCT GC (dDAT ORF position 410-429) and the 3′ primer combination and next with 5′-CCC TCT TTC CCT ATG CAG TG (dDAT ORF position 805-824) and the 3′ nested primer combination. The resulting PCR product, which had a shorter length than the expected size of dDAT, was cloned and sequenced.

Southern blot analysis was performed using both control and fmn genomic DNA with the entire dDAT cDNA ORF as a probe. PvuII digestion resulted in a 4.5 kb band of expected size in control DNA, but a 6.5 kb band in fmn, suggesting that the insertion is ∼2 kb in length.

Mechanical stimulation. fmn and control flies (male, 2-4 d of age), raised in LD conditions, were collected and placed in glass tubes in activity monitors. The monitor was first maintained in LD, then transferred to DD by wrapping monitors in aluminum foil at the end of the light period. To apply stimuli, monitors were placed on a firm, stable laboratory stone table in a small quiet room. Two small plastic cushions, 7 mm thick (mild stimuli) or 21 mm thick (moderate and strong stimuli), were placed under the center of both longitudinal sides of the monitors. Mechanical stimuli were delivered at different times of the subjective night [circadian time (CT) 14, 16, 18, 20, 22] by quickly removing both cushions, allowing the monitors to drop onto the stone table. For the mild and moderate stimuli, only one round of stimulation was applied; for strong stimuli, the monitors were dropped consecutively six times.

Activity was monitored every 15 s, and data were used only when there was no activity 5 min before the stimulus. When a fly began to move within 1 min after the stimulus and when its movement continued for at least 1 min, the fly was scored as responding to the stimulus; otherwise, it was scored as no response. For flies that responded to a mechanical stimulus, we measured the duration of the response. Once a fly stopped moving and the inactive period continued for at least 5 min, the response was considered to have ended. To calculate response times, data from strong stimulations were used, but similar results were obtained with mild and moderate mechanical stimulation.

Rest (sleep) deprivation. fmn and control flies were placed in glass tubes in an activity monitor, as described in the above stimulation experiments. Manual mechanical stimulation was applied to the monitor at 5 min intervals for the indicated number of hours. The movements of flies were observed by real time monitoring, so that the strength of stimulations could be adjusted to be sufficient to keep all flies awake. Rest deprivations began at CT 16 (6 h), CT 18 (4 h), and CT 20 (2 h) and ended at CT 22. Rest, as defined by 5 min intervals with zero activity, was calculated for each 2 h period. Rest rebound was calculated as the increase in rest observed in treated flies, relative to nontreated control populations of the same genotype, within a 6 h period (CT 22-CT 4) after rest deprivation.

Determination of life span. In preparation for studies of longevity, w and w; fmn male and female flies were separated under CO₂ anesthesia and transferred to vials containing fly medium. For each genotype, ∼200 flies were housed in six vials, each containing ∼35 flies. Flies were maintained in a 12 h light/dark cycle and transferred to new vials twice a week until all flies died. The number of dead flies were counted at each transfer.

Results

fmn mutants exhibit increased locomotor activity

In the course of analyzing the nature of sleep-like behavior in Drosophila circadian mutants (Hendricks et al., 2003a), we discovered by serendipity a mutant in a y w; tim⁰¹ laboratory stock that exhibited high levels of activity at all times of day. This mutant has been named fumin, meaning sleepless in Japanese. We outcrossed fmn mutants to flies from a Cantonized w¹¹¹⁸ stock (CS10) (Tamura et al., 2003) for seven generations and then examined locomotor activity and rest phenotypes in the descendents. Activity was monitored in individual flies in a 12 h light/dark cycle and in constant dark conditions (see Materials and Methods for a description of activity recording and the definition of rest/activity parameters). Records for three individual control and fmn flies are shown in Figure 1a (3 d of LD followed by 3 d of DD). Control w flies displayed a substantial amount of inactivity (5 min intervals without any locomotor activity) both in LD and DD. In contrast, fmn mutants were active almost continuously throughout the day. Although fmn flies displayed high levels of locomotor activity at all times of the cycle, they nonetheless showed rhythmic circadian changes in activity levels for at least the first 2 d of DD, as shown by the averaged DD records in Figure 1b. We note, however, that free-running activity rhythms are not as robust as those seen in the wild type because of the relatively high baseline level of activity in the mutant at all times of subjective day (Fig. 1b). fmn populations show rhythmic, circadian eclosion (data not shown), indicating that fmn is not a clock mutant. Flies in the fmn line show a suppression of activity by light, which is relieved when flies are put into constant conditions (Fig. 1b), but this phenotype may not be caused by fmn, because it is seen in several other lines having a similar genetic background.

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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.

On average, fmn mutants showed a twofold to threefold increase in total daily activity in LD and DD compared with control flies (Fig. 1c). In previous studies (Hendricks et al., 2000; Shaw et al., 2000), Drosophila activity events were recorded in 10-30 min intervals, and rest (sleep) was defined as those intervals with no activity. In the present study, we used 1 min recording intervals with a moving window analysis package we developed (Hendricks et al., 2003a) and defined rest as a 5 min interval with no activity. Using such a definition, w fmn flies showed significantly less rest than w controls (Fig. 1d). Interestingly, however, the activity index, defined as the total daily activity divided by the length of the daily active period, was similar in fmn and control flies (Fig. 1e). This indicates that fmn flies are hyperactive because they have a longer period of daily activity rather than being more active during any given activity period. This increased activity is a result of decreased rest period durations and increased lengths of activity bouts in fmn, both in LD and DD conditions (supplemental Fig. 1, available at www.jneurosci.org as supplemental material and data not shown). The average durations of activity bouts for fmn and control flies, respectively, were 82.9 ± 3.0 and 28.2 ± 3.0 in LD and 536.4 ± 271 and 17.8 ± 1.4 in DD.

fmn flies are fertile and have normal longevity

Sleep is widely believed to be essential for viability, and two different Drosophila sleep mutants have decreased life span (Shaw et al., 2002; Cirelli et al., 2005). Thus, we determined whether fmn affects life span. Those studies indicated that both male and female fmn flies have normal life span compared with genetic background controls (Fig. 2). Furthermore, the mutant has no obvious morphological, developmental, or fertility phenotypes, although fertility has not been examined quantitatively. Thus, the mutation appears to have a relatively selective effect on activity and rest and no significant impact on development, fertility, or longevity.

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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.

Drosophila fmn is an allele of the DAT gene

Chromosome segregation analysis showed that fmn is an autosomal recessive mutation mapping to the second chromosome. Meiotic mapping, using several dominant genetic markers, showed that fmn is located approximately halfway between Lobe (cytogenetic position 51A4) and Black cell (position 54F6) (see Materials and Materials and Methods). We used recombinant chromosomes carrying fmn and the L marker to further define the position of the behavioral mutation relative to several P-element insertions within the 51A to 54F interval. The results indicated that fmn mapped near cytogenetic interval 53, close to and to the left of the P-element insertion 12056, which is located in 53C9-10 (Fig. 3a). Perusal of the published Drosophila genome and annotated cDNA sequences (Adams et al., 2000) showed that the Drosophila homolog of the mammalian dopamine transporter (dDAT), a gene for which loss in mice leads to hyperactivity (Giros et al., 1996), resides within region 53C7-8 (Porzgen et al., 2001). Thus, we examined dDAT as a candidate for fmn.

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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).

Using a probe representing the whole dDAT open reading frame, we detected a 3.2 kb transcript by Northern blot analysis in samples of control w fly head polyA⁺ RNA. In contrast, a truncated dDAT mRNA of 1.8 kb was detected in fmn (Fig. 3b, left). Furthermore, a probe representing the 3′ end of the dDAT ORF did not detect an mRNA in fmn but did hybridize to the 3.2 kb mRNA in wild-type samples (Fig. 3b, right), indicating that the 3′ end of dDAT is deleted in fmn.

To define the nature of the fmn mutation, we cloned dDAT cDNA from fmn flies using the 3′ rapid amplification of cDNA end protocol. Sequencing of the fmn dDAT cDNA revealed that there was no splicing at the exon 6-intron 6 junction, as a result of an insertion of the 5′ portion of a roo transposon (Kaminker et al., 2002) into intron 6. The insertion is located 70 bp downstream of the splice donor junction (Fig. 3c), and apparently it results in transcriptional termination and polyadenylation of the dDAT mRNA at a site corresponding to sequence within the inserted segment. As a result of an in-frame stop codon in intron 6, the dDAT protein is truncated at amino acid 343 in fmn, whereas wild-type dDAT is 632 amino acids in length. Southern blot analysis and PCR amplification of genomic DNA showed that the size of the transposon insertion in fmn is ∼2 kb (data not shown), indicating that it is not a full-length 9 kb roo element.

To verify that the fmn activity phenotype is caused by loss of dDAT activity, we performed phenotypic rescue of fmn, using a UAS-dDAT transgene and a pan-neuronal ELAV-GAL4 driver. fmn mutants carrying both ELAV-GAL4 and UAS-dDAT showed a significant but partial phenotypic reversion (i.e., they exhibited decreased daily activity and prolonged sleep periods) (Fig. 3d,e, column 4 vs 8). Together with the molecular analysis, these results show that fmn is a mutation in the dDAT gene.

fmn mutants exhibit an altered arousal threshold

Given the decreased rest of fmn flies, we wondered whether arousal might be altered in the mutant. To determine whether fmn flies showed altered arousal threshold, we examined their sensitivity to mechanical stimuli. In these experiments, mechanical shocks were applied to inactive fmn or control flies, during the subjective night, while they were housed within an activity monitor (see Materials and Methods). Positive responses were defined as flies inactive before the stimulus that were activated for at least 1 min after the stimulus among all flies that were inactive for the 5 min before the stimulus. As shown in Figure 4a, fmn flies were hyper-responsive to mechanical stimuli compared with w control flies, with the greatest difference observed at the weakest stimulus tested. Only 15% of control flies responded to mild stimuli, but in the same experiments, 49% of fmn mutants showed a response. A similar differential effect was seen for moderate stimuli (responses by 48% of control and 88% of fmn), whereas all mutant and control flies responded to strong stimuli. In addition, we measured the durations of the activity bouts induced by mechanical stimulation, and those experiments showed that the responses of fmn flies were lengthened relative to controls. As illustrated by the response times (Fig. 4b-d), control flies showed stimulus-induced activity with a median bout length of 5 min, whereas fmn mutants remained active for a median time of 10 min, with many flies showing activity periods of >30 min.

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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).

Rest deprivation causes attenuated rebound in fmn mutants

Wild-type flies show a prominent rest (sleep) rebound, during which there is more rest after deprivation (Andretic and Shaw, 2005; Ho and Sehgal, 2005). Because the fmn mutation decreased rest, we expected that rebound might also be affected. To address this issue, we examined rebound parameters in control and mutant flies after various durations of rest deprivation. Flies were deprived of rest for 2-6 h during the subjective night by manual mechanical stimulation of the activity monitors housing the flies, and then activity levels were measured after deprivation. The mechanical stimulation was sufficient to completely or nearly completely deprive both w and fmn flies of rest (Fig. 5a,b). After rest deprivation for 2, 4, or 6 h during the subjective night, w control flies showed increased rest relative to control nondeprived w flies, in particular at the beginning of the day when flies are normally active (Fig. 5a). For the wild type, significant differences were observed between treated and control flies with regard to amount of rest for all three durations of deprivation (2, 4, or 6 h), both during and after deprivation at CT 0, indicating significant rest rebound. Although treated fmn flies rested significantly less than control flies during deprivation (CT 16, 18, 20), there was no significant rebound observed after deprivation at CT 0 or later time points (Fig. 5b). It thus appears that altered synaptic dopamine, as a consequence of lack of dopamine transporter activity, has perturbed the homeostatic regulation of the rest/activity profile in Drosophila.

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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.

Discussion

Footnotes

References