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J Neurosci. Author manuscript; available in PMC 2009 Sep 11.
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PMCID: PMC2742176
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Octopamine Regulates Sleep in Drosophila through PKA Dependent Mechanisms

Abstract

Sleep is a fundamental process, but its regulation and function are still not well understood. The Drosophila model for sleep provides a powerful system to address the genetic and molecular mechanisms underlying sleep and wakefulness. Here we show that a Drosophila biogenic amine, octopamine, is a potent wake-promoting signal. Mutations in the octopamine biosynthesis pathway produced a phenotype of increased sleep, which was restored to wild type levels by pharmacological treatment with octopamine. Moreover, electrical silencing of octopamine-producing cells decreased wakefulness while excitation of these neurons promoted wakefulness. Since protein kinase A (PKA) is a putative target of octopamine signaling and is also implicated in Drosophila sleep, we investigated its role in the effects of octopamine on sleep. We found that decreased PKA activity in neurons rendered flies insensitive to the wake-promoting effects of octopamine. However, this effect of PKA was not exerted in the mushroom bodies (MB), a site previously associated with PKA action on sleep. These studies identify a novel pathway that regulates sleep in Drosophila.

Keywords: Drosophila, Octopamine, Sleep, Locomotion, Norepinephrine, Biogenic Amine, Arousal

Introduction

Sleep is a core process that spans genetically diverse eukaryotes from mammals to arthropods (Tobler, 2005). Disrupting sleep in any of these organisms is detrimental to their performance, memory and health (Rechtschaffen, 1998). Extreme loss of sleep can even lead to death (Shaw et al., 2002). Thus, it must serve a very important function. Its conservation over evolution supports this claim for another reason: the need to sleep must outweigh selection pressure to eliminate it as a risk to predation. However, the function of sleep is unknown and the molecular regulation underlying it is poorly understood.

Sleep can be monitored through electroencephalograms (EEG) and electromyograms (EMG), but when such electrophysiological recordings are technically difficult, as in the case of Drosophila, it is monitored through analysis of behavior. Drosophila show a sleep state characterized by changes in position, increased arousal threshold, and periods of inactivity which can last several hours (Hendricks et al., 2000; Shaw et al., 2000). Although little is known about the regulation of this sleep state, effects of some neurotransmitters have been described. Thus, GABA and serotonin promote sleep, the latter by acting through the 5-HT1A receptor expressed in the mushroom body (Yuan et al., 2006). The only arousal-promoting signal identified in Drosophila is dopamine (Andretic et al., 2005; Kume et al., 2005).

In mammals, dopamine and norepinephrine are associated with states of arousal (Aston-Jones and Bloom, 1981). However, results regarding the effects of norepinephrine on total sleep and wake amounts have been mixed (Hunsley and Palmiter, 2003; Ouyang et al., 2004), in part because of differences in experimental protocols and also perhaps due to the effects of norepinephrine manipulation on dopamine levels (Schank et al., 2006). The insect equivalent of norepinephrine, octopamine, is synthesized and regulated through pathways that are distinct from those that produce dopamine. However, although octopamine is known to play a role in memory formation, larval locomotion, wing beating, ovulation and aggression (Roeder, 2005), it has not been examined for effects on sleep. Here we demonstrate a novel role for octopamine in the regulation of sleep and wake in Drosophila. Feeding octopamine to flies leads to a PKA-dependent decrease in total sleep, while removal of octopamine from the food is followed by a sleep rebound. In addition, flies mutant for octopamine show an increase in total sleep, which can be restored to control levels with the administration of octopamine. We show that electrical excitation of octopamine-producing cells decreases total sleep, whereas electrical silencing of these cells increases sleep. Other parameters of sleep such as sleep latency and arousal threshold are also altered. Lastly, we demonstrate an activity-promoting role for the octopamine precursor, tyramine, which is independent of the effects of octopamine.

Methods

Fly Strains Used

Wildtype fly strains: w;RC1;RC1 (isogenized chromosome 1 from the w1118 stock; isogenized chromosomes 2 and 3 from the RC1 strain), w1118, Canton S, Iso31 (Isogenic w1118 strain). Octopamine production mutants: Tdc2RO54 (Tyrosine decarboxylase 2 mutant), TβHnM18/FM7 (Tyramine Beta Hydroxylase mutant). Gal4 lines used: Tdc2-Gal4 (neuronal Tdc2 expression pattern), Tdc1-Gal4 (nonneuronal Tdc1 expression pattern), ElavGeneSwitch (panneuronal expression during adulthood), MBSwitch (mushroom body specific expression during adulthood). Upstream Activating Sequence (UAS) lines used:, UAS-B16B (NaChBac) (bacterial Na+ channel), UAS-Kir2.1 (inward rectifying K+ channel), UAS-BDK33 (PKAr) (Drosophila inhibitory subunit of PKA with mutated cAMP binding site), UAS-Tdc2, UAS-Tdc1. The following lines were ordered from the Bloomington Stock center: Tdc2-Gal4 (9313), Tdc1-Gal4 (9312), UAS-NaChBac (9466), UAS-Tdc2 (9315), UAS-Tdc1 (9314), UAS-GFPnls (7032), Iso31 (5905). UAS-Kir2.1 and UAS-B16B were a gift from Dr. B. White. ElavGeneSwitch (Osterwalder et al., 2001), MBSwitch (Mao et al., 2004) and UAS-BDK33 (Rodan et al., 2002) were previously used in the lab (Hendricks et al., 2001; Joiner et al., 2006). The wildtype isogenic line w;RC1;RC1 was a gift from W. Joiner. Tdc2RO54 lines and w1118 background line were a gift from Dr. G. Schupbach and Dr. J. Hirsh. The TβHnM18/FM7, TβHm6/FM7, and Canton S background control were a gift from Dr. E. Kravitz.

All Gal4 and UAS lines were outcrossed 7 times into the w;RC1;RC1 or Iso31 background. The w;RC1;RC1 background was chosen for the Tdc2-Gal4 expressing Kir2.1 because it shows lower levels of nighttime sleep (Suppl. Table 1) and thus affords the potential to avoid a ceiling effect.

Sleep Analysis

Sleep analysis was performed as previously described (Joiner et al., 2006). All flies were kept on a 12:12 LD 25°C schedule unless otherwise noted. Female and male flies 4–8 days old were placed in 65mm × 5mm tubes containing 5% sucrose and 2% agar and entrained for 24–36 hours prior to the sleep recording. Baseline sleep was determined by monitoring activity for at least 3 days with no disruptions in an LD cycle. Locomotor activity was monitored using the DAMS/Trikinetics system (Waltham, MA) as described previously (Joiner et al., 2006). Sleep was defined as a 5 minute bout of inactivity as described previously (Shaw, 2003; Joiner et al., 2006) . Latency to sleep was defined as the time in minutes from the moment lights were turned off to the first bout of sleep. Sleep consolidation scores were generated based on the amount of fragmentation seen in sleep, as measured by brief awakenings and the length of sleep bouts.

Arousal Threshold

Arousal threshold was measured at three times of the night (2 hours after lights off, 6 hours after lights off and 10 hours after lights off). Increasing levels of mechanical stimulation were applied to determine the minimum stimulus for arousal. The levels were then labeled weak, medium, and strong, with weak being the lowest level of stimulation and strong being the maximum. Animals were scored based on their response to these three levels (Hendricks et al., 2000).

Feeding Octopamine and Tyramine

Wildtype (w;RC1;RC1, Iso31) female animals were loaded into monitors as described above and given 24–36 hours to acclimate. One day of baseline data was collected, and then at the lights-on transition the flies were transferred either to tubes containing 5% Sucrose/2% Agar plus 10mg/ml octopamine or onto sucrose- agar alone. 10mg/ml orally administered octopamine was previously shown to be optimal for rescuing egg-laying (Monastirioti et al., 1996). A dose-response curve of Iso31 flies on octopamine is shown in Suppl. Fig. 3a. The animals were left for 3 days on or off octopamine and removed at the lights-on transition. Rebound was determined and analyzed as previously described for sleep deprivation (Joiner et al., 2006). The males were examined in the same manner; however 10mg/ml octopamine was fatal for them over three days so the octopamine concentration was reduced to 5mg/ml (data not shown). Other concentrations of octopamine were tested, but 10mg/ml was found to be the optimal amount for 3 days for wildtype females (Suppl. Fig 3a). A blue food assay was performed as described (Edgecomb et al., 1994) to ensure that the animals were eating the food (data not shown). Due to the lethality seen with 10mg/ml octopamine, the TβHnm18 females and controls were placed on 7.5mg/ml octopamine.

Tyramine was also fed to wildtype flies. Similar to what was seen with octopamine, tyramine at higher concentrations produced increased lethality so wildtype male flies were fed 5mg/ml tyramine in 5%sucrose/2%agar. Females were placed on 10mg/ml tyramine in 5%sucrose/2%agar. We tested other amounts of tyramine and determined that 10mg/ml was the optimal concentration (data not shown).

Mianserin was also used to address octopamine signaling. Based upon the work of Maqueira et al., who used this compound to block cAMP increases due to octopamine in vitro (Maqueira et al., 2005), it was used at 0.2mg/ml. All mianserin experiments were done with Iso31 control flies. Following the same protocol outlined above for octopamine we placed flies on either mianserin alone, mianserin + 10mg/ml octopamine, control food or control + 10mg/ml octopamine.

Hydroxyurea Analysis

Ablation of mushroom bodies with hydroxyurea (HU) was performed as previously described (de Belle and Heisenberg, 1994). First instar larvae of Iso31 flies were collected and either placed on a yeast paste and water mixture, or a yeast paste and 50mg/ml HU mixture for 4 hours at 25°C. They were then washed with water and placed in regular food vials till adulthood. 4–8 days post-eclosion animals were loaded into monitor tubes as described above. They were given three days of acclimation and then transferred onto 7.5mg/ml octopamine, as high lethality was observed at 10mg/ml. After three days the animals were then transferred back onto normal 5% sucrose agar tubes. Sleep analysis was then performed as described above. Following the completion of the sleep analysis fly heads were dissected in 4% paraformaldehyde and fixed for 30 minutes, mounted on slides and analyzed for loss of the alpha and beta lobes of mushroom bodies.

PKA inhibition studies

For PKA inhibition studies we crossed the ElavGeneSwitch transgene into UAS-BDK33 flies. 10mg/ml of octopamine was used as described above. Since the GeneSwitch construct can be turned on during adulthood using the drug RU486, we placed half the animals on 5% sucrose/2% Agar tubes containing either 500 µM RU486 dissolved in ethanol or ethanol alone (1%) for three days. Half of each group was then transferred to octopamine-containing food- either 5% sucrose/ 2% Agar + 500 µM RU486 + octopamine or 5% sucrose/2% Agar + ethanol (1%) + octopamine. Both groups were also simultaneously fed 10 mg/ml octopamine for 3 days. At the lights-on transition at the end of this period, animals were transferred off of octopamine onto 5%sucrose/2% agar containing either 500 µ RU486 or ethanol (1%). Sleep analysis was performed as described above.

Statistics

To compare multiple groups, 2-way ANOVA was used to determine significance for total sleep, nighttime sleep, daytime sleep, sleep bout number for both day and night and latency to sleep. For non-Gaussian distributed data we used the Kruskal Wallis Test; this included sleep bout length (daytime and nighttime), consolidation score, activity per waking minute and peak activity. Statistical significance is denoted by asterisks. * = p≤.01, ** = p≤.001, *** = p≤.0001.

Results

Mutants with reduced octopamine have increased sleep

To address a role for octopamine and its precursor, tyramine, in Drosophila sleep, we analyzed two known genes that affect biosynthesis of these amines (Monastirioti et al., 1996; Cole et al., 2005; Certel et al., 2007). As shown in the flow diagram in Figure 1A, the tyrosine decarboxylase 2 (Tdc2) enzyme synthesizes tyramine (precursor to octopamine) from tyrosine; thus, its disruption results in low levels of octopamine and tyramine (Cole et al., 2005). In contrast, tyramine beta-hydroxylase (TβH) synthesizes octopamine from tyramine and therefore its loss reduces octopamine, but increases tyramine levels 10-fold (Fig.1a) (Monastirioti et al., 1996). Since synthesis of both amines requires Tdc2, the cellular distribution of Tdc2 should reflect cells that potentially produce both tyramine and octopamine. We determined the expression pattern of Tdc2 by using the well-known UAS-Gal4 binary system to express green fluorescent protein (GFP) under the control of the Tdc2 promoter (Fig. 1b). Expression of GFP was seen in discrete unilateral subsets of cells located along the ventral medial line of the brain, as well as in discrete bilateral clusters of cells in the lateral protocerebrum region and surrounding the oesophagus cavity.

Figure 1
The octopamine biosynthesis pathway and its distribution in the fly brain

We then assayed sleep in flies mutant for each of the two genes in the octopamine biosynthesis pathway. Baseline sleep levels were examined in flies carrying a point mutation in the Tdc gene (Tdc2RO54) or a lesion, created by imprecise excision of a P transposable element, in the TβH gene (TβHnm18). Under baseline conditions, we found that male and female animals of both mutants displayed increased levels of sleep (Fig. 2a,b; 3a,b). All male data are shown in Supplemental Table 1 while female data are depicted in Figures 2 through 5. The increase in sleep in Tdc2RO54 and TβHnm18 mutants occurred largely during the day (Fig.2a,b; 3a,b), perhaps because flies are already sleeping maximally during the night. The increase in total sleep was accompanied by a decreased latency to sleep (the time from lights off until the animal’s first bout of sleep) in the Tdc2RO54 mutants (Fig. 2b), suggesting an increase in homeostatic drive to sleep. The TβHnm18 mutation did not have a significant effect on latency although there was a slight decrease in males (Suppl. Table 1). The Canton S background strain that the TβHnm18 mutants were crossed into displays rapid onset of sleep compared to other wild type control lines used in this study (Suppl. Table 1), which may occlude any decrease in latency caused by the mutation.

Figure 2
Baseline sleep phenotype of Tdc2RO54 mutants, which have decreased levels of octopamine and tyramine
Figure 3
Baseline sleep phenotype of TβHnm18 mutants, which have decreased levels of octopamine and increased levels of tyramine

Analysis of sleep architecture indicated that the increase in sleep in Tdc2RO54 mutants was due to an increase in sleep bout number (Suppl. Table 2) while the increase in TβHnm18 mutants occurred from an increase in bout length (Suppl. Table 2). Since both mutants affect octopamine similarly, this difference in sleep architecture is most likely due to differences in levels of tyramine. We also determined the arousal threshold in both mutants by measuring their response to a stimulus of increasing intensity. The arousal threshold during sleep was higher in both sets of mutants than in wildtype flies (Fig 2c and and3c).3c). This suggests that these animals are in a deeper state of sleep than their controls.

An increase in sleep could result from the animals being sick and unable to move. To address this possibility, we measured locomotor activity in Tdc2RO54 and TβHnm18. We found that Tdc2RO54 and TβHnm18 had peak activity levels that were not significantly different from those of controls (data not shown). However the TβHnm18 flies showed significantly increased waking activity (Fig. 3d), as measured by total activity while awake; indeed, despite their increased total sleep time, the TβHnm18 mutants showed a hyperactive phenotype when awake. In contrast, the average rate of movement was significantly decreased in Tdc2RO54 flies as compared to the wild type controls (Fig. 3d). Since activity in the two mutants is affected in opposite directions, it is unlikely that the increased sleep phenotype of the mutants is secondary to effects on activity. More likely, the loss of octopamine (common to both mutants) underlies the decrease in sleep while differences in tyramine levels account for effects on locomotor activity. In fact, our data are consistent with previously published data indicating that the increased (10 fold) levels of tyramine in the TβHnm18 flies cause an increase in locomotor behavior when awake (Hardie et al., 2007).

It was previously shown that expression of Tdc1 (the nonneuronal form of Tdc) in Tdc2 producing cells rescues the Tdc2RO54 mutant locomotor phenotype (Hardie et al., 2007). We found the same to be true for sleep. When Tdc1 was expressed in Tdc2 producing cells in a Tdc2RO54 background we found we were able to rescue the baseline sleep phenotype (Fig. 2e), as well as sleep architecture and latency to sleep (data not shown).

Altering excitability of octopamine/tyramine producing cells affects sleep

If octopamine and/or tyramine are released in the brain to regulate sleep then blocking or increasing their release should also affect sleep. Thus, we sought to determine if electrical manipulation of the cells producing tyrosine decarboxylase, which should affect the release of octopamine and tyramine, produces a change in sleep. The Tdc2-Gal4 line mentioned above, which expresses Gal4 in cells producing octopamine and tyramine, was crossed to flies carrying transgenes for ion channels under the control of a UAS element recognized by Gal4 (UAS-NaChBac or UAS-Kir2.1) (Baines et al., 2001; White et al., 2001; Cole et al., 2005; Nitabach et al., 2005). The UAS-NaChBac transgene is derived from a gene encoding a bacterial Na+ channel, which has the characteristics of high open probability and low inactivation, thus driving membrane voltage to a more depolarized and easily excited state. Expression of the Na+ channel in Tdc2-positive cells resulted in a decrease in sleep of 56.5%, corresponding to a loss of ~346 minutes (Fig.4a,b). The loss of sleep was specific to the nighttime, with no significant sleep loss during the daytime, which may be indicative of normally high octopamine activity during the day. This hypothesis is consistent with mammalian studies where the noradrenergic cells of the locus coeruleus fire primarily during the active period (Aston-Jones and Bloom, 1981). We also found a corresponding increase in sleep latency in these flies. They took, on average, 74 minutes longer to fall asleep after lights off (Fig.4b). Flies expressing NaChBac in Tdc2-positive cells also showed a decreased arousal threshold, suggesting that they are easily awakened during the night (Fig.4c). Bout analysis indicated that nighttime sleep bouts were shorter in duration (Suppl. Table 2). In addition there was a significant increase in daytime bout number (Suppl. Table 2) which may reflect increased homeostatic drive resulting from the reduced sleep at night. However, the animals were unable to maintain long sleep bouts even under these conditions.

Figure 4
Baseline sleep phenotype produced by depolarizing Tdc2-positive neurons

We also expressed a hyperpolarizing K+ channel transgene under the control of the Tdc2-Gal4 driver and found that this produced an increase in total sleep (Fig.5a,b). Kir2.1 is an inward rectifying K+ channel that has a high open probability and no inactivation. Expression of this channel hyperpolarizes neurons and decreases membrane resistance, thus making it more difficult for membrane potential to reach threshold for firing action potentials (Baines et al., 2001). Consistent with a previous report, we found that locomotor activity decreased when Tdc2-Gal4 was used to express the inward rectifying K+ channel (UAS-Kir2.1) (data not shown). Analysis of sleep parameters, however, revealed that it was actually an increase in sleep that accounted for the phenotype (Fig.5a,b). Flies expressing UAS-Kir2.1 in Tdc2 cells showed, on average, a 174 minute increase in sleep and also displayed changes in several sleep measures such as latency and arousal threshold. These flies also showed a decrease in latency to sleep (Fig. 5b) and an increased arousal threshold during sleep, requiring more stimulation to wake up compared to controls (Fig. 5c). In addition, they showed a trend toward longer bouts of sleep during the night, though the major increase in sleep came from the increased number of sleep bouts during the day (Suppl. Table 2). The relative lack of an increase in nighttime sleep length may result from a ceiling effect of sleep at night.

Figure 5
Baseline sleep phenotype produced by hyperpolarizing Tdc2-positive neurons

Although sleep levels and architecture are clearly affected in flies expressing sodium or potassium channels in Tdc2 neurons, it is possible that altered activity levels contribute to the overall phenotype. Thus, we also examined peak activity levels and activity while awake. The Tdc2-Gal4 females expressing NaChBac showed significantly lower peak activity (Suppl. Table 1) as well as decreased activity while awake compared to the outcrossed background control (w;Tdc2-Gal4/ NaChBac:+ = 1.7 ± .06 S.E.M. Iso31 = 2.09 ± .06 S.E.M. p≤.01 Kruskal Wallis Test). However, it is unlikely that a decrease in waking activity underlies a reduced sleep phenotype. We also found significantly reduced waking activity in animals expressing the K+ channel under the control of the Tdc2 driver (Suppl. Table 1). This decrease in waking activity is similar to the decreased waking activity seen with the Tdc2RO54 mutant, where levels of both octopamine and tyramine are low (w;Tdc2-Gal4/Kir2.1;RC1 = 2.14 ± .06 S.E.M. w;RC1;RC1 = 3.87 ± .97 S.E.M. p≤.0001 Kruskal Wallis Test). Thus, reducing electrical activity in octopamine-producing cells has the same effect on activity and sleep as a mutation (Tdc2R054) that decreases levels of octopamine.

To ensure that the effect on sleep caused by loss of octopamine signaling is specific to neuronal Tdc2 and not to a global loss of Tdc, we made use of a Tdc1-Gal4 driver that is expressed in non-neuronal cells. Expression of the UAS-B16B transgene (NaChBac channel) under the control of this driver produced no significant change in sleep (Suppl. Fig.1). Thus, we conclude that the sleep phenotype observed in the Tdc2R054 mutant or produced by manipulations of Tdc cells is specific to the neuronal form of Tdc, Tdc2. Unfortunately the UAS-Kir2.1 channel proved to be lethal with Tdc1-Gal4.

Given that the NaChBac channel increases excitability of Tdc2 cells and thereby presumably stimulates release of octopamine/tyramine, we asked whether overexpressing Tdc2 would have the same effect. As predicted, we found that overexpression of Tdc2 in Tdc2 producing cells resulted in a 300 minute decrease in nighttime sleep (Suppl. Fig 2). As with the NaChBac channel this appears to be a nighttime specific sleep loss. There was also a decrease in sleep with the overexpression of Tdc1 in Tdc2 producing cells (data not shown).

Oral administration of octopamine reduces sleep in flies

Since the Tdc2RO54 and TβHnm18 mutants change levels of octopamine and tyramine, it is important to dissociate the effects of the two to identify the transmitter responsible for the sleep phenotype. As noted earlier, Tdc2RO54 decreases levels of both tyramine and octopamine while the TβHnm18 decreases octopamine but increases tyramine. To determine if a change in octopamine is sufficient to regulate sleep, we placed a wildtype isogenic line, Iso31, on octopamine-containing food for three days. It was previously shown that animals fed 10mg/ml octopamine have increased levels of this neurotransmitter, particularly in the brain (Barron et al., 2007). Supporting this finding, ingested octopamine rescues the egg laying phenotype displayed by TβHnm18 and Tdc2RO54 mutants (Monastirioti et al., 1996; McClung and Hirsh, 1999; Cole et al., 2005). We found that flies fed 10 mg/ml octopamine had approximately 200 minutes less nighttime sleep than control flies maintained on sucrose-agar alone (Fig.6a) (Dose response curve is shown in Suppl. Fig 3a). Thus, similar to the UAS-NaChBac effect, this was a nighttime specific effect. Following the removal of octopamine, these flies showed a corresponding sleep rebound of ~70 minutes (Control = 289.90 ± 13.92 S.E.M. N=40; 10mg/ml octopamine = 367.53 ± 16.91 S.E.M. N=40 p≤0.001 2-way ANOVA). In addition to the effect it had on wild type flies, a lower concentration of orally administered octopamine (7.5mg/ml octopamine) was able to restore the sleep phenotype of the TβHnm18 mutant to control levels (Fig. 6b). This concentration of octopamine produced no significant change in sleep in the Canton S strain, which is the background of the TβHnm18 mutants.

Figure 6
Oral administration of octopamine decreases sleep in Iso31 flies and TβHnm18 mutant flies

To exclude the possibility that the sleep phenotype was due to some non-specific toxicity associated with octopamine, we attempted to block the effect by inhibiting octopamine signaling. Thus, we co-administered 0.2mg/ml Mianserin, which acts by inhibiting octopamine-induced cAMP increase (Fig. 6c) (Maqueira et al., 2005). Co-administration of mianserin almost completely blocked the effect of feeding 10mg/ml octopamine to flies. This demonstrates that the loss in sleep produced by octopamine feeding is due to the ingestion of octopamine itself and not due to toxic, non-physiological effects. In addition, these data suggest that the effects of octopamine on sleep are mediated by beta receptors.

Wildtype flies fed tyramine did not show significant changes in sleep amount (Suppl. Fig.3b). In addition, neither octopamine nor tyramine produced a significant change in activity while awake (Supp. Fig. 3c).

Octopamine acts through neuronal PKA to decrease sleep

Cyclic AMP-dependent protein kinase (PKA) signaling plays an important role in sleep in Drosophila (Hendricks et al., 2001) (Joiner et al., 2006). It is also known to be coupled to some of the octopamine and tyramine G-protein coupled receptors (Evans and Maqueira, 2005). Indeed, the blocker of octopamine used above inhibits cAMP signaling. In order to directly address whether the effect of octopamine on sleep is through PKA-dependent pathways, we expressed the regulatory subunit of PKA (PKAr), which inhibits activity of PKA, under the control of an ElavGeneSwitch driver (Osterwalder et al., 2001; Joiner et al., 2006). The use of the ElavGeneSwitch driver allowed us to inducibly express the regulatory subunit in all neurons. We found that expression of PKAr in adult neuronal tissue rendered the flies insensitive to the sleep reducing effects of octopamine (Fig. 7a), supporting the idea that octopamine is acting through a PKA dependent pathway to promote arousal.

Figure 7
Effects of octopamine are mediated by PKA and are independent of the Mushroom Body

Since the mushroom body (MB) is implicated in the effects of PKA on sleep (Joiner et al., 2006), we sought to determine if this is also the site of octopamine action on sleep. Thus, we ablated the mushroom bodies with hydroxy urea as previously described (de Belle and Heisenberg, 1994) and then treated these flies with octopamine. The sensitivity to octopamine was intact despite the absence of the MBs (Fig. 7b). As expected, the MB ablation itself reduced sleep, but there was a further decrease produced by feeding octopamine (Fig. 7b). The decrease was 87 minutes, which was comparable to the amount of sleep lost when Iso31 flies were fed 7.5mg/ml octopamine (82 minutes). To verify that the MBs were ablated, we followed up the behavioral analysis of all flies with anatomical analysis of the brain. All flies that still contained some alpha and beta lobes were eliminated from analysis.

We also examined the effects of octopamine on flies expressing PKAr under control of an inducible mushroom body GeneSwitch driver (MBSwitch) and found that these flies were still sensitive to octopamine (data not shown). Our inability to block effects of octopamine on sleep by inhibiting PKA signaling in MBs supports the finding that elimination of MBs by hydroxyurea does not block the wake-promoting effects of octopamine.

Discussion

Biogenic amines play many important roles in mammals, with several having significant effects on sleep:wake states. Thus, dopamine, serotonin, and norepinephrine are all important for regulating states of arousal. We hypothesized that, like its counterparts in mammals, the invertebrate neurotransmitter octopamine would be important for arousal in Drosophila. That prediction was supported by the data reported here. We find that decreases in levels of octopamine increase sleep, while increasing octopamine causes a decrease in sleep. In addition, while the mammalian data have been contradictory with respect to the role norepinephrine plays in total sleep, we find that octopamine decreases total sleep time. The mammalian data are complicated in part because perturbations of the norepinephrine pathway result in changes in the levels of dopamine (Ouyang et al., 2004). The use of Drosophila allows us to examine specifically the role of octopamine without perturbing dopamine signaling.

By modulating the excitability of octopamine-producing cells we were able to manipulate the output of these cells. In mammals one can record from specific cell populations to determine when the cells fire action potentials. Though this assay is difficult to do in flies, we were able to electrically modulate the cells through expression of K+ and Na+ ion channels. We found that when octopamine-producing cells were more depolarized (expression of a Na+ channel), the animal was awake more and unable to stay asleep, whereas when the cells were hyperpolarized (expression of a K+ channel), the animals slept more.

Based largely upon larval crawling assays, octopamine and tyramine were previously implicated in locomotor behavior (Saraswati et al., 2004) (O'Dell, 1994; Gong et al., 2004) (Scholz, 2005). Specifically, larvae move slower through quadrants when they have decreased octopamine levels (the TβHnm18 and Tdc2RO54 mutants). More recent work showed that adult Tdc2RO54 flies also have a decrease in locomotor activity due to the lack of tyramine (Hardie et al., 2007). Our data showing differences in activity in the Tdc2RO54 and the TβHnm18 mutants support the claim that tyramine plays an important role in locomotion. Thus, while increased levels of tyramine in Tbh mutants increase activity, decreased levels in Tdc mutants decrease locomotor activity. However, both mutations increase sleep, which is most likely due to the loss of octopamine. In addition to overall sleep, we find that other sleep parameters such as latency to sleep and arousal threshold are affected in flies carrying these mutations. We infer that tyramine plays a role in locomotion, but octopamine specifically affects arousal states.

Studies of other invertebrate species support a role for octopamine in arousal (Corbet, 1991). In fact, octopamine agonists are potential natural pesticides because they cause insect species to “walk off” the leaves (Roeder, 1999). As in Drosophila, changes in octopamine levels affect behavior in honey bees, as demonstrated through feeding and injection of octopamine as well as through analysis of endogenous levels of octopamine. Fussnecker et al. showed that injections of octopamine promote flying in honeybees (Fussnecker et al., 2006). In addition, octopamine and tyramine regulate other behaviors in honeybees such as hive maintenance and foraging (Schulz and Robinson, 1999; Wagener-Hulme et al., 1999; Schulz and Robinson, 2001; Barron et al., 2002) . Octopamine and tyramine also modulate sensory input in honeybees (Kloppenburg and Erber, 1995; Scheiner et al., 2002). In the locust, octopamine mediates heightened arousal in response to new visual stimuli (Bacon et al., 1995). Bacon et al. found that a specific subset of octopamine producing neurons in the brain of the locust fires during the presentation of new visual stimuli causing dishabituation of the descending contralateral movement detector (DCMD) interneuron. Interestingly, application of endogenous octopamine can mimic this state of heightened arousal. Our study suggests that octopamine serves to promote arousal in Drosophila. It is possible that the increased arousal we see with too much octopamine, or decreased arousal with too little, is a result of improper gating of sensory stimuli, but without electrophysiological data we are unable to draw any conclusions. Note also that the Tdc2 cells important for sleep and arousal in the fly brain have not been identified yet.

In previous studies, octopamine was fed to flies in order to rescue or verify a phenotype of the TβHnm18 flies. The ability of octopamine to rescue egg laying in TβHnm18 mutants was assayed in this fashion, as TβHnm18 flies are unable to release eggs. Animals were placed on different levels of octopamine, and 10mg/ml octopamine over a period of 6 days provided maximal rescue (Monastirioti et al., 1996). Using the same concentration, we found that a steady increase in octopamine levels led to a decrease in nighttime sleep. Based upon the specific effect on nighttime sleep, we speculate that octopamine levels are already high during the daytime, thereby precluding any effects of an increase. This analysis is supported by the Na+ channel data in which a significant decrease in total sleep was found only during the nighttime sleep periods. We speculate that normally, activity of these cells is low at night, and so expression of the Na+ channel causes them to fire more and release octopamine at an abnormal time, thereby producing a decrease in sleep. Similar results, indicating nighttime specific effects, were obtained with overexpression of Tdc2. Work in other insects also supports the idea of modulated octopamine release. Pribbenow and Erber demonstrated that honeybees who are already in a heightened arousal state of antennae scanning, do not change scanning frequency in response to octopamine administration,, but in animals scanning at a low frequency, injections of octopamine significantly increase scanning (Pribbenow and Erber, 1996).

Our data suggest that the effects of octopamine are mediated through PKA-dependent signaling. In mammals, there are 9 different adrenergic receptors, some of which signal through PKA (Hoffman and Lefkowitz, 1996). The α1 adrenergic receptor is the only receptor associated with a wake-promoting effect in that the agonist methoxamine causes an increase in waking (Hilakivi and Leppavuori, 1984; Monti et al., 1988). However, the antagonist has no effect on total sleep (Benington et al., 1995; Berridge and Espana, 2000). It is important to note that the a1 receptor in mammals is thought to be coupled to phospholipase C and Gq (Ramos and Arnsten, 2007). The β adrenergic receptors (which are coupled to cAMP and PKA) probably do not have specific effects on sleep in mammals since, contrary to known effects of norepinephrine, the agonist increases sleep and the antagonist decreases sleep (Monti et al., 1988). Studies in Drosophila may be better able to identify biogenic amine receptors relevant for sleep because of the ease of genetic manipulation. Many G-protein-coupled receptors in Drosophila display activity that allows their bona fide classification as octopamine receptors (Evans and Maqueira, 2005). Our data here suggest that receptors sensitive to mianserin are likely to be involved in regulating fly sleep. Since mianserin inhibits cAMP signaling, these data not only further support a role for PKA, but also implicate beta receptors in octopamine action. We note that none of these receptors is known to display a circadian cycling profile.

Given that PKA was previously shown to regulate sleep in Drosophila, we are starting to see a link between the various molecules that affect Drosophila sleep. Interestingly though, octopamine does not appear to act through the MBs, a structure known to mediate effects of PKA on sleep and also to express a class of octopamine receptors. Since flies lacking MBs still have substantial amounts of sleep, it is clear that other parts of the fly brain can drive sleep. The current study shows that even PKA can affect sleep in regions outside the MB. Defining the site of action of sleep-regulating molecules such as octopamine should help to identify these other brain regions.

Supplementary Material

Supp1

Supplemental Figure 1. Total sleep in Tdc1-Gal4 female flies expressing UAS-NaChBac:

A. Total sleep is not significantly different between Tdc1-Gal4;NaChBac/+;+ flies and sibling controls. (Tdc1-Gal4;NaChBac/+;+= 774 ± 48 S.E.M. N=32 Tdc1-Gal4;Cyo/+;+ (sibling controls) = 886 ± 78 S.E.M. N=32 2-way ANOVA)

Supp2

Supplemental Figure 2. Overexpression of Tdc2 causes decreases in sleep:

A. Female flies carrying a UAS-Tdc2 transgene under the control of Tdc2-Gal4 were assayed for sleep. Baseline sleep was measured for six days in 16 animals of each genotype and total nighttime sleep was quantified. Tdc2 × Tdc2 flies (grey line) show significantly less sleep than controls (black solid line) (w;UAS-Tdc2/ Tdc2-Gal4;+ = 322 ± 69 S.E.M. N=16 w;Tdc2-Gal4/+;+ = 676 ± 6 S.E.M. N=16 p≤.0001 2-way ANOVA)

Supp3

Supplemental Figure 3. A. Dose response curve for effects of octopamine on sleep:

As the concentration of octopamine increases there is a corresponding decrease in nighttime sleep. (0 oct. 647 ± 22 S.E.M. n=18, 5mg/ml oct. 597 ± 19 S.E.M. n=8, 7.5 mg/ml oct. 514 ± 21 S.E.M. n=24, 10mg/ml oct. 420 ± 31 S.E.M. n=30, 12 mg/ml oct. 343 ± 33 S.E.M. n=21) B. Effect of tyramine on nighttime sleep. 10 mg/ml tyramine did not significantly alter nighttime sleep amounts in Iso31 flies. (Iso31 n=32, Iso31 + Tyramine n=32) C and D. Waking activity was not significantly different in flies fed either 10mg/ml octopamine or 10mg/ml tyramine.

Supp4

Supplemental Table 1:

Sleep and activity in flies mutant for the octopamine pathway.

Supp5

Supplemental Table 2:

Sleep bout analysis in flies mutant for the octopamine pathway

Acknowledgements

We thank Drs. Jay Hirsh, Ed Kravitz, Trudi Schupbach, Ben White and Bill Joiner for providing flies used in this study. We also thank Bill Joiner, Kyunghee Koh, Max Kelz, Eliot Friedman, and Natalia Nedelsky for critical reading of the manuscript and K. Luu for assisting with animal maintenance. A.C. was supported by a training grant to the Center for Sleep and Respiratory Neurobiology at the University of Pennsylvania and by an NRSA from the NIMH. This work was also supported in part by a program project grant from the NIA.

Contributor Information

Dr. Amanda Crocker, Dept. of Neuroscience Stemmler Hall Rm 232 University of Pennsylvania, Philadelphia PA 19104.

Dr. Amita Sehgal, Dept. of Neuroscience Stemmler Hall Rm 232 Howard Hughes Medical Institute University of Pennsylvania, Philadelphia PA 19104.

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