Octopamine regulates sleep in drosophila through protein kinase A-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, whereas excitation of these neurons promoted wakefulness. Because 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, a site previously associated with PKA action on sleep. These studies identify a novel pathway that regulates sleep in Drosophila.

Figure 1.

The octopamine biosynthesis pathway and its distribution in the fly brain. A, Octopamine and tyramine are derivatives of tyrosine. Tyrosine is converted to tyramine by Tdc. Tyramine is then converted to octopamine by TβH. B, Expression pattern of the Tdc2–Gal4 line as visualized with a GFP reporter. Octopamine is produced in a subset of neurons in the brain and thoracic ganglion, respectively. Expression was characterized by crossing Tdc2–Gal4 with nuclear UAS–GFPnls.

Figure 2.

Baseline sleep phenotype of Tdc2^RO54 mutants, which have decreased levels of octopamine and tyramine. A, Three days of baseline sleep recording in Tdc2 females. The Tdc2^RO54 line (gray dashed line) shows significantly more sleep than its control w¹¹¹⁸ (black solid line). Dark bars and white bars on top indicate nighttime and daytime, respectively. n = 8 for Tdc2^RO54 and w¹¹¹⁸. CT, Circadian time; ZT, Zeitgeber time. B, Total sleep is significantly increased in Tdc2^RO54 mutant females (mean ± SEM; Tdc2^RO54, 1115 ± 19, n = 23; w¹¹¹⁸, 895 ± 32, n = 32; p ≤ 0.0001, two-way ANOVA). Latency to sleep is significantly lower in Tdc2^RO54 mutants (mean ± SEM; Tdc2^RO54, 20.6 ± 4.6, n = 23; w¹¹¹⁸, 47.5 ± 8.7, n = 32; p ≤ 0.001, two-way ANOVA). C, Arousal threshold during sleep in the Tdc2^RO54 mutants. The animals were given three levels of stimulation to determine whether they were arousable. All animals that responded to the first stimulation were also aroused on stronger stimulation. Compared with wild type, the Tdc2 mutant line had a higher percentage of flies that did not respond to any of the three levels of stimulation. In addition, fewer Tdc2^RO54 flies responded to the weaker stimuli (Tdc2^RO54, n = 23; w¹¹¹⁸, n = 32). D, Rescue of the baseline sleep phenotype in Tdc2^RO54 with expression of the Tdc1 gene. Total sleep was quantified, and Tdc2^RO54 mutant females were significantly different from both wild-type and rescued animals. There was no significant difference between rescued animals and controls (mean ± SEM; w¹¹¹⁸, 895 ± 33, n = 32; Tdc2^RO54, 1238 ± 33, n = 16; p ≤ 0.0001; UAS–Tdc1; Tdc2–Gal4, Tdc2^RO54, 871 ± 33; n = 16).

Figure 3.

Baseline sleep phenotype of TβH^nm18 mutants, which have decreased levels of octopamine and increased levels of tyramine. A, Five days of baseline sleep recording in TβH females. The TβH^nm18 line (gray dashed line) shows significantly more sleep than its control, Canton S (black solid line). Dark bars and white bars on top indicate nighttime and daytime, respectively. Sixteen animals are shown for each group. CT, Circadian time; ZT, Zeitgeber time. B, Total sleep is significantly increased in the TbH^nm18 mutants (mean ± SEM; TβH^nm18, 1176 ± 17, n = 46; Canton S, 884 ± 20, n = 52; p ≤ 0.0001, two-way ANOVA). C, Arousal threshold for the TβH^nm18 mutants. The animals were given three levels of stimulation to determine whether they were arousable. All animals that responded to the first stimulation were also aroused on stronger stimulation. Compared with wild type, the Tbh mutant line had a higher percentage of flies that did not respond to any of the three levels of stimulation. In addition, fewer Tdc2^RO54 flies responded to the weaker stimuli (TβH^nm18, n = 46; Canton S, n = 52). D, Activity per waking minute, which quantifies activity during the time the animal is awake, was significantly increased in the TβH^nm18 mutants (mean ± SEM; TβH^nm18, 1.84 ± 0.13; Canton S, 1.57 ± 0.05; p ≤ 0.01, Kruskal–Wallis test) but significantly decreased in the Tdc2^RO54 mutants (mean ±SEM; Tdc2^RO54, 1.2 ± 0.09; w¹¹¹⁸, 1.5 ± 0.08; p ≤ 0.01, Kruskal–Wallis test).

Figure 4.

Baseline sleep phenotype produced by depolarizing Tdc2-positive neurons. Female flies carrying a UAS–NaChBac transgene under the control of TDC2–Gal4 were assayed for sleep. A, Six days of baseline sleep recording of eight animals each. w;Tdc2–Gal4/NaChBac;+ flies (gray dashed line) show significantly less sleep then Iso31 controls (black solid line). Dark bars and white bars indicate nighttime and daytime, respectively. CT, Circadian time; ZT, Zeitgeber time. B, Nighttime sleep is significantly decreased in w;Tdc2–Gal4/NaChBac;+ flies (mean ± SEM; w;Tdc2–Gal4/NaChBac;+, 267 ± 31, n = 33; Iso31, 613 ± 9, n = 58; p ≤ 0.0001, two-way ANOVA). The latency to sleep is significantly longer in w;Tdc2–Gal4/NaChBac;+ flies (mean ± SEM; w;Tdc2–Gal4/NaChBac;+, 107 ± 20, n = 33; Iso31, 34 ± 3, n = 58; p ≤ 0.01, two-way ANOVA). C, Arousal threshold in the w;Tdc2–Gal4/NaChBac;+ flies. The animals were given three levels of stimulation to determine whether they were arousable. All the w;Tdc2–Gal4/NaChBac;+ flies were more arousable at each stimulation level, and there was a much lower percentage of animals that did not respond at all (w;Tdc2–Gal4/NaChBac;+, n = 33; Iso31, n = 58).

Figure 5.

Baseline sleep phenotype produced by hyperpolarizing Tdc2-positive neurons. Female flies carrying a UAS–Kir2.1 transgene under the control of Tdc2–Gal4 flies were assayed for sleep. A, Four days of baseline sleep recording of 16 animals for each group. w;Tdc2–Gal4/Kir2.1;RC1 flies (gray dashed line) show significantly more sleep then their controls, w;RC1;RC1 (black solid line). Dark bars and white bars indicate nighttime and daytime, respectively. CT, Circadian time; ZT, Zeitgeber time. B, Total sleep is significantly increased in w;Tdc2–Gal4/Kir2.1;RC1flies (mean ± SEM; w;Tdc2–Gal4/Kir2.1;RC1, 829.19 ± 14.14, n = 47; W+;RC1;RC1, 655.35 ± 15.30, n = 47; p ≤ 0.0001, two-way ANOVA). The latency to sleep is significantly lower in Tdc2 × Kir2.1 flies (mean ± SEM; w;Tdc2–Gal4/Kir2.1;RC1, 13.86 ± 1.19, n = 47; w;RC1;RC1, 45.68 ± 8.40, n = 47; p ≤ 0.001, two-way ANOVA). C, Arousal threshold in w;Tdc2–Gal4/Kir2.1;RC1 flies. The animals were given three levels of stimulation to determine whether they were arousable. All animals that responded to the first stimulation were aroused on stronger stimulation. Compared with controls, an increased percentage of w;Tdc2–Gal4/Kir2.1;RC1 flies did not respond to any of the three levels of stimulation. At the strongest stimulation, more control flies responded than w;Tdc2–Gal4/Kir2.1;RC1 flies (w;Tdc2–Gal4/Kir2.1;RC1, n = 47; w;RC1;RC1, n = 47).

Figure 6.

Oral administration of octopamine decreases sleep in Iso31 flies and TβH^nm18 mutant flies. Female flies of the Iso31 strain were treated with octopamine and assayed for sleep. A, The sleep profile shows 1 d of baseline sleep, followed by administration of 10 mg/ml octopamine (gray dashed line) for 3 d. Octopamine was then removed and sleep was assayed for 2 more days. Control flies (black line) were fed normal food during this time. Arrows indicate the time when food was changed. The data are from 16 animals for each group. There was a significant decrease in nighttime sleep for the Iso31 flies on 10 mg/ml octopamine (mean ± SEM; control, 612.04 ± 8.47, n = 40; 10 mg/ml octopamine, 409.33 ± 19.77, n = 40; p ≤ 0.0001, two-way ANOVA). CT, Circadian time; ZT, Zeitgeber time. B, Sleep in TbH^nm18 mutant flies is restored to control levels through the administration of octopamine. Before octopamine administration, TβH^nm18 flies showed a significant increase in total sleep time; after administration of octopamine, sleep in TβH^nm18 flies was not significantly different from that of control (Canton S) flies not on octopamine (Canton S, 796 ± 55 min; Canton S plus 7.5 mg/ml octopamine, 792 ± 54 min; n = 16 for each group). C, Coadministration of mianserin blocks the effect of octopamine on nighttime sleep. When 10 mg/ml octopamine was administered along with 0.2 mg/ml mianserin, there was no longer a significant drop in nighttime sleep (compare with columns on the right). Sleep in flies treated with mianserin alone was not significantly different from that of controls [mean ± SEM; mianserin, 654 ± 8, n = 32; mianserin plus 10 mg/ml octopamine, 627 ± 15, n = 32; Iso31 (Control), 647 ± 22, n = 32; 10 mg/ml octopamine, 450 ± 35, n = 32; p ≤ 0.001, two-way ANOVA].

Figure 7.

Effects of octopamine are mediated by PKA and are independent of the mushroom body. A, Total nighttime sleep in control (Iso31) and ElavGeneSwitch flies crossed with UAS–BDK33 (PKAr). The control on 7.5 mg/ml octopamine shows a significant decrease in nighttime sleep, whereas flies expressing PKAr under the control of ElavGeneSwitch do not show a decrease in sleep in response to octopamine [mean ± SEM; Control (Iso31), 623 ± 11, n = 24; control plus octopamine, 489 ± 22, n = 24; p ≤ 0.001, two-way ANOVA; ElavGeneSwitch, 665 ± 7, n = 51; ElavGeneSwitch plus octopamine, 667 ± 11, n = 51]. B, Iso31 flies subjected to hydroxyurea treatment to ablate the mushroom body still show sensitivity to the sleep-reducing effects of octopamine. Hydroxyurea-treated flies show a significant decrease in sleep compared with controls; they also show an additional decrease in sleep when placed on 7.5 mg/ml octopamine (mean ± SEM; control, 583 ± 11, n = 24; control plus 7.5 mg/ml octopamine, 491 ± 12, n = 24; p ≤ 0.01, two-way ANOVA; HU, 469 ± 32, n = 24; HU plus 7.5 mg/ml octopamine, 382 ± 30, n = 22; p ≤ 0.01, two-way ANOVA).