Go signaling in mushroom bodies regulates sleep in Drosophila

Abstract

Study objectives: Sleep is a fundamental physiological process and its biological mechanisms are poorly understood. In Drosophila melanogaster, heterotrimeric Go protein is abundantly expressed in the brain. However, its post-developmental function has not been extensively explored.

Design: Locomotor activity was measured using the Drosophila Activity Monitoring System under a 12:12 LD cycle. Sleep was defined as periods of 5 min with no recorded activity.

Results: Pan-neuronal elevation of Go signaling induced quiescence accompanied by an increased arousal threshold in flies. By screening region-specific GAL4 lines, we mapped the sleep-regulatory function of Go signaling to mushroom bodies (MBs), a central brain region which modulates memory, decision making, and sleep in Drosophila. Up-regulation of Go activity in these neurons consolidated sleep while inhibition of endogenous Go via expression of Go RNAi or pertussis toxin reduced and fragmented sleep, indicating that the Drosophila sleep requirement is affected by levels of Go activity in the MBs. Genetic interaction results showed that Go signaling serves as a neuronal transmission inhibitor in a cAMP-independent pathway.

Conclusion: Go signaling is a novel signaling pathway in MBs that regulates sleep in Drosophila.

Keywords: Drosophila; Go; Mushroom bodies; RNAi; cAMP.

Figure 1

Pan-neuronal expression of Go^WT and Go^GTP causes sleep increase in Drosophila. (A) The total amount of sleep increases significantly in ElavGeneSwitch > Go^WT and ElavGeneSwitch > Go^GTP female flies after feeding with RU486 as compared with controls (Student t-test; **P < 0.001). (B) Responses to moderate (left) and strong (right) mechanical stimulation in drug-treated ElavGeneSwitch > Go^GTP female flies and controls (Student t-test; **P < 0.001). (C) Sleep bout number is reduced in ElavGeneSwitch > Go^GTP female flies after RU486 treatment (Student t-test; *P < 0.05). (D) Average sleep bout duration of drug-treated ElavGeneSwitch > Go^GTP female flies increased significantly. The data, which is not normally distributed, is represented by box plots. Horizontal lines in the boxes indicate the median; upper and lower boxes represent 75% and 25%, respectively; vertical lines above and below the boxes represent 95% and 5%. Asterisks represent statistically significant groups determined by the Mann-Whitney U test; **P < 0.001. n = 16 for each genotype. Error bars indicate the SEM.

Figure 2

Go^GTP functions in mushroom bodies to regulate sleep. (A) Expression of Go^GTP with DDC-GAL4 or GAD-GAL4 had no marked effect on sleep. White bars represent the untreated group; gray bars represent a 12 hour induction at 32°C. (B) Increased sleep in flies expressing Go^GTP with MB247 or OK107 GAL4 (**p < 0.001 by one-way ANOVA). The genotypes of flies were: w¹¹¹⁸/y w;;MB247/+, w¹¹¹⁸/y w;;MB247/UAS-Go^GTP, w¹¹¹⁸/y w;; UAS-Go^GTP/+, w¹¹¹⁸;;OK107/+, w¹¹¹⁸;;OK107/UAS-Go^GTP, and w¹¹¹⁸;; UAS-Go^GTP/+ (from left to right). For parental controls, each GAL4 line was crossed to w¹¹¹⁸ (the background of UAS- Go^GTP), UAS-Go^GTP was crossed to w¹¹¹⁸and y w (the background of OK107 and MB247). (C) Sleep profile at 30-min intervals for RU486 treated (black line) and untreated (gray line) P{MBSwitch} > Go^GTP female flies. The bar below the panel indicates 12 h light (white) and 12 h dark (black) period. (D) Expression of Go^GTP in mushroom bodies with P{MBSwitch} significantly increases sleep (**P < 0.001 by Student t-test). For each group in (A) (B), n = 32; in (C) (D), n = 15-16. Error bars indicate the SEM.

Figure 3

Inhibition of endogenous Go signaling in mushroom bodies alters sleep. (A) Western blotting of endogenous Go levels in pan-neuronal GoRNAi-expressing and control fly brains. The genotype of each group is given above the lane. (B) Daily sleep amount in flies expressing PTX or GoRNAi in mushroom bodies with MB247 GAL4 and controls (**P < 0.001 by one-way ANOVA), n = 16 for each group. The genotypes of flies were: w¹¹¹⁸/y w;;MB247/+, w¹¹¹⁸/y w;;MB247/UAS-PTX, w¹¹¹⁸/y w;; UAS-PTX/+, w¹¹¹⁸/y w; UAS-Dcr-2/+; MB247/UAS-Go^RNAi, w¹¹¹⁸/y w; UAS-Dcr-2/+; UAS-Go^RNAi/+ (from left to right). (C) The sleep profile for PTX-induced (black line) and control (gray line) P{MBSwitch} > PTX female flies in LD and DD conditions. The bar below the x-axis indicates day (white), night (black), subjective day (gray), and subjective night (black) periods. n = 16 for each group. (D) Daily sleep was reduced after RU486 induction in P{MBSwitch} > PTX female flies in either LD or DD (*P < 0.05, **P < 0.001 by Student t-test). The genotypes are P{MBSwitch} > PTX (left) and PTX/+ (right). n = 16 for each group. Error bars indicate the SEM.

Figure 4

Go signaling in mushroom bodies is necessary for sleep regulation. (A) GFP expression in brains of HU-untreated (left) and treated (right) P{MBSwitch} > mCD8:GFP flies. (Scale bar, 100 μm) (B) The brains of flies expressing mCD8:GFP with 30Y (left) and MB-GAL80;30Y (right). (Scale bar, 100 μm) (C) Histograms show average daily sleep amount of RU486-induced P{MBSwitch} > PTX and P{MBSwitch} > Go^GTP flies with or without hydroxyurea treatment. Results were normalized to the mean value of the sleep amount in the control without RU486 treatment (*P < 0.05, **P < 0.001 by Student t-test), n = 16 for each group. (D) Sleep of w¹¹¹⁸; UAS-Dcr-2/+; UAS-Go^RNAi/+, w¹¹¹⁸; UAS-Dcr-2/+; UAS-Go^RNAi/30Y, w¹¹¹⁸; 30Y/+, w¹¹¹⁸; UAS-Dcr-2/MB-GAL80; UAS-Go^RNAi/30Y, and w¹¹¹⁸; MB-GAL80/+;30Y/+ flies (from left to right) (**P < 0.001 by one-way ANOVA). n = 16-32 for each group. Error bars indicate the SEM.

Figure 5

The role of Go signaling in sleep regulation is independent of the cAMP pathway. (A) Histograms show the average daily sleep amount in RU486-induced groups normalized to the mean value of the sleep amount in their sibling controls without RU486 treatment. ElavGeneSwitch/UAS-Gs^GTP (left); ElavGeneSwitch/UAS-Gi^GTP (middle); and rut²⁰⁸⁰;;ElavGeneSwitch/UAS-Gs^GTP (right). Statistical significance (**P < 0.001 by Student t-test) is indicated. (B) Histograms are the ratios of daily sleep amount in the RU486 induced group to the mean daily sleep amount in the RU486 untreated comparison groups. From left to right: rut²⁰⁸⁰; ElavGeneSwitch/Go^GTP, rut¹; ElavGeneSwitch/Go^GTP, dnc¹; ElavGeneSwitch/Go^GTP, and rut¹; P{MBSwitch}/Go^GTP. Asterisks represent statistically significant groups (**P < 0.001 by Student t test). n = 16. (C) Histograms indicate the ratios of daily sleep amount in the RU486 induced group to the mean daily sleep amount in the RU486 untreated comparison groups. From left to right: ElavGeneSwitch/PKAmc*, ElavGeneSwitch/PKAr, Go^GTP/+; ElavGeneSwitch/PKAmc*, Go^GTP/+; ElavGeneSwitch/PKAr, and Go^GTP/+; ElavGeneSwitch/CREB-2b. Asterisks represent the level of statistical significance; (**P < 0.001 by Student t-test). For each group, n = 16. Error bars indicate the SEM.