A subset of cholinergic mushroom body neurons requires Go signaling to regulate sleep in Drosophila

Abstract

Study objectives: Identifying the neurochemistry and neural circuitry of sleep regulation is critical for understanding sleep and various sleep disorders. Fruit flies display sleep-like behavior, sharing essential features with sleep of vertebrate. In the fruit fly's central brain, the mushroom body (MB) has been highlighted as a sleep center; however, its neurochemical nature remains unclear, and whether it promotes sleep or wake is still a topic of controversy.

Design: We used a video recording system to accurately monitor the locomotor activity and sleep status. Gene expression was temporally and regionally manipulated by heat induction and the Gal4/UAS system.

Measurements and results: We found that expressing pertussis toxin (PTX) in the MB by c309-Gal4 to block Go activity led to unique sleep defects as dramatic sleep increase in daytime and fragmented sleep in nighttime. We narrowed down the c309-Gal4 expressing brain regions to the MB α/β core neurons that are responsible for the Go-mediated sleep effects. Using genetic tools of neurotransmitter-specific Gal80 and RNA interference approach to suppress acetylcholine signal, we demonstrated that these MB α/β core neurons were cholinergic and sleep-promoting neurons, supporting that Go mediates an inhibitory signal. Interestingly, we found that adjacent MB α/β neurons were also cholinergic but wake-promoting neurons, in which Go signal was also required.

Conclusion: Our findings in fruit flies characterized a group of sleep-promoting neurons surrounded by a group of wake-promoting neurons. The two groups of neurons are both cholinergic and use Go inhibitory signal to regulate sleep.

Keywords: Go signaling; Mushroom body; cholinergic neurons; sleep.

Figure 1

Blocking Go signaling in c309 neurons leads to sleep increase. (A) Procedure for sleep recording and heat induction. White and black bars show light/dark (L/D) periods. Fly sleep was monitored for 2 days before (light gray) and after (dark gray) heat shock induction. (B) Sleep profiles for c309-Gal4/ tub-Gal80^ts;UAS-PTX/+ flies and their parental flies tub-Gal80^ts/+;UAS-PTX/+ during the sleep recording procedure. Diagonal crossing lines represent heat shock period. (C) Sleep profiles for c309-Gal4/tub-Gal80^ts;UAS-PTX/+ are averaged for the preheat or the post-heat recording days. (D-H) Sleep parameters are analyzed and averaged in the preheat or the post-heat recording days for c309-Gal4/tub-Gal80^ts;UAS-PTX/+ flies and their parental controls c309-Gal4/+ and tub-Gal80^ts/+;UAS-PTX/+flies: daily sleep amount (D), change in sleep amount calculated by subtracting sleep before heat induction from sleep after heat induction (E), number of sleep bouts (F), average duration of sleep bout during the day or night (G), and average duration of wake bouts during the day or night (H). n = 30-36 in each group. Data were analyzed by one-way analysis of variance in (E) and paired Student t-test in other panels. Data are presented as the mean ± standard error of the mean. ***P < 0.001; n.s, no significant difference, P > 0.05.

Figure 2

Elevating Go signaling in c309 neurons decreases sleep. (A) Sleep profiles before or after temporally expressing Go^GTP using c309-Gal4 with tub-Gal80^ts. (B-F) Sleep parameters before (light gray bar) or after heat induction (dark gray bar) were analyzed for c309-Gal4/UAS-Go^GTP;tub-Gal80^ts/+ flies and their parental controls c309-Gal4/+ and UAS-Go^GTP/+;tub-Gal80^ts/+flies: daily sleep amount (B), change in sleep amount during daytime and nighttime (C), number of sleep bouts (D), average duration of sleep bouts during daytime and nighttime (E), and average duration of wake bouts during daytime and nighttime (F). n = 30-36 in each group. Data were analyzed by one-way analysis of variance in (C) and with paired Student t-test in other panels. Data are presented as means ± standard error of the mean. ***P < 0.001; n.s, no significant difference, P > 0.05.

Figure 3

c309-Gal4 is expressed in MB α/β core neurons in adult stage flies. (A) Procedure of heat induction of mCD8::GFP for examining the expression pattern. (B-C) In the MB region, c309-Gal4 has restricted expression, which could be eliminated by MB-Gal80. (D-F) Projection view of the MB lobe area (D) and a single horizontal section of the tip of the α lobe (E-F). FasII signal (red) shows the neuropils of MB neurons. GFP signal (green), representing c309-Gal4 expressing neurons, is only observed in the core area of MB α/β lobe. Scale bars represent 50 μm in B and C, and 10 μm in D-F.

Figure 4

MB-Gal80 eliminates PTX- and Go^GTP- induced sleep effects in c309-Gal4. (A) Sleep profiles are comparable before and after heat induction when MB-Gal80 is introduced. (B-E) Temporally expression of PTX or Go^GTP with c309-Gal4,MB-Gal80 leads to no significant change in sleep amount (B), number of sleep bouts (C), sleep bout duration in daytime (D) or sleep bout duration in nighttime (E). Light gray bars and dark gray bars indicate sleep parameters for 2 light-dark (LD) days before and after heat induction, respectively. Data were analyzed by paired Student t-tests; n = 30-36 in each group. Data are presented as means ± standard error of the mean. ***P < 0.001, paired Student t-test.

Figure 5

Inhibiting Go signal in c309-related Gal4 lines regulates sleep in different ways. (A) Schematic diagram of the P-element insertion sites of several Gal4 lines in the scabrous promoter region. Arrows above the triangles indicate insertion orientation. The numbers below the line represent insertion sites on the second chromosome. (B-D) Heat-induced expression patterns are shown for Gal4 lines with the existence of tub-Gal80^ts. c309 (B), NP2122-Gal4 (D) and NP4289-Gal4 (E) drive strong expression in the MB α/β core area, whereas Sca^109–68-Gal4 (C) drives weak expression in the MB. Scale bar represents 50 μm. (F) Temporal expression of PTX with NP2122- or NP4289- Gal4, but not with Sca^109–68-Gal4, leads to increases in sleep amount. Numbers on bars indicate the number of flies in each group. Data were analyzed by paired Student t-test. ***P < 0.001.

Figure 6

Inhibition of the endogenous Go signal in multiple MB-Gal4 lines decreases sleep. (A-C) Heat-induced expression patterns are shown for respective Gal4 lines with tub-Gal80^ts. From left to right are projection views of the central brain, the MB lobes, the MB lobes co-stained with FasII and a single horizontal section at the tip of α lobe. (A) D52H-Gal4 drives strong expression in MB α/β surface and γ area, moderate expression in the MB α/β posterior and weak expression in the α/β core areas. (B) R15E01-Gal4 drives strong expression in MB α/β surface, posterior, core, and γ areas. (C) NP3061-Gal4 drives strong expression in MB α/β surface, and core area and moderate expression in MB α/β posterior area. c, core; p, posterior; s, surface. Scale bar represents 10 μm. (D and E) Temporal expression of PTX with these Gal4s leads to significantly decreased sleep amount. The numbers on bars indicate the number of flies in each group. Data were analyzed using paired Student t-tests. ***P < 0.001.

Figure 7

c309-MB neurons are cholinergic, and reducing the ACh signal in these neurons affects sleep. (A-B) c309 expression in the MB is blocked by Cha-Gal80 but not GAD1-Gal80 in adult stage flies. Scale bar represents 50 μm. (C) Cha-Gal80, but not GAD1-Gal80, eliminates sleep defects induced by temporally expression of PTX or Go^GTP mediated by c309-Gal4. n = 25-39 in each group. (D) Continuously expressing vAChT^RNAi by c309-Gal4 leads to reduced sleep, which is abolished by MB-Gal80 or Cha-Gal80. Sleep amount is increased in D52H > vAChT^RNAi flies. Numbers on the bars indicate the number of flies in each group. ***P < 0.001 in paired Student t-tests (C) or one-way analysis of variance (D). n.s., no significant difference.