A single pair of leucokinin neurons are modulated by feeding state and regulate sleep–metabolism interactions
Fig 1
Lk is required for metabolic regulation of sleep.
(A, B) IHC using antibody against Lk. White arrows indicate LHLK cell bodies, and yellow arrows indicate SELK cell bodies. Lk staining in flies expressing Lk-RNAi results in loss of Lk protein level in LHLK neurons (B) compared to UAS-Lk-RNAi/+ control (A). The brains were counterstained with nc82 (bruchpilot, gray). Scale bar = 100 μm. Color-coded images reflect fluorescence intensity, where minimum = 0 and max = 4,025. Scale bar for color-coded images = 10 μm. (C) Sleep is significantly reduced in starved UAS-Lk-RNAi/+ (n ≥ 20, p < 0.0001) and dcr2,Lk-GAL4/+ (n ≥ 25, p < 0.0001) control flies, while no significant differences are observed in Lk-GAL4>UAS-dcr2,Lk-RNAi flies (n = 13, p > 0.99). Two-way ANOVA (F [2, 141] = 10.87). White bars in column graphs represent amount of sleep during the day (ZT 0–12), while colored bars represent night sleep (ZT 12–24). (D) Sleep profile representative of (C). Flies are placed in food tubes during day 1 (fed, gray), then transferred to agar during day 2 (starved, blue). White/black bars represent lights on and off, respectively. (E, F) Lk staining in Lkc275 mutants reveals reduced protein levels compared to w1118 control (E). (G) Sleep is significantly reduced in starved w1118 controls (n = 63, p < 0.0001) and flies harboring one copy of Lkc275 (n = 68, p < 0.0001), while no significant differences are observed in Lkc275 mutants (n = 58, p = 0.90). Two-way ANOVA (F [2, 372] = 53.57). (H) Sleep profile representative of data in (G). (I, J) Lk protein levels are reduced in Lk−/−(GAL4) flies compared to w1118 control (J). (K) Lk−/− (GAL4) (n ≥ 47; p = 0.31) mutant flies and Lk+/−(GAL4) (n = 68, p = 0.35) fail to suppress sleep in response to starvation, while w1118 control flies suppress sleep (n = 75, p < 0.0001). Two-way ANOVA (F [2, 376] = 57.03). (L) Sleep profile representative of data in (K). All columns represent mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001. Underlying data can be found in S1 Data. ANOVA, analysis of variance; dcr2, dicer-2; GAL4, galactose-responsive transcription factor; IHC, immunohistochemistry; LHLK, Lateral Horn leucokinin; Lk, leucokinin; max, maximum; nc82, neuropil marker; RNAi, RNA interference; SELK, subesophageal ganglion leucokinin; UAS, upstream activation sequence; ZT, Zeitgeber time.