A single pair of leucokinin neurons are modulated by feeding state and regulate sleep–metabolism interactions
Fig 5
AMPKα in LHLK neurons is required for starvation-induced sleep suppression.
(A) Knockdown of AMPKα in Lk neurons (Lk-GAL4, n = 32, p = 0.14) abolishes starvation-induced sleep suppression, while control flies Lk-GAL4/+ (n = 27, p < 0.0001) and AMPKα-RNAi/+ (n ≥ 58, p < 0.0001) suppress sleep. During the fed state, sleep is significantly reduced in Lk-GAL4>UAS-AMPKα-RNAi compared to controls Lk-GAL4/+ (p < 0.0001) and AMPKα-RNAi/+ (p < 0.0001). Two-way ANOVA (F [2, 230] = 22.70). (B) Sleep profile representative of (A). 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. (C) Flies expressing UAS-AMPKα-RNAi in brain Lk neurons (tsh-GAL80;Lk-GAL4) fail to suppress sleep in response to starvation (n ≥ 56, p = 0.16) compared to control flies that suppress sleep (tsh-GAL80;Lk-GAL4/+, n ≥ 45, p < 0.0001) and AMPKα-RNAi/+ (n ≥ 58, p < 0.0001). Sleep while fed is significantly reduced in tsh-GAL80;Lk-GAL4>UAS-AMPKα-RNAi compared to tsh-GAL80;Lk-GAL4/+ (p = 0.0004) and AMPKα-RNAi/+ (p < 0.0001). Two-way ANOVA (F [2, 364] = 23.12). (D) Sleep profile representative of (C). (E) In vivo Ca2+ imaging during the fed state shows an increase in the average ratio of GCaMP/mCherry in LHLK neurons expressing UAS-AMPKα-RNAi;UAS-Gerry (gray and white, n = 10) compared to flies harboring Gerry alone (gray; n = 8, p = 0.01, t = 2.86). Unpaired t test. (F) During the starved state, no significant differences in LHLK Ca2+ activity are observed in Lk-GAL4>UAS-AMPKα-RNAi;UAS-Gerry flies (blue and white, n = 8) compared to flies harboring Gerry alone (blue, n = 8, p = 0.44, t = 0.78), Unpaired t test. Fluorescence intensity scale represents the ratio range of GCaMP6m/mCherry ranging from 2 (max) to 0 (minimum). Scale bar = 10 μm. Error bars for GCamp6m/mCherry ratio during the fed versus starved state indicate SEM; *p < 0.05; **p < 0.01; ***p < 0.001. Underlying data can be found in S1 Data. AMPK, 5′ adenosine monophosphate-activated protein kinase; ANOVA, analysis of variance; GAL4, galactose-responsive transcription factor; GCaMP6m, GFP-calmodulin and M13 peptide sequence; GFP, green fluorescent protein; LHLK, Lateral Horn leucokinin; Lk, leucokinin; max, maximum; RNAi, RNA interference; tsh, teashirt; UAS, upstream activation sequence; UAS-Gerry, GCaMP6m-mCherry; ZT, Zeitgeber time.