LHLK neurons are required for the metabolic regulation of sleep.

(A) Whole-brain and ventral nerve cord confocal reconstruction of Lk-GAL4>CD8::GFP. GFP-expressing neurons (green). Immunostaining for anti-Lk (red) reveals colocalization of LHLK and SELK neurons (yellow). The brain and ventral nerve cord are counterstained with nc82 (magenta). Scale bar = 100 μm. (B) GFP expression in the ventral nerve cord (ABLK neurons) of flies carrying Lk-GAL4>CD8::GFP is blocked by the tsh-GAL80 transgene. (C) Blocking synaptic release in Lk neurons with TNT impairs starvation-induced sleep suppression (n = 29, p = 0.60), while impTNT controls suppress sleep (n ≥ 27, p = 0.0002). No differences were observed between genotypes during the fed state (p = 0.06). Two-way ANOVA (F [1, 109] = 4.88). (D) Starvation-induced sleep suppression is absent in tsh-GAL80;Lk-GAL4 flies (n = 41, p = 0.12), while controls expressing impTNT suppress sleep (n = 33, p TNT and impTNT flies (p = 0.21). Two-way ANOVA (F [1, 144] = 22.53). (E) TNT expression in Apt-GAL4 neurons abolishes starvation-induced sleep suppression (n ≥ 18, p = 0.48) and shows a significant increase in sleep during the fed state (p = 0.01), compared to impTNT that suppresses sleep (n ≥ 34, p Lk-RNAi in Apt-GAL4 neurons (Apt-GAL4>UAS-dcr2,Lk-RNAi, n ≥ 12) blocks starvation-induced sleep suppression (p = 0.877), while UAS-Lk-RNAi/+ (n = 79, p n = 13, p UAS-Lk under control of Apt-GAL4 in the Lkc275 mutant background restores starvation-induced sleep suppression (n = 15, p = 0.002) compared to Lkc275 flies and mutant control flies UAS-Lk/+;Lkc275 (n = 16; p = 0.98) or Apt-GAL4/+;Lkc275 (n = 16, p = 0.88). Control flies, Apt-GAL4/+;Lkc275/+ (n ≥ 16, p UAS-Lk/+;Lkc275/+ (n = 21, p p p p S1 Data. ANOVA, analysis of variance; Apt, Apterous; CD8::GFP; membrane-tethered GFP, LK-GAL4>CD8:GFP;tshGAL80; dcr2, dicer-2; GAL4, galactose-responsive transcription factor; GFP, green fluorescent protein; impTNT, inactive variant of tetanus toxin; LHLK, Lateral Horn leucokinin; Lk, leucokinin; nc82, neuropil marker; RNAi, RNA interference; TNT, tetanus toxin; tsh, teashirt; UAS, upstream activation sequence.