A single pair of leucokinin neurons are modulated by feeding state and regulate sleep–metabolism interactions
Fig 6
Lk targets the IPCs to promote wakefulness during starvation.
Expression pattern for Lkr−/−>UAS-mCD8::GFP (A), R65C07-GAL4 (C), R67D01-GAL4 (F), and Dilp2-GAL4 (I). The brains were counterstained with nc82 (magenta). Scale bar = 100 μm. (B) Lkr−/−(GAL4) mutants fail to suppress sleep in response to starvation (n ≥ 147, p = 0.09) compared to control w1118 flies (n = 155, p < 0.0001), Lkr+/− (n ≥ 134, p < 0.0001), and UAS-Lkr/+ (n = 26, p < 0.0001). Expression of UAS-Lkr in neurons labeled by Lkr−/−(GAL4) (Lkr−/−(GAL4); UAS-Lkr) restores starvation-induced suppression (n = 28, p = 0.0003). There were no significant differences during the fed state between control (UAS-Lk/+) and rescue flies (p = 0.10) or Lk−/− (p = 0.24). Two-way ANOVA (F [4, 965] = 13.05). (D) Blocking synaptic release in Lkr-expressing neurons that label the dFSB (R65C07-GAL4>UAS-TNT) does not affect starvation-induced sleep suppression (n = 48, p < 0.0001), similar to impTNT controls (n = 50, p < 0.0001). Sleep while fed is reduced in R65C07-GAL4>TNT flies compared to control (p = 0.0025). Two-way ANOVA (F [1, 109] = 11.17). (E) No impairments in starvation-induced sleep suppression were observed when knocking down Lkr in Lkr-expressing neurons (R65C07-GAL4>UAS-dcr2,Lkr-RNAi, n ≥ 45, p < 0.0001). Control UAS-Lkr-RNAi/+ (n = 27, p < 0.0001) and R65C07-GAL4/+ (n = 32, p < 0.0001) suppress sleep in response to starvation. Two-way ANOVA (Fcolumn [1, 205] = 130.8). (G) Expression of TNT in R67D01-GAL4, which labels the IPCs, impairs starvation-induced sleep suppression (n = 31, p = 0.47), while impTNT control flies suppress sleep (n = 48, p < 0.0001). Two-way ANOVA (F [1, 154] = 37.02). (H) Starvation-induced sleep suppression is absent with Lkr knockdown in Lkr-expressing neurons (R67D01, n = 41, p = 0.53). Control UAS-Lkr-RNAi/+ (n = 25, p < 0.0001) and dcr2,R67D01-GAL4 (n ≥ 53, p < 0.0001) suppress sleep in response to starvation. Two-way ANOVA (F [2, 232] = 13.86). (J) Knocking down Lkr in the IPCs (Dilp2-GAL4>UAS-dcr2,Lkr-RNAi, n ≥ 30, p = 0.67) results in flies that fail to suppress sleep in response to starvation, while dcr2,Dilp2-GAL4/+ control flies suppress sleep (n = 25, p = 0.0016). Two-way ANOVA (F [1, 108] = 4.1). (K) Sleep profile representative of (J). 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. All columns are mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001. Underlying data can be found in S1 Data. ANOVA, analysis of variance; CD8::GFP, membrane-tethered GFP; LK-GAL4>CD8:GFP;tshGAL80; dcr2, dicer-2; dFSB, dorsal fan-shaped body; Dilp2, Drosophila insulin-like peptide 2; GAL4, galactose-responsive transcription factor; GFP, green fluorescent protein; impTNT, inactive variant of tetanus toxin; IPC, insulin-producing cell; Lk, leucokinin; Lkr, leucokinin receptor; nc82, neuropil marker; RNAi, RNA interference; TNT, tetanus toxin; tsh, teashirt; UAS, upstream activation sequence; ZT, Zeitgeber time.