1. Volume 19, Issue 12, Pages 2441-2450 (June 2017)
Drosophila Neuropeptide F Signaling Independently Regulates Feeding and Sleep- Wake Behavior 
Brian Y. Chung, Jennifer Ro, Sabine A. Hutter, Kylie M. Miller, Lakshmi S. Guduguntla, Shu Kondo, Scott D. Pletcher 
Cell Reports 
Volume 19, Issue 12, Pages (June 2017)
DOI: /j.celrep
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2. Cell Reports 2017 19, 2441-2450DOI: (10.1016/j.celrep.2017.05.085)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1
Acute Activation of the NPF-NPFR1 Signaling Axis Increases Wakefulness
(A and B) NPF-expressing cell activation reduces sleep behavior and is reversible, illustrated as a sleep profile depicting sleep amounts in 30 min binned intervals (A) and a stacked bar chart showing total sleep amount (B). n = 80–111.
(C and D) Characterization of a loss-of-function NPF allele, NPFSK2.
(C) Schematic showing the position of the 11-bp deletion within the NPF coding region.
(D) Representative images of control (D1) and NPFSK2 adult male brains immunostained against NPF (magenta) (D2). The white scale bars represent 100 μm.
(E and F) NPF-expressing cell activation effects on wakefulness depend on intact NPF signaling, illustrated as a sleep profile (E) and a boxplot quantifying total sleep amount (F). n = 32.
(G and H) NPFR-expressing cell activation increases wake behavior, illustrated as a sleep profile (G) and as a boxplot quantifying total sleep amount (H). n = 16–63. For all boxplots, the squares within the box indicate mean, the box range represents SEM, the whiskers denote 10–90 percentiles, and the dots indicate individual data values. All of the error bars indicate SEM.
See also Figure S1.
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4. Figure 2 NPF Signaling Regulates the Physiological State of the Fly
(A and B) Loss of NPF signaling suppresses starvation-induced sleep loss.
(A) Sleep profiles of starved and fed control flies carrying the NPFSK2 allele compared to control genotypes. n = 14–15.
(B) Percent change in 12 hr dark period sleep during the first day of starvation compared to prior 12 hr dark fed baseline period.
(C) NPF cell activation increases liquid food consumption in adult male flies. n = 11 experimental replicates.
(D) NPF cell activation decreases whole-male adult fly triacylglyceride levels. n = 12 experimental replicates.
(E) NPF-expressing cell activation renders adult male flies sensitive to starvation. All of the error bars indicate SEM.
See also Figures S2 and S3.
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5. Figure 3
A Subset of Circadian Pacemaker Cells Largely Regulates NPF Effects on Wakefulness, but Not Feeding
(A and B) Repressing activation of NPF signaling in cry-expressing cells largely reverts the sleep loss phenotype, illustrated as a sleep profile (A) and as boxplots quantifying total sleep amount (B). n = 59–128.
(C) NPF-GAL4 and cry-GAL80 expression patterns reveal NPF expression in a subset of the PER+ LNd circadian pacemaker neurons. Representative expression patterns of adult brain images for NPF-GAL4 (C1) and NPF-GAL4 and cry-GAL80 (C2) using membrane GFP as a reporter are shown (top). The white arrows denote the location of LNd neurons in the adult brain. High magnification images of the regions indicated by the white arrows in the top panels demonstrating co-localization of NPF (green) and PER (blue) within LNd neurons are shown (bottom). The white scale bars in the images denote 25 μm.
(D) Repressing activation of NPF signaling in cry-expressing cells does not alter adult feeding behavior. n = 19–21 experimental replicates. All of the error bars indicate SEM.
See also Figure S4.
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6. Figure 4
A Mosaic Population Reveals that NPF Regulation of Feeding and Wake Behaviors Can Be Dissociated
(A) Increased prevalence of short-sleeping flies in a mosaic NPF activation population (red; n = 183) compared to a non-heat shock control population (blue; n = 41).
(B) Characterization of a mosaic population reveals distinct neuronal centers important for NPF control of wakefulness. The inset image shows the numbering system used to catalog NPF+ loci. Frequency represents the percentage of short-sleeping mosaic flies where GFP expression was observed (brain images characterized, n = 41). Some flies exhibited expression in more than one neuronal locus and across both hemispheres. “FSB” is short for fan-shaped body.
(C and D) Feeding behavior does not correlate with sleep-wake behavior in a mosaic NPF activation population.
(C) Representative image (left), matching sleep profile (middle), and number of feeding events (right) from two individual males from the mosaic population. The bold white arrows highlight the location of LNds. The error bar indicates SD.
(D) Scatterplot of individual fly phenotypes (n = 60), with 29°C light-dark (LD) sleep amount plotted on the y axis and subsequent 29°C LD feeding events plotted on the x axis. p = as determined by ANOVA.
(E) NPFR-GAL4 exhibits prominent expression in established homeostatic centers of the adult brain. The white scale bar represents 100 μm.
Cell Reports  , DOI: ( /j.celrep )
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