Overexpression of Zebrafish NPY Increases Sleep and Arousal Threshold (A–G) Overexpression of zebrafish NPY following a heat shock at 3 p.m. resulted in decreased locomotor activity (A and B) and increased sleep (C and D), due to more sleep bouts (E) and shorter sleep (F) and wake (G) bouts. Yellow bars indicate heat shock (HS). Pre-HS and post-HS quantify data for day 5 before and after HS. Mean ± SEM from four experiments is shown. (H) Representative stimulus-response curve for Tg(hsp:npy) animals compared to WT siblings following HS. Data points represent mean ± SEM. Dashed lines mark ETP50 value for each genotype. Tg(hsp:npy) animals had an ETP50 value of 24.2 versus 8.2 for WT siblings (293% increase, p < 0.05 by extra sum-of-squares F test). (I and J) Overexpression of NPY reduced the response of Tg(hsp:npy) animals to the stimulus compared to WT siblings during both awake (I) and sleep (J) states. Stimulus intensities of 2.3, 3.0, and 4.0 arbitrary units (a.u.) were tested. A dose-dependent response was observed for WT animals, but not their Tg(hsp:npy) siblings. Bar graphs show mean ± SEM. n, number of animals. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 by two-tailed Student’s t test. See also Figures S1 and S2 and Movie S1.

Loss of npy Reduces Daytime Sleep (A) Sequences of WT and mutant zebrafish NPY proteins. The mature peptide is indicated with a red box. Altered amino acids in the mutant are shaded gray. (B–M) npy−/− animals were more active (B and C), and slept less (E and F), than their npy+/+ and npy+/− siblings during the day, due to fewer sleep bouts (H) and longer wake bouts (L). There was no difference in activity (B and D), sleep (E and G), sleep bout number (I) or wake bout length (M) at night, or in sleep bout length during the day (J) or night (K). Mean ± SEM from seven experiments is shown. n, number of animals. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 by one-way ANOVA with Holm-Sidak test.

Entrained npy Mutants Sleep Less in Constant Light Larvae were entrained in 14:10-hr LD cycles for the first 4 days and nights of development, and then behaviorally monitored for 24 hr in LD and then for 48 hr in LL. npy−/− animals were more active (A–C) and slept less (D–F) than their npy+/− and npy+/+ siblings during subjective day and night, due to fewer (G and H) and shorter (I and J) sleep bouts and longer wake bouts (K and L). Mean ± SEM from three experiments is shown. n, number of animals. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 by one-way ANOVA with Holm-Sidak test. See also Figure S3.

Functional Evidence that NPY Promotes Sleep by Inhibiting NE Signaling (A–D) Tg(hsp:npy);dbh−/− and dbh−/− animals were less active (A and B) and slept more (C and D) than dbh+/− siblings during the day before and after HS. Tg(hsp:npy);dbh+/− animals were less active and slept more than dbh+/− siblings during the day after HS. NPY overexpression in Tg(hsp:npy);dbh−/− animals did not further decrease locomotor activity or increase sleep compared to dbh−/− siblings. Yellow bars indicate HS. Pre-HS and post-HS quantify data on day 5 before and after HS. (E–J) npy+/+, npy+/−, and npy−/− siblings were treated with either DMSO or prazosin. DMSO-treated npy−/− animals were more active (E and F) and slept less (H and I) than their DMSO-treated npy+/− and npy+/+ siblings during the day, but there was no difference at night (E, G, H and J). Prazosin decreased activity (E and F) and increased sleep (H and I) to a similar extent for npy−/−, npy+/−, and npy+/+ siblings during the day. Arrows indicate behavioral artifacts due to addition of water. Mean ± SEM for 2 (A–D) or 4 (E–J) experiments is shown. n.s., not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001 by two-way ANOVA with Holm-Sidak test. See also Figures S6 and S7 and Table S2.

A genetic screen identifies a sleep-promoting role for NPY and zebrafish npy is widely expressed in the brain. Related to Figure 1. (A) Histogram depicting the total amount of sleep during the night after heat shock for ~1200 human genes tested in the larval zebrafish genetic screen. Larvae overexpressing human NPY had a Z-score of 1.8. Overexpression of human NPY decreased locomotor activity (B-D) and increased sleep (E-G) compared to EGFPoverexpressing controls during the night following heat shock (indicated by yellow bar). (H) Amino acid sequence alignment of human (Hs), mouse (Mm) and zebrafish (Dr) NPY mature peptide sequences. Colors indicate residues with similar properties. (I-L) ISH using an npyspecific probe reveals discrete yet widespread nuclei of npy expression in a 6-dpf zebrafish. Images show 4 different focal planes, with the most dorsal image at left. The full image stack of npy expression throughout the brain mapped onto the Z-brain atlas is shown in Movie S1. (M,N) Schematic drawings illustrate relative positions of different npy-expressing populations in ventral (M) and lateral (N) views. npy is expressed in the olfactory bulb (a), telencephalon (b), preoptic area (c), posterior tuberculum (d), Intermediate lateral hypothalamus (e), caudal medial hypothalamus (f), pretectum (g), torus semicircularis (h), tectum (i), locus coeruleus (j), medial rhombomere (k) and subpallium (l). a, anterior; p, posterior; d, dorsal; v, ventral. Scale bar: 100 μm. Mean (B,E) and mean ± SEM (C,D,F,G) are shown. n=number of animals. *P<0.05, **P<0.01 by two-tailed Student’s t test.

Specific expression of kalta4 in npy-expressing neurons and TUNEL labeling of apoptotic cells. Related to Figure 4. (A) Double FISH showing kalta4 and npy co-expression using probes specific for kalta4 and npy in different populations of npy-expressing neurons. (B-D) Tg(npy:kalta4);Tg(uas:nfsb-mcherry) animals were treated with DMSO (B) or 10 mM MTZ (C,D) from 72-90 hpf, and then fixed and processed for TUNEL. TUNEL labeling was observed in npyexpressing neurons of animals treated with MTZ (C,D), but not in animals treated with DMSO (B). Note that mCherry fluorescence is weaker in MTZ-treated animals because the neurons are undergoing apoptosis. Leftmost panels show schematic brain diagrams with npy expression domains colored as in Figure S1, and boxes indicate exemplar regions shown in the fluorescent images. a, anterior; p, posterior. Scale bar: 10 μm.