Neural signatures of sleep in zebrafish

Abstract

Slow-wave sleep and rapid eye movement (or paradoxical) sleep have been found in mammals, birds and lizards, but it is unclear whether these neuronal signatures are found in non-amniotic vertebrates. Here we develop non-invasive fluorescence-based polysomnography for zebrafish, and show-using unbiased, brain-wide activity recording coupled with assessment of eye movement, muscle dynamics and heart rate-that there are at least two major sleep signatures in zebrafish. These signatures, which we term slow bursting sleep and propagating wave sleep, share commonalities with those of slow-wave sleep and paradoxical or rapid eye movement sleep, respectively. Further, we find that melanin-concentrating hormone signalling (which is involved in mammalian sleep) also regulates propagating wave sleep signatures and the overall amount of sleep in zebrafish, probably via activation of ependymal cells. These observations suggest that common neural signatures of sleep may have emerged in the vertebrate brain over 450 million years ago.

Extended Data Fig. 1 |. Sleep deprivation by the continuous-swim paradigm.

a, Left, evolutionary tree depicting current description of SWS and PS/REM sleep in amniotes. Right, schematic showing lateral view of major brain regions and neurochemistry that is implicated in sleep. Ob, olfactory bulbs; Am, amygdala; Hi, hippocampus; Te, telencephalon; Mi, midbrain; Cb, cerebellum; Md, medulla. b, Schematic showing the sleep deprivation (SD) setup (see Methods). c, d, Behavioural actimetry verifies that the sleep rebound induced by sleep deprivation causes decreasing wake and increasing sleep amounts as a function of number of days of sleep deprivation. Trial-averaged traces across 24 h after zebrafish were released following 1 day (n = 11, purple (c) or red (d)), 2 days (n = 11, blue (c) or orange (d)) or 3 days (n = 11, green (c) or yellow (d)) of sleep deprivation, against non-sleep-deprived sibling controls (n = 12, black). Mean activity is expressed as seconds of activity per 10-min bin (bold line) ± s.e.m. (shaded), per condition. Mean sleep amount is expressed as minutes of sleep per 10-min bin (bold) ± s.e.m. (shaded), per condition. The black triangle depicts the day-to-night transition, the white triangle depicts the night-to-day transition and the orange triangle shows the time of feeding. Red box in c indicates the equivalent time at whichCa²⁺ imaging was conducted. e, Box plots showing the spread of total amounts of activity and sleep between non-sleep-deprived and sleep-deprived conditions, separated out by day and night. Blue lines denote median and black circles present the mean value for the group. *P < 0.05, one-way ANOVA with 0.05 alpha level and Tukey’s post hoc multi-comparison testing. f, Scatter plot of average ITI from desynchronized and synchronized brain activities. *P < 0.05, two-sided Wilcoxon rank-sum test, n = 10 fish per condition. g, Bar chart showing proportion of samples that displayed SBS within 2 h of release from non-sleep-deprived, 1-day, 2-day and 3-day sleep deprived conditions.

Extended Data Fig. 2 |. Hypnotics produce distinct neural dynamics, including synchronous activity, travelling waves and broad silencing.

a–e, Left, representative activity from 3-h recordings in which drugs were added after 1 h (red triangle) to freely swimming zebrafish. Activity traces are expressed as the number of seconds of activity per 10-min time bin, and plotted as mean (solid line) ± s.e.m. (shaded) per dose of promethazine (a, n = 24 fish), MS222 (b, n = 24 fish), gaboxadol (c, n = 24 fish), zolpidem (d, n = 24 fish) and eserine (e, n = 24 fish) from low to high doses (black < purple < blue < green; see Supplementary Table 1 for concentrations), showing the induction of dose-responsive behavioural sleep. Middle, representative time-aligned ΔF/F traces of20 randomly selected dorsal pallium neurons (red triangle indicates the addition of drug after 10 min). Promethazine induced mild synchronization of dorsal pallium neurons, MS222 and gaboxadol induce total silencing, and zolpidem and eserine induced travelling waves. Right, neural signature across all time points represented by the first independent component (top) or replotted on two-dimensional phase space following PCA (bottom, grey lines and black lines depict wake versus sleep trajectories, respectively). See Supplementary Information for reproducibility information.

Extended Data Fig. 3 |. fPSG of SBS and PWS.

a, b, Dorsal pallium signature of wake (a) versus SBS (b) with time-aligned ΔF/F traces of 20 randomly selected masks. Neural signature plotted in two-dimensional phase space following PCA. c–e, Typical light-sheet images of a single z-slice through brain and trunk muscle during wake (c) and during SBS (d, e). Scale bar, 100 μm. f, g, Dorsal pallium signature of wake (f) versus PWS (g) with time-aligned ΔF/F traces of the 20 masks depicted in the left image. Pre- versus post-PMT-wave synchronicity indices: ITI, 2.88 ± 2.14 s versus 2.96 ± 1.35 s, P = 0.792, Wilcoxon rank-sum test, n = 5 fish; coherence index, 1.32% (n = 1,024 spikes) versus 1.08% (n = 1,065 spikes); P > 0.05, χ² test. Neural signature plotted in two-dimensional phase space following PCA. Scale bars, 50 μm. h, Subpanels h1–h6 show selected frames from a typical fPSG volume acquisition of PWS in which z-planes have been compressed into a maximum projection. Frames depict preceding events up until, and through, the PMT-wave transition to sleep. Arrow in h1 indicates muscle rostrocaudal wave; arrows in h2 indicates coordinated ependymal cell activation; arrow in h3 indicates anterior hindbrain activation; arrow in h4 indicates wave initiation; arrow in h6 indicates propagation to the dorsal pallium (see Supplementary Information for detailed description). Scale bar, 100 μm. See Supplementary Information for reproducibility information.

Extended Data Fig. 4 |. Heart rate analysis.

a–c, Heart rate analysis before and after carbachol treatment (a) (*P < 0.05, two-sided paired t-test, n = 4 fish), mepyramine treatment (b) (*P < 0.05; two-sided paired t-test, n = 4 fish) and with or without sleep deprivation (c) (*P < 0.05; two-sided Student’s t-test, n = 7 non-sleep-deprived and 7 sleep-deprived fish). d–f, Histograms comparing the distribution of interbeat interval before (black bars) and after (red bars) treatment with carbachol (d)or mepyramine (e), and with (red bars) or without (black bars) sleep deprivation (f). Three representative histograms are shown for each condition. g, Representative images from heart rate capture showing ventricular contraction and the drawing of a mask (red) to measure fluorescence changes from the green fluorescent heartbeat. h, An illustrative plot of fluorescence variation measured by the mask, overlaid with the result of running peak analysis (red triangles) from which the heart rate and interbeat interval are derived.

Extended Data Fig. 5 |. Brain-wide imaging with additional hypnotics.

a, Brain-wide single-plane imaging of promethazine-stimulated zebrafish. Promethazine induces bilaterally synchronous burst dynamics in the dorsal pallium (red bar). Left, masks used to extract Ca²⁺ transient traces shown in the middle panel. Dynamics are summarized by PCA (right). b, Brain-wide single-plane imaging of eserine-stimulated zebrafish. Eserine induces PMT waves. Left, masks used to extract Ca²⁺ transient traces shown in the middle panel. Dynamics are summarized by PCA (right). c, Brain-wide single-plane imaging of carbachol-stimulated zebrafish pre-incubated with methoctramine. Methoctramine prevents carbachol-induction of the PMT wave. Left, masks used to extract Ca²⁺ transient traces shown in the middle panel. Dynamics are summarized by PCA (right). d, Brain-wide single-plane imaging of eserine-stimulated zebrafish pre-incubated with methoctramine. Methoctramine prevents the eserine-induced PMT dynamics. Left, masks used to extract Ca²⁺ transient traces shown in the middle panel. Dynamics are summarized by PCA (right). e, Brain-wide single-plane imaging of zolpidem-stimulated zebrafish. Zolpidem induced multiple PMT waves. Left, masks used to extract Ca²⁺ transient traces shown in the middle panel. Dynamics are summarized by PCA (right). Red triangles show the point at which the drug was added. f, PCA by singular value decomposition of the fPSG PMT signature seen in Fig. 3e. Distinct steady states for wake (grey lines) and PWS sleep (black lines) can be separated, as can marked trajectories of muscle and brain travelling waves (blue lines). See Supplementary Information for reproducibility information. Scale bars, 100 μm.

Extended Data Fig. 6 |. Ependymal cell activation is tightly correlated to the PMT wave and can be induced by the neuropeptide melanin concentrating hormone.

a, Dorsal view of a single slice through the optic tectum of Et(EP:mCherry) fish. Inset dashed box indicates location along the anteroposterior axis. Scale bar, 50 μm. b, Slice through the optic tectum of a 7-dpf Et(EP:mCherry);Tg(α-tubulin:gal4;UAS:GFP) triple transgenic fish. Arrows, mature ependymal cells (magenta) co-expressing α-tubulin (green). c, Slice through the optic tectum of a 7-dpf Et(EP:mCherry);Tg(GFAP:gal4;UAS:GFP) triple transgenic fish. Mature ependymal cells largely do not overlap with GFAP-positive cells. d–g, Ventricular cells activated before the PMT wave are EP:mCherry-positive ependymal cells. Confocal z-slice through a 7-dpf zPSG;EP:mCherry double transgenic fish before stimulation (d), ependymal cell activation (e) and PMT wave (f). g, Mean ± s.e.m. ΔF/F trace of five randomly selected double-positive cells showing the transient activation of ependymal cells before the PMT wave. Arrows indicate the time from which images shown in d–f are taken. Scale bar, 50 μm. h–m, MCH peptide induces activation of ependymal cells. Representative maximum projection through SPIM volume acquisition before (h, j) and 5 min after intracerebroventricular injection with carrier (i) or MCH peptide (k). l, Mean normalized fluorescence between carrier (n = 9 fish) and MCH (n = 12 fish) groups from the peritectoventricular zone (dashed lines) before and after injection. m, Box plot of normalized fluorescence percentage change values between carrier- and MCH-injected groups with black circle depicting mean. *P < 0.05; two-sided Student’s t-test. Scale bar, 100 μm. n, Pigment contraction is tightly coupled to discrete activations during PMT-wave dynamics. Representative frames selected from a single z-recording of a pigmented zebrafish during a PMT wave; (1) colour-coded selection of all pigments covering the optic tectum as a representative sample; (2) during darkness and baseline activity, pigments are spread out and cover more pixel area; (3, 4) pigments contract with ependymal cell activation and the PMT wave; (5) when the wave abates, pigments re-spread. Scale bar, 100 μm. o, Quantification of pixel area plotted with representative colour from subpanel (1) in n at the different time points shown in subpanels (1) to (5) in n. *P < 0.05, repeated-measures ANOVA, n = 10 pigments. See Supplementary Information for pigment dynamics description and reproducibility information.

Extended Data Fig. 7 |. Characterization of GFAP:epNTR and EP-mCherry transgenic fish.

a, Dorsal view of maximum projection of a 7-dpf Tg(GFAP:epNTR-tagRFPT) zebrafish used to ablate GFAP-positive cells. Scale bar, 100 μm. b, Maximum projection of a 7-dpf Et(EP:mCherry) zebrafish expressing mCherry in putative ependymal cells. Scale bar, 50 μm. c, Single z-slice of dorsal pallium of Et(EP:mCherry) fish. mCherry cells located on the ventricular surface of the telencephalic neuroepithelium and present in the midline. Scale bar, 50 μm. d, Single z-slice of optic tectum parenchyma of Et(EP:mCherry) fish. mCherry cells are located all along the ventricular surface comprising the ependyma. Long thin processes invade the mesencephalic parenchyma, which suggests the morphology of non-neuronal glial cells and (in particular) the specialized ependymal cell type, tanycytes. e, Single z-slice of hindbrain of Et(EP:mCherry) fish. mCherry cells are located all along the midline ventricular surface, representing the ependyma stretching to spinal cord central canal. Long thin processes suggest the morphology of non-neuronal cells. f, g, Actimetry shows GFAP-cell-ablated fish are irreversibly immobile without recovering activity during daytime. Mean activity is expressed as seconds of activity per 10-min bin (bold) ± s.e.m. (shaded), per condition. Mean sleep amount is expressed as minutes of sleep per 10-min bin (bold) ± s.e.m. (shaded), per condition. The black triangle depicts the day-to-night transition, the white triangle depicts the night-to-day transition and the orange triangle shows the time of feeding. Box plots showing total activity and sleep for sibling controls versus GFAP-cell-ablated larva. Blue line denotes median and black circles indicate the mean value (g). *P < 0.05, two-sided Student’s t-test, n = 18 sibling control and 18 GFAP:epNTR fish from two independent experiments. h, Photograph at 4× magnification of a restrained 7-dpf zebrafish receiving an intracerebroventricular injection of MCH solution with phenol red solution (pale red colour in the photograph). The ventricle is slightly expanded as a result of injection. Scale bar, 500 μm. i, j, Behavioural actimetry shows MCH-injected larvae are less mobile and sleep more during the first day and night after injection, but mostly recover by the second day. Box plots showing total activity and sleep for carrier- versus MCH-injected larva. Blue line denotes median and black asterisks indicate the mean value (j). P > 0.05, two-sided Student’s t-test, n = 6 carrier- and 6 MCH-injected fish.

Extended Data Fig. 8 |. Characterization of MCH:epNTR, H6408 and mchr1a mchr1b double mutants.

a, Schematic of the location of MCH-positive cells in the lateral hypothalamus of the zebrafish larvae. b, Confocal maximum projection shows MCH cells from the Tg(−5kbMCH:epNTR-tagRFPT) transgenic fish before treatment with the ablation agent metronidazole (MTZ). Faint signal of projections to the pituitary are seen (dashed region and asterisk). Scale bar, 50 μm. c, MCH cells are ablated in Tg(−5kbMCH:epNTR-tagRFPT) larva treated with metronidazole. d–f, Tg(−5kbMCH:epNTR-tagRFPT) larva stained with MCH antibody show lateral hypothalamic location and projection to the pituitary gland (d) (green) co-localizing to tagRFP-T expression(e, magenta; f, white). g, Alignment of wild-type MCHR1a protein sequence and predicted truncated mutant protein resulting from CRISPR deletion. h, Sequencing confirmation of loss of TATG sequence from exon 2 of mchr1a gene from genotyping of homozygous MCHR1a mutant. i, Alignment of wild-type MCHR1b protein sequence and predicted truncated mutant protein resulting from TILLING mutant with early stop codon. j, Sequencing confirmation of TGG to TGA substitution from exon 1 of mchr1b gene from genotyping of homozygous MCHR1b mutant. k–m, MCHR-antagonist (H6408)-treated fish display increased activity and disrupted onset of ependymal cell and PMT-wave dynamics. k, Top, timeline of experiment and addition of pharmacological agents during the 2-h Ca²⁺ imaging experiment (red triangle indicates the addition of carbachol). Bottom, proportion of activation of ependymal cells (open circle) and/or PMT wave (filled circle), witnessed in the imaging session between carrier control (n = 8 fish) and H6408 (n = 7 fish) is plotted against the mean latency to activation (in min) ± s.e.m. *P < 0.05 χ² test. l, Top, actimetry of wild-type zebrafish (n = 20) treated with carrier versus MCHR antagonist (H6408; n = 20) in 10-min bins over a 24-h period, showing mean seconds of activity per bin (bold) ± s.e.m. (shaded). Bottom, sleep depicted as minutes of sleep per 10-min bin (bold) ± s.e.m. across a 24-h period. Black triangle depicts the day-to-night transition, white triangle depicts the night-to-day transition and the orange triangle shows the time of feeding. m, Box plots showing spread of total activities and sleep between between carrier (E3)-stimulated versus H6408-treated groups, separated further by day and night. Blue lines denote median and black circles indicate the mean value. *P < 0.05, two-sided Student’s t-test. See Supplementary Information for reproducibility information.

Extended Data Fig. 9 |. fPSG comparison of induced SBS in non-sleep-deprived and sleep-deprived fish.

a, b, Representative fPSG recording of non-sleep deprived (a) and sleep-deprived zebrafish (b) during normal night phase. Activity traces from fEOG (top), fEEG (middle) and fEMG (middle). fEEG and fEMG are ΔF/F traces from 14 masks covering broad brain regions (as labelled), with integration of neuronal activity across 1-s bins. Heart rate was measured immediately before imaging, and interbeat interval distribution is shown with coefficient of variation (CoV) (bottom). Transient synchronous bursting was detected (red boxes for example) between infrequent muscle twitches and broad brain arousal events. See Supplementary Information for reproducibility information.

Extended Data Fig. 10 |. SPIM of wake-maintained larvae reveal SBS and PWS signatures during normal night.

a, Schematic of wake-maintenance apparatus adopting randomized mechano-acoustical stimulus during the day phase to consolidate wake, and thus sleep in the night phase for imaging. b, Representative actimetry of wild-type zebrafish not treated with the stimulus (n = 12 fish) with actimetry of wild-type fish treated with the stimulus (n = 12 fish) in 10-min bins over a 24-h period, showing mean seconds of activity per bin (bold) ± s.e.m. (shaded). Bottom, sleep depicted as minutes of sleep per 10-min bin (bold) ± s.e.m. (shaded) across the 24-h period. The black triangle depicts the day-tonight transition, the white triangle depicts the night-to-day transition and the orange triangle shows the time of feeding. c, d, fPSG recording of SBS (c) and PWS (d) during normal night phase. Red box shows transient synchrony in dorsal pallium, indicative of SBS. Activity traces from fEOG (top), fEEG (middle) and fEMG (bottom). fEEG and fEMG are ΔF/F traces from 14 masks covering broad brain regions (as labelled), with integration of neuronal activity across 1-s bins. See Supplementary Information for reproducibility information.

Extended Data Fig. 11 |. SPIM imaging reveals spontaneous SBS and PWS during the night phase.

a, Schematic of protocol to sham mount versus extended agarose mount for 1–3 days to test the degree of wake stimulation resulting from agarose restraint. b, Behavioural actimetry shows that while initially agarose-mounted animals display sleep behaviour upon release, sleep–wake cycles return to normal; this indicates that long-term restraint is not traumatic or lethal to zebrafish larvae. Actimetry of sham-mounted (n = 36 fish) versus mounted (n = 31 fish) wild-type zebrafish, in 10-min bins over a 24-h period showing mean seconds of activity per bin (bold) ± s.e.m. (shaded). Bottom, sleep depicted as minutes of sleep per 10-min bin (bold) ± s.e.m. (shaded), across the 24-h period. The black triangles depict the day-to-night transition, the white triangles depict the night-to-day transition and the orange triangle shows the time of feeding. c, d, fPSG recording of SBS(c) and PWS (d) during normal night phase. Red box shows transient synchrony in dorsal pallium, indicative of SBS between muscle twitches. Activity traces from fEOG (top), fEEG (middle) and fEMG (bottom). fEEG and fEMG are ΔF/F traces from 14 masks covering broad brain regions (as labelled), with integration of neuronal activity across 1-s bins. See Supplementary Information for reproducibility information.

Fig. 1 |. Sleep deprivation produces slow bursting activity in the zebrafish dorsal pallium.

a, Dorsal view of a brain from Tg(a:nk;5×UAS:GCaMP6F-p2a-nls-mCherry) (see Methods for details of zebrafish lines) zebrafish at seven days post-fertilization (only GCaMP is shown), superimposed on its body plan (transmitted light). Orange ovals depict anterolateral dorsal pallium, at which high spontaneous awake activity occurs. Scale bar, 100 μm. A, anterior; L, left; R, right; P, posterior. b, Single z-slice image of the telencephalic region (GCaMP (green) and nuclear-localized mCherry (red)) from dashed box in a, at the level of the neuromast fiduciary marker (white arrowhead). Scale bar, 50 μm. c, d, Left, representative images showing 20 masks on non-sleep-deprived (c) or sleep-deprived (d) brains. Right, magnified clips of Ca²⁺ imaging in the dorsal pallium (DP) from the region in dashed box in the leftmost panel. Scale bars, 50 μm. e, f, Time-aligned ΔF/F traces of the 20 neurons from non-sleep-deprived fish (e) marked in c and sleep-deprived fish(f) marked in d. Desynchronous versus synchronous indices, ITI: 3.84 ± 0.14 s versus 12.26 ± 2.91 s; P = 0.00018, Wilcoxon rank-sum test n = 10 fish per condition; coherence index, 6.32% (n = 40,175 spikes) versus 18.84% (n = 41,161 spikes); P < 0.05, χ² test.

Fig. 2 |. Hypnotics induce heterogeneous effects on telencephalic activity.

a, Left, schematic of experimental pipeline. Compounds are first validated as hypnotic by screening freely behaving fish, before being applied to agar-restrained fish for Ca²⁺ imaging. Middle, time-aligned ΔF/F traces of 20 randomly selected dorsal pallium neurons from a carrier-stimulated fish (red triangle indicates addition after 10 min). Right, neural signature across all time points represented by the first independent component (IC) (top) or replotted following principal components analysis (PCA) (bottom). LH, left hemisphere; RH, right hemisphere. b, c, Left, three-hour actimetry recordings, in which a range of drug dosages were added after one hour (red triangle). Activity traces are expressed as the number of seconds active per ten-minute time bin, and plotted as trial-averaged mean (solid line) ± s.e.m. (shaded) per dose of mepyramine (b, n = 24 fish), and carbachol (c, n = 24 fish) from low to high (black < purple < blue < green; see Supplementary Table 1 for concentrations), showing induction of dose-responsive behavioural sleep. Middle and right plots show representative time-aligned ΔF/F traces, independent component analysis and PCA (grey and black lines depict wake and sleep dynamics, respectively) for respective drugs. Wake versus mepyramine SBS synchronicity indices: ITI, 4.42 ± 0.38 s versus 16.55 ± 6.79 s, P = 0.041, Wilcoxon rank-sum test, n = 6fish and coherence index 6.06% (n = 5,177 spikes) versus 31.18% (n = 7,215 spikes), P < 0.00001, χ² test. See Supplementary Information for reproducibility information.

Fig. 3 |. Fluorescence-based polysomnography reveals SBS and PWS in zebrafish.

a, Schematic diagram of the light-sheet microscope platform (based on the openSPIM design; see ‘fPSG’ in ‘Sample preparation and Ca²⁺ imaging’ in Methods) used to take time-lapse recordings of synchronous x–y frames as single slices or volumes customized with temperature control and drug perfusion apparatus. b, Single z-plane (dorsal view) of a zPSG transgenic fish at seven days post-fertilization, co-expressing a trunk muscle gal4 and pan-neuronal gal4 used to drive expression of UAS:GCaMP7a. Broad brain regions are indicated as telencephalon (Tel), optic tectum (TeO), rhombencephalon (Rh) and spinal cord (Sc). Scale bar, 100 μm. c, Depiction of parameters that can be measured, based on fluorescent markers of zPSG fish that form an fPSG. d, e, Left, broad fPSG region-of-interest masks on a maximum projection image to measure GCaMP activity before, during and after initiation of SBS (d) or PWS (e). Middle, Brain and muscle activity traces (ΔF/F) from broad regions defined by masks (middle panels show fEEG and bottom panels show fEMG), with integration of activity across 1 s (d) or 2.5 s (e) bins. Asterisks denote muscle contractions. Time trace of eye movements (fEOG) is shown in the top panels. Right, heart rate (fECG), measured before and after recording of SBS (d) or PMT wave (e) (top), and distribution of interbeat intervals (bottom). *P < 0.05. Two-sided paired t-test, n = 4 fish per condition. A–m, anteromedial; di., distal; NL, neuronal layer; prox., proximal.

Fig. 4 |. MCH signalling is required for sleep and generation of PWS dynamics.

a–f, MCH2 neuron-ablated (a–c) and MCHR mutant (d–f) fish display increased activity, decreased sleep and disrupted onset of ependymal cell and PMT-wave dynamics. a, d, Top, timeline of a two-hour Ca²⁺ imaging experiment with addition of the PWS inducer (red triangle). Bottom, proportion of sibling (sib.)-treated control (n = 10 fish) versus MCH2-ablated (n = 14 fish) groups (a) or mchr1a^+/+mchr1b^+/+ (mchr1a/b^+/+) controls (n = 10 fish) versus mchr1a^−/−mchr1b^−/− (mchr1a/b^−/−) double mutants (n = 6 fish) (d) with ependymal (open circle) and/or PMT-wave (filled circle) activation within the imaging session, plotted against the mean latency to activation (min) ± s.e.m. *P < 0.05 χ² test. b, e, Top, actimetry of sibling-treated control (n = 42) versus MCH2-neuron-ablated (n = 44) fish (b) or mchr1a^+/+mchr1b^+/+ (n = 67) versus mchr1a^−/−mchr1b^−/− double mutant (n = 72) fish (e) in 10-min bins over a 24-h period, showing mean seconds of activity per bin (bold) ± s.e.m. (shaded). Bottom, sleep depicted as minutes per 10-min bin ± s.e.m. (shaded). Black or white triangles indicate day-to-night or night-to-day transitions, respectively, and orange triangles show time of feeding. c, f, Box plots showing spread of total activities and sleep between sibling-treated control versus MCH2-neuron-ablated (c) or mchr1a^+/+mchr1b^+/+ versus mchr1a^−/−mchr1b^−/− double (f) groups separated out by day and night. Blue lines and black circles denote the median and mean, respectively. Two-sided Student’s t-test.