Research ArticleNEUROPHYSIOLOGY

A sleep-like state in Hydra unravels conserved sleep mechanisms during the evolutionary development of the central nervous system

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Science Advances  07 Oct 2020:
Vol. 6, no. 41, eabb9415
DOI: 10.1126/sciadv.abb9415
  • Fig. 1 A sleep-like state in Hydra.

    (A) H. vulgaris (strain 105). The white bar indicates 1 mm. (B) Two-cell layers in Hydra. Ect, ectoderm; End, endoderm. (C and D) Behavioral recording and data processing (see Materials and Methods). LED, light-emitting diode. (E) Diurnal behaviors in Hydra under LD cycles. ZT, zeitgeber time (lights-on at ZT0; lights-off at ZT12). The last feeding was >24 hours (ZT8) before loading into the imaging chamber (ZT10). Data represent mean ± SEM (n = 32). (F) Light-induced reversibility of the quiescent state. The size of pixel changes between 5-s frames, and fraction movement in 2-min bins was traced in a single animal subject to a light pulse at ZT16. (G and H) Inverse correlation of quiescent bout length and latency to the light-induced arousal at ZT16 (n = 16 to 89). Box plots range from Q1 to Q3 quartile; crosses and horizontal lines indicate mean and median values, respectively; whiskers extend to minimum or maximum values of 1.5× interquartile range. (I and J) Daily sleep profiles represent mean ± SEM (n = 32). Box plots represent sleep amount, averaged sleep bout length (ABL), and total number of sleep bouts. (K) Quantification of Hydra movements using averaged pixel change between frames (n = 32). n.s., not significant. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Aligned ranks transformation analysis of variance (ANOVA), Wilcoxon rank sum test [(H); Sleep and # Sleep bouts in (J); left in (K)]; by Kruskal-Wallis ANOVA, Dunn’s multiple comparisons test [ABL in (J)]; or by Mann-Whitney U test [right in (K)].

  • Fig. 2 Homeostasis in Hydra sleep.

    (A and B) Sleep rebound after MSD for the last 6 hours in the D phase (A, blue), but not in the L phase (B, orange), under LD cycles. Box plots represent sleep amount and latency to sleep onset after light transitions (n = 13 to 16). Yellow and blue bars indicate light and dark phases, respectively. *P < 0.05 by one-tailed Student’s t test (Sleep) or by Mann-Whitney U test (Sleep latency). (C and D) Sleep rebound after sleep deprivation by a 6-hour shift to high ambient temperature in the D phase (C, green), but not in the L phase (D, purple), under LD cycles (n = 51). Green and purple colors indicate data from sleep-deprived animals during the D and L phase, respectively. **P < 0.01, ***P < 0.001, and ****P < 0.0001 by repeated-measures ANOVA, Friedman’s test (Sleep min/last 6 hours) or by Wilcoxon matched-pairs signed rank test, one-tailed (Sleep min/12 hours). (E) Comparison of the amount of sleep loss by high ambient temperature in the D phase (green) or L phase (purple). n.s., not significant by unpaired t test with Welch’s correction, two-tailed. (F) Comparison of the amount of rebound sleep after nighttime (green) or daytime (purple) sleep deprivation. ****P < 0.0001 by Mann Whitney test, two-tailed.

  • Fig. 3 Pharmacological manipulations of Hydra sleep.

    (A) Effects of melatonin administration on daily sleep amount, ABL, L sleep latency, the number of sleep bouts, and waking activity (averaged pixel change per awake-bout frame) under LD cycles (n = 12 to 26). (B) Effects of neurotransmitter administration (100 μM) on LD sleep (n = 15 to 50). DA, dopamine; GABA, γ-aminobutyric acid; H, histamine; 5-HT, serotonin; OA, octopamine; ACh, acetylcholine; NE, norepinephrine; Glu, glutamate. (C and D) Effects of GABA transaminase inhibitor (EOS) (n = 18 to 28) or GABA transporter inhibitor (NipA) on LD sleep (n = 9 to 21). (E and F) Effects of a DA precursor (l-DOPA) (n = 9 to 17) or a tyrosine hydroxylase inhibitor (3IY) on LD sleep (n = 14 to 26). *P < 0.05, **P < 0.01, ***P < 0.01, and ****P < 0.0001 by one-way ANOVA, Holm-Sidak’s multiple comparisons test [Sleep in (A) and (E); # Sleep bouts in (B to (D)]; by Kruskal-Wallis ANOVA, Dunn’s multiple comparisons test [Sleep in (C) and (D); ABL in (A), (B), and (E); Latency in (A) and (C) to (F); # Sleep bouts in (A) and (F); Waking activity in (A) and (C) to (E)]; by Welch’s ANOVA, Dunnett’s T3 multiple comparisons test [# Sleep bouts in (E)]; or by Aligned ranks transformation ANOVA, Wilcoxon rank sum test [Sleep in (B) and (F); ABL in (C), (D), and (F); Latency and Waking activity in (B)].

  • Fig. 4 Genetic mechanisms underlying Hydra sleep.

    (A) Microarray analysis of differentially expressed genes (DEGs) in mechanically sleep-deprived Hydra (MSD). Mean and SD were set to 0 and 1, respectively, and expression levels were normalized accordingly. (B) Pairwise comparison of log2-transformed fold changes (FCs) in gene expression by MSD (88 genes, correlation coefficient R = 0.92). Data represent mean ± SEM (n = 2 for microarray; n = 3 for quantitative PCR). (C) Diagram of the DEG homologs. The numbers of homologous genes were indicated. (D) Whole-mount in situ hybridization of Hydra PRKG1. A sense probe in the thumbnail served as a negative control. White bars indicate 250 μm (left) and 100 μm (right), respectively. (E and F) Effects of a PRKG1 inhibitor (KT5823) (n = 25 to 66) or a PRKG1 activator (8-pCPT-cGMP) (n = 9 to 26) on LD sleep. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by one-way ANOVA, Holm-Sidak’s multiple comparisons test [Sleep and # Sleep bouts in (F)]; by Kruskal-Wallis ANOVA, Dunn’s multiple comparisons test [Latency in (E) and (F); Waking activity in (E)]; or by Aligned ranks transformation ANOVA, Wilcoxon rank sum test [Sleep and # Sleep bouts in (E); ABL in (E) and (F)].

  • Fig. 5 Sleep regulation by ornithine metabolism.

    (A) Effects of the pan-neuronal depletion of individual DEGs on daily sleep amount in Drosophila (n = 15 to 219). Gene names in black/bold or gray colors indicate RNA interference (RNAi) lines that display significant sleep phenotypes compared to both transgenic controls (Gal4 control and RNAi control) or to only either one of the two, respectively. (B) Biochemical pathways of the urea cycle. (C and D) Effects of dietary ornithine on Drosophila sleep in LD cycles (n = 36 to 52). Raw data were collected individually from mated female, virgin female, and male flies. Yellow and blue colors indicate light and dark phases, respectively. *P < 0.05, **P < 0.01, and ***P < 0.001 by Kruskal-Wallis ANOVA, Dunn’s multiple comparisons test (sleep amount); by Mann-Whitney U test (latency in female and virgin); or by Welch’s t test (latency in male). (E) Whole-mount in situ hybridization of Hydra OAT. A sense probe in the thumbnail served as a negative control. White bars indicate 250 μm (left) and 100 μm (right), respectively. (F and G) Effects of ornithine administration (n = 10 to 33) or an OAT inhibitor (L-canaline) (n = 19 to 30) on Hydra sleep in LD cycles. *P < 0.05, **P < 0.01, and ***P < 0.001 by Kruskal-Wallis ANOVA, Dunn’s multiple comparisons test [Sleep and # Sleep bouts in (G); ABL and Latency in (F) and (G); Waking activity in (F)] or by Aligned ranks transformation ANOVA, Wilcoxon rank sum test [Sleep in (F); Waking activity in (G)].

  • Fig. 6 Attenuation of cell proliferation by sleep deprivation in Hydra.

    (A and B) Experimental scheme of cell proliferation assay in sleep-deprived Hydra. Mechanical or pharmacological sleep deprivation was applied for 36 hours before immunostaining. BrdU incorporation was quantified from each body part. (C) Representative images of BrdU and propidium iodide (PI) labeling. The white bar indicates 50 μm. HCS, Hydra culture solution (control for MSD and 3IY); MSD, mechanical sleep deprivation; 3IY, 3-iodo-tyrosine (a tyrosine hydroxylase inhibitor; 3 mM); DMSO, dimethyl sulfoxide (vehicle control for KT5823, 0.1%); KT5823, a PRKG1 inhibitor (3 μM). (D) Quantitative analyses of BrdU incorporation. Fluorescence signals from anti-BrdU staining were quantified from six regions of interest per animal (n = 5 to 6 animals per condition). Data were normalized to each control group. **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Kruskal-Wallis ANOVA, Dunn’s multiple comparisons test (upper body), Aligned ranks transformation ANOVA, Wilcoxon rank sum test (the others). ††P < 0.01 and ††††P < 0.0001 by Mann-Whitney U test.

Supplementary Materials

  • Supplementary Materials

    A sleep-like state in Hydra unravels conserved sleep mechanisms during the evolutionary development of the central nervous system

    Hiroyuki J. Kanaya, Sungeon Park, Ji-hyung Kim, Junko Kusumi, Sofian Krenenou, Etsuko Sawatari, Aya Sato, Jongbin Lee, Hyunwoo Bang, Yoshitaka Kobayakawa, Chunghun Lim, Taichi Q. Itoh

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