Fig. 4

Melatonin Is Required for Circadian Regulation of Sleep and May Promote Sleep via Adenosine Signaling

(A–C) Larvae were raised in LD and tested in DD. aanat2^−/− larvae, but not sibling controls, lack circadian sleep oscillations. Black and hatched boxes represent subjective night and day, respectively. There is no significant difference in amount of sleep for aanat2^−/− larvae during each subjective day (B) and night (C) (p > 0.22 by Tukey’s test). (D–G) Larvae were treated with DMSO vehicle or 50 μM NECA on day 6. On night 6, vehicle-treated aanat2^−/− larvae are more active (D and F) and sleep less (E and G) than vehicle-treated aanat2^+/− larvae. In contrast, NECA-treated aanat2^−/− larvae exhibit the same amount of activity (D and F) and sleep (E and G) as NECA-treated aanat2^+/− larvae. (H–K) Larvae were treated with vehicle control or 10 μM pyrilamine on day 6. Pyrilamine decreases locomotor activity (H and J) and increases sleep (I and K) for aanat2^−/− and aanat2^+/− larvae to a similar extent. Bar graphs represent mean  ± SEM. n, number of larvae. p < 0.01 compared to aanat2^−/− (B and C), aanat2^−/− DMSO (F and G), or for the indicated comparisons (J and K) by Dunnett’s (B, C, F, and G) or Tukey’s (J and K) test. Arrows indicate transient artifacts due to pipetting of compounds. See also Figure S4.