Rapamycin rescued age-related night sleep fragmentation in an S6K-dependent manner.

<p>(A) Rapamycin treatment (9 d) did not significantly affect day/night activity or wakefulness, but significantly increased night sleep duration, reduced night sleep fragmentation, and increased the length of night sleep periods (<i>w^Dah</i>, age 10 d, <i>n</i> = 21/20 control/rapamycin). (B) Average activity count data (30 min bins) under 12∶12 h LD. (C) Acute rapamycin treatment of 45-d-old flies for 3 d did not significantly affect day/night activity or wakefulness, but significantly increased night sleep duration, reduced night sleep fragmentation, and increased the length of night sleep periods (<i>w^Dah</i>, <i>n</i> = 64/64). Rapamycin-mediated night sleep, bout number, and bout length were independent of (D) 4E-BP (<i>n</i> = 19/19) and (E) reduced autophagy (a = <i>da-Gal4/UAS-ATG5-RNAi</i>, <i>n</i> = 20/17 or genetic controls c1 = <i>da-Gal4/+</i>, <i>n = </i>20/21 and c2 = <i>UAS-ATG5-RNAi/+</i>, <i>n</i> = 23/19). Flies with reduced autophagy responded to rapamycin as controls in sleep (<i>p</i> = 0.81), bout number (<i>p</i> = 0.82), and night bout length (<i>p</i> = 0.42) (GLM). (F) Ubiquitous expression of constitutively active S6K blocked the rescue of night sleep fragmentation by rapamycin (c1 = <i>da-Gal4/+</i>, <i>n = </i>20/21, and c2 = <i>UAS-S6K^STDETE/+</i>, <i>n = </i>20/18, <i>S6K = da-Gal4/UAS-S6K^STDETE</i>, <i>n = </i>20/17). Flies expressing a constitutively active form of S6K significantly differed from controls in the response to rapamycin (sleep <i>p</i> = 0.01, bout number <i>p</i> = 0.03, night bout length <i>p</i> = <0.0001, GLM). Kruskal Wallis test with Dunn's multiple comparisons of selected pairs. ***<i>p</i><0.001, **<i>p</i><0.01, and *<i>p</i><0.05. Error bars represent s.e.m. Day behaviours of (D–F) are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001824#pbio.1001824.s006" target="_blank">Figure S6B–D</a>.