Flies lacking <i>inc</i> or <i>Cul3</i> are hyper-arousable.

<p>(A) Schematic shows arousal threshold paradigm. At ZT16, flies were rotated off horizontal and back, and the number of sleeping flies to wake up within 5 min were counted (see methods). (B) Graph shows arousal threshold (percent flies awoken by a rotational stimulus 2 h after lights-off) in <i>inc^f00285</i> (red), <i>inc^mw</i> (dark blue), and <i>iso31</i> (grey). (C) Graph shows arousal threshold in <i>elav-Gal4;Cul3-RNAi</i> (purple), <i>elav-Gal4</i> heterozygotes (dark grey), and <i>Cul3-RNAi</i> heterozygotes (light grey). n>55 sleeping male flies for all genotypes. Error bars are SEM. * p<0.01.</p

<em>Cul3</em> and the BTB Adaptor <em>Insomniac</em> Are Key Regulators of Sleep Homeostasis and a Dopamine Arousal Pathway in Drosophila

<div><p>Sleep is homeostatically regulated, such that sleep drive reflects the duration of prior wakefulness. However, despite the discovery of genes important for sleep, a coherent molecular model for sleep homeostasis has yet to emerge. To better understand the function and regulation of sleep, we employed a reverse-genetics approach in <em>Drosophila</em>. An insertion in the BTB domain protein <em>CG32810/insomniac</em> (<em>inc</em>) exhibited one of the strongest baseline sleep phenotypes thus far observed, a ∼10 h sleep reduction. Importantly, this is coupled to a reduced homeostatic response to sleep deprivation, consistent with a disrupted sleep homeostat. Knockdown of the INC-interacting protein, the E3 ubiquitin ligase <em>Cul3</em>, results in reduced sleep duration, consolidation, and homeostasis, suggesting an important role for protein turnover in mediating INC effects. Interestingly, <em>inc</em> and <em>Cul3</em> expression in post-mitotic neurons during development contributes to their adult sleep functions. Similar to flies with increased dopaminergic signaling, loss of <em>inc</em> and <em>Cul3</em> result in hyper-arousability to a mechanical stimulus in adult flies. Furthermore, the <em>inc</em> sleep duration phenotype can be rescued by pharmacological inhibition of tyrosine hydroxylase, the rate-limiting enzyme for dopamine biosynthesis. Taken together, these results establish <em>inc</em> and <em>Cul3</em> as important new players in setting the sleep homeostat and a dopaminergic arousal pathway in <em>Drosophila</em>.</p> </div

The INC-interactor CUL3 is necessary for proper sleep behavior.

<p>(A) Western blot shows results of an α-V5 immunoprecipitation from S2 cell supernatant transfected with <i>Cul3-V5</i>, <i>inc-HA</i>, and/or <i>inc-V5</i>, and blotted with α-HA to detect INC-HA. INC interacts with both CUL3 and itself under these conditions. Sleep duration (B), latency to sleep after lights-off (C), and average sleep bout length (ABL) (D) with <i>Cul3-RNAi</i> knockdown and <i>UAS-Cul3</i> rescue in post-mitotic neurons in males (n>26 male flies for all conditions). (E) Pan-neuronal knockdown with <i>Cul3-RNAi</i> results in reduced <i>Cul3</i> transcript levels as compared with heterozygous controls (n = 3×20 male heads/genotype). (F) Sleep duration and (G) ABL in <i>30Y-Gal4</i> knockdown of <i>inc</i> and <i>Cul3</i>. (H) Graph shows the percent change in sleep (%Δsleep) after 12 h of mechanical sleep deprivation (red), 6 h of sleep recovery (light blue), and 9 h of sleep recovery (dark blue) in <i>elav-Gal4;;UAS-dcr2/Cul3-RNAi</i> (n = 76 females), <i>elav-Gal4;;UAS-dcr2/+</i> (n = 31 females), and <i>Cul3-RNAi/+</i> (n = 54 females) showing that the homeostatic response to sleep loss is disrupted in flies lacking <i>Cul3</i>. Error bars are SEM. ** p<0.01, * p<0.05 with Student's t test.</p

A reverse-genetics screen identifies novel sleep genes.

<p>(A) Flow chart shows gene selection at each stage of the screen. (B) The sleep duration and (C) average sleep bout length (ABL) distributions from the reverse-genetics screen.</p

3IY rescues sleep homeostasis defects in <i>Cul3-RNAi</i> and <i>inc^f00285</i> but not <i>inc^mw</i>.

<p>The graph shows the percent change in sleep (%Δsleep) after 12 h of mechanical sleep deprivation (red), 6 h of sleep recovery (light blue), and 9 h of sleep recovery (dark blue) in <i>iso31</i>, <i>elav-Gal4;Cul3-RNAi/UAS-dcr2 (Cul3-RNAi)</i>, <i>inc^f00285</i>, and <i>inc^mw</i> with 2 mg/mL 3IY (light green), 10 mg/mL 3IY (dark green) or no 3IY. n≥41 females for all conditions. * p<0.05. Error bars are SEM.</p

<i>inc</i> effects on sleep require a dopamine sleep-regulatory pathway.

<p>(A) Graph shows sleep duration in <i>inc^f00285;DAT^fmn</i> (tan), <i>inc^mw;DAT^fmn</i> (tan), <i>inc^f00285</i> (red), <i>inc^mw</i> (dark blue), <i>DAT^fmn</i> (light purple), and <i>iso31</i> (grey) males showing a non-additive effect on sleep duration between <i>inc</i> and <i>DAT^fmn</i> (n>38 males for all genotypes). (B) Schematic shows the dopamine synthesis pathway. <i>3-iodo-tyrosine</i> (3IY) and <i>α-methyl-p-tyrosine methyl ester</i> (AMPT) are inhibitors of <i>tyrosine hydroxylase</i> (TH). (C–D) Graphs show sleep duration in <i>inc^f00285</i> (red), <i>inc^mw</i> (dark blue), <i>DAT^fmn</i> (light purple), and <i>elav-Gal4;UAS-Cul3-RNAi/UAS-dcr2</i> (<i>Cul3-RNAi</i>, dark purple) after consumption of food laced with TH antagonists 2 mg/mL 3IY (<b>C;</b> green) or 1 mg/mL AMPT (D; brown; n≥31 males for all conditions). (E–F) Graphs show effects of 2 mg/mL 3IY (green), 1 mg/mL AMPT (brown), 2 mg/mL L-DOPA (yellow), and 5 mg/mL L-DOPA (orange) on sleep duration in <i>iso31</i> flies. L-DOPA is the product of TH, and the precursor to dopamine. A non-significant effect of 3IY or AMPT on the reduction in sleep caused by L-DOPA (blue) demonstrates pathway specificity for these TH inhibitors (n≥31 males for all conditions). Error bars are SEM. * p<0.001 with Student's t test.</p

Flies lacking <i>inc</i> exhibit reduced behavioral sleep homeostasis.

<p>The graph shows the percent change in sleep (%Δsleep) after 12 h of mechanical sleep deprivation (red), 6 h of sleep recovery (light blue), and 9 h of sleep recovery (dark blue) in <i>inc^f00285</i> (n = 45 females), <i>inc^mw</i> (n = 68 females), and iso31 (n = 41 females) showing that the homeostatic response to sleep loss is disrupted in <i>inc</i> mutants. Error bars are SEM. * p<0.05 with Student's t test.</p

Sleep architecture is altered in <i>inc</i> mutants.

<p>(A) Average sleep bout length (ABL), (B) total number of sleep bouts, (C) latency to sleep after lights-off, (D) and waking activity in <i>inc^f00285</i> (red, n = 44 males), <i>inc^f00285</i> isogenic control (WT, dark grey, n = 47 males), <i>inc^mw</i> (blue, n = 72 males), and <i>inc^mw</i> isogenic control (WT, light grey, n = 32 males). Error bars are SEM. * p<0.005 with Student's t test for all data except sleep bout length. Sleep bout length is not normally distributed; therefore, Mann-Whitney U Test was used.</p

Sleep is reduced throughout the day in <i>inc</i> mutants.

<p>(A) <i>inc</i> mRNA transcript levels are reduced in <i>inc^f00285</i> (n = 4×20 male heads/genotype). (B) Western blot shows INC protein is detectable in wild type (<i>iso31</i>), but not <i>inc^f00285</i> or <i>inc^mw</i> male heads (n = 30 male heads/genotype). (C) Sleep duration in <i>inc^f00285</i> (red, n = 44 males), <i>inc^f00285</i> isogenic control (WT, dark grey, n = 47 males), <i>inc^mw</i> (blue, n = 72 males), and <i>inc^mw</i> isogenic control (WT, light grey, n = 32 males). (D) Sleep duration per 30 min bins. Data is shown as 30 min periods averaged from 4 days 12 h∶12 h light∶dark (ZT0 is lights-on, ZT12 is lights-off) in <i>inc^f00285</i> (red, n = 88 males), <i>inc^f00285</i> isogenic control (WT, dark grey, n = 47 males), <i>inc^mw</i> (blue, n = 72 males), and <i>inc^mw</i> isogenic control (WT, light grey, n = 32 males), showing that <i>inc</i> mutant flies exhibit reduced sleep throughout a 24 h day and anticipatory locomotor activity before lights-on and –off. Error bars are SEM. * p<0.005 with Student's t test.</p