Effect of constant and acute light during the scotophase on GEFS+ and control sleep.

<p><b>(A)</b> Sleep profiles of control (<i>n</i> = 83) and GEFS+ (<i>n</i> = 95) flies under constant light/light (LL) exposure. <b>(B)</b> Total sleep and <b>(C)</b> sleep latency during subjective night under LD and LL conditions; Rank Sum Tests. <b>(D)</b> Sleep and <b>(E)</b> activity profiles of control (<i>n</i> = 52) and GEFS+ (<i>n</i> = 59) flies subjected to a 1 hr scotophase light pulse; repeated measures ANOVA on Ranks. <b>(F)</b> Sleep latencies of control and GEFS+ flies after normal lights off (12 hr and 36 hr) and the scotophase pulse (42 hr); ANOVA on Ranks, Dunn’s vs 12 hr control. Data presented as averages with SEM (<b>A</b>, <b>D</b> and <b>E</b>) or boxplots with means (“X”) (<b>B</b>, <b>C</b> and <b>F</b>); ***p < 0.001.</p

Pharmacologic suppression of GABA_A receptor function differentially affects sleep in control and GEFS+ flies.

<p><b>(A)</b> Sleep profiles of control (<i>n</i> = 63, 60, 62, 60) and GEFS+ (<i>n</i> = 62, 61, 63, 62) flies fed vehicle or various concentrations of CBZ starting at ZT 8 (arrow). <b>(B)</b> CBZ feeding decreased nighttime sleep; ANOVA on Ranks, Dunn’s within genotype compared to vehicle-fed flies. <b>(C)</b> The percent change of nighttime sleep normalized within genotype to vehicle-fed flies revealed that GEFS+ mutants were more resistant to CBZ as compared to control flies at each CBZ concentration; Rank Sum Tests. <b>(D)</b> CBZ feeding increased sleep latency; ANOVA on Ranks, Dunn’s within genotype compared to vehicle-fed flies. Data are presented as averages with SEM <b>(A)</b> or boxplots with means (“X”) <b>(B-D)</b>; *p < 0.05, **p < 0.01, ***p < 0.001.</p

Sleep deprivation reduces heat-induced seizure susceptibility of the GEFS+ mutant.

<p>Percentage of seizing flies for untreated and 24 hr sleep-deprived GEFS+ mutants when exposed to 40°C; two-way repeated measures ANOVA, Holm-Sidak Multiple Comparisons. Data presented as averages of three independent experiments (<i>n</i> = 16, 28, 25 for untreated and <i>n</i> = 14, 27, 27 for deprived) with SEM; ***p < 0.001.</p

Exaggerated Nighttime Sleep and Defective Sleep Homeostasis in a <i>Drosophila</i> Knock-In Model of Human Epilepsy

<div><p>Despite an established link between epilepsy and sleep behavior, it remains unclear how specific epileptogenic mutations affect sleep and subsequently influence seizure susceptibility. Recently, Sun <i>et al</i>. (2012) created a fly knock-in model of human generalized epilepsy with febrile seizures plus (GEFS+), a wide-spectrum disorder characterized by fever-associated seizing in childhood and lifelong affliction. GEFS+ flies carry a disease-causing mutation in their voltage-gated sodium channel (VGSC) gene and display semidominant heat-induced seizing, likely due to reduced GABAergic inhibitory activity at high temperature. Here, we show that at room temperature the GEFS+ mutation dominantly modifies sleep, with mutants exhibiting rapid sleep onset at dusk and increased nighttime sleep as compared to controls. These characteristics of GEFS+ sleep were observed regardless of sex, mating status, and genetic background. GEFS+ mutant sleep phenotypes were more resistant to pharmacologic reduction of GABA transmission by carbamazepine (CBZ) than controls, and were mitigated by reducing GABA_A receptor expression specifically in wake-promoting pigment dispersing factor (PDF) neurons. These findings are consistent with increased GABAergic transmission to PDF neurons being mainly responsible for the enhanced nighttime sleep of GEFS+ mutants. Additionally, analyses under other light conditions suggested that the GEFS+ mutation led to reduced buffering of behavioral responses to light on and off stimuli, which contributed to characteristic GEFS+ sleep phenotypes. We further found that GEFS+ mutants had normal circadian rhythms in free-running dark conditions. Interestingly, the mutants lacked a homeostatic rebound following mechanical sleep deprivation, and whereas deprivation treatment increased heat-induced seizure susceptibility in control flies, it unexpectedly reduced seizure activity in GEFS+ mutants. Our study has revealed the sleep architecture of a <i>Drosophila</i> VGSC mutant that harbors a human GEFS+ mutation, and provided unique insight into the relationship between sleep and epilepsy.</p></div

GEFS+ mutation affects sleep/wake behavior.

<p><b>(A)</b> The 24 hr activity profiles, <b>(B)</b> 12 hr LD activity counts, and <b>(C)</b> 24 hr sleep profiles of virgin females (☿), mated females (♀), and males (♂) for knock-in controls (<i>n</i> = 85, 93, 95) and GEFS+ mutants (<i>n</i> = 88, 87, 94). <b>(D)</b> Nighttime 12 hr sleep/activity parameters of mated females for control (<i>n</i> = 93), GEFS+ heterozygotes (<i>n</i> = 44), and GEFS+ homozygotes (<i>n</i> = 87). Data are presented as averages with SEM for <b>(A, C)</b> or boxplots with means (“X”) for <b>(B, D)</b>. ANOVA on Ranks, Dunn’s compared to control; *p < 0.05, ***p < 0.001.</p

GEFS+ mutants lack homeostatic sleep regulation.

<p><b>(A)</b> The 24 hr sleep profiles of baseline day and recovery day following 24 hr sleep deprivation in control (<i>n</i> = 81) and GEFS+ mutants (<i>n</i> = 86). <b>(B)</b> The percentage of time asleep over the 24 hr period and <b>(C)</b> subjective sleep latencies for baseline and recovery days; ANOVA on Ranks, Dunn’s compared to baseline data within genotype. <b>(D)</b> Cumulative sleep loss during 24 hr sleep deprivation and recovery. Sleep debt is presented relative to baseline sleep for each genotype. <b>(E)</b> Percent change in 24 hr sleep compared between before and after sleep deprivation; Rank Sum Test. Data presented as averages with SEM <b>(A, D)</b> or boxplot with means (“X”) <b>(B, C, E)</b>; ***p < 0.001.</p

<i>Rdl</i> GABA_A knockdown in PDF-positive neurons differentially influences sleep latency in GEFS+ mutants.

<p><b>(A, B)</b><i>Rdl</i> knockdown in PDF neurons of control and heterozygous GEFS+ mutants; +<i>/Rdl-RNAi</i> (<i>n</i> = 55), <i>pdf-GAL4/Rdl-RNAi</i> (<i>n</i> = 49), <i>GEFS+/+;</i> +<i>/Rdl-RNAi</i> (<i>n</i> = 56), <i>GEFS+/+; pdf-GAL4/Rdl-RNAi</i> (<i>n</i> = 38). <b>(A)</b><i>Rdl</i> knockdown in PDF neurons reduced sleep to the same extent in both control and GEFS+ flies. <b>(B)</b><i>Rdl</i> knockdown in PDF neurons specifically increased sleep latencies in heterozygous GEFS+ mutants (but not in control flies), restoring the GEFS+ short sleep latencies to control levels; ANOVA on Ranks, Dunn’s Multiple Comparisons. To determine the extent of change caused by <i>Rdl</i> knockdown, differences within a genotype were calculated by subtracting experimental data to the averages of RNAi only controls; Rank Sum Test. All data presented as boxplots with means (“X”); **p < 0.01, ***p < 0.001.</p