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

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

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

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

<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

A Novel Role for Ecdysone in <i>Drosophila</i> Conditioned Behavior: Linking GPCR-Mediated Non-canonical Steroid Action to cAMP Signaling in the Adult Brain

<div><p>The biological actions of steroid hormones are mediated primarily by their cognate nuclear receptors, which serve as steroid-dependent transcription factors. However, steroids can also execute their functions by modulating intracellular signaling cascades rapidly and independently of transcriptional regulation. Despite the potential significance of such “non-genomic” steroid actions, their biological roles and the underlying molecular mechanisms are not well understood, particularly with regard to their effects on behavioral regulation. The major steroid hormone in the fruit fly <i>Drosophila</i> is 20-hydroxy-ecdysone (20E), which plays a variety of pivotal roles during development via the nuclear ecdysone receptors. Here we report that DopEcR, a G-protein coupled receptor for ecdysteroids, is involved in activity- and experience-dependent plasticity of the adult central nervous system. Remarkably, a courtship memory defect in <i>rutabaga</i> (Ca²⁺/calmodulin-responsive adenylate cyclase) mutants was rescued by <i>DopEcR</i> overexpression or acute 20E feeding, whereas a memory defect in <i>dunce</i> (cAMP-specific phosphodiestrase) mutants was counteracted when a loss-of-function <i>DopEcR</i> mutation was introduced. A memory defect caused by suppressing dopamine synthesis was also restored through enhanced DopEcR-mediated ecdysone signaling, and rescue and phenocopy experiments revealed that the mushroom body (MB)—a brain region central to learning and memory in <i>Drosophila</i>—is critical for the DopEcR-dependent processing of courtship memory. Consistent with this finding, acute 20E feeding induced a rapid, DopEcR-dependent increase in cAMP levels in the MB. Our multidisciplinary approach demonstrates that DopEcR mediates the non-canonical actions of 20E and rapidly modulates adult conditioned behavior through cAMP signaling, which is universally important for neural plasticity. This study provides novel insights into non-genomic actions of steroids, and opens a new avenue for genetic investigation into an underappreciated mechanism critical to behavioral control by steroids.</p></div

DopEcR is required for the 30-minute courtship memory induced by 1-hour courtship conditioning.

<p>(A) Thirty-minute courtship memory in wild-type flies (control) and flies heterozygous (<i>DopEcR^PB1</i>/+), homozygous (<i>DopEcR^PB1</i>/<i>DopEcR^PB1</i>), and hemizygous (<i>DopEcR^PB1</i>/Df(3L)ED4341) for <i>DopEcR</i>. <i>DopEcR^PB1</i> homozygotes and hemizygotes were defective for 30-minute courtship memory. (B) Time course of courtship memory in <i>DopEcR^PB1</i> homozygotes. Significant memory was observed immediately after conditioning, but not 15 or 30 minutes after conditioning. (C) A defect in 30-minute courtship memory in flies that ubiquitously express the <i>DopEcR</i> RNAi after eclosion, in response to RU486 stimulation of the <i>tub</i>-GS-Gal4 driver. The presence or absence of courtship memory was evaluated by applying the Mann–Whitney U-test to naïve and conditioned males. Statistical significance is shown above each bar as NS, no significant difference, **, <i>P</i><0.01 or ***, <i>P</i><0.001. Sample numbers for naïve and conditioned flies are shown under each graph. PIs were analyzed using Krustal-Wallis One-Way ANOVA, followed by Dunn's pairwise test for multiple comparisons. #, <i>P</i><0.05; ##, <i>P</i><0.01. Error bars (s.e.m.).</p

The mushroom body is critical for the DopEcR-dependent processing of courtship memory.

<p>(A) Rescue of the <i>DopEcR^PB1</i> memory defect by expression of wild-type <i>DopEcR</i> transgene using the <i>DopEcR</i>-Gal4 driver. (B) Rescue of <i>DopEcR^PB1</i> memory defect by expression of wild-type <i>DopEcR</i> transgene under control of the MB-specific c772, c739 and 201 y drivers. Note that MB-Gal4 lines drive reporter expression in different subsets of MB neurons (see text). (C) Courtship memory defect induced by MB-specific expression of the <i>DopEcR</i> RNAi using the c772 and c739 drivers. The presence or absence of courtship memory was evaluated by applying Mann–Whitney U-test to naïve and conditioned flies. Statistical significance is shown above each bar. NS, no significant difference. **, <i>P</i><0.01; ***, <i>P</i><0.001. Sample numbers for naïve and conditioned flies are shown under each graph. PIs were analyzed using Student's t-test or Krustal-Wallis One-Way ANOVA, followed by Dunn's pairwise test for multiple comparisons. #, <i>P</i><0.05; ###, <i>P</i><0.001. Error bars (s.e.m.).</p