Sleep-enhancement does not require core circadian clock genes and circadian clock neurons.

<p>Sleep profiles of (a, left) <i>per</i>⁰, (a, middle) <i>tim</i>⁰ and (a, right) <i>Clk</i>^jrk after 4 days of social interaction. (b) Sleep analysis revealed that daytime sleep of socialized <i>tim</i>⁰ and <i>Clk</i>^jrk males is significantly greater as compared to solitary controls. In <i>per</i>⁰ flies, although daytime sleep of socialized flies does not show any change, its nighttime sleep is significantly increased as compared to solitary controls (<i>p <</i> 0.0005, Student’s <i>t</i>-test; <i>n</i> = 32 and 19 for socialized and control groups respectively). Change in sleep in <i>tim</i>⁰ and <i>Clk</i>^jrk flies shows a statistically significant increase in daytime sleep (<i>p</i> < 0.001 for <i>tim</i>⁰ and <i>p</i> < 0.005 for <i>Clk</i>^jrk, Student’s <i>t</i>-test; <i>n</i> = 22 and 17 for socialized and control groups respectively for <i>tim</i>⁰, and <i>n</i> = 27 and 23 for <i>Clk</i>^jrk) of socialized males from both the genotypes as compared to solitary controls. (c, left) Sleep profiles of <i>cryGAL4>UASdti</i> and (c, right) <i>PdfGAL4>UAShid</i> males following pair-wise social interaction. (d) Sleep analysis revealed that day as well as nighttime sleep of socialized <i>cryGAL4>UASdti</i> males is significantly greater as compared to that of solitary controls (<i>p</i> = 0.06 for daytime and <i>p <</i> 0.05 for nighttime; <i>n</i> = 16 for each group). Also, socialized <i>PdfGAL4>UAShid</i> males show an increase in day as well as nighttime sleep (<i>p <</i> 0.0005 for both; <i>n</i> = 17 and 13 for socialized and control groups respectively) as compared to solitary controls. Sleep profiles of (e, left) <i>PdfGAL4>UASKir2</i>.<i>1</i> males and (e, right) <i>PdfGAL4</i>/+ males following 4 days of pair-wise social interaction. (f) Sleep analysis showed that day as well as nighttime sleep of socialized <i>PdfGAL4>UASKir2</i>.<i>1</i> males is significantly greater (<i>p <</i> 0.05 for daytime and <i>p <</i> 0.005 for nighttime; <i>n</i> = 12 and 14 for socialized and control groups respectively) as compared to that of solitary controls. Also, socialized <i>PdfGAL4</i>/+ males show a significant increase (<i>p <</i> 0.05; <i>n</i> = 13 and 14 for socialized and control groups respectively) in daytime sleep as compared to solitary controls. Other details are same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150596#pone.0150596.g001" target="_blank">Fig 1</a>.</p

Olfactory cues mediate sleep-enhancement.

<p>(a) Sleep profiles of <i>norpA</i> males following 4 days of pair-wise social interaction in 12:12 h light/dark cycles (LD12:12). (b) Sleep profiles of <i>CS</i> and <i>Iso31</i> males following pair-wise social interaction for 4 days in constant darkness (DD). (c) Sleep analysis revealed that daytime sleep is significantly increased in socialized <i>norpA</i> males as compared to solitary controls (<i>p <</i> 0.0005 for daytime and <i>p <</i> 0.02 for nighttime, Student’s <i>t</i>-test; <i>n</i> = 21 and 24 for socialized and control groups respectively). Daytime sleep of socialized <i>CS</i> (<i>p <</i> 0.02, Student’s <i>t</i>-test; <i>n</i> = 14 and 16 for socialized and control groups respectively) and <i>Iso31</i> (<i>p <</i> 0.01, Student’s <i>t</i>-test; <i>n</i> = 29 and 18 for socialized and control groups respectively) males is also significantly increased as compared to that of solitary controls. (d, left) Sleep profiles and (d, right) change in sleep of <i>Gr66aGAL4>UAShid</i> and <i>Gr33aGAL4>UASdti</i> males following 4 days of pair-wise social interaction. Daytime sleep of socialized <i>Gr66aGAL4>UAShid</i> males (<i>p <</i> 0.0001, Student’s <i>t</i>-test; <i>n</i> = 16 and 14 for socialized and control groups respectively) and socialized <i>Gr33aGAL4>UASdti</i> males (<i>p <</i> 0.0001, Student’s <i>t</i>-test; <i>n</i> = 32 and 19 for socialized and control groups respectively) is significantly increased in comparison to that of solitary controls. Nighttime sleep of <i>Gr33aGAL4>UASdti</i> males is also significantly increased (<i>p <</i> 0.0005, Student’s <i>t</i>-test) in comparison to that of that of solitary controls. (e, left) Sleep profiles and (e, right) change in sleep of <i>Orco</i> males following 4 days of pair-wise social interaction. Sleep analysis revealed that day as well as nighttime sleep of socialized <i>Orco</i> males is comparable (<i>p ></i> 0.05; <i>n</i> = 27 and 20 for socialized and control groups respectively) to solitary controls, whereas daytime sleep of socialized <i>Iso31</i> males is significantly greater as compared to that of solitary controls (<i>p <</i> 0.05; <i>n</i> = 13 and 10 for socialized and control groups respectively), daytime sleep-enhancement in <i>Iso31</i> flies is significantly greater (<i>p <</i> 0.05) than that of <i>Orco</i> flies. (f, left) Sleep profiles and (f, right) change in sleep of <i>Or83bGAL4>UASKir2</i>.<i>1</i> males following 4 days of pair-wise social interaction. (f, right) Day as well as nighttime sleep of socialized <i>Or83bGAL4>UASKir2</i>.<i>1</i> males is comparable (<i>p ></i> 0.05) to that of solitary controls (e, right), whereas socialized parental (<i>Or83bGAL4/+</i>, <i>p <</i> 0.05 and <i>UASKir2</i>.<i>1/+</i>, <i>p <</i> 0.005; <i>n</i> = 13–16 per group per genotype) males show a statistically significant increase in sleep as compared to solitary controls. Daytime sleep-enhancement in parental control flies is significantly greater (<i>p <</i> 0.0001 for both) than that in silenced flies. Horizontal lines with asterisks above a pair of bars indicate statistically significant difference in sleep-enhanced in the experimental versus control genotypes. Other details are same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150596#pone.0150596.g001" target="_blank">Fig 1</a>.</p

Sleep in males during social interaction.

<p>(a) Sleep profiles of males across 4 days of social interaction. (b) Sleep profiles of females across 4 days of social interaction. (c) Change in sleep bars (mean ± SEM) for males (<i>n</i> = 16 and 24, for socialized and control groups respectively) and females (<i>n</i> = 14 and 22, for socialized and control groups respectively), where white and dark bars represent sleep during day as well as nighttime. Sleep of socially interacting males is not significantly different (<i>p</i> = 0.99 for daytime and <i>p</i> = 0.50 for nighttime, Student’s <i>t</i>-test) from that of solitary controls. Sleep of socially interacting females is also not significantly different (<i>p</i> = 0.98 for daytime and <i>p</i> = 0.76 for nighttime, Student’s <i>t</i>-test) from that of solitary controls. (d, e) Sleep latency of socially interacting (d) males and (e) females is comparable (<i>p ></i> 0.05, Student’s <i>t</i>-test) to that of solitary controls. Other details are same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150596#pone.0150596.g001" target="_blank">Fig 1</a>.</p

Sleep-enhancement in two other strains of <i>D</i>. <i>melanogaster</i>.

<p>(a, left) Sleep profiles of <i>Oregon R</i> (<i>OR</i>) males following 4 days of pair-wise social interaction. (a, right) Daytime and nighttime sleep latency is significantly lower (<i>p <</i> 0.05, Student’s <i>t</i>-test) as compared to that of solitary controls. (b, left) Sleep profiles of <i>Iso31</i> males following 4 days of pair-wise social interaction. (b, right) Although sleep latency of socialized males is also decreased, it did not reach statistically significant levels (<i>p ></i> 0.05, Student’s <i>t</i>-test). (c) Daytime sleep of socialized <i>OR</i> males (<i>p <</i> 0.0001, Student’s <i>t</i>-test; <i>n</i> = 16 for each group) and <i>Iso31</i> males (<i>p <</i> 0.0005, Student’s <i>t</i>-test; <i>n</i> = 15 for each group) is significantly greater as compared to that of solitary controls. Other details are same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150596#pone.0150596.g001" target="_blank">Fig 1</a>.</p

Sleep-enhancement in males due to pair-wise social interaction.

<p>(a, left) Sleep profiles of <i>Canton S</i> (<i>CS</i>) males following pair-wise social interaction with other males for (1D) 1 day, (2D) 2 days, (3D) 3 days or (4D) 4 days. In the sleep profiles, black circles and dark broken lines represent sleep of flies subjected to pair-wise social interaction, whereas grey circles and grey solid lines represent sleep of solitary controls. (a, right) Bar graphs show change in sleep of socialized males as compared to solitary controls and there is an increase in daytime sleep (white bars) in socialized males as compared to solitary controls (<i>p</i> = 0.09 for 1D, <i>p</i> = 0.06 for 2D, <i>p <</i> 0.0005 for 3D and 4D, ANOVA followed by post hoc multiple comparison by Tukey’s test). Nighttime sleep (dark bars) of socialized males does not differ from that of solitary controls. (b) Sleep profiles of <i>CS</i> females following pair-wise social interaction with other females for (1D) 1 day, (2D) 2 days, (3D) 3 days or (4D) 4 days. Both daytime and nighttime sleep does not differ between socialized and solitary control females (<i>p</i> > 0.05, ANOVA followed by post hoc by Tukey’s test). (c) Following 4 days of social interaction, daytime sleep latency of males is significantly reduced (<i>p <</i> 0.01, Student’s <i>t</i>-test) as compared to solitary controls. Although nighttime sleep latency also shows a similar decrease, it did not reach statistically significant levels (<i>p</i> > 0.05, Student’s <i>t</i>-test). In case of females, both daytime and nighttime sleep latency does not differ between socialized and solitary individuals. (d) While there is a statistically significant increase in sleep bout length during nighttime (<i>p</i> < 0.05, Student’s <i>t</i>-test) in socialized males as compared to solitary controls, bout number does not different significantly among socialized and solitary control males. Data is presented as mean ± SEM (standard error of means) and <i>n</i> = 16 for each group of males and females. Asterisks over each bar indicate statistically significant difference between socialized and solitary control flies unless mentioned otherwise, where <i>p <</i> 0.05 is represented by single asterisk, <i>p <</i> 0.005 by two asterisks and <i>p <</i> 0.0005 by three asterisks. Other details about the bar graphs are same as in 1a.</p

Social Experience Is Sufficient to Modulate Sleep Need of <i>Drosophila</i> without Increasing Wakefulness

<div><p>Organisms quickly learn about their surroundings and display synaptic plasticity which is thought to be critical for their survival. For example, fruit flies <i>Drosophila melanogaster</i> exposed to highly enriched social environment are found to show increased synaptic connections and a corresponding increase in sleep. Here we asked if social environment comprising a pair of same-sex individuals could enhance sleep in the participating individuals. To study this, we maintained individuals of <i>D</i>. <i>melanogaster</i> in same-sex pairs for a period of 1 to 4 days, and after separation, monitored sleep of the previously socialized and solitary individuals under similar conditions. Males maintained in pairs for 3 or more days were found to sleep significantly more during daytime and showed a tendency to fall asleep sooner as compared to solitary controls (both measures together are henceforth referred to as “sleep-enhancement”). This sleep phenotype is not strain-specific as it is observed in males from three different “wild type” strains of <i>D</i>. <i>melanogaster</i>. Previous studies on social interaction mediated sleep-enhancement presumed ‘waking experience’ during the interaction to be the primary underlying cause; however, we found sleep-enhancement to occur without any significant increase in wakefulness. Furthermore, while sleep-enhancement due to group-wise social interaction requires Pigment Dispersing Factor (PDF) positive neurons; PDF positive and CRYPTOCHROME (CRY) positive circadian clock neurons and the core circadian clock genes are not required for sleep-enhancement to occur when males interact in pairs. Pair-wise social interaction mediated sleep-enhancement requires dopamine and olfactory signaling, while visual and gustatory signaling systems seem to be dispensable. These results suggest that socialization alone (without any change in wakefulness) is sufficient to cause sleep-enhancement in fruit fly <i>D</i>. <i>melanogaster</i> males, and that its neuronal control is context-specific.</p></div