Figure 1.
Sleep in male flies with up-regulated npf.
Sleep during the nighttime was significantly increased when npf expression was enhanced (n=60 for controls, and 62 for yw; npf-Gal4/UAS-npf). (A) Daily sleep profile that showed increased sleep during the nighttime in npf over-expressing line. (B) Average daily sleep profile over 4 days that revealed increased sleep during the night. (C) Statistical analysis of sleep. Black and gray histograms indicate the controls, and blue histograms indicate the up-regulated npf line. Black and gray lines indicate controls, and the blue line represents the up-regulated npf line. Error bars indicate the SEM. White bars indicate lights on; black bars indicate lights off. ***, p<0.001; *, p<0.05; n.s., no significant differences.
Figure 2.
Sleep in male flies with up-regulated npfr1.
Sleep during the nighttime was significantly increased when npfr1 expression was enhanced (n=61 for controls, and 51 for yw; npfr1-Gal4/UAS-npfr1). (A) Daily sleep profile that showed increased sleep during the nighttime and the daytime in an npfr1 over-expressing line. (B) Average daily sleep profile over 4 days that revealed increased sleep during the night and the day. (C) Statistical analysis of sleep. Labeling is the same as that described in Figure 1.
Figure 3.
Analysis of sleep quality and quantity in male flies with up-regulated npf and npfr1.
(A–B) Analysis of sleep episode duration for npf and npfr1. (C–D) Analysis of sleep episode number for npf or npfr1. (E–F) Analysis of sleep latency for npf and npfr1. Labeling is the same as that described in Figure 1.
Figure 4.
Analysis of sleep in male flies with up-regulated npfr1 during adulthood.
Larvae and newly emerged adults were reared at 18°C, and newly emerged flies were entrained for three LD cycles at 18oC and then transferred to 30°C to block Gal80, in order to activate Gal4-driven gene expression.
(A) Total sleep was increased in npfr1 over-expression flies (n=15 for both the controls and n=13 for npfr1-Gal4/UAS-npfr1;tub-Gal80(ts)). (B) Daily sleep profile showed increased sleep during nighttime in an npfr1 over-expressing line. (C) Sleep increase during nighttime was mainly derived from increased sleep episode duration. Error bars and significant difference are as described above.
Figure 5.
The homeostatic sleep response regulated by NPF signal in males.
(A) Sleep rebound after 12 hour sleep deprivation in wild type flies (yw) is depicted. (B) npf expression was reduced after 12 hour sleep deprivation. (C, D and E) NPF regulated sleep homeostasis, in which C and D are controls for E. Sleep rebound was reduced when npf expression was enhanced (n=19-22 for controls, and n=20 for yw; npf-Gal4/UAS-npf). (F, G and H) NPFR1 regulated sleep homeostasis, in which F and G are controls for H. Sleep rebound was reduced when npfr1 expression was enhanced (n=21-24 for controls, and n=23 for yw; npfr1-Gal4/UAS-npfr1). Error bars and significant differences are as described above. (I) Sleep loss is reduced after sleep deprivation in npf and npfr1 up-regulated lines. (J) Sleep gain/lost ratio is reduced after sleep deprivation in npf and npfr1 up-regulated lines. Error bars and significant difference are as described above.
Figure 6.
Brain npf levels in male and female flies.
(A) npf levels in female flies with the presence of males (FM) and female flies without the presence of males (FF): (B) npf levels in male flies with the presence of females (MF) and male flies without the presence of females (MM). Error bars and significant differences are as described above.
Figure 7.
Male specific expression of NPF in male brain.
(A) anti-NPF labeled NPF localization in male brains in which D1 is male-specific. (B) NPF localization in female brains in which D1 is not detected. (C) NPF oscillation in male-specific D1 neurons. The arrows in the images represent the staining intensity of NPF at ZT0, ZT 4, ZT8, ZT12, ZT16 and ZT20. The ZT8 and ZT20 are compared with ZT16 for statistical difference analysis (*, p < 0.05). Scale bar: 5 µm for neuron figures.