Sleep pressure induces PDFR expression in mature l-LNvs.

(A) Normalized FRET ratio during PDF application in l-LNvs from starved (n = 10) and fed (n = 16) Pdf-GAL4>UAS-Epac1 flies. (B)The amplitude of l-LNv responses to PDF is observed in 5-day-old Pdf-GAL4>UAS-Epac1 flies following 21–24 h of starvation. Data are shown for 8 h bins (ANOVA F_[7,71] = 9.08, p = 9.8^E-8; ROI = 7–20). (C) Sleep in Cs flies on sleep restriction day 2 and recovery. (D) Normalized FRET ratio during PDF application in l-LNvs recorded from Pdf-GAL4>UAS-Epac1 flies during sleep restriction sleep deprivation and bang controls (n = 12–25 ROI/genotype). (E) Immunohistochemistry of PDF (red) and myc (green) in 5-day-old sleep-restricted P[acman] Pdfr-myc70 flies. (F) The amplitude of l-LNv responses to PDF in l-LNvs during baseline, sleep restriction, and recovery (ANOVA F_[4,81] = 8.00, p = 1.94^E-4, n = 9–24 ROI/condition). (G) Sleep rebound in Dcr2; c929-GAL4/UAS-Pdfr^RNAi flies and parental controls (ANOVA F_[2,147] = 6.37, p = 2.22^E-3; n = 43–55). *p < 0.05, modified Bonferroni test. Data underlying this figure can be found in S5 Data. FRET, Förster Resonance Energy Transfer; l-LNv, large ventral lateral neuron; n.s., not significant; PDF, pigment dispersing factor; PDFR, pigment dispersing factor receptor; ROI, region of interest.