FIGURE 3.

R59E08-Gal4-expressing PB interneurons promote sleep. (A) Maximum-intensity projection of the confocal brain or ventral nerve cord (VNC) images of flies expressing UAS-mCD8::GFP under the control of R59E08-Gal4 (left two panels) or R59E08-Gal4 with tsh-Gal80 (right two panels). Transgene tsh-Gal80 efficiently suppressed R59E08-Gal4 driven GFP expression in the VNC, while did not in the brains. Scale bars indicate 100 μm. (B,C) Sleep profiles in 60-min intervals (B) or total daily sleep (C) for control flies (R59E08-Gal4 × tsh-Gal80, black circles or bars, n = 12) or flies expressing dTrpA1 in R59E08-Gal4 brain neurons (R59E08-Gal4 × tsh-Gal80; UAS-dTrpA1, blue circles or bars, n = 11) in DD. The behavior was monitored as described in Figure 2. Data are presented as mean ± SEM. *p < 0.05 vs. control; two-way repeated measures ANOVA followed by simple main effect test. (D) Horizontal movements of three representative control flies (R59E08-Gal4 × tsh-Gal80, black circles) or flies expressing dTrpA1 in R59E08-Gal4 brain neurons (R59E08-Gal4 × tsh-Gal80; UAS-dTrpA1, blue circles) in a glass tube at 29°C during the late subjective day (CT 8 to CT 12). Positions of the cotton plug and the food were at the top and the bottom in each plot, respectively. The behavior of flies was recorded at 2 frames/sec using an infrared video camera in DD conditions. The fly position in a glass tube was calculated for each image using an ImageJ plugin. (E,F) Sleep profiles in 60-min intervals (E) or total daily sleep (F) for control flies (R59E08-Gal4 × w¹¹¹⁸, black circles or bars, n = 16) or flies expressing dTrpA1 in R59E08-Gal4 except for cholinergic neurons (R59E08-Gal4 × UAS-dTrpA1; Cha-Gal80, blue circles or bars, n = 16) in DD. Data are presented as mean ± SEM. *p < 0.05 vs. control; two-way repeated measures ANOVA followed by simple main effect test. (G) Maximum-intensity projection of the confocal brain images of flies expressing UAS-mCD8::GFP under the control of R59E08-Gal4 (left panel) or R59E08-Gal4 with Cha-Gal80 (right panel). Scale bars indicate 100 μm. (H) Responsiveness to mechanical stimuli of sleeping flies with activation of R59E08-Gal4-expressing neurons. Sleeping control (R59E08-Gal4 × w¹¹¹⁸, black bar) or experimental (R59E08-Gal4 × UAS-dTrpA1, blue bar) flies were mechanically stimulated during the subjective night at 29°C. Response rate was calculated by subtracting the percentage of flies spontaneously aroused from that of flies awakened by stimulation (see Materials and Methods for details). Numbers of examined sleeping flies: 65 (R59E08-Gal4 × w¹¹¹⁸) and 130 (R59E08-Gal4 × UAS-dTrpA1). *p < 0.05; chi-square test.