Figure 2. Behavioral effects of acutely activating R23E10 neurons.

(A–D) Effects on sleep and responsiveness following the activation of R23E10 neurons (blue, UAS-CsChrimson/+;R23E10-Gal4/+ with ATR, n = 102) compared to those in control flies (black, no ATR feeding, n = 118). Error bars and shading indicate standard errors of the mean (SEMs) and asterisks indicate significance (****p<0.0001, ns = not significant, t-tests). (A) Mean sleep duration (min/hr) during the period 24 hr before red light activation (left), then during the next 24 hr when red light is delivered for 12 hr during the day (pink shading, right). (B) Comparison of the 12-hr day period without red light (baseline) to the period of red-light activation in terms of sleep duration and bout length. (C) Mean activity (mm/s) for R23E10 activation for the time periods in (A). Spikes in activity show timing of hourly vibration stimuli. (D) Comparison between the 12-hr day period without red light (baseline) and the period of red-light activation for peak responsiveness and sleep intensity. (E) Left, average stimulus response for UAS-CsChrimson/+;R23E10-Gal4/+ with ATR (blue, n = 50) compared to control flies (black, no ATR feeding, n = 48) during red-light activation (Figure 2A–D red shading) in flies that moved in the minute prior to the stimulus (i.e. awake flies). Right, summary histogram (average ± SEM). **p<0.01, t-test. (F) Example activity trace of flies responding to stimuli 15 min apart (gray dashed lines): 1 min CsChrimson activation (red shading) prior to the stimulus is alternated with trials without red light (left panel). One minute of dFB activation is sufficient to decrease responsiveness (right panel, UAS-CsChrimson/+;R23E10-Gal4/+ with ATR n = 83, control n = 80, *p<0.05, t-test). See also Figure 2—figure supplements 1, 2 and 3.

Figure 2—figure supplement 1. Measuring sleep intensity.

(A) Schematic showing fly activity prior to and in response to a mechanical stimulus (orange dashed line). Flies can be inactive (grey shading) or active (green shading) prior to stimulus delivery. Flies that are inactive for longer than 5 min are classified as asleep, and the proportion of sleeping flies that respond to the stimulus (green shading) compared to those not responding (orange shading) is a measure of sleep intensity. (B) Sleep intensity for w2202 flies during the day (yellow) and night (grey) for inactivity bins of 5 min (n = 250, error bars are SEM). (C) During the night, flies are less likely to respond to a stimulus than they are during the day and are therefore sleeping more intensely. (D) Sleep intensity during red-light activation of dFB neurons in UAS-CsChrimson/+;R23E10-Gal4/+flies fed ATR (blue, n = 102) compared to non-ATR-fed controls (black, n = 118). ****p<0.0001, ***p<0.001, t-test corrected for multiple comparisons (using the Bonferroni correction).

Figure 2—figure supplement 2. Acute effects in awake flies.

(A) Mean speed of all flies for the minute prior to stimulation during acute CsChrimson activation. n.s. represents not significantly different in a t-test. (B) Average stimulus response for UAS-CsChrimson/+;R23E10-Gal4/+with ATR (blue, n = 83) compared to control flies (black, no ATR feeding, n = 80) in flies that moved in the minute prior to the stimulus (i.e. awake flies), with the red light off. (C) Average stimulus response for the same flies as those in (B) that moved in the minute prior to the stimulus (i.e. awake flies), with the red light on. (D) Summary histogram of mean peak responsiveness data in (B) and (C) (average ± SEM). *p<0.05, t-test.

Figure 2—figure supplement 3. 1 Hz optogenetic activation of the dFB.

(A)-(D) Effects on sleep and responsiveness following 1 Hz activation of R23E10 neurons (blue, UAS-CsChrimson/+;R23E10-Gal4/+ with ATR, n = 115) compared to control flies (black, no ATR feeding, n = 115). Error bars and shading indicate SEM and asterisks indicate significance (*p<0.05, ****p<0.0001, t-tests). (A) Mean sleep duration (min/hr) during the 24 hr before red-light activation, then during the next 24 hr when red light is delivered for 12 hr during the day (red shading). (B) Comparison of the 12-hr day period without red light (baseline) to the period of red-light activation in terms of sleep duration and bout length. (C) Mean activity (mm/s) for R23E10 activation for the same time periods as in (C). Spikes in activity show the timing of hourly vibration stimuli. (D) Comparison between the 12-hr day period without red light (baseline) and the period of red-light activation in terms of peak responsiveness and sleep intensity. Error bars and shading indicate SEM and asterisks indicate significance (**p<0.01, ****p<0.0001, t-tests).