Immobility was scored by thresholding maximal velocity on 10-s epochs, as previously described (24). Sleep was computed using the so-called 5-min rule, according to which all immobility bouts longer than 300 s were counted as sleep bouts (including the first 300 s). During mechanical sleep deprivation, velocity measurements subsequent to stimuli were masked to avoid false positive of fly movement (24). Specifically, data in the 6 s following the onset of each rotation were not considered for sleep scoring. Sleep rebound shown in Fig. 5 (D and H) was expressed as the difference between the sleep amount measured during rebound and the expected sleep amount. Expected sleep amounts were inferred by a linear regression between the reference baseline sleep and sleep during the rebound period in the relevant control population. Formally, the homoeostatic rebound Hi of an individual i was expressed asEmbedded Image(1)Embedded Image(2)where Embedded Image is the predicted sleep after treatment (ZT ∈ [0, 3]), R is the measured sleep after treatment (ZT ∈ [0, 3]), B is the measured sleep before treatment (ZT ∈ [0, 3]), and α and β are the coefficients of the linear regression RC = α + βBC on the control group C.Embedded Image(3)Embedded Image(4)

Behavioral state (“quiescence,” “micromovement,” and “walking”) was defined for each consecutive minute of behavior (B) according to the following ruleEmbedded Image(5)where Vmax is the maximum velocity, Tv is the validated threshold under which immobility is scored, X is the position along the tube, and Td is a threshold of 15 mm on the distance moved above which walking is scored. Td was defined empirically on the basis of the observation of a bimodal distribution of the total distance moved in a minute.

Because of the different amounts of food and cotton wool in each tube, the space available inside each experimental tube may be slightly different between individual animals. To compare the position of flies with respect to the boundary of their respective experimental environments, the animal longitudinal position was expressed relatively to the food (position = 0) and the cotton wool (position = 1) edgesEmbedded Image(6)where Qn is the quantile function.

First and last percentiles were used instead of minimum and maximum to avoid the possible effect of spurious artifactual detections beyond physical limits of the tube.

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