BioProject

To gain insight into the dynamic molecular processes that are altered during prolonged wakefulness and during sleep. We performed an RNA expression profiling study examining temporal changes in the brain of Drosophila in relationship to the duration of prior sleep or wakefulness. Our experimental design allowed us to determine whether genes identified as differentially regulated between sleep and wakefulness were up- or down-regulated in these states. Because stimulation of the experimental animal during the normal sleep period is used to prolong wakefulness in most experimental paradigms, the interpretation of the effects of prolonged wakefulness is confounded by the effect of the perturbation stimulus itself on the animal’s biology. We controlled for this effect in our experimental paradigm by examining gene expression changes in response to identical stimulation but during the animal’s normal wakefulness. The design of our study also allowed us to control for circadian variation in gene expression, since we compared sleeping and sleep deprived flies at the same diurnal time. Keywords: sleep deprivation, time course, stress response Overall design: Gene expression was measured in brains of virgin Canton-S female Drosophila kept awake during their normal consolidated sleep period by sleep deprivation (Deprived) and in brains of time-matched control flies allowed to sleep normally (SLEEP). Activity of populations of flies was monitored by measuring the number of times infrared beams were interrupted. Rest/activity patterns of all populations of flies evaluated were within previously established normal ranges for this genotype, gender, and age. Sleep deprivation was produced by manually stimulating the population monitors that housed the flies and was verified by both visual observation of the population of flies and by analysis of population activity. RNA was isolated from a pool of 139-193 brains dissected from flies after sleep deprivation durations of 0, 2, 4, or 6 hours starting at the beginning of the consolidated sleep period (ZT 14), and from brains of groups of undisturbed control flies sacrificed at the same diurnal time as the respective experimental groups.The inclusion of a 0-hr time point allowed us to determine the temporal direction of change for genes that were differentially expressed between the SLEEP and SD groups. For each group at each time-point, there were 3 biological replications. Gene expression was evaluated using Drosophila 1.0 Affymetrix gene chips (Affymetrix, Santa Clara, CA). Normalization was carried out using the robust multi-array average (RMA) method (Irizarry et al., 2003) to form one expression measure for each gene on each array. Differences in gene expression between the SLEEP and SD groups at 2, 4, and 6 hours were analyzed using a one-way ANOVA based on the Fs statistic (Cui et al., 2005). P-values were calculated by permuting model residuals 1000 times and subsequently adjusted using a false discovery rate (FDR) algorithm (Storey, 2002). At a FDR of 10%, 578 genes were determined to be differentially expressed between SLEEP and SD groups, and at an FDR of 5%, 252 genes were differentially expressed. We used the more stringent 5% FDR for subsequent analysis. In addition, to control for the effect of handling the flies during the sleep deprivation, we manually stimulated the flies during their active period from ZT10-ZT14. Gene expression in brains isolated from these active period stimulation (APS) flies was compared to gene expression in brains isolated from unperturbed flies sacrificed at the same diurnal time (WAKE). We chose to identify genes differentially expressed between APS and WAKE by analyzing only the subset of expression data from the active period stimulation experiment for the 252 genes identified in the sleep deprivation experiment since this was our primary analysis. Here, we used the ANOVA analysis without correcting for multiple testing due to the small number of tests (252) (P<0.01) and to avoid underestimating the effects of manual stimulation.