SummaryThe diversity of cell types and regulatory states in the brain, and how these change during aging, remains largely unknown. We present a single-cell transcriptome atlas of the entire adult Drosophila melanogaster brain sampled across its lifespan. Cell clustering identified 87 initial cell clusters that are further subclustered and validated by targeted cell-sorting. Our data show high granularity and identify a wide range of cell types. Gene network analyses using SCENIC revealed regulatory heterogeneity linked to energy consumption. During aging, RNA content declines exponentially without affecting neuronal identity in old brains. This single-cell brain atlas covers nearly all cells in the normal brain and provides the tools to study cellular diversity alongside other Drosophila and mammalian single-cell datasets in our unique single-cell analysis platform: SCope (http://scope.aertslab.org). These results, together with SCope, allow comprehensive exploration of all transcriptional states of an entire aging brain.

The Q-system is a repressible binary expression system for transgenic manipulations in living organisms. Through protein engineering and in vivo functional tests, we report here new variants of the Q-system transcriptional activator, including QF2, that allows the Q-system to drive strong and ubiquitous expression for the first time in all tissues. Our new QF2, GAL4QF and LexAQF chimeric transcriptional activators substantially enrich the toolkit available for transgenic regulation in Drosophila.

Sleep is under homeostatic control, whereby increasing wakefulness generates sleep need and triggers sleep drive. However, the molecular and cellular pathways by which sleep need is encoded are poorly understood. In addition, the mechanisms underlying both how and when sleep need is transformed to sleep drive are unknown. Here, using ex vivo and in vivo imaging, we show in Drosophila that astroglial Ca 2+ signaling increases with sleep need. We demonstrate that this signaling is dependent on a specific L-type Ca 2+ channel and is required for homeostatic sleep rebound. Thermogenetically increasing Ca 2+ in astrocytes induces persistent sleep behavior, and we exploit this phenotype to conduct a genetic screen for genes required for the homeostatic regulation of sleep. From this large-scale screen, we identify TyrRII, a monoaminergic receptor required in astrocytes for sleep homeostasis. TyrRII levels rise following sleep deprivation in a Ca 2+ -dependent manner, promoting further increases in astrocytic Ca 2+ and resulting in a positive-feedback loop. These data suggest that TyrRII acts as a gate to enable the transformation of sleep need to sleep drive at the appropriate time. Moreover, our findings suggest that astrocytes then transmit this sleep need to the R5 sleep drive circuit, by upregulation and release of the interleukin-1 analog Spätzle. These findings define astroglial Ca 2+ signaling mechanisms encoding sleep need and reveal dynamic properties of the sleep homeostatic control system.