Female Mice Show Stronger Time‐of‐Day Modulation of Astrocytic Ca 2+ Activity in the Sleep‐Regulatory Ventrolateral Preoptic Nucleus

ABSTRACT Astrocytes actively contribute to sleep regulation through intracellular calcium (Ca 2+ ) signaling. Yet, whether astrocytic dynamics within sleep‐promoting hypothalamic nuclei vary across the nycthemeral cycle in a sex‐dependent manner remains unknown. The ventrolateral preoptic area (VLPO) is a key sleep‐promoting nucleus whose neuronal circuitry has been extensively characterized. However, the local astrocytic Ca 2+ activity remains poorly defined. Here, we investigated astrocytic Ca 2+ signaling in the VLPO of male and female mice across the nycthemeral cycle. Using two‐photon Ca 2+ imaging in acute VLPO‐containing brain slices prepared at Zeitgeber Time (ZT)‐2, corresponding to the onset of the rest period, and ZT‐14, corresponding to the beginning of the active period, we combined single‐event analyses with graph‐based network approaches to characterize astrocytic activity across scales. At the level of individual events, spontaneous astrocytic Ca 2+ dynamics exhibited marked state dependence and sexual dimorphism. In males, Ca 2+ events were smaller and faster at ZT‐14 than at ZT‐2 (shorter duration and accelerated rise and decay time). In contrast, in females, ZT‐14 was characterized by increased event amplitude and frequency, consistent with upregulated Ca 2+ signaling during the active phase. At the network level, functional connectivity remained stable in males. Conversely, females exhibited robust network remodeling at ZT‐14, including increased astrocyte recruitment, higher node degree of correlations, and a marked rise in the number and proportion of highly connected astrocytes. Together, these findings reveal sex‐specific astrocytic signaling strategies in the VLPO across the nycthemeral cycle and underscore the need to incorporate sex as a biological variable in astrocyte‐based sleep research.