Female Mice Show Stronger Time-of-Day Modulation of Astrocytic Ca²⁺ Activity in the Sleep-Regulatory Ventrolateral...

Astrocytes actively contribute to sleep regulation through intracellular calcium (Ca²⁺) 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 Ca²⁺ activity remains poorly defined. Here, we investigated astrocytic Ca²⁺ signaling in the VLPO of male and female mice across the nycthemeral cycle. Using two-photon Ca²⁺ 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 Ca²⁺ dynamics exhibited marked state dependence and sexual dimorphism. In males, Ca²⁺ 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 Ca²⁺ 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.