Circadian timekeeping enables organisms to anticipate and adapt to daily environmental cycles. In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus is recognised as the primary circadian clock; however, it is now well established that additional, autonomous clocks are distributed throughout the brain and peripheral tissues. Importantly, circadian organisation is not solely a neuronal property. Glial cells express clock genes and actively contribute to circadian function, yet their roles outside the SCN remain poorly understood. Here, we focus on the dorsal vagal complex (DVC), a brainstem structure that plays key roles in satiety and metabolic regulation. Previously, we identified robust clock gene expression across both neuronal and non-neuronal cell populations of the DVC. This prompted us to ask how glial cells – specifically astrocytes and ependymocytes – contribute to circadian timekeeping and oscillator coupling within the DVC.

To address this, we combined ex vivo, molecular, and in vivo approaches. Circadian rhythms were monitored using PER2::LUC bioluminescence recordings from organotypic DVC slices cultured and imaged for up to one week on an EM-CCD equipped Olympus LV200 system. To probe glial–glial and glial–neuronal communication, we applied TAT-GAP19 to block connexin-43 (Cx43) hemichannels and fluoroacetate to inhibit astrocytic metabolism, and quantified rhythm robustness and phase relationships between putative oscillators. Complementary in vivo studies were performed by measuring spatial and temporal expression of clock genes and glial markers using RNAscope and immunohistochemistry across the 24-hour cycle. These were augmented by, NanoString transcriptomic profiling to determine how neuronal clock disruption (neuronal Bmal1 knockout) impacts glial and neuronal gene expression in the DVC. Finally, we employed time-restricted feeding paradigms at distinct phases of the day to assess how physiologically relevant feeding cues reshape neuronal and glial clocks in vivo, analysed across four circadian time points.

We identify robust clock gene expression in multiple DVC glial populations, including astrocytes within the nucleus tractus solitarius (NTS), glial cells forming a specialised area postrema–nucleus of the solitary tract glial border, and ependymal cells lining the wall of the central canal. PER2::LUC recordings reveal that, ex vivo, ependymal rhythms are stably anti-phased relative to putatively neuronal oscillators. In vivo, these oscillators are phase-aligned under ad libitum feeding but become differentially shifted by scheduled feeding. Under time-restricted feeding, a phase difference between ependymal and neuronal rhythms re-emerges, albeit smaller than that observed ex vivo. Notably, pharmacological blockade of Cx43 hemichannels reduces this phase disparity in culture, implicating glial communication pathways in oscillator coupling.

Together, our findings demonstrate that, analogous to the SCN, glial cells make a substantive contribution to circadian timekeeping within the DVC. Glial–neuronal communication is critical for phase alignment and likely involved in entrainment to feeding schedules. Given that clock genes act as transcriptional regulators, it is plausible that glial clock activity shapes downstream gene expression, glial physiology, and sensitivity to metabolic cues. These data support a more universal role for glia in mammalian circadian systems and motivate future studies to define the physiological consequences of selectively disrupting glial clocks in brainstem circuits controlling feeding and metabolism.