A whole-brain single-cell atlas of circadian neural activity in mice.

The mammalian brain comprises numerous anatomical regions with distinct functions despite their extensive connectivity. How spontaneous neural activity is coordinated across regions over the circadian cycle remains elusive. We used tissue clearing and whole-brain c-Fos immunostaining on 144 mouse brains collected over 2 days under constant darkness. Time-series analysis revealed brainwide circadian rhythmicity at single-cell resolution, with 79% of the 642 anatomically defined regions oscillating in diverse circadian phases that delineate functional specializations. Voxelwise analyses further highlighted distinct subregions, suggesting intricate spatiotemporal coordination within regions. Additionally, brain circadian time could be accurately inferred from global c-Fos patterns using omics-derived prediction methods. This whole-brain circadian atlas enhances our understanding of neural coordination and provides a resource for integrating time-of-day information into functional and pharmacological research. Editor's summary: Although circadian rhythms have been well studied in a few specific regions, their brainwide organization remains poorly understood. To quantify spontaneous circadian neural activity at single-cell resolution, Yamashita et al. used tissue clearing and whole-brain immunostaining on a large number of mouse brains over two full circadian cycles. Circadian rhythmicity was present in many brain regions. The activity of most regions peaked during the animals' active phase. However, sleep centers, visual areas, the dentate gyrus, and the cerebellum all peaked during the inactive phase. A closer look revealed distinct circadian phases even within individual regions, highlighting temporal heterogeneity. These findings will be useful for relating physiological and behavioral experimental data to the time-of-day–driven internal regulatory forces. —Peter Stern INTRODUCTION: Neural activity across different brain regions underlies essential physiological and behavioral functions. These activities are coordinated in space and time, and circadian rhythms are a fundamental temporal regulator of such activity, influencing sleep, metabolism, hormone secretion, and cognition. Although the suprachiasmatic nucleus (SCN) has been extensively studied as the master pacemaker, how spontaneous neural activity is coordinated across the entire brain over the circadian cycle has remained elusive. Previous approaches, including electrophysiological recordings, in situ hybridization, immediate early gene labeling, circadian gene reporters, and calcium imaging, have typically been restricted to limited regions and lack spatial continuity, making it difficult to achieve a systematic view. RATIONALE: To overcome these limitations, we used tissue clearing and three-dimensional whole-brain c-Fos immunostaining. c-Fos is notable for its rapid and broad induction across the brain, making it suitable for spatially comprehensive mapping of neural activity. By sampling brains every 4 hours over 2 days under constant darkness, we aimed to generate a whole-brain atlas of circadian neural activity at single-cell resolution and to identify how different regions and subregions contribute to the temporal organization of brain function. RESULTS: Each brain contained between 0.4 and 3.0 million c-Fos–positive cells. Time-series analysis of 144 brains revealed that 79% of 642 anatomically defined regions showed significant circadian rhythmicity. Most regions peaked during the late subjective night, corresponding to the active phase in nocturnal mice, whereas some, including sleep-promoting nuclei such as the ventrolateral preoptic area, peaked during the subjective day. Visual regions peaked during the daytime, in antiphase to auditory regions at night, highlighting functional specialization. The hippocampal memory system showed notable internal diversity: CA1 and CA3 peaked during the active phase, whereas the dentate gyrus peaked during the inactive phase, nearly in antiphase. This inversion aligns with reports of dentate gyrus recruitment during sleep stages, suggesting phase-specific contributions to memory processing. Voxelwise analysis further revealed distinct subregional dynamics, including heterogeneous patterns in the SCN and dorsomedial nucleus of the hypothalamus, and a gradual peak time shift along the dorsoventral axis within CA1, highlighting continuous spatiotemporal variation even within single structures. In addition, we demonstrated that whole-brain c-Fos activity patterns could accurately predict circadian time using computational approaches adapted from omics data, confirming that brain-wide rhythms collectively encode temporal information. CONCLUSION: Our study establishes a comprehensive atlas of circadian neural activity at the whole-brain scale. By combining tissue clearing with large-scale time-series sampling and systematic quantitative analysis, we provide a global view of how neural activity rhythms are organized across hundreds of regions and subregions. The open-access database we developed allows users to explore these rhythms by region or voxel and to upload custom regions of interest for analysis. It is designed to be compatible with gene expression, connectivity, and cell-type resources, enabling integrative analyses that link circadian activity with molecular and anatomical data. Thus, this resource not only advances chronobiology but also provides a temporal framework across neuroscience, linking time-of-day dynamics to studies of diverse brain functions, pharmacology, and disease. Whole-brain single-cell atlas of circadian neural activity.: Time-series sampling of mouse brains under constant darkness, followed by tissue clearing and whole-brain c-Fos immunostaining, enabled the creation of a brainwide atlas of circadian neural activity at single-cell resolution. Analysis of 144 brains revealed significant rhythmicity in 79% of 642 regions, with diverse peak time patterns across brain regions and subregions, all accessible through an open-access database. [ABSTRACT FROM AUTHOR]