Circadian cycles studied at single-cell level across the whole brain

Amazing stuff!

"Although circadian rhythms have been well studied in a few specific regions, their brain-wide 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."

From the editor's summary and abstract:

"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. 

Structured Abstract

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."

In Science Journals | Science

A whole-brain single-cell atlas of circadian neural activity in mice (no public access)

Here is an almost 17 minutes long YouTube video about this paper. (Caveat: I did not watch it)

Whole-brain single-cell atlas of circadian neural activity.

The circadian cycle of Deep-sea shrimp is based on the tides

Amazing stuff!

"... In the depths of the sea, creatures still sync their internal schedules—but they do it to the tides instead, according to a new study.

Hydrothermal vent ecosystems are full of life that never sees the light of day. Given that most animals examined to date have light-based clocks, researchers wondered if the denizens of the deep have figured out a daily schedule without light—or if they have any clock at all.

So they collected shrimp from a vent, kept them in the dark, and analyzed gene expression patterns every 4 hours for a total of 72 hours. That revealed roughly 12.4-hour cycles instead of the 24-hour ones seen in humans—timing that matches up perfectly with tidal fluctuations. ..."

From the abstract:

"Biological clocks are a ubiquitous feature of all life, enabling the use of natural environmental cycles to track time. Although studies on circadian rhythms have contributed greatly to the knowledge of chronobiology, biological rhythms in dark biospheres such as the deep sea remain poorly understood.

Here, based on a free-running experiment in the laboratory, we reveal potentially endogenous rhythms in the gene expression of the deep-sea hydrothermal vent shrimp Rimicaris leurokolos.

Oscillations with approximately 12 h periods, probably reflecting tidal influence, greatly prevail over others in the temporal transcriptome, indicating R. leurokolos probably depends on a circatidal clock consisting of at least some components independent from the circadian clocks. The tidal transcripts exhibit an antiphased expression pattern divided into two internally synchronized clusters, correlated with wide-ranging biological processes that occur in the nucleus and cytoplasm, respectively.

In addition, the tidal transcripts showed great similarities with genes in fruit flies and mice exhibiting approximately 12 h ultradian rhythms, indicating that the tide probably had a broad impact on the evolution of approximately 12 h oscillations found across the Metazoa. These findings not only provide new insights into the temporal adaptations in deep-sea organisms but also highlight hydrothermal vent organisms as intriguing models for chronobiological studies, particularly those linked to approximately 12 h ultradian rhythms."

ScienceAdviser

Circatidal control of gene expression in the deep-sea hot vent shrimp Rimicaris leurokolos

Fig. 1 Deep-sea chronobiology study on the hydrothermal vent shrimp Rimicaris leurokolos.

Even bacteria button up for winter. Photoperiodism might have evolved before circadian clocks

Amazing stuff!

"As winter approaches ... chronobiologists have shown that at least one kind of bacterium also prepares for the cold season—the first such seasonal response known among microbial life ...

When chilled, microbes increase the proportion of unsaturated fats in their membranes to keep them from freezing. ...

Not only did they make their membranes more fluid, but they also altered gene activity, the team reports. In anticipation of winter, when photosynthesis slows, the cyanobacteria revved up genes for conserving energy. When days got longer, they increased the activity of genes that protect against damage from sunlight. ..."

"... “To me, the most exciting thing about these results are their ramifications for the evolution of biological timekeeping,” he said. “I think we ‘chronobiologists’ have always assumed that daily (circadian) clocks evolved before organisms could measure day/night-length and thereby anticipate the changing seasons. But the facts that
(1) photoperiodism evolved in such ancient and simple organisms, and
(2) our gene expression results implicate stress response pathways that probably evolved very early in life on Earth, suggest that photoperiodism might have evolved before circadian clocks. This is because either long days or long nights are differentially stressful for photoautotrophs like cyanobacteria, and therefore these conditions would be expected to trigger stress responses differentially. As a result of these selective pressures, photoperiodic time measurement might have evolved even before bona fide circadian clocks appeared. ...” ..."

From the editor's summary and abstract:

"Editor’s summary

Long-lived plants and animals clearly regulate their physiology according to seasonal changes in day length to appropriately adjust their physiology. Jabbur et al. found that cyanobacteria can do the same even though individuals only live for a few hours, a time shorter than a single daily photoperiod. Exposure to short photoperiods characteristic of winter stimulated these cyanobacteria to adjust their membrane lipids and gene expression to accommodate cold conditions. These responses required a functional circadian clock. Thus, population-based sensing of photoperiod appears to have evolved early, perhaps as a refinement of stress pathways....

Abstract

Photoperiodic time measurement is the ability of plants and animals to measure differences in day versus night length (photoperiod) and use that information to anticipate critical seasonal transformations, such as annual temperature cycles. This timekeeping phenomenon triggers adaptive responses in higher organisms, such as gonadal stimulation, flowering, and hibernation. Unexpectedly, we observed this capability in cyanobacteria—unicellular prokaryotes with generation times as short as 5 to 6 hours. Cyanobacteria exposed to short, winter-like days developed enhanced resistance to cold mediated by desaturation of membrane lipids and differential programs of gene transcription, including stress response pathways. As in eukaryotes, this photoperiodic timekeeping required an intact circadian clockwork and developed over multiple cycles of photoperiod. Therefore, photoperiodic timekeeping evolved in much simpler organisms than previously appreciated and enabled genetic responses to stresses that recur seasonally."

Researchers Discover the Evolution of Seasonal Anticipation in Cyanobacteria (original news release)

ScienceAdvisor

In a first for microbes, bacteria seem to track—and button up for—the cold

Bacteria can anticipate the seasons: Photoperiodism in cyanobacteria

Lead scientist Maria Luísa Jabbur sitting in front of petri dishes