Researchers led by Dr Marco Ferrari and Dr Marco Brancaccio (UK DRI at Imperial) have developed a live imaging platform capable of processing hundreds of samples at a time, to investigate how circadian rhythms in neurons and glial cells are organised in brain tissue. The new study, published in Advanced Science, also identifies a previously unknown role for calcium in synchronising the brain’s ‘master clock', offering new insight into the network mechanisms that underpin daily biological rhythms.
What was the challenge?
Circadian clocks in cells coordinate daily rhythms in gene expression, physiology, and behaviour. In mammals, these rhythms depend on an area of the brain called the suprachiasmatic nucleus (SCN), a small region in the hypothalamus that acts as the master circadian pacemaker. The SCN generates daily rhythms through the coordinated activity of different cell types within the brain tissue.
Much of our current understanding of this time-keeping process comes from slower, traditional methods, or from highly specialised, custom-built microscopes that are available in only a handful of laboratories investigating chronobiology – the study of biological rhythms - worldwide. These approaches can typically only collectively measure activity in a single sample at a time, and can miss rhythmic activities in single cells, as well as any interactions between cells within the tissue. These limitations mean investigating how brain tissue maintains the correct daily biological rhythms, and how these break down in disease, can be challenging.
What did the team do and what did they find?
To overcome this, the researchers developed a new, large scale, live imaging platform called ClockCyte. The platform enables continuous, long-term monitoring of circadian rhythms in up to 144 brain tissue samples simultaneously, across multiple fluorescent channels and over several weeks.
The researchers used mouse SCN tissue as a model to investigate a previously uncharacterised signal: calcium ions travelling along the long processes, called axons, that connect neurons. They discovered that in the SCN, these axonal calcium signals follow a 24-hour rhythm, but they behave very differently from calcium inside the cell body. While inside the cell, calcium moves through the tissue slowly in waves lasting several hours, axonal calcium rhythms are shaped in coordinated pulses sweeping instantly through the tissue. This suggests that the axons may be the site of key synchronisation signals, critically important for the organisation of coordinated circadian activities in brain tissue.
Bmal1 is the essential ‘clock gene’ which dictates many circadian processes in mammals. The researchers removed this gene from SCN neurons to investigate how clock genes contribute to the axonal rhythmic signals over time. They found that removing Bmal1 gradually disrupted the network organisation of axonal calcium. The tissue lost its structured pattern of connectivity early on, and the timing of signals across the circuit became less consistent. Surprisingly, some individual nerve fibres continued to produce rhythmic signals for over ten days, but because they were no longer in step with one another, the overall daily rhythm collapsed.
Circadian rhythms are central to brain health but studying them in real time has been slow and technically challenging. We developed ClockCyte to change that - enabling live tracking of how disease alters daily rhythms and investigating how clock mechanisms in neurons and glia might be therapeutically leveraged directly in brain tissue and at speed.
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What is the impact?
The ClockCyte platform could extend the study of circadian brain rhythms well beyond a handful of chronobiology laboratories. By adapting a widely commercially available imaging system, as well as creating dedicated freely available analysis tools, the team has created a setup that is accessible, capable of analysis at scale, and directly compatible with standardised laboratory and industry workflows. It produces more consistent, reproducible data than custom-built equipment, and will help different research groups compare their findings directly.
Moreover, the modular design of this platform means that ClockCyte could be adapted to study other neural circuits and other tissues beyond the brain, that keep their own internal rhythms. This could lead to future studies into how circadian rhythms contribute to health and disease across the body, with important potential applications in dementia research, as the disruption of daily sleep-wake cycles is common in people with Alzheimer's and other neurodegenerative conditions. By using ClockCyte to study tissue from models of these diseases over long periods, researchers could track in real time how neural circuits break down, and assess whether treatments could successfully restore healthy rhythmic function.
Dr Brancaccio explained:
“Circadian rhythms are central to brain health but studying them in real time has been slow and technically challenging, which has limited this research to a handful of specialised laboratories worldwide. We developed ClockCyte to change that - enabling live tracking of how disease alters daily rhythms and investigating how clock mechanisms in neurons and glia might be therapeutically leveraged directly in brain tissue and at speed. From the outset, the goal was not just to solve our own challenges, but to open up this space to others and accelerate collaborative discovery.”
Reference: M.Ferrari, N.Ness, J.Acosta, and M.Brancaccio, “A High-Throughput Live Imaging Platform to Investigate Circuit-Dependent Regulation of Circadian Rhythms in Brain Tissue.” Advanced Science (2026): e75427. https://doi.org/10.1002/advs.75427
