Pinpointing the cells that keep the body's master circadian clock ticking

A new mouse model helps researchers study the roles of cell types in keeping time inside the body

August 7, 2020

Science Daily/UT Southwestern Medical Center

UT Southwestern scientists have developed a genetically engineered mouse and imaging system that lets them visualize fluctuations in the circadian clocks of cell types in mice. The method, described online in the journal Neuron, gives new insight into which brain cells are important in maintaining the body's master circadian clock. But they say the approach will also be broadly useful for answering questions about the daily rhythms of cells throughout the body.

"This is a really important technical resource for advancing the study of circadian rhythms," says study leader Joseph Takahashi, Ph.D., chair of the department of neuroscience at UT Southwestern Medical Center, a member of UT Southwestern's Peter O'Donnell Jr. Brain Institute, and an investigator with the Howard Hughes Medical Institute (HHMI). "You can use these mice for many different applications."

Nearly every cell in humans -- and mice -- has an internal circadian clock that fluctuates on a roughly 24-hour cycle. These cells help dictate not only hunger and sleep cycles, but biological functions such as immunity and metabolism. Defects in the circadian clock have been linked to diseases including cancer, diabetes, and Alzheimer's, as well as sleep disorders. Scientists have long known that a small part of the brain -- called the suprachiasmatic nucleus (SCN) -- integrates information from the eyes about environmental light and dark cycles with the body's master clock. In turn, the SCN helps keep the rest of the cells in the body in sync with each other.

"What makes the SCN a very special kind of clock is that it's both robust and flexible," says Takahashi. "It's a very strong pacemaker that doesn't lose track of time, but at the same time can shift to adapt to seasons, changing day lengths, or travel between time zones."

To study the circadian clock in both the SCN and the rest of the body, Takahashi's research group previously developed a mouse that had a bioluminescent version of PER2 -- one of the key circadian proteins whose levels fluctuate over the course of a day. By watching the bioluminescence levels wax and wane, the researchers could see how PER2 cycled throughout the animals' bodies during the day. But the protein is present in nearly every part of the body, sometimes making it difficult to distinguish the difference in circadian cycles between different cell types mixed together in the same tissue.

"If you observe a brain slice, for instance, almost every single cell has a PER2 signal, so you can't really distinguish where any particular PER2 signal is coming from," says Takahashi.

In the new work, the scientists overcame this problem by turning to a new bioluminescence system that changed color -- from red to green -- only in cells that expressed a particular gene known as Cre. Then, the researchers could engineer mice so that Cre, which is not naturally found in mouse cells, was only present in one cell type at a time.

To test the utility of the approach, Takahashi and his colleagues studied two types of cells that make up the brain's SCN -- arginine vasopressin (AVP) and vasoactive intestinal polypeptide (VIP) cells. In the past, scientists have hypothesized that VIP neurons hold the key to keeping the rest of the SCN synchronized.

When the research team looked at VIP neurons -- expressing Cre in just those cells, so that PER2 luminesced green in VIP cells, while red elsewhere -- they found that removing circadian genes from the neurons had little overall effect on the circadian rhythms of the VIP neurons, or the rest of the SCN. "Even when VIP neurons no longer had a functioning clock, the rest of the SCN behaved essentially the same," explains Yongli Shan, Ph.D., a UTSW research scientist and lead author of the study. Nearby cells were able to signal to the VIP neurons to keep them in sync with the rest of the SCN, he says.

When they repeated the same experiment on AVP neurons, however -- removing key clock genes -- not only did AVP neurons themselves show disrupted rhythms, but the entire SCN stopped synchronously cycling on its usual 24-hour rhythm.

"What this showed us was that the clock in AVP neurons is really essential for the synchrony of the whole SCN network," says Shan. "That's a surprising result and somewhat counterintuitive, so we hope it leads to more work on AVP neurons going forward."

Takahashi says other researchers who study circadian rhythms have already requested the mouse line from his lab to study the daily cycles of other cells. The mice might allow scientists to hone in on the differences in circadian rhythms between cell types within a single organ, or how tumor cells cycle differently than healthy cells, he says.

"In all sorts of complex or diseased tissues, this can let you see which cells have rhythms and how they might be similar or different from the rhythms of other cell types."

https://www.sciencedaily.com/releases/2020/08/200807111938.htm

Stress hormone helps control the circadian rhythm of brain cells

November 8, 2019

Science Daily/University of Copenhagen The Faculty of Health and Medical Sciences

Researchers have shown how the brain's circadian rhythm in rats is, among other things, controlled by the stress hormone corticosterone -- in humans called cortisol. This has been shown by means of a completely new method in the form of implanted micropumps.

As day turns into night, and night turns into day, the vast majority of living organisms follow a fixed circadian rhythm that controls everything from sleep needs to body temperature.

This internal clock is found in everything from bacteria to humans and is controlled by some very distinct hereditary genes, known as clock genes.

In the brain, clock genes are particularly active in the so-called suprachiasmatic nucleus. It sits just above the point where the optic nerves cross and sends signals to the brain about the surrounding light level. From here, the suprachiasmatic nucleus regulates the rhythm of a number of other areas of the body, including the cerebellum and the cerebral cortex.

However, these three areas of the brain are not directly linked by neurons, and this made researchers at the University of Copenhagen curious. Using test rats, they have now demonstrated that the circadian rhythm is controlled by means of signalling agents in the blood, such as the stress hormone corticosterone.

'In humans, the hormone is known as cortisol, and although the sleep rhythm in rats is the opposite of ours, we basically have the same hormonal system', says Associate Professor Martin Fredensborg Rath of the Department of Neuroscience.

He explains that recent years have seen an increasing, scientific focus on research on clock genes, one reason being that previous research on clock genes have found a correlation between depression and irregularities in the body's circadian rhythms.

New Method with Medical Micropumps

In the study with the stress hormone corticosterone, the researchers removed the suprachiasmatic nucleus in a number of rats. As expected, this removed the circadian rhythm of the animals.

Among other things, the body temperature and activity level of the rats went from circadian oscillations to a more constant state. The same was true of the otherwise rhythmic hormone production.

However, the circadian rhythm of the cerebellum was restored when the rats were subsequently implanted with a special programmable micropump, normally used to dose medication in specific quantities.

In this case, however, the researchers used the pump to emit carefully metered doses of corticosterone at different times of the day and night, similar to the animals' natural rhythm.

'Nobody has used these pumps for anything like this before. So technically, we were onto something completely new', says Martin Fredensborg Rath.

For that reason, the researchers spent the best part of a year carrying out a large number of control tests to ensure that the new method was valid.

Interaction Between Neurons and Hormones

As mentioned, the new method paid off. With the artificial corticosterone supplement, researchers were again able to read a rhythmic activity of clock genes in the cerebellum of the rats, even though their suprachiasmatic nucleus had been removed.

'This is hugely interesting from a scientific point of view, because it means that we have two systems -- the nervous system and the hormonal system -- that communicate perfectly and influence one another. All in the course of a reasonably tight 24-hour programme', says Martin Fredensborg Rath.

With the test results and the new method in the toolbox, the researchers' next step is to study other rhythmic hormones in a similar manner, including hormones from the thyroid gland.

https://www.sciencedaily.com/releases/2019/11/191108102850.htm

New Evidence of Age-Related Decline in the Brain's Master Circadian Clock

July 25, 2011

Science Daily/University of California - Los Angeles

A new study of the brain's master circadian clock -- known as the suprachiasmatic nucleus, or SCN -- reveals that a key pattern of rhythmic neural activity begins to decline by middle age. The study, whose senior author is UCLA Chancellor Gene Block, may have implications for the large number of older people who have difficulty sleeping and adjusting to time changes.

"Aging has a profound effect on circadian timing," said Block, a professor of psychiatry and biobehavioral sciences and of physiological science. "It is very clear that animals' circadian systems begin to deteriorate as they age, and humans have enormous problems with the quality of their sleep as they age, difficulty adjusting to time-zone changes and difficulty performing shift-work, as well as less alertness when awake. There is a real change in the sleep-wake cycle.

"The question is, what changes in the nervous system underlie all of that? This paper suggests a primary cause of at least some of these changes is a reduction in the amplitude of the rhythmic signals from the SCN."

The SCN, located in the hypothalamus, is the central circadian clock in humans and other mammals and controls not only the timing of the sleep-wake cycle but also many other rhythmic and non-rhythmic processes in the body.

The SCN keeps the system of multiple distributed circadian oscillators in synchrony, but disruptions in the SCN lead to disrupted sleep, as well as dysfunction in memory, the cardiovascular system, and the body's immune response and metabolism.

The SCN, Block said, can be imagined as a heavy pendulum that controls many light pendulums (oscillators), with rubber bands between them.

"If the central clock weakens, it's effectively like making those rubber bands thinner and weaker," Block said. "When the SCN ages and those rubber bands become weaker, it becomes hard for the SCN to synchronize all of these other oscillators."

http://www.sciencedaily.com/releases/2011/07/110719093808.htm