New Signal Found in System Controlling Circadian Activity

For the past 65 million years, indeed, ever since they first ventured out into a world without dinosaurs, rodents have been scurrying across the globe in search of food and mates. Still, the world is a dangerous place for these often tiny creatures. Though many rodents move only under cover of night, straying from their burrows just a few minutes too early could be costly.

Photo by Graham Ramsay

Charles Weitz (left) and Sebastian Kraves have identified a factor that helps control the remarkably precise switch from rest to activity in rodents.

“They are probably subject to predation if they are out wandering around before the sun goes down,” said Charles Weitz, the Robert Henry Pfeiffer professor of neurobiology at HMS. It turns out, rodents such as mice and hamsters are programmed to rest for 12 to 14 hours each day. So powerful is this drive that caged lab animals living in total darkness will remain relatively inactive for the entire rest period, at the end of which they will suddenly jump on their running wheels and start furiously exercising. Biologists have marveled over the phenomenon for decades. What has intrigued them is the precision with which the mice switch from rest to activity, even in the absence of external cues such as light.

“It happens during every circadian cycle, every day at approximately the same time, with an accuracy of one minute,” said Sebastian Kraves, HMS research fellow in neurobiology. He and Weitz report in the February Nature Neuroscience that they have identified a protein that plays a critical role in regulating these animals’ baffling circadian behavior. The protein is not the first activity-controlling protein to be found, but its discovery affords a much fuller view of the complex system underlying this and possibly other circadian behaviors.

Watching the Clock
Researchers have known that the rodents’ precise pattern of wheel-running behavior is under the control of a master circadian clock located just behind the eyes. In 2002, Weitz and colleagues, working in hamsters, discovered that this clock, situated in the suprachiasmatic nucleus (SCN), releases a factor, TGF-alpha, that inhibits circadian locomotor activity (Focus, Jan. 11, 2002). They suspected that the animals’ pattern of rest followed by abrupt onset of activity was not the product of TGF-alpha alone because individual factors are often subject to perturbation.

“In order to create such a sharp transition between rest and activity—no running-wheel activity and then, bam, this very sharp active zone that starts within one minute every day even without any environmental signal—you probably have to have overlapping signals that are rhythmically released,” said Weitz. In fact, soon after the discovery of TGF-alpha, researchers elsewhere stumbled upon another locomotor-dampening factor, prokineticin-2. Weitz was convinced more players were needed.

He and Kraves had an important clue. Workers in the field had shown that the SCN sends locomotor-inhibiting messengers to a particular spot in the nearby hypothalamus, near the third ventricle. They decided to scour the region for receptors, this time in mice, and work their way back to the SCN to find the missing proteins. Kraves detected a molecule, GP130, that combines with two others to create a receptor complex that intercepts messages from eight different cytokines.

It turns out, two of the eight are expressed by the SCN. One of them, cardiotrophin-like cytokine (CLC), exhibited an intriguing feature. Kraves and Weitz expected that activity-controling proteins would be expressed in a rhythmic pattern that corresponded to the behavioral pattern of rest and movement. CLC did just that. What is more, it appeared, like TGF-alpha, to be an inhibitory factor. It came on early in the rest period, reached a peak toward the end, and then plummeted just as the animals suddenly became active.

But were these CLC-expressing cells actually clock cells? Weitz and others had spent much of the 1990s trying to figure out how cells of the SCN actually measure time—using which specific core protein components—and had discovered a handful of clock genes. It turns out, many of these core clock genes are turned on in the CLC cells. In fact, he and Kraves found that the CLC gene was itself under the control of a central time-keeping component, CLOCK-BMAL1.

Putting Cytokine Through Its Paces
The next step was to see if CLC could actually inhibit locomotor activity. Switching to hamsters, they infused CLC into the third ventricle close to its putative GP130-containing receptors. As long as the cytokine was present, the animals refrained from moving. CLC was thought to work through the GP130-containing receptor, but it was not clear if this receptor was actually responsible for delivering the activity-quieting message. To find out, Kraves infused another GP130-stimulating cytokine, CNTF, into the third ventricle. Again, the hamsters remained at rest. Finally, he blocked the GP130 receptor using anti-GP130 antibodies. The animals hopped onto their wheels two hours earlier than normal.

The fact that they did not jump on earlier in the rest period is probably due to the activity of the other locomotor-inhibiting factors, TGF-alpha and prokineticin-2. And there may be more. “Three factors have been postulated to perform similar functions so far,” said Kraves. “What is interesting is their apparent temporal specificity. Prokineticin-2 peaks early in the rest phase while CLC peaks very late. TGF-alpha is overlapping with both and that may allow for the very precise temporal control of the structure of the locomotor activity.”

He and Weitz compare this pattern to what happens in developing embryos. Multiple signaling molecules are released at various times and places to form overlapping spatial gradients that ultimately create the precise spatial borders of developing organs and tissues. The sharp transition from rest to activity may be a temporal equivalent of those spatial borders and could be due to the overlapping, rhythmic release of CLC, TGF-alpha, and prokineticin-2. Though speculative, the idea is testable and, in this sense, modern circadian biology may be turning a page in its history. In the 1990s, circadian biologists like Weitz were preoccupied with understanding how individual clock cells in the SCN actually work. Now he and his colleagues are looking at ensembles of clock cells in the SCN, such as those secreting CLC, TGF-alpha, and prokineticin-2, and asking how they communicate to bring about particular circadian behaviors.

There is a third episode waiting to be written. Until recently, it was assumed that the SCN was the only circadian clock in the body. It now appears that there are clusters of clock cells in other structures in the brain and even in organs such as the lungs, heart, and liver (Focus May 3, 2002). “How do these other clock structures communicate—what are the rules?” Weitz asked. “Are they communicating back to the SCN? And what are they really doing for the animal?” The clock is already ticking on the search for answers. “We have lots of experiments along this line,” said Weitz.

—Misia Landau

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