20,000 neurons keep steady time when working together
Alexis Webb enters a small room at Washington University in St. Louis with walls, floor and ceiling painted dark green, shuts the door, turns off the lights and bends over a microscope in a black box draped with black cloth. Through the microscope, she can see a single nerve cell on a glass cover slip glowing dimly.
The glow tells her the isolated nerve cell is busy keeping time.
Webb, a graduate student in the neuroscience program, working with Erik Herzog, Ph.D., associate professor of biology in Arts & Sciences; Nikhil Angelo, an undergraduate biology major; and James Huettner, Ph.D., associate professor of cell biology and physiology in the School of Medicine, has demonstrated that individual cells isolated from the biological clock can keep daily time all by themselves.
However, by themselves, they are unreliable. The neurons get out of synch and capriciously quit or start oscillating again.
The biological clock, a one-square millimeter area of the brain called the suprachiasmic nucleus, or SCN, just above the roof of the mouth and atop the crossing of the optic nerves, comprises about 20,000 neurons.
These cells, remarkably, contain the machinery to generate daily, or circadian, rhythms in gene expression and electrical activity. But the individual cells are sloppy and must communicate with one another to establish a coherent 24-hour rhythm, says Herzog.
These features make the SCN a flexible clock that can reset to stay in synch in an ever-changing environment. The underlying sloppiness is probably what allows us to adjust to local time when we cross time zones and to vary our sleep cycles with the season, say the WUSTL researchers.
The research is being published the week of Sept. 7 in the online Early Edition of the Proceedings of the National Academy of Sciences.
"We've known for more than 15 years that unicellular organisms like cyanobacteria can keep 24-hour time, and isolated cells from the marine snail eye can as well," says Herzog. "But nobody was sure whether individual cells in vertebrates are circadian pacemakers."
The SCN includes many kinds of neurons that make different neurochemicals and connections within the SCN and to other parts of the brain.
"Some scientists felt that all of the cells in the SCN would be intrinsically rhythmic and that there was nothing special about any of them," says Herzog. "Some thought that none of the cells would be rhythmic and that the rhythm arose instead from their network interactions, and a third group thought specialized SCN neurons would be rhythmic and the others wouldn't be at all capable. Our experiments proved all three hypotheses wrong."
Capturing the rhythm
Webb digested slices of mouse SCN with enzymes to isolate individual neurons and then plated the cells sparsely on a dish. "The neurons will actually attach to the glass and grow," says Webb. "And as long as you give them all of the nutrients they need, they'll live for months."
The cells had been genetically engineered to glow whenever they expressed the time-keeping gene Period 2. (The cells came from transgenic mice where the Period 2 gene had been linked to one found in firefly tails.)
The rhythmically waxing and waning glow was detected by a camera designed to capture the light from distant stars and so sensitive that it will register the passage of even a single cosmic ray.