Fish catch zzzz
By Tim Stoddard
The gentle bubbling sound from hundreds of fish tanks in Irina Zhdanova’s laboratory could lull a person to sleep. The silvery striped zebrafish inside the shoe-box-size aquariums, however, are nodding off for other reasons: Zhdanova is investigating how the hormone melatonin regulates sleep in the fish, research that may someday help insomniacs get a good night’s rest.
The humble zebrafish, common in pet stores, has achieved biology stardom in recent years. It is now the organism of choice for studying human development, genetics, and a wide range of diseases. Zhdanova, a MED associate professor of anatomy and neurobiology, studies the biology of sleep and the role of melatonin in circadian rhythms, the daily cycles in physiological processes such as wakefulness and sleep. She recently discovered that zebrafish and humans have a lot in common when it comes to regulating their internal clocks: the diurnal fish have a pineal gland in their brains that secretes melatonin, which lulls them into a sleep-like stupor and affects the timing of the sleep-wake cycle.
In a recently renovated laboratory on the Medical Campus, Zhdanova and her colleagues are putting the finishing touches on a zebrafish facility containing several hundred tanks. It eventually will house tens of thousands of zebrafish, with adjacent rooms for microscopes and other equipment for studying the fish. The zebrafish lab is the first to be built at BU and for now is dedicated exclusively to Zhdanova’s research, but she’s eager to help other BU researchers build their own facilities.
A fish earns its stripes
Zebrafish research still is a relatively new field, but Zhdanova says its popularity “in the United States and abroad is exploding.” Zebrafish are in vogue for many reasons: they’re easier to keep than frogs, mice, and monkeys, and researchers can maintain large numbers of the one-inch-long fish within close quarters. They are prolific, laying about 200 eggs a week, and their clear embryos develop quickly and are ideal for observing developing organs. Researchers have sequenced nearly the entire zebrafish genome and are already studying human diseases in the fish. “Of course, nonhuman primates are the best models for sleep research, because they are our closest relatives,” Zhdanova says, “but maintaining them is expensive and labor-intensive, and a lot of ethical issues are rightfully involved in using them.” Because of this, Zhdanova’s research team focuses primarily on zebrafish, but also extends its most promising work at a separate facility with parallel studies in rhesus monkeys.
Specifically, Zhdanova wants to better understand how melatonin works at the molecular level to affect sleep, circadian rhythms, and cognitive function. Researchers have known about melatonin for nearly 50 years, but it’s still unclear how it interacts with certain brain structures and, perhaps, with other tissues in the body. The hormone can shift a person’s circadian clock forward or backward, tricking the body into thinking the previous night has been extended or the coming night has arrived early. “It’s nontoxic and its effect is very subtle,” she says. “It does not work like typical hypnotics that completely knock you out. We know a lot about the effects of melatonin, but we still don’t know how it works to promote sleep and maintain it.”
Zebrafish may give researchers a window on how and where melatonin works. “The beauty of these fish, in addition to many other things, is that they are transparent during development,” Zhdanova says. “Through the egg you can see the entire embryo. Within 48 hours after fertilization it is already swimming, and the larvae are also transparent. Under the microscope you can see all the structures of the body and the brain, especially if some are highlighted by fluorescent proteins or dyes.” Melatonin appears to interact differently with different cells, and Zhdanova wants to identify the different proteins that bind melatonin in the brain and in other tissues. To do this, she inserts a gene in the fish’s DNA that produces a fluorescent green protein when a nearby gene is activated. In this way, she’ll be able to see where the glowing melatonin goes in the fish, and which genes in the zebrafish genome encode the melatonin receptors.
More important, though, she can conduct these studies while the fish are alive and swimming. With a video monitoring system, she can monitor their behavior over time as the levels of melatonin rise and fall in their bodies.
Before Zhdanova began working with zebrafish four years ago, she was interested primarily in sleep in higher vertebrates. Born in Kiev, Ukraine, she trained as a medical doctor, earned her Ph.D. in behavioral physiology, and studied psychiatric diseases such as manic depression in St. Petersburg. She was impressed that “99 percent of these diseases are correlated with altered sleep patterns that might reflect their biological roots” and became interested in the complicated biochemistry involved in regulating sleep. She came to Boston for a postdoc at an MIT sleep lab and investigated the role of melatonin in sleep.
Zhdanova at first worked mainly with elderly patients who had age-related insomnia, and she showed that low doses of melatonin helped them fall asleep and sleep through the night. But as a “hobby project” in 2000, she and an undergraduate student developed an automated system for recording the behavior of zebrafish. Video cameras traced the fish’s movements, providing a comprehensive record of their activity day and night. She wanted to know if the fish respond to melatonin the way people do. “These larvae can actually breathe through their skin,” she says, “and can absorb a lot of things from the water, including melatonin. The interesting thing we saw was that melatonin had a very similar effect in the zebrafish as it does in monkeys and humans. It would slow them down, but they were not anesthetized. If you disturbed them even a little, they would wake up. Since then, I’ve loved zebrafish.”
In subsequent studies, Zhdanova proved that melatonin does in fact promote sleep in zebrafish and that it somehow affects several tissues at once, slowing down the heart and lowering body temperature. Her other research was showing that low doses of melatonin administered at night to children with insomnia stemming from severe neurological diseases also helped them get to sleep.
The goal now is to better understand how melatonin works, in the hope of someday finding safe and effective medicines for treating insomnia in people. “The combination of the two model organisms in our labs is in many respects ideal for doing this,” Zhdanova says. “We have two diurnal species that are at very different evolutionary stages. Molecular biology, genetics, drug discovery, and drug testing are excellent things to do in zebrafish, for example, because you can have excellent statistics. We can record simultaneously the behavior of 80 or 100 or 160 different fish. That’s impossible to do in humans, and it’s very difficult to do in monkeys.” But when a promising drug pans out in zebrafish, Zhdanova can then test it in monkeys.
The hope, she says, is that this powerful new model organism will shed light on a poorly understood behavior. “We still don’t really know what sleep is,” she says. “Its physiological function is still an enigma.”