Friday, January 21, 2005

ClockNews #12: Zebrafish Research at BU

MED researcher probes sleep at new zebrafish facility

"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

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
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

The video-monitoring system was invented by Dr.Greg Cahill (at U. of Texas at Houston, I believe). The tiny fish larvae are placed in a 96-well plate and a camera is positioned above. A special piece of software was developed to analyze daily rhythms of activity (random swimming around the well) for each individual fish. Behavioral output such as gross motor activity has its pros and cons, but works perfectly well for the kind of research that Dr.Zhdanova is doing.

Better sleep

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 same property of permeability of the zebrafish skin to melatonin was used by another researcher, Dr.Keith Barrett (then at The Howard Hughes Center for Biological Timing at Northwestern University in Chicago, IL), to develop a method for monitoring physiological circadian rhythms in zebrafish. He also placed fish larvae in a 96-well plate, but has modified the plate somewhat, allowing the water/medium to flow through each well at a steady rate (via tiny input and output tubes). The medium coming out of each well was collected by a fraction collector and each hour the test tubes were automatically moved and the medium would start flowing into the next set of test tubes. This way, each tube contained the medium collected from a single well (fish) over a period of one hour.

As the zebrafish produce melatonin, and the melatonin permeates the skin, it naturally leaks out into the medium. Thus, Keith only had to assay the samples for melatonin concentrations in order to monitor daily rhythms of melatonin synthesis and release in zebrafish. While a great system, overcoming the problems of a gross behavioral output, it is unsuited for Dr.Zhdanova's work as she is supplementing melatonin into the medium in the first place.

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." "


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