Biomedicine

Sleep with the Fishes

Zebrafish larvae are a surprisingly compatible stand-in for humans as researchers test the next generation of insomnia drugs.

There’s a new guinea pig in the search for sleep-related drugs: the zebrafish. Researchers at Harvard University have developed a screening tool that tests the effects of thousands of compounds on zebrafish behavior in an effort to discover new pathways that govern sleep. The research, published this week in the journal Science, may result in new drugs to treat insomnia and other sleep-related disorders.

Sleepy head: Harvard scientists are using zebrafish as a model to find drug candidates for insomnia and other sleep disorders. Pictured above is the head portion of a zebrafish larva. The zebrafish brain is labeled in green.

Alexander Schier and his colleagues at Harvard developed an automated system to assess 60,000 distinct sleep-related behaviors in zebrafish, a tropical fish often used in scientific research. After screening 5,600 small molecules on the larvae, the team discovered 463 significant sleep-altering compounds, many of which have been known to have similar effects in humans.

“We didn’t expect as much conservation of the effects of drugs between humans and zebrafish,” says Schier, professor of molecular and cellular biology. “This was a proof of principle that many of the pathways found in humans are conserved in fish.”

Schier says such behavioral similarities may make zebrafish an ideal model for studying how and why humans sleep, mysteries that are largely unsolved. It’s still unclear what molecular mechanisms control sleep and wakefulness. Pinpointing these pathways, and finding drugs to block or promote them, is a major focus for many pharmaceutical companies–sleep drugs generate $7 billion in annual profits in the United States. However, the drug development process is tedious and expensive. Schier believes that testing drug candidates in zebrafish could be a cheap and straightforward alternative to conventional drug screening.

Typically, to test a drug, researchers first study its effects in cultured cells, looking to see if the drug binds successfully to a target receptor or molecule. They then advance the drug to animal experiments, testing behavioral effects in live subjects. But drugs that have certain effects in cultured cells often have unexpected side effects–or no effect–in a live animal.

“The advantage of zebrafish is that you can keep large numbers of animals in a very small space, and raise many, many animals relatively cheaply,” says Schier. Unlike flies and worms, which are often used in the early stages of pharmaceutical research, fish are vertebrates. “Much can be found in zebrafish that is relevant to mammals,” he says.

To screen the drugs, researchers pipetted single zebrafish larvae into a tiny well of a 96-well tray. Each well was injected with a drug, with one drug tested on 10 different larvae. They placed the tray in a recording chamber with infrared and white LED lights and a camera connected to computer software. After lining the tray up with a corresponding grid on the computer screen, researchers programmed the timing of light to simulate day and night. The camera recorded each fish’s activity over two days, and video tracking software plotted out each fish’s movements per second.

Z’s for zebrafish: Zebrafish larvae (above) are naturally transparent. Scientists hope to one day study the effects of sleep drugs on the brain and spinal cord, which can be seen in the image above as a long white structure stretching left to right.

Using clustering algorithms, Schier and his colleagues grouped fish into 60,000 distinct behavioral profiles, depending on various constraints. “When you turn off the light, how often are they active? When they are inactive, how long? That’s what we observe in the fish,” says Schier. “You can measure many different parameters, and that allows you to profile different drugs.”

Anti-inflammatories, such as cytokines, nonsteroidal anti-inflammatory drugs, and cyclosporine, had a surprising effect. Normally, these drugs induce sleep when taken to combat infection such as the flu. However, Schier found that when given to normal, healthy zebrafish, these compounds, or immunomodulators, made fish more active during the day.

“In disease, immunomodulators have been implicated in sleep,” says Schier. “We propose that maybe there’s some baseline function for these immunomodulators during normal sleep and wake cycles.”

Such findings could help researchers identify new molecular players involved in sleep and wakefulness. Irina Zhdanova, associate professor of anatomy and neurobiology at Boston University Medical School, studies the physiological mechanisms of circadian rhythms and sleep in zebrafish. Zhdanova says there are many sleep-related drugs on the market with substantial side effects; these effects might be avoided with better screening tools.

“The huge scope of drugs tested [by Schier’s group] shows that zebrafish-based tests can be effectively used to at least prescreen multiple classes of existing drugs and new candidate substances,” says Zhdanova. “[That is] certainly very helpful.”

In the future, Schier says, zebrafish could also be used as a model for testing drugs for human psychiatric diseases like schizophrenia and autism. The idea is to identify genes associated with the human disease, and try to engineer the same genetic defect in zebrafish. Researchers could then look for certain behavioral changes as a result, such as a fish’s sensitivity to touch, or its reaction to visual cues.

“Hopefully there would be a connection between the gene affected, and change in behavior, and one would try to correct the change in behavior by adding particular drugs,” says Schier. “That’s a bit science fiction at the moment, but it is possible.”

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