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Awake All Night
Scientists use sleep deprivation in flies to study the function of sleep
By Kate Dalke

Featured article.

We've all pulled an all-nighter. And we've all paid for our sleepless night the next day. Despite its importance to our lives and our inability to get enough of it, sleep is one big mystery to researchers. No one knows the purpose of sleep.

The fruit fly Drosophila melanogaster.

Some of the first evidence that sleep is essential to life came from studies of rats in the early eighties. Rats that were deprived of sleep for about two weeks died. The result was dramatic and seemed important, but the researchers did not know why the rats died.

"At the end of the day we didn't know what went wrong," says Paul J. Shaw, a sleep researcher at the Neurosciences Institute in San Diego, California.

Now, using genetic and genomic strategies, fruit fly researchers have begun to dissect the biology of sleep. In a new study, scientists have identified two genes that regulate the balance of sleep in flies, both of which have human counterparts. They are the cycle gene, which regulates the fly's circadian clock, and the heat-shock gene Hsp83, which protects the body's proteins against stresses. The study confirms that sleep is vital for the fruit fly's survival.

"Identifying two genes that affect the regulation of sleep is a kind of foot in the door—especially when you don't know where to start," says Ralph J. Greenspan, one of the researchers on the study at the Neurosciences Institute.

‘I think we have a hook now, although the pathway we've identified is extremely complex.’

At the institute, Shaw and colleagues investigated what happens when a fly does not get enough sleep one night and then tries to make it up the next night. The state of a balanced sleep-wake cycle is called homeostasis. The unfortunate truth is that most humans and flies never fully recover lost sleep—only about 30 to 40 percent.

Some mutant flies, the researchers discovered, make up unusual amounts of sleep after being deprived. In the study, flies with mutations in either of the two genes made up three times the amount of lost sleep during the next day. In addition, both types of mutants died after sleep deprivation for 10-12 hours, while normal flies did not.

Another finding of the study was that heat shock proteins could protect the flies against the harmful effects of not sleeping. To activate the heat shock protein, researchers heated cycle mutant flies prior to sleep deprivation. Fewer of these flies died compared to typical sleep-deprived cycle mutants.

"I think we have a hook now, although the pathway we've identified is extremely complex," says Shaw. He and his colleagues write in Nature that the 'protective' quality of the heat-shock proteins "provides a first hint about the functional targets of sleep and its molecular mechanisms."

The new research builds upon previous studies of sleep in fruit flies. Two years ago, scientists first discovered that fruit flies do indeed sleep. Sleepy flies and sleepy humans are a lot alike. They sack out at night for about eight hours, caffeine keeps them awake, and they are hard to rouse while dozing.

Fruit flies even have their own bedtime routine. Around dusk, flies in the laboratory eat something, groom themselves and then go looking for peace and quiet. "They turn away from food and other flies to find a more private place to rest," says Joan C. Hendricks, who studies Drosophila sleep at the University of Pennsylvania in Philadelphia. She co-authored one of two papers published in 2000 describing sleep in fruit flies.

Male fruit flies need not apply for sleep studies. Researchers prefer female flies because their male counterparts are more likely to take mid-day siestas. Female fruit flies tend to sleep at night and this makes them more consistent models for study.

The sleepless machines. A prototype of the fly agitators now used at the University of Wisconsin-Madison. View larger

To keep flies awake for studies, researchers have come up with some creative technologies. A machine called the Sleep Nullifying Apparatus rhythmically tilts flies back and forth, displacing sleepy flies ten times per minute. This automated technology is a far cry from the original method of sleep deprivation—scientists gently handling fruit flies or tapping on their glass jars.

"You have to find the right technique" for keeping flies awake, says Ciara Cirelli, a sleep researcher at the University of Wisconsin-Madison. Her laboratory has shiny, metallic fly agitators that continually rotate to prevent flies from dosing.

Cirelli was at the Neurosciences Institute two years ago and was part of a team that compared gene expression in flies and rats. The researchers found that the same minority of genes—less than one percent—in both species were differentially expressed between the cycles of sleeping and waking.

This finding was made using 'differential display,' a time-consuming and labor-intensive method of examining gene expression. More recently, sleep researchers have adopted genomic strategies to identify genes of interest. The Drosophila genome is available on a microchip, and researchers can do genome-wide surveys of genes involved in sleep.

A place to rest. Fruit fly monitors at the University of Wisconsin-Madison keep flies in a stable environment before sleep deprivation. Each monitor holds 32 tubes, which are each home to one fly.

At the University of Wisconsin, Cirelli is using microarrays to profile gene expression in fruit flies, rats and humans. They have discovered unique patterns of gene expression in rats with long-term sleep loss that do not resemble short-term deprivation or spontaneous wakefulness. With the microarrays, they have found an enzyme, Arylsulfotransferase, whose level of expression in rodents is proportional to the amount of sleep deprivation.

To learn about human sleep, the University of Wisconsin team is analyzing gene expression in individuals who died of fatal familial insomnia. Patients with this disease appear to eventually die of sleep loss. The researchers are analyzing gene expression in post-mortem tissue from the cerebral cortex. With its patient population, the study may be unique in that scientists can investigate genes involved in sleep loss in humans.

Cirelli also heads a project to create mutant flies that get by on little or no sleep. Her laboratory is screening 100 mutant flies per week to identify those that need only two hours per night (compared to the typical eight hours). The project, which is funded by the US Department of Defense, has a goal of screening 5,000 different mutants.

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Shaw, P.J. et al. Stress response genes protect against lethal effects of sleep deprivation in Drosophila. Nature 417, 287-291 (May 16, 2002).
Cirelli, C. How sleep deprivation affects gene expression in the brain: a review of recent findings. J Appl Physiol 92 394-400 (January 2002).
Greenspan, R.J. et al. Sleep and the fruit fly. Trends Neurosci 24, 142-145 (March 2001).
Shaw, P.J. et al. Correlates of sleep and waking in Drosophila melanogaster. Science 287, 1834-1837 (March 10, 2000).
Hendricks, J.C. et al. Rest in Drosophila is a sleep-like state. Neuron 25, 129-138 (January 2000).

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