Insomniac fish help understand the genetics behind sleep disorders

Scientists at the Stanford University School of Medicine have hooked a fish that suffers from insomnia in their quest to understand the genetics behind sleep disorders.

The findings, to be published in the Oct. 16 issue of the Public Library of Science-Biology, show that even zebrafish - a common aquarium pet - can have a genetic mutation linked to sleep problems. The work represents a milestone in sleep research by Emmanuel Mignot, MD, PhD, who also uncovered the genetic cause of narcolepsy in dogs.

Because most fish lack eyelids, many people have wondered whether fish can even nod off. The paper from Mignot's team provides proof that they do, and that zebrafish are a powerful new animal model for studying sleep disorders.

Zebrafish are all the rage among developmental biologists because, compared with mice, they are inexpensive to breed. And unlike cheaper fruit fly and worm models, fish have a backbone - thereby better representing the human nervous system. And their babies reveal many details because they are see-through.

"The fact that zebrafish larvae are transparent means you can look directly at their neuronal network, even in living fish," said Mignot, professor of psychiatry and behavioral sciences. "The idea is to try to use this as an entry point to understand the neurobiology of sleep regulation."

Mignot's laboratory found the gene responsible for narcolepsy in Dobermans and Labradors in 1999, helping reveal how the disorder occurs in humans. Narcolepsy affects about one in 2,000 people, with few recognizing their sleepiness as a medical problem. Symptoms include not only cataplexy - the sudden weakening of muscles that can result in a person's collapse - but also daytime drowsiness and irregular sleep at night. Mignot found that neurons in the brain's hypothalamus, a region controlling such basic behaviors as hunger and sex, secrete a neuropeptide called hypocretin.

Narcoleptic dogs have neurons that lack a working receptor for hypocretin, but later investigations found that it doesn't work the same way in humans. People with narcolepsy instead have an abnormally low amount of the neuropeptide in their spinal fluid.

Fish provide a cheaper, easier and faster way to find mutants to offer insight into how the wiring of hypocretin neurons in the brain affects sleep, said senior research associate Philippe Mourrain, PhD, who led the zebrafish research team. "The only way to answer these questions is to use a genetic model," Mourrain said. "We were lucky enough to isolate a mutant in zebrafish with the same kind of mutation that has been isolated in narcoleptic dogs."

The researchers' first task entailed precisely characterizing how normal zebrafish snooze. The paper's first author, postdoctoral scholar Tohei Yokogawa, PhD, trained infrared lights and cameras on aquariums to monitor zebrafish in the dark. After watching hours of footage, Yokogawa noted that zebrafish drooped their tail fin when motionlessness and spent most of the night just under the water's surface or at the tank's bottom.

To find out if the droopy-tailed fish were asleep, Yokogawa checked to see if they experienced sleep rebound, the drive to try to catch up on lost sleep, after being sleep-deprived. So first he had to make sure the fish stayed awake. Tapping on the aquarium walls and using an underwater speaker didn't work, but he found a gentle electrical pulse kept fish active. He then created a computerized system to stimulate a fish each time it started to doze off. Once a sleep-deprived fish returned to a peaceful, dark aquarium, it compensated for lost rest with longer napping.

Unfortunately, some of the researchers had to stay awake along with the fish. "Originally, we didn't have the automated sleep-deprivation system, so I manually sleep-deprived them, becoming sleep-deprived myself," Yokogawa added.

The new model has already provided insights into the function of sleep-regulating molecules and brain circuits in zebrafish. Compared with normal zebrafish, the sleep mutants with neurons lacking hypocretin receptors experienced something akin to insomnia rather than narcolepsy. Although in dogs the loss of the receptor results in full narcolepsy, in zebrafish only nighttime activity was affected. Overall sleep decreased 30 percent in mutant fish, and when they finally did drift off, they remained asleep only half as long as normal fish.

Future research will search for fish mutants that might oversleep or lack sleep completely, in hopes of discovering new regulatory molecules and brain networks passed on through evolution to humans. "Many people ask the questions, 'Why are we sleeping?' and, 'What is the function of sleep?'" Mignot said. "I think it is more important to figure out first how the brain produces and regulates sleep. This will likely give us important clues on how and maybe why sleep has been selected by natural evolution and is so universal."

The Howard Hughes Medical Institute and the McKnight Foundation funded the study. The other authors are graduate students Wilfredo Marin, Jian Zhang, Guillaume Pézeron; research associate Juiette Faraco, PhD; postdoctoral scholar Lior Applebaum, PhD; and professor Frédéric Rosa, PhD, at the Ecole Normale Supérieure in Paris.