It took a decade’s worth of work—and probably a few sleepless nights—but for the first time, researchers have identified sleep patterns in the brains of tiny zebrafish, and those patterns look remarkably similar to the brain activity in sleeping humans.
As scientists report today in the journal Nature, evidence of similar sleep patterns in both fish and mammals may offer clues about the evolution of sleep in our common ancestors, which could in turn help us better understand the biological function of nodding off.
“Sleep is a huge mystery in neuroscience,” says William Joiner, a biologist at the University of California, San Diego, who studies sleep in fruit flies but was not involved with this research. Plenty of work has asked why we do it, and “people really haven’t settled on a good answer.”
For the new study, the team used advanced imaging techniques to watch as zebrafish fell asleep, and they found that the tiny fish cycle between sleep states similar to what we have in humans: rapid eye movement, or REM, sleep and non-REM sleep. This pattern has been seen before in a wide range of mammals, birds, and lizards, but this is the first time it’s been spotted in a fish.
Based on our understanding of the evolutionary relationships between fish and mammals, the team suggests that REM-like sleep states evolved more than 450 million years ago, making this type of sleep a deeply held biological phenomenon.
“We share a backbone, but we share much more than that,” says study coauthor Philippe Mourrain, a neuroscientist at Stanford University. “It makes it easier to understand sleep and what it does in ourselves.”
Other experts say that the methods the authors used set a new standard in studying sleep, with Joiner calling the paper a “technological tour de force.” But not everyone is convinced that it actually reveals much about sleep evolution.
“I have my doubts that you can draw a straight lineage from fish to mice and birds and reptiles and humans,” says Paul Franken, a neuroscientist at University of Lausanne in Switzerland who studies sleep in mice.
Sleeping with the fishes
Scientists already knew that zebrafish could sleep simply from watching their behavior. But the gold standard to study sleep is using physiology, Franken says.
Lead author Louis C. Leung, a neuroscientist at Stanford University, built the microscope responsible for the complex imaging done for the study. Most body activity is choreographed by an intricate network of nerve cells, or neurons. When neurons are active, calcium levels rise inside, so researchers genetically engineered the zebrafish to include a protein that would flash fluorescent green when it detected calcium, indicating an area of the body is active.
Then, the real work began. The team focused on zebrafish that were just two weeks old, because the fish are transparent at this age. This allowed the researchers to observe the brain and other activity inside the body without cutting into the animal or implanting electrodes, Leung says.
They immobilized the tiny fish by plopping it in a gelatin-like substance under the microscope and then started looking at key physiological components: brain activity, heart rate, muscle activity, and eye movement.
Almost immediately, the patterns of active and nonactive neurons started to stand out, revealing the “fingerprint” of activity similar to REM and non-REM sleep cycles.
“It just about took my breath away,” Leung says.
To confirm the activity patterns were really sleep, the researchers then prevented the fish from taking “naps,” creating some very sleepy fish. When they tested the sleep-deprived fish, they found the same neuronal patterns, but more of them. In addition, when the fish were in a non-REM-like state, their heart rates halved and body muscles relaxed.
Compared to the sleeping states, the awake brain in the zebrafish was very noisy, with chaotically flashing neurons, Leung says.
A sleepy common ancestor
For many organisms, environmental factors like temperature influence the duration and intensity of sleeping—humans sleep longer in cooler temperatures, for instance. Mammals have to thermoregulate, adjusting their own body temperatures to stay warm or to cool off, and thermoregulation has long been associated with sleep. But because the zebrafish sleep state is similar to ours, it suggests this type of sleep existed before the rise of thermoregulation, the team argues.
However, it’s hard to relate the results of this study to mammals, because there is so much evolutionary time between them and fish, says Jerry Siegel, a sleep scientist at University of California in Los Angeles. Sleep is almost ubiquitous among animals, he acknowledges, but it varies a lot in mammals.
“You can’t just say sleep is sleep,” he says. Just among mammals, the amount of sleep required ranges from three to 20 hours a day. REM sleep can be non-existent, as it is in many cetaceans. Or it may constitute a large portion of sleep, lasting up to 8 hours in mammals like the platypus.
In addition, the sleep signatures were found in very young fish, Siegel says, and these results don’t necessarily apply to adults. Across the animal kingdom, infants sleep differently from their parents.
The future of sleep?
Other experts are more optimistic, especially about the techniques used in the paper. The neural signatures “didn’t have to be there in fish, but he found them,” says Paul Shaw, a sleep scientist at Washington University in Saint Louis who was not part of the study. “I do think it’s surprising. It’s super cool!”
Shaw and others specifically praise the detailed imaging used to watch sleep happen on such a scale.
“Seeing is believing, and that’s what I really like about this technology,” Shaw says. “You don’t have to infer sleep [in this study].”
The advance could be particularly valuable for health professionals seeking to design new drugs to combat the growing epidemic of sleep deprivation in many countries. Better sleep-enhancing drugs could provide some relief for people who struggle to drift off. By implementing these techniques in the future, we can potentially better screen drugs to see if they activate the right cells, so that patients wake up feeling refreshed, Leung says.
“That you can see the individual neurons in a live animal, and watch how it responds to different drugs, is incredible,” Franken says. “This is a big advancement.”