Inside a series of tubes in a bright, warm room at Harvard Medical School, hundreds of fruit flies are staying up late. It has been days since any of them have slept: The constant vibrations that shake their homes preclude rest, cling as they might to the caps of the tubes for respite. Not too far away in their own tubes live other sleepless flies, animated with the calm persistence of those consigned to eternal day. A genetic tweak to certain neurons in their brains keeps them awake for as long as they live.
They do not live long. The shaken flies and the engineered flies both die swiftly — in fact, the engineered ones survive only half as long as well-rested controls. After days of sleeplessness, the flies’ numbers tumble, then crash. The tubes empty out. The lights shine on.
We all know that we need sleep to be at our best. But profound sleep loss has more serious and immediate effects: Animals completely deprived of sleep die. Yet scientists have found it oddly hard to say exactly why sleep loss is lethal.
Sleep is primarily seen as a neurological phenomenon, and yet when deprived creatures die, they have a puzzlingly diverse set of failures in the body outside the nervous system. Insufficient sleep in humans and lab animals, if chronic, sets up health problems that surface over time, such as heart disease, high blood pressure, obesity and diabetes. But those conditions are not what slays creatures that are 100% sleep deprived within days or weeks.
What does sleep do that makes it deadly to go without? Could answering that question explain why we need sleep in the first place? Under the pale light of the incubators in Dragana Rogulja’s lab at Harvard Medical School, sleepless flies have been living and dying as she pursues the answers.
On a cold morning this winter, Rogulja leaned over a tablet in her office, her close-cropped dark hair framing a face of elfin intensity, and flicked through figures to explain some of her conclusions. Rogulja is a neuroscientist and a developmental biologist by training, but she is not convinced that the most fundamental effect of sleep deprivation starts in the brain. “It could come from anywhere,” she said, and it might not look like what most people expect.
She has findings to back up that intuition. Publishing today in the journal Cell, she and her colleagues offer evidence that when flies die of sleeplessness, lethal changes occur not in the brain but in the gut. The indigo labyrinths of the flies’ small intestines light up with fiery fuchsia in micrographs, betraying an ominous buildup of molecules that destroy DNA and cause cellular damage. The molecules appear soon after sleep deprivation starts, before any other warning signs; if the flies are allowed to sleep again, the rosy bloom fades away. Strikingly, if the flies are fed antioxidants that neutralize these molecules, it does not matter if they never sleep again. They live as long as their rested brethren.
The results suggest that one very fundamental job of sleep — perhaps underlying a network of other effects — is to regulate the ancient biochemical process of oxidation, by which individual electrons are snapped on and off molecules in service to everything from respiration to metabolism. Sleep, the researchers imply, is not solely the province of neuroscience, but something more deeply threaded into the biochemistry that knits together the animal kingdom.
More Fatal Than Starvation
The first studies to investigate total sleep deprivation had a maniacal quality to them. In Rome in 1894, Maria Mikhailovna Manaseina, a Russian biochemist, made a presentation at the International Congress of Medicine about her experiments on 10 puppies. She and her lab assistants had kept the dogs awake and in constant motion 24 hours a day; within about five days, all the puppies had died. Sleep deprivation seemed to kill puppies much more quickly than starvation, she reported: “The total absence of sleep is more fatal for the animals than the total absence of food.”
Autopsies revealed that the puppies’ tissues were in bad repair, particularly in the brain, which was rife with hemorrhages, damaged blood vessels and other gruesome features. Sleep, Manaseina concluded, is not a useless habit. It does something profound for brain health.
More all-day, all-night dog walking followed. In 1898 Lamberto Daddi, an Italian researcher, published detailed drawings of the brains of dogs that had been sleep-deprived; he reported apparent degenerative damage in the brain, similar to that seen in dogs that had faced other stressors. Around the same time, the psychiatrist Cesar Agostini kept dogs in cages rigged with bells that jangled horribly whenever they tried to lie down and sleep, and in the 1920s researchers in Japan did something similar with cages studded with nails.
The studies, aside from their consistent cruelty, had a similar weakness: They had no valid controls. The dogs had died and their tissues looked abnormal — but was that truly because they had not slept? Or was it because nonstop walks and stimulation are inherently stressful? Separating the effects of sleeplessness from being kept on your feet until it killed you seemed impossible.
The Turntable Cage
It took decades for scientists to return to the question in a serious way. In the 1980s, Allan Rechtschaffen, a sleep researcher at the University of Chicago celebrated for his pioneering work on narcolepsy, began to design experiments that could separate the effects of overstimulation from those of sleeplessness. He devised a rat cage in the form of a turntable suspended over water. A divider ran down the middle, so animals could live on either side while the turntable floor beneath them spun freely. Into the device the experimenters put pairs of rats, one of which was destined to be denied sleep. Whenever that rat tried to rest, the scientists spun the table, nudging both rats awake and sometimes pushing them into the water.
This setup ensured that although both rats fell into the water equally often, the control rat could still catch some winks whenever the rat denied sleep was active. In fact, control rats managed to sleep about 70% as much as they normally would, suffering only mild sleep deprivation. The unluckier experimental rats got less than 9%, almost total sleep loss.
Both sets of rats were disturbed the same number of times. Both suffered the stress of falling into the water and having to clamber back out, dripping. But only the severely sleep-deprived rats began to decline. Their fur grew rough and disheveled, and it went from white to a mangy yellow. They developed lesions on their skin. They lost weight. After around 15 days on average, they died. Rechtschaffen had discovered a way to show that sleep loss itself really did kill.
For the graduate students running these experiments, the days were long. “The lab was in an apartment building, so you’d have a bedroom next to an animal testing room,” said Ruth Benca, a professor of psychiatry at the University of California, Irvine who worked with Rechtschaffen for some years. “They had bedrooms next to the rooms where their animals were being deprived so they could monitor around the clock.”
The work was challenging in other ways as well. “They were tough, tough experiments to do, psychologically, to put an animal through that,” said Paul Shaw, one of Rechtschaffen’s later graduate students and now a professor of neuroscience at Washington University in St. Louis. “The last seven days of the experiment, you’re working with this cloud over your head.” When his rats were just a day or two from death, the experimental protocol called for him to let them sleep and observe their electroencephalograms, or EEGs. Shaw recalls that as the monitor exploded with life, announcing the animals’ long-awaited slumber, he felt a weight fall from his shoulders. “To this day I can see it,” he said, speaking of the EEG readout. “I could put it in a frame up on my wall, and it could make me happy every time.”
But the work was also thrilling. “You have to believe in the outcome to do it. There’s no other way,” Shaw said. He arrived at the lab after students who had pioneered these experiments received their degrees and left, but he still heard their stories at meetings, where they reminisced about the excitement. “No one wanted to get their Ph.D.,” he recalled, because if they could stay, “they all thought that tomorrow, they’d discover the function of sleep.”
Confusing Causes of Death
Rechtschaffen’s experimental successes should have finally enabled scientists to see how insufficient sleep kills, which might have led to bigger insights into what makes sleep so indispensable. But when the researchers performed autopsies on the animals, what they found mostly just added to the confusion. There were few consistent differences between the control rats and those that died from lack of sleep, and no sign of what had killed them. The deprived rats were thin and had enlarged adrenal glands, but that was about it. “No anatomical cause of death was identified,” the researchers concluded.
Observations of the animals’ behavior showed something more interesting. “Animals [chronically] sleep-deprived under these carefully controlled conditions would increase their food intake two and three times normal amounts, and lose weight,” said Carol Everson, a professor of medicine and neurobiology at the Medical College of Wisconsin who was one of Rechtschaffen’s graduate students. “We did all sorts of metabolic studies to try to find out if there was an impairment we could detect.”
There was a strong feeling in the sleep field, however, that answers about sleep’s most basic functions would be found in the brain. John Allan Hobson, a prominent Harvard Medical School sleep researcher, had just published a paper in Nature with the title “Sleep is of the brain, by the brain and for the brain.” As Shaw recalled, “This captured the zeitgeist of the entire sleep community.”
Indeed, the vast preponderance of sleep research today still centers on the brain and on subjects like cognitive impairment. Sleep loss does alter metabolism in humans — there are connections to diabetes and metabolic syndrome — but public health researchers are often the only ones who concern themselves with it. Those looking to understand the fundamental purpose of sleep rarely seek answers in metabolism or other chemical processes.
Reactive Oxygen Species
The neurons involved in regulating sleep are a focus of Rogulja’s work. But the fact that sleep loss impairs circulation, digestion, the immune system and metabolism made her curious about whether these were downstream effects of neurological problems, or if they were independent. “It seems like it can’t be all about the brain,” she said.
She knew about the Rechtschaffen experiments — “real classics” — and that there had been few follow-ups. Once it was established that total sleep loss kills, using deprivation to study sleep’s purpose had fallen by the wayside. In the intervening decades, however, fruit flies had become a major model organism in the sleep field, because their genetics are widely understood and easy to manipulate and they are inexpensive to keep in the lab. Many sleep discoveries first made in flies have been verified in mammals. With the rise of flies as proven test subjects, when Rogulja became curious about terminal sleep deprivation, it again seemed like a plausible thing to study.
When the postdoctoral researcher Alexandra Vaccaro arrived at Rogulja’s lab in 2016, the two came up with a plan. First, from other laboratories they obtained flies genetically engineered to have temperature-sensitive channels in certain neurons. Above 28 degrees Celsius, the channels opened and stayed open, keeping the neurons activated and the flies awake. With the channels closed, the flies enjoyed normal 110-day life spans. With the channels open, they started dying of total sleep deprivation after only 10 days or so, and they were all dead within 20 days.
Intriguing patterns emerged as Vaccaro performed tests. If she closed the channels and allowed the flies to sleep on day 10, they recovered and lived as long as controls. But if she deprived them again five or 10 days later, they died: Whatever damage had accrued during their initial sleeplessness had apparently not yet been repaired. It took a full 15 days of sleeping normally before they could be sleep-deprived again without immediately dying.
When Vaccaro dissected flies at various levels of deprivation, their tissues all seemed unharmed, with one very marked exception: Their guts were thick with reactive oxygen species (ROS), molecules with an oxygen atom that bears a spare electron. Some ROS are produced in the normal course of organisms’ respiration, metabolism and immunological defense, sometimes for specific functions and sometimes as byproducts. But if ROS are not swept up by antioxidant enzymes, they become extremely dangerous, because that unbalanced oxygen rips electrons away from DNA, proteins and lipids. Indeed, after ROS appeared a week into the flies’ sleep deprivation, markers of oxidative damage soared — a sign that cells were in crisis.
ROS levels peaked on the 10th day of deprivation. When flies were allowed to start sleeping normally, it took about 15 days for their ROS levels to get close to baseline again — the same time it took for flies to be able to withstand renewed deprivation.
Rogulja and Vaccaro had not expected such a clear result within mere months of starting the project. It was so easy to see that it made them instantly skeptical. When Rogulja showed preliminary data at a meeting of Pew Biomedical Scholars, their excitement unnerved her a little. “It’s never like that,” she said, preferring to be cautious about the findings.
As a result, over the last three years Vaccaro and Rogulja, along with the postdoctoral researcher Yosef Kaplan Dor, have been working to poke holes in this apparent connection between oxidation and sleep loss. They deprived flies of sleep by a more traditional method — shaking the tube containing them every two seconds — and checked to see whether levels of ROS correlated with levels of sleep loss; they did. The team looked at flies with mutations that promoted sleep or wakefulness; the sleep-deprived flies had ROS in their guts. Conversely, no ROS showed up in the guts of a strain of mutant flies known to tolerate a lack of sleep.
The strangest, most exciting period of the project may have been when the researchers decided that if oxidation from ROS was killing the flies, perhaps they should give the flies antioxidants. It sounded like a zany health food experiment, but Vaccaro searched out antioxidants known to work in flies, then fed them to the insects. To the researchers’ surprise, the lethally sleep-deprived flies reached a normal fly life span. The same thing happened when they raised levels of antioxidant enzymes in the gut (but, tellingly, not when they did it in the nervous system).
“I cannot imagine having more fun in science,” said Rogulja of that summer. “My whole family, and the whole lab, we would all gather around in the morning, once we started giving them these antioxidants: ‘They’re alive!’ And not only were they alive, they looked good.”
Vaccaro and a technician in the lab, Keishi Nambara, along with collaborators in the laboratory of Michael Greenberg at Harvard, performed a pared-down version of the fly experiment with mice. They kept the mice awake for up to five days in a cage with a rotating bar that gently pushed the animals to make them move. In the animals’ guts, the telltale glow of ROS appeared.
A Gut-Level Problem
For Shaw, the team’s new paper is very interesting. “It’s super exciting to see they’ve harnessed the power of genetics,” he said. “We gave up on the whole project of sleep-depriving flies [mechanically] till they die because they’re long, hard experiments to do,” and it’s difficult to control for stress. Because the study uses both genetic and mechanical means of sleep deprivation, it sidesteps that issue. “It’s fantastic, fantastic. … I was very impressed,” he said. “I thought it was very well controlled.”
Just what the findings mean still needs to be explored. They suggest that sleep is vitally important to the body’s regulation of oxidation, particularly in the gut, and that this is likely to have widespread consequences in the body. As Rogulja and Vaccaro write in their new paper: “Prevention of death by a single means would argue that the gradual collapse of nearly all major bodily functions derives from a common origin.” In the flies they studied, antioxidants were the single means.
Their findings dovetail with a stream of previous reports that have linked oxidation and insufficient sleep, in particular those of Everson, who grew interested in metabolism while in Rechtschaffen’s lab. Everson felt early on that while the brain is a regulator of sleep, there’s more to sleep than neurology. In sleep-deprived rats, she observed signs of immunological failures and bacteria in tissues that should have been sterile. Then in 2016, she and her colleagues reported that they had found oxidation in the livers, lungs and small intestines of sleep-deprived rats. Markers of inflammation are often found floating around in tissues after sleep deprivation, Everson said, but their source has never been clear. If oxidation is out of control somewhere in the body, the resulting crisis of cellular damage could cause that boost.
Everson also found that the guts of sleep-deprived rats grew leaky, releasing bacteria into the animals’ bloodstreams. But from what Rogulja and her colleagues have seen, the flies’ guts do not seem to leak. ROS also did not seem to be rising in any of the other tissues they examined. And although the flies sometimes ate more when they were sleep-deprived, the ROS level in their guts looked the same regardless.
It’s unclear how all these puzzle pieces concerning oxidation in rats and flies might fit together, and Giorgio Gilestro, a sleep researcher at Imperial College London, notes that while these experiments make it clear that the ROS are killing the flies, that doesn’t necessarily mean the same thing killed the rats. A small study of humans who lost sleep showed that the makeup of their gut microbiomes, the bacteria that live in the intestines, shifted after insufficient sleep, an intriguing if preliminary finding drawing another link between sleep and the gut.
Still, perhaps the most pressing issue is that no one knows where the ROS are coming from, and why they accrue in the gut. What process — metabolic or otherwise — is generating them? Does sleep deprivation cause ROS to be overproduced? Or does it interfere with some process that normally clears them away? And why would ROS be linked to sleep anyway? Rogulja is planning experiments to explore some aspects of these questions.
Behind all this is the astonishing, baffling breadth of what sleep does for the body. The fact that learning, metabolism, memory, and myriad other functions and systems are affected makes an alteration as basic as the presence of ROS quite interesting. But even if ROS is behind the lethality of sleep loss, there is no evidence yet that sleep’s cognitive effects, for instance, come from the same source. And even if antioxidants prevent premature death in flies, they may not affect sleep’s other functions, or if they do, it may be for different reasons.
The flies that never sleep and their glowing guts remind us that sleep is profoundly a full-body experience, not merely a function of the mind and brain. In their deaths may lie some answers as to why sleeplessness kills and — potentially, tantalizingly — what sleep does to link disparate systems throughout the body. Shaw, for one, is interested to see what happens next in Rogulja’s lab. “It’s a super important question,” he said, “and they’ve come up with a way to address it.
Corrections: June 10, 2020
Dragana Rogulja was originally described as a developmental neuroscientist by training; her doctorate is in developmental biology.
The description of the turntable experiment in the Rechtschaffen laboratory was revised for accuracy.