‘If it’s alive, it sleeps.’ Brainless creatures shed light on why we slumber | Science

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A version of this story appeared in Science, Vol 374, Issue 6567.

Dive among the kelp forests of the Southern California coast and you may spot orange puffball sponges (Tethya californiana)—creatures that look like the miniature pumpkins used for pies. No researchers paid them much mind until 2017, when William Joiner, a neuroscientist at the University of California (UC), San Diego, decided to look into whether sponges take naps.

That’s not as silly a question as it seems. Over the past few years, studies in worms, jellyfish, and hydra have challenged the long-standing idea that sleep is unique to creatures with brains. Now, “The real frontier is finding an animal that sleeps that doesn’t have neurons at all,” says David Raizen, a neurologist at the University of Pennsylvania (UPenn) Perelman School of Medicine. Sponges, some of the earliest animals to appear on Earth, fit that description. To catch one snoozing could upend researchers’ definition of sleep and their understanding of its purpose.

Scientists have often defined sleep as temporary loss of consciousness, orchestrated by the brain and for the brain’s benefit. That makes studying sleep in brainless creatures controversial. “I do not believe that many of these organisms sleep—at least not the way you and I do,” says John Hogenesch, a genome biologist at Cincinnati Children’s Hospital Medical Center. Calling the restful, unresponsive state seen in jellyfish and hydra “sleeplike” is more acceptable to him.

But others in the field are pushing for a much more inclusive view: that sleep evolved not with modern vertebrates as previously assumed, but perhaps a half-billion years ago when the first animals appeared. “I think if it’s alive, it sleeps,” says Paul Shaw, a neuroscientist from Washington University in St. Louis. The earliest life forms were unresponsive until they evolved ways to react to their environment, he suggests, and sleep is a return to the default state. “I think we didn’t evolve sleep, we evolved wakefulness.”

If that’s true, sleep in humans, rodents, and other vertebrates is a highly evolved behavior—one adapted to each organism’s needs and lifestyle. Gleaning insights into its basic function from those species could be difficult. Earlier evolving creatures, with fewer cell types, less complicated molecular pathways, and simpler behaviors may reveal sleep in its most fundamental form.

So, some sleep researchers have turned to invertebrates such as fruit flies and roundworms—and most recently to sponges and another early-evolving group, placozoans. Already, their work is driving home two key new insights: that sleep’s benefits extend far beyond the brain, and that muscles, the immune system, and the gut can all have a say in when and how sleep occurs. That work “might change our focus from studying sleep’s role in complex cognitive processes to how it impacts basic cellular function,” says Alex Keene, a neurogeneticist at Texas A&M University, College Station.

A new picture of what controls sleep might also lead researchers to new treatments for sleep disorders, says Amita Sehgal, a neuroscientist at UPenn’s Chronobiology and Sleep Institute. “The hope is that what we learn will be relevant to understanding why some people can’t sleep and also how disrupted sleep might affect their health and performance.”

The earliest studies of sleep defined it by how it changes human behavior: We lie down, close our eyes, remain motionless, and become oblivious to the outside world. Also obvious are the consequences of skipping sleep: We lose our ability to function, struggling to focus in a meeting or dozing off at the wheel.

By the 1950s and ’60s, researchers were converging on a definition of sleep based on polysomnography, a combined measure of brain activity, eye movement, and muscle tone that became a gold standard. Neuroscientists figured out how to capture brain activity from electrodes on the surface of the head and discovered that human sleep has two major stages: rapid eye movement (REM), a more active stage in which dreaming occurs; and non-REM, defined by slow, synchronous waves of electrical firing.

Behavioral and physiological tests have revealed how varied sleep can be in the animal world. Cows and other large grazing mammals sleep standing up. Some marine mammals sleep while swimming and some seabirds catnap while flying, letting one half of the brain doze while the other keeps working. Bats sleep about 20 hours a day; wild elephants as few as two. Most of the animals studied with electrical recording techniques have at least two stages of sleep, though the brain activity characterizing these stages can vary. The color changes of the octopus as it sleeps suggest it, too, has several sleep stages.

The signs of sleep

By looking broadly for behaviors characterizing sleep in humans and other organisms, researchers are finding that most animals, even very simple ones, have a restful state. How well each creature satisfies these criteria is controversial, but the work is expanding our understanding of the role and control of sleep, even in humans.

Many creatures, from sponges to fish to humans, show various signs of sleep.
N. Desai/Science

By the turn of the 21st century, evidence of sleep outside mammals prompted researchers to start to work down the animal tree of life to evolutionarily older species. They had to confront the question of how to define sleep in these simpler species. A sleeping jellyfish looks a lot like a waking one, after all, and is nearly impossible to outfit with electrodes. Researchers must instead recognize where and when simple creatures seek respite and find a behavior that ceases when they sleep. Studies must also poke or otherwise bother the animals to make sure they are unresponsive—and to see whether being forced to stay awake has consequences.

In 2017, Michael Abrams and two other California Institute of Technology graduate students devised such tests for Cassiopea, known as the upside-down jellyfish because it tends to stay near the shallow sea floor, pulsing with its tentacles pointing up so more light reaches the photosynthetic microorganisms it relies on for energy. The students observed that at night, this motion slowed from 60 pulses per minute to 39.

To see whether the jellyfish were really asleep, they built a false bottom to the aquarium and lowered it—essentially “pulling the rug out” from under the creatures. At night, the groggy jellyfish were slower to react and swim to the new bottom than in the day. And when the team disturbed the jellyfish by pulsing currents of water over them, the animals were less active the next day—as if having to recuperate from sleep loss. Finally, the drug melatonin, an over-the-counter sleep remedy, slowed their pulses to nighttime speeds. All this without a real brain: Jellyfish have a ring of nerve cell clusters around the rim of their bells.

Recently, researchers caught another brainless creature napping: Hydra vulgaris, a stationary freshwater relative of jellyfish. Taichi Itoh, a chronobiologist at Kyushu University, and colleagues filmed these centimeter-long animals as they wiggled their tentacles during 12-hour periods of light and dark in the lab. In the dark, the hydra were less active. Other researchers probing sleep in simpler animals have also adopted definitions based on behavioral changes such as reduced responsiveness.

More recently, however, a few are advocating a shift to molecular criteria such as whether an organism has genes that are part of sleep-promoting pathways in mammals and other species known to sleep. For example, Itoh’s team reported last year that more than 200 genes changed their activity in sleep-deprived hydra. Several of these genes are involved with sleep in fruit flies, they noted.

“We are moving from a behavioral or physiological definition to a cellular and molecular definition,” says Philippe Mourrain, a neurobiologist at Stanford University. “As we define more and more what sleep is [on those levels], we will have an idea of its function.”

There’s no question that sleep benefits the brain in creatures that have one. It helps the brain consolidate memories and flush out toxic waste. It may also help the brain stay plastic by pruning and strengthening connections between nerve cells.

But if animals without brains need sleep, those functions can’t be the whole story, Sehgal says. “Given that sleep is so widely conserved, it likely serves a fundamental function to preserve basic physiological processes.”

Some recent clues from brainless animals suggest sleep factors into energy balance and metabolism. Raizen’s team has found the much-studied roundworm Caenorhabditis elegans only naps when metabolic demands are high. The larvae go limp for 1 or 2 hours when they are molting and replacing their exoskeletons, or when excess heat or ultraviolet (UV) light causes stress. An enzyme called salt-inducible kinase 3 provides a direct link between sleep and metabolism. Known to help regulate sleep in mammals, it also mobilizes fat stores in C. elegans to boost the worm’s energy levels. In hydra, too, Itoh’s team has found a gene that both regulates metabolism and influences sleep.

A fish with transparent skin.
The see-through fish Danionella translucida may help reveal how the body controls sleep.James Jaggard, Philippe Mourrain, Adam Douglass, and Adriadne Penalva

Deprivation studies also point to sleep’s metabolic role. Sehgal has found fruit flies carrying a mutation that reduces their sleep are less able to metabolize nitrogen, which means they may have trouble breaking down and rebuilding proteins and processing waste. The result is a buildup of charged molecules called polyamines that can damage DNA and RNA, Sehgal’s team reported on 2 October on bioRxiv. “When we are sleep-deprived, it’s not just brain function that is affected,” she says.

Sleep deprivation also appears to attack the gut in fruit flies and mice, by leading to a buildup of harmful molecules known as reactive oxygen species, Harvard Medical School biologist Dragana Rogulja and colleagues reported last year in Cell. Somehow, that accumulation leads to early death in both species, the team found. Rogulja suspects the gut, among the first organs to evolve in multicellular animals, was one of the original beneficiaries of sleep, and that “a lot of [sleep’s] additional roles evolved as animals become more complex.”

To get to the essence of why animals sleep, however, requires studying it in species so simple they don’t even have a gut. Raizen decided to look at placozoans—round, flat, transparent creatures no bigger than a sesame seed that have just two layers of cells, each outfitted with whiplike projections called cilia. Placozoans lack nerve cells; their cells communicate via chemical secretions that control cilia movements. Outside of parasites that live attached to other life forms, placozoans “are the simplest animals on Earth,” says Carolyn Smith, a neuroscientist at the National Institute of Neurological Disorders and Stroke who has studied them for more than 10 years.

Placozoans use their cilia to crawl randomly along rocks at the tideline until they detect microalgae and stop to graze. They slow down at night, notes Bernd Schierwater, an evolutionary biologist at the University of Veterinary Medicine Hannover. He thinks that slowdown represents “the first [evolutionary] step toward sleep—getting a rhythm for rest” to recharge for the next feeding cycle. That may be enough of a respite for an animal that lacks energy-hungry nerve cells, he says.

A placozoa, a simple multi-celled organism similar in appearance to an amoeba.
Researchers want to know whether early-evolving placozoans sleep.Wolfgang Jakob/Schierwater Lab

Smith thought the idea of a sleeping placozoan was silly—until the jellyfish and hydra studies drove home that sleep was not just for brainy creatures. At times, placozoans rotate in place, which Smith suspects may also represent a kind of sleep. Because these creatures cringe when exposed to UV light, it might be possible to test whether they become unresponsive to UV light in this state, she notes. She provided Raizen with some animals to run such tests. But he couldn’t keep them alive in the lab—they are very finicky eaters—and eventually he gave up.

Joiner had similar problems with his puffball sponges. He and his collaborator, marine biologist Greg Rouse of the Scripps Institution of Oceanography, babied the sensitive creatures, which require a constant supply of fresh seawater laden with the microbes they eat. Joiner stopped by the ocean daily on his way to work to the collect seawater. The researchers stuck tubs housing the sponges in an incubator to control light levels and temperature. Then they mounted a digital camera over the tubs to watch for subtle contractions in the sponges’ bodies as they pumped water through their chambers to filter out food.

Eventually, with the help of magnetic stirrers in the tubs, they were able to keep the sponges healthy enough that they started to contract—about once every 3 hours. That was exciting, Joiner says, because it gave the researchers a reliable behavior they could monitor for sleep-related changes.

Studies in another sponge species have suggested the animals have cycles of rest that may allow them to reorganize and rejuvenate their cells after pumping water equivalent to 1000 times their volume daily. Marine biologists Sandie Degnan and Bernard Degnan at the University of Queensland, St. Lucia, saw hints of daily rhythms in the contractions in Amphimedon queenslandica, found on the Great Barrier Reef. And after sequencing its genome—the first for a sponge—they reported in 2017 that a few genes associated with circadian rhythm in other organisms turned on and off in 24-hour cycles. In unpublished work, graduate student Davide Poli has observed that select regions of the sponges’ bodies seem to stop pumping throughout the day, as if working in shifts. That might be “behavior that can be approximated to sleep,” he says.

Future studies could use glutamate or other substances that stimulate contractions to keep sponges pumping nonstop for hours or even weeks to test whether their health declines, says Sally Leys, a marine biologist at the University of Alberta, Edmonton. “That could indicate that by being a multicellular animal, you depend on having periods during which the tissues can repair and regenerate.”

If cells throughout the body benefit from sleep, it makes sense that those cells would have some say in when sleep happens. And the search for sleep’s far-flung control switches could lead researchers to new treatments for sleep disorders, which affect 60 million people in the United States alone.

Paul Ketema, a neuroscientist at UC Los Angeles, has studied Bmal1, a ubiquitous protein that regulates gene expression and is known to help sleep-deprived mice stay awake. Until now, researchers had assumed the brain made and used Bmal1 for that task. But Ketema and his colleagues discovered sleep-deprived mice depend instead on Bmal1 produced in the muscles. He suspects the protein may be part of a pathway that somehow helps link muscle exertion to levels of sleepiness. And he’s hopeful a Bmal1 drug targeted to muscles may one day counter the negative effects of all-nighters. “I am one of those people who thought sleep was all about the brain,” he laughs. “That is not only an erroneous viewpoint,” it’s also one many of his colleagues still hold, he says.

Other mouse studies have shown the gastrointestinal tract, the pancreas, and fat tissue generate signaling molecules called neurohormones that appear to affect the onset and duration of sleep. Understanding feedback from these organs to the brain “could suggest new pharmacological approaches, with drugs that target organs other than the brain,” Sehgal says.

Mourrain’s team at Stanford is developing a way to watch that feedback process play out cell by cell in the thumbnail-size, transparent fish Danionella translucida. By using fluorescent tags and other markers that track the activity of certain molecules in the fish’s brain and body, his team will observe how different types of cells control sleep—and benefit from it—over time.

When Mourrain started to study sleep in fish 15 years ago, “there was a lot of pushback that fish don’t sleep,” he recalls. That changed about 2 years ago, when he and his colleagues developed polysomnography techniques for fish and discovered that, like people, they cycle between quiet and active sleep states. “It was a tipping point for our field,” Mourrain says, convincing skeptics that fish could be good stand-ins for mammals in sleep studies.

Will the orange sponges be the next creatures to stun the sleep skeptics? Maybe not right away. Joiner and Rouse couldn’t keep their sponges healthy long enough to carry out reliable experiments. After months of fits and starts, they paused the work to rethink the set up. Then, COVID-19 hit and shut the experiment down. Joiner doesn’t have the staff to start it back up.

But another sponge may step in instead to show that even the simplest animals truly sleep. In emails, Itoh coyly referred to his lab’s work on these simple creatures. Mum’s the word for now, it seems. “These are ongoing projects,” he wrote. “Please look forward to them.”



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