Clinical studies are underway that may address whether these observations apply to IH. People with IH aren’t sleep-deprived goats, but it is possible that some of their sleepiness comes from substances produced by intestinal bacteria or from heightened levels of inflammatory cytokines. People with sleep apnea—who may superficially resemble those with IH—have elevated markers of inflammation, although this is difficult to disentangle from obesity, which often contributes to sleep apnea.

  ROMANCES GONE ASTRAY

  By the time Factor S was being characterized, several rival sleep molecules had appeared. Two examples demonstrate how sleep’s biochemical complexity has defied simple dissection.

  In the 1960s, a wave of enthusiasm washed over the sleep research field for the neurotransmitter serotonin. The French neuroscientist Michel Jouvet, who discovered the areas of the brain that control REM sleep in cats, described his and others’ research on serotonin as like a love story: a romantic encounter, leading to a honeymoon, followed by divorce.19

  Jouvet and others found that depleting serotonin by inhibiting its synthesis caused profound insomnia in cats. Anticipating a central role for serotonin in sleep, the Swiss neuroscientist Werner Koella suggested renaming it “somnotonin.” But electrical probing of neurons that produce serotonin showed that their activity decreased during sleep and increased during time awake—the opposite of what Jouvet and Koella had predicted. Decades later, the insomnia of serotonin depletion in the cats was found to come from hypothermia.20 It illustrates how sleep is bound together with metabolism and regulation of body temperature. Researchers still think that serotonin is central to the regulation of sleep, but one molecule is unlikely to be the golden thread coordinating a complex set of brain functions.

  Another sleep-associated molecule that did not withstand close scrutiny deserves highlighting: DSIP (delta sleep-inducing peptide). The rationale for pursuing DSIP was the opposite of that for Factor S. Instead of depriving animals of rest, Swiss researchers thought they could synchronize animals’ brains and see if they produce something that can cause sleep in another animal. They exploited the discovery that electrical stimulation of the thalamus produced deep slow-wave, or delta, sleep—hence DSIP’s name.

  The Swiss researchers set up a system in which rabbits were electrically submerged into sleep while relevant substances were continuously extracted from their blood via hemodialysis. In the late 1970s, they had accumulated enough of the “sleep potion,” as the New York Times called it, to decipher biochemically.21 It also turned out to be a peptide, nine amino acids long.

  In the 1980s, DSIP was cited as the most extensively tested sleep molecule. DSIP was different from Factor S, but the two molecules were perceived as competitors. Although a variety of neurochemical effects were attributed to DSIP, doubts began to emerge early on.22 A peptide would be unlikely to last long enough in the blood or cross the blood-brain barrier if administered peripherally.

  In their long trek toward purifying DSIP, the Swiss team was supported by several pharmaceutical companies. DSIP appeared promising enough that Hoffmann-La Roche filed for a patent on it for treatment of addiction withdrawal.23 As clinical studies proceeded, DSIP’s previously reported properties became difficult to explain, and Roche abandoned commercial development of DSIP in the late 1980s. Some of the observed benefits probably came from a placebo effect. DSIP enjoyed an afterlife at a few Swiss clinics, where people recovering from addiction were willing to pay for it,24 and in Russia, where derivatives of DSIP were tested in clinical trials.25

  Nobody ever isolated a gene encoding DSIP or a receptor for it. In 2006, Vladimir Kovalzon, a Russian sleep researcher who had studied DSIP at length, suggested that its original discoverers had gotten the peptide sequence wrong. He called DSIP a “still unresolved riddle.”26

  A IS FOR ADENOSINE

  For one sleep regulatory factor, there was no need to go hunting for it. The first letter of the genetic alphabet had been in front of us for decades. Adenosine is what’s left when phosphates are removed from ATP (adenosine triphosphate), the carrier of chemical energy in cells. In the brain, extracellular adenosine is like discarded wrapping paper and boxes outside a house: a sign that gifts were opened inside. When brain cells send messages to one another, they pack ATP into vesicles, together with neurotransmitters, such as dopamine, glutamate, or GABA. Upon delivery, ATP is left over and gets quickly converted into adenosine. Outside the brain, adenosine has other functions, regulating blood pressure and inflammation. It is found on emergency room crash carts because it can calm a dangerously racing heart.

  Adenosine’s sleep-inducing effects were observed in the 1950s by scientists in London, who were injecting drugs directly into the brains of live cats.27 They were testing many substances—acetylcholine, serotonin, and others, too. At that point, they were really just stabbing in the dark; it was difficult to know the physiologically relevant amounts.

  In the early 1970s, the neurochemist Henry McIlwain observed that cells released adenosine when they were stimulated electrically. Others showed that when enough adenosine is around, it will inhibit brain cells’ firing, sort of like GABA. However, adenosine has its own set of receptors—four, with two types in the brain—and acts through different biochemical mechanisms.

  For adenosine to modulate alertness and sleepiness makes intuitive sense, because caffeine, something many are familiar with on a daily basis, interferes with adenosine’s access to its receptors. Adenosine seemed to fulfill Pieron’s hypothesis that a product of cellular metabolism accumulates in the brain.

  Some popular books, such as Matthew Walker’s Why We Sleep, say that adenosine is responsible for sleep pressure, the force that drives us to feel drowsy after a long time awake, and that adenosine builds up with time awake.28 This is a nice explanation, but adenosine’s actual accumulation in the brain has been difficult to demonstrate, and adenosine is not the only element of sleep pressure.

  In the 1990s, scientists at Harvard Medical School proposed this role for adenosine, showing that its concentration progressively increased in the brains of cats kept awake for several hours.29 These experiments weren’t as cruel as before; the cats were kept awake by petting them or by having them play with plastic toy lizards.30

  The Harvard group originally thought that prolonged wakefulness would elevate adenosine levels throughout the brain.31 However, in animals, accumulation of adenosine has only been confirmed in one region, the basal forebrain, which is part of the network that modulates alertness. At other sites, adenosine levels appear to stay stable or fall off over time awake. Adenosine may be broken down or recycled too quickly to accumulate noticeably in extracellular spaces.

  In humans, the question was tested—just once. Neuroscientists rarely have a chance to peer inside the human brain directly, so they take advantage of the opportunities they have, which come when epilepsy patients are in the hospital for seizure diagnosis. While monitoring the brain for seizure activity, surgeons can leave in place electrodes that are hollowed out, with space to collect a small amount of fluid for chemical analysis. When investigators at UCLA did this, they did not see increases in adenosine concentration. The levels actually fluctuated or decreased over time, even in patients who were kept awake overnight to amplify the likelihood of a seizure.32

  “We couldn’t say much about the basal forebrain, because we were restricted to probing areas that were clinically relevant for the patients’ epilepsy,” said Jamie Zeitzer, lead author of a 2006 report on the UCLA experiments. “However, we could say that an increase in extracellular adenosine seems not to be a global phenomenon.” “Technically speaking, adenosine doesn’t accumulate,” he added in an email. “Increased extracellular concentrations are due to a mismatch between release and reuptake, where the latter cannot keep up with the former. Adenosine, though, is rapidly removed from the extracellular space.”

  Despite inconclusive experiments in humans, plenty of evidence supports adenosine’s role as a messenger of sleepiness. Even if it’s difficult to detect adenosine’s accumulation, having more available to the nervous system has a measurable effect. For example, people who have a less active form of an enzyme that breaks down adenosine displayed deeper sleep, with fewer awakenings at night.33 Those with the less active form didn’t sleep longer overall, but they were more susceptible to sleep deprivation.34 Experiments with mice showed that signals from adenosine appear to be important for the deeper slumber that occurs after insufficient sleep.35

  Still, the lack of observable accumulation leads to a more fundamental question: where does sleepiness live? In a substance outside cells or within the cells themselves? “It’s simpler to conceive of it as one thing, but I don’t think it is,” Zeitzer said.

  Besides adenosine, other molecules have been seen to accumulate in response to sleep deprivation in humans, such as beta-amyloid, the neurotoxic protein fragment connected with Alzheimer’s disease plaques.36 This doesn’t mean beta-amyloid is the embodiment of sleepiness, but it does indicate that sleep deprivation impairs the machinery that flushes beta-amyloid out of the brain.

  Recent experiments suggest an alternative way of viewing how the brain is responding to the lack of sleep. German researchers have observed that when volunteers went without sleep overnight, one type of adenosine receptor increased in density throughout the brain. That is, the stress of sleep deprivation pushed brain cells to produce more receptor proteins and to keep more of them on their surfaces. When study participants were able to go to sleep, adenosine receptor density went back down again.37 Incidentally, caffeine mainly works through a different adenosine receptor—so caffeine and sleep deprivation are on parallel tracks. With extended wakefulness, the brain is becoming more sensitive to adenosine. But the molecules determining that sensitivity—the physical embodiment of sleepiness—are embedded in our cell membranes, rather than flowing between them.

  SLEEPY FLIES AND SLEEPY MICE

  Pieron’s and Pappenheimer’s ideas continue to inspire modern sleep researchers. But by the twenty-first century, it became possible to use genetic tools, or to comprehensively scan proteins and other molecules in the brains of sleep-deprived animals, rather than having to rely on ingenious schemes to distill the essence of sleepiness. There is also more of an emphasis on the distinction between acute sleep loss (easier to study) and chronic sleep deprivation (pervasive in society).

  Using Drosophila fruit flies, sleep researchers at the University of Pennsylvania have isolated a sleep-inducing factor called nemuri.38 Hirofumi Toda, a postdoc in Amita Sehgal’s laboratory, randomly inserted thousands of individual genes into flies, in such a way that each gene would be overproduced in flies’ brains in response to a drug. After screening thousands of genes in this way, they found only one that increased total sleep time. “I had been fascinated with the idea of a somnogen ever since I started working on sleep,” said Sehgal, who had previously studied circadian rhythm genes in flies (along with many others). “We had considered trying to get at this biochemically, but then decided to focus on the genetics approach that Drosophila is so well-suited for.”

  The nemuri gene encodes a secreted peptide that is also involved in defending flies against bacterial infection. The protein is difficult to detect in unperturbed flies, but its production is turned on in response to infection, stress, or sleep deprivation, either as a result of caffeine or physical agitation. In a way, the sleepy flies recapitulate Pappenheimer’s experiments with sleep-deprived goats’ CSF, even though the molecule in the goats came from bacteria, rather than being produced by the animals’ cells. Nemuri may correspond to the inflammation-related cytokines Krueger identified as inducing sleepiness during infections in mammals.

  Sehgal has argued that nemuri meets criteria for a homeostatic factor, one that helps maintain balance and whose production increases when an animal needs more sleep. Supporting this idea, when the nemuri gene is disrupted in flies, they took longer to recover from sleep deprivation; the mutant flies were also easier to arouse and took longer to return to sleep afterward.

  Looking more closely at nemuri might give us clues about the elusive sleep-promoting peptide in hypersomnia patients. Flies that overproduced nemuri slept over three hours per day more than control flies. The flies were also more difficult to arouse by jostling them, a situation possibly analogous to sleep inertia. Unfortunately, there is not a gene in humans whose sequence looks similar enough to make it a counterpart for nemuri.

  Several alternative peptides identified in vertebrate brains could be somnogen candidates. For example, scientists working with zebrafish—a vertebrate, if not a mammal—have identified a secreted sleep-promoting factor called neuropeptide VF.39 The relationship between any of these peptides and what Jenkins and his labmates glimpsed in hypersomnia patients’ cerebrospinal fluid is still unclear. Several candidates identified by other scientists could match the criteria they laid out. If or when the identity of the “sleepy stuff” comes to light, the same questions will come up as for adenosine and Factor S: how and where does it function in healthy people? Does it become more abundant in response to sleep deprivation, inflammation, or other stresses?

  Another recent tour de force of molecular biology comes from Japan, and it raises just as many questions about the neurochemical basis of hypersomnia and sleepiness. The leader of this effort, Masashi Yanagisawa, has a flair for epic projects, and he seems to see himself as the scientific heir of Pieron and Pappenheimer. Intent on “deciphering the neural substrate for sleepiness,” Yanagisawa set out on producing mutant mice on an industrial scale and established a facility at the University of Tsukuba that would allow his team of geneticists to do so. They sprinkled genetic flaws across mouse sperm and then systematically screened more than eight thousand of the progeny for altered sleep behavior.40 They found a line of mutant mice that sleep several hours longer than normal laboratory mice. The mice are named, unsurprisingly, sleepy.

  The sleepiness in Yanagisawa’s mutant mice doesn’t appear to come from something secreted outside cells like adenosine or a peptide. Rather, it derives from the altered activity of an enzyme called SIK3, which is located inside brain cells. SIK3 is a kinase, a type of enzyme that adds a negatively charged phosphate group to another protein, usually altering its activity. Among the arrays of signaling proteins inside cells, a kinase enzyme selectively turns up a few dials or adjusts a few control levers. Intricate chains of kinases modifying other proteins manage many basic processes in cells, such as cell division.

  In the sleepy mice, the SIK3 enzyme isn’t gone, but it is missing a stretch that another kinase operates on. Sleepiness can’t be distilled down to one particular molecule and is not represented by SIK3 itself. Instead, sleepiness is a pattern of all the phosphate alterations SIK3 makes on many different proteins, which are nestled in the synapses of the brain. In fact, mice that are sleep deprived and the sleepy mice have patterns of phosphate alterations that overlap and look similar.41

  Yanagisawa and colleagues have proposed that their sleepy mice may be a potential animal model for idiopathic hypersomnia in humans.42 The mice didn’t seem to have alterations in wakefulness or arousal—they responded normally to caffeine or the mental stimulation of being placed in a new cage. They did have an increased need for sleep, along with an exaggerated response to sleep deprivation. But do the brains of people with IH look biochemically like people who are sleep deprived, like the sleepy mouse brains did? Just like the situation with adenosine, this question may be difficult to answer directly.

  Our picture of the molecules that make up facets of sleepiness will continue to evolve, since adenosine, various peptides, and patterns of phosphate modifications are probably not the only embodiments of sleepiness. Chronic sleep disruption leaves imprints all over the brain.43 One example: recent research in zebrafish and mice indicates that transient DNA damage, marked by the accumulation of DNA repair proteins, is a component of sleep pressure.44 The challenge will be to show how these findings apply to people with IH and other sleep disorders.

  CHAPTER 7

  MY FAVORITE MISTAKE

  “Serendipity” is a category used to describe discoveries in science that occur at the intersection of chance and wisdom.

  —Samantha Copeland, 2019

  In the summer of 2008, while Anna Sumner was still getting back on her feet, something unexpected happened. She developed bronchitis and lost her voice, which is a problem for someone whose professional role occasionally requires her to argue in court. Most of Anna’s legal work for corporate clients was conducted behind the scenes, but around this time, she was defending the owner of a home for rescued dogs against noise complaints in Gwinnett County, near Atlanta.1

  To treat her bronchitis, Anna’s internist prescribed the antibiotic clarithromycin, known commercially as Biaxin. After taking it, she couldn’t sleep. After three nights of insomnia, she became frightened. “This had never happened to me before,” Anna said. “I was concerned that it was some bizarre individual reaction to the medication.”

  She frantically called the neurologist Lynn Marie Trotti, who had become her main sleep specialist. Rye was in the United Kingdom attending a sleep research conference, but Trotti was not filling in as a one-off substitute. She had first come to Emory after finishing medical school in 2003. During her fellowship year after residency, she had worked with Rye on restless leg syndrome, saw several of his patients, and took over care for some of them. We will come back to Trotti, since she became more central to hypersomnia research at Emory over the next several years. But first: clarithromycin.