eBriefing

Sleep Regulation and Dysregulation: From Molecules to Flies to Man

Sleep Regulation and Dysregulation
Reported by
Alan Dove

Posted August 31, 2007

Presented By

New York Academy of Sciences

Overview

Circadian rhythms are governed, not only by external environmental factors, but also by intrinsic physiological regulators. A meeting held at the Academy on June 27, 2007, examined the biochemical and molecular regulation of the neural networks that control sleep/wake cycles in organisms ranging from flies to man. The focus was on how disorders of sleep affect us at both the cellular level, and at the larger level of the endocrine and metabolic systems.

Mike Young of the Rockefeller University and Michael Rosbash of Brandeis University discussed respectively, circadian rhythms and the effects of human sleeping drugs in fruit flies. Martha Gillette of the University of Illinois reviewed the complex interactions between sleep cycles and hormone levels. James Krueger of Washington State University proposed a new model of sleep regulation. Marcos Frank of the University of Pennsylvania focused on why we sleep. Eric Nofzinger of the University of Pittsburgh School of Medicine discussed the current state of clinical sleep science.


Sponsorship

This conference and eBriefing were made possible with support from:

Web Sites

American Academy of Sleep Medicine
The American Academy of Sleep Medicine aims to set the clinical standards for the field of sleep medicine, advocating for recognition, diagnosis and treatment of sleep disorders, educating professionals dedicated to providing optimal sleep health care, and fostering the development and application of scientific knowledge.

Brain Basics: Understanding Sleep
The National Institute of Neurological Disorders and Stroke put together this primer on sleep and the brain.

Journal of Sleep Research
The Journal of Sleep Research, owned by the European Sleep Research Society, is an international journal that encourages important research papers presenting new findings in the field of sleep and wakefulness (including biological rhythms and dreaming). The Journal reflects the progress in this rapidly expanding field, promoting the exchange of ideas between scientists at a global level.

National Center on Sleep Disorders Research
Contains information on research, professional education, patient and public information, and communications.

Sleep
The official publication of the Associated Professional Sleep Societies, LLC. A joint venture of the American Academy of Sleep Medicine and the Sleep Research Society.


Journal Articles

Michael Young

Boothroyd CE, Wijnen H, Naef F, et al. 2007. Integration of light and temperature in the regulation of circadian gene expression in Drosophila. PLoS Genet. 3: e54. Full Text

Meyer P, Saez L, Young MW. 2006. PER-TIM interactions in living Drosophila cells: an interval timer for the circadian clock. Science 311: 226-229.

Wijnen H, Young MW. 2006. Interplay of circadian clocks and metabolic rhythms. Annu. Rev. Genet. 40: 409-448.

Michael Rosbash

Abruzzi KC, Belostotsky DA, Chekanova JA, et al. 2006. 3′-end formation signals modulate the association of genes with the nuclear periphery as well as mRNP dot formation. EMBO J. 25: 4253-4262.

Abruzzi K, Denome S, Olsen JR, et al. 2007. A novel plasmid-based microarray screen identifies suppressors of rrp6Delta in Saccharomyces cerevisiae. Mol. Cell Biol. 27: 1044-1055. Full Text

Tardiff DF, Lacadie SA, Rosbash M. 2006. A genome-wide analysis indicates that yeast pre-mRNA splicing is predominantly posttranscriptional. Mol. Cell. 24: 917-929. Full Text

Martha Gillette

Barnes JW, Tischkau SA, Barnes JA, et al. 2003. Requirement of mammalian Timeless for circadian rhythmicity. Science 302: 439-442.

Tischkau SA, Mitchell JW, Pace LA, et al. 2004. Protein kinase G type II is required for night-to-day progression of the mammalian circadian clock. Neuron 43: 539-549. Full Text

Tischkau SA, Weber ET, Abbott SM, et al. 2003. Circadian clock-controlled regulation of cGMP- protein kinase G in the nocturnal domain. J. Neurosci. 23: 7543-7550. Full Text

James Krueger

Cambras T, Weller JR, Angles-Pujoras M, et al. 2007. Circadian desynchronization of core body temperature and sleep stages in the rat. Proc. Natl. Acad. Sci. USA 104: 7634-7639.

Garcia-Garcia F, Ponce S, Brown R, et al. 2005. Sleep disturbances in the rotenone animal model of Parkinson disease. Brain Res. 1042: 160-168.

Kreuger J. Tripping on the edge of consciousness. The Human Side of Science series. (PDF, 122 KB) Full Text

Winner C. 2006. The secrets of sweet oblivion. Washington State Magazine (Spring) Full Text

Yasuda T, Yoshida H, Garcia-Garcia F, et al. 2005. Interleukin-1beta has a role in cerebral cortical state-dependent electroencephalographic slow-wave activity. Sleep 28: 177-184.

Marcos Frank

Dadvand L, Stryker MP, Frank MG. 2006. Sleep does not enhance the recovery of deprived eye responses in developing visual cortex. Neuroscience 143: 815-826.

Frank MG, Jha SK, Coleman T. 2006. Blockade of postsynaptic activity in sleep inhibits developmental plasticity in visual cortex. Neuroreport 17: 1459-1463.

Jha SK, Jones BE, Coleman T, et al. 2005 Sleep-dependent plasticity requires cortical activity. J. Neurosci. 25: 9266-9274. Full Text

Eric Nofzinger

Buysse DJ, Thompson W, Scott J, et al. 2007. Daytime symptoms in primary insomnia: a prospective analysis using ecological momentary assessment. Sleep Med. 8: 198-208.

Germain A, Nofzinger EA, Meltzer CC, et al. Diurnal variation in regional brain glucose metabolism in depression. Biol. Psychiatry 2007 Jan 8; [Epub ahead of print].

Nofzinger EA, Nissen C, Germain A, et al. 2006. Regional cerebral metabolic correlates of WASO during NREM sleep in insomnia. J. Clin. Sleep Med. 2: 316-322.

Speakers

Michael Young, PhD

The Rockefeller University
e-mail | web site | publications

Michael Young is the Richard and Jeanne Fisher Professor and vice president for academic affairs at the Rockefeller University. From 1991 until 2001 he was head of the Rockefeller Unit of the National Science Foundation Science and Technology Center for Biological Timing. Young has received numerous honors and awards including the Pittendrigh/Aschoff Award from the Society for Research on Biological Rhythms in 2006. He became a fellow of the American Academy for Microbiology and a member of the National Academy of Sciences in 2007. Young received his PhD in genetics from the University of Texas, Austin.

Michael Rosbash, PhD

Brandeis University
e-mail | web site | publications

Michael Rosbash studies both RNA processing and circadian rhythm regulation. Together with fellow Brandeis University researcher Jeffrey Hall, he cloned the period gene in Drosophila in 1984, and later proposed a negative feedback model of circadian regulation with period at its center. Rosbash is now a professor of biology at Brandeis, an adjunct professor of molecular biology at Massachusetts General Hospital, and an investigator in the Howard Hughes Medical Institute. He is a member of the National Academy of Sciences, the American Academy of Arts and Sciences, and has received numerous awards, including the NIH Research Career Development Award and the Caltech Distinguished Alumni Award.

Martha Gillette, PhD

University of Illinois
e-mail | web site | publications

Martha Gillette received her doctorate from the University of Toronto in 1976 and joined the faculty at the University of Illinois at Urbana-Champaign (UIUC) in 1978. She is a professor in the Departments of Cell and Developmental Biology and Molecular and Integrative Physiology, the Colleges of Medicine and Liberal Arts and Sciences, and the Neuroscience Program at UIUC. Gillette has served as head of Cell and Developmental Biology since 1998. She has been an affiliate with the Beckman Institute for Advanced Science and Technology (Neurotech Group) since 1988 and the Institute for Genomic Biology (Genomics of Neural & Behavioral Plasticity Theme), since its inception in 2003. Her many accomplishments were acknowledged by the University of Illinois with an appointment in 2004 as alumni professor of cell and developmental biology.

James M. Krueger, PhD

Washington State University
e-mail | web site | publications

James Krueger received his PhD in physiology from the University of Pennsylvania in 1974. From 1974 to 1978 he served as a research fellow and then an instructor in the Harvard Medical School Department of Physiology; from 1978 to 1981 he was a research associate in the same department. In 1981, he joined Chicago Medical School's Department of Physiology and Biophysics, first as an assistant professor. He worked as an associate and full professor from 1985 to 1997 at the Department of Physiology and Biophysics at the University of Tennessee. He joined the Washington State University faculty in 1997 and in 2007 was named a WSU Regents Professor.

Marcos Frank, PhD

University of Pennsylvania
e-mail | web site | publications

Marcos Frank is an assistant professor in the Department of Neuroscience at the Mahoney Institute of Neurological Sciences at the University of Pennsylvania. He was a postdoctoral fellow in the laboratory of Michael P. Stryker at the University of California, San Francisco. Frank's lab is interested in the function of sleep in developing and adult animals.

Eric Nofzinger, MD

University of Pittsburgh School of Medicine
e-mail | web site | publications

Eric Nofzinger is a professor of psychiatry and the director of the Sleep Neuroimaging Research Program at the University of Pittsburgh School of Medicine. He is currently the president of the United States Sleep Research Society. Nofzinger received his MD from the Ohio State University School of Medicine in Columbus, Ohio, in 1987. He completed residency training in psychiatry in 1991 and a postgraduate National Institute of Mental Health (NIMH) extramural research fellowship in sleep research at the University of Pittsburgh School of Medicine in 1993. In addition to his clinical practice of sleep disorders medicine, Nofzinger has developed methods to define the brain mechanisms of human sleep disorders using functional neuroimaging methods such as positron emission tomography (PET).


Alan Dove

Alan Dove is a science writer and reporter for Nature Medicine, Nature Biotechnology, and Bioscience Technology. He also teaches at the NYU School of Journalism, and blogs at http://dovdox.com.

We all have at least one good story about it: a time when we needed it really badly, or went without it for a long stretch, or got caught doing it someplace inappropriate. When the urge strikes, though, there's no stopping it: eyes flutter, the scene goes gray, and we surrender to it entirely. Ah, sleep.

Why do we sleep? Where do dreams come from? What causes insomnia?

For a process that's so essential to life, though, sleep remains bafflingly mysterious, even to scientists who have devoted their careers to studying it. Indeed, after centuries of investigation, the field is still pondering some of the same fundamental questions. Why do we sleep? Where do dreams come from? What causes insomnia, and how can it be cured?

Armed with sophisticated new tools, researchers are finally starting to find answers to some of these questions. On June 22, 2007, the New York Academy of Sciences and Takeda Pharmaceuticals sponsored a conference on the subject, featuring leading sleep researchers from several institutions.

Flies and fat

Mike Young of the Rockefeller University discussed circadian rhythms in fruit flies. Though insects are far removed from mammals evolutionarily, many of the same basic mechanisms govern their sleep cycles. Because flies are easy to manipulate genetically, Young and his colleagues have been able to use the system to uncover several of these mechanisms.

Mutations in a key clock-regulating gene in the flies derails a complex gene expression program, sending the animals' sleep schedules into chaos. The dysfunction persists even when the mutant flies receive normal light cues about the length of the day.

Michael Rosbash of Brandeis University has taken his own sleep studies on flies in a different direction, examining the effects of human sleeping drugs on the insects. Besides the circadian clock, evolution has also conserved another feature of sleep regulation: the inhibitory neurotransmitter γ-aminobutyric acid (GABA). Flies with a GABA-receptor mutation have much shorter sleep latency than wild-type flies, meaning they fall asleep faster.

Giving wild-type flies carbamazepine, a common epilepsy drug, lengthens sleep latency and decreases sleep duration. Mutant flies on the drug also have shorter sleep duration, but their sleep latency is unaffected, showing that sleep initiation and sleep maintenance can be uncoupled. The investigators now hope to use the system to identify other compounds that target the two processes separately, potentially leading to a new generation of more selective sleep drugs.

Martha Gillette of the University of Illinois reviewed the complex interactions between sleep cycles and hormone levels. Among the long list of hormones that oscillate in response to normal sleep/wake cycles, Gillette highlighted two particularly topical ones: the opposing hunger regulators leptin and ghrelin.

Leptin, which inhibits hunger, is high at night and low during the day, while ghrelin, which stimulates eating, rises in the early morning and falls after meals. Recent studies have revealed that sleep restriction produces a lower leptin peak, more ghrelin, and more hunger. Epidemiological studies also show that people who sleep for shorter durations are fatter, on average, than those who sleep longer.

Staying up, feeling down

James Krueger of Washington State University challenged a long-accepted model of sleep regulation. Traditionally, sleep researchers have imagined a hierarchical system, with specific sleep centers dictating the onset and maintenance of sleep through a defined chain of command. Instead, Krueger advocates a swarm-like model, in which sleep originates and spreads through small local areas throughout the brain.

Krueger believes that sleep can be replicated in culture, provided that the neurons are grown at a density that promotes interaction. In a series of biochemical experiments, he and his colleagues have found exactly this phenomenon in distinct columns of neurons in rats' cerebral cortices.

Marcos Frank of the University of Pennsylvania focused on a more fundamental—but still unanswered—question in sleep research: why do we sleep? He noted that dozens of theories have littered the field over the years.

To help clean things up, Frank and his colleagues have been testing the link between sleep and learning. In an elegantly designed series of experiments with cats, the investigators have found that sleep is critical for neuronal plasticity, or rewiring, especially in groups of neurons that have received the most stimulation during waking hours.

Eric Nofzinger of the University of Pittsburgh School of Medicine provided an update on the current state of clinical sleep science. Using fluorodeoxyglucose PET scans (FDG-PET), Nofzinger and his colleagues have identified several critical areas of the brain that undergo a deactivation process during sleep, including the thalamus and the prefrontal cortex.

Earlier studies have highlighted malfunctions in the prefrontal cortex in depressed patients, and Nofzinger's team found that these patients also deactivate this area less during sleep. In another study, subjects with insomnia showed significantly elevated activity in several brain regions, validating a common complaint in these patients, "that they can't shut their brains off, that their brains seem to be too active."

After the presentations, a wide-ranging panel discussion reviewed and extended the topics the speakers had covered. Though sleep research is still filled with unanswered questions, some of the new results could help inform novel strategies for treating everything from insomnia and depression to obesity. At least, that's the dream.


Alan Dove is a science writer and reporter for Nature Medicine, Nature Biotechnology, and Bioscience Technology.

Speakers:
Mike Young, The Rockefeller University
Michael Rosbash, Brandeis University
Martha Gillette, University of Illinois

Highlights

  • In flies, both light and temperature cues calibrate the circadian clock, which regulates sleep.
  • Insects may be ideal models for studying the mechanisms of new sleep drugs.
  • Sleep cycles regulate the daily cycles of numerous hormones.
  • Inadequate sleep may exacerbate or even cause many cases of obesity.

Clocking out

As diurnal mammals, we naturally get tired after sunset, fall asleep, then wake up more or less at sunrise, several hours later. Nocturnal animals invert the cycle, but still follow a 24-hour pattern. Nor is this global biological clock simply a light sensor; it keeps ticking even in Alaskans lit by the midnight sun, and New York's subway rats living in permanent midnight.

What regulates sleep, and what does sleep regulate?

So what regulates sleep? Like many basic questions in sleep research, this one has baffled scientists for centuries. But using cleverly designed animal experiments, sophisticated molecular analysis, and careful epidemiological studies, the field is finally starting to answer it.

In recent years, researchers have also started pondering the corollary: what does sleep regulate? Tests on a wide range of animals show that the levels of several critical hormones cycle in lock-step with sleep, and a new generation of sensitive tools is helping sleep scientists understand this process.

When flies sleep, do they dream of walking?

Mike Young of the Rockefeller University began with a stimulating discussion about sleeping flies. Though the subject may seem far removed from clinical sleep studies, investigators have found striking parallels between insect activity patterns and human sleep cycles. Flies, however, are much easier to study and manipulate in large numbers.

In a typical experiment, the scientists place Drosophila melanogaster in individual chambers, each with a light-emitting diode (LED) on one side and a phototransistor on the other. When the fly moves, a computer records the phototransistor's changing signal. Over the past 30 years, researchers have used systems such as this to show that normal flies have about the same circadian activity cycle as every other animal, and mutations in specific genes can alter or eliminate this sleep/wake clock.

Using gene expression chips, Young and his colleagues tracked the oscillations of gene expression in the fly head. They found that in normal flies the expression of numerous genes oscillates in a complex pattern during a 24-hour day. In flies with mutations in one of the key clock-regulating genes, the pattern goes completely haywire, even when the flies are given light cues. "This is not something that can be driven by light/dark cycles ... so we can't drive rhythmicity in the absence of a circadian clock," says Young.

Besides light, temperature tends to change over the course of a day in the fly's natural environment, so the researchers exposed flies to two days of regular shifts between 18°C and 25°C. Gene chips showed another large set of gene expression cycles after this treatment. Interestingly, though, clockless flies show essentially the same gene expression cycle in this experiment, "indicating that most of the rhythmicity that we see in response to a temperature cycle is driven independently of the presence of a circadian clock," says Young.

Both light and temperature regulate flies' sleep cycles.

The two systems are not entirely independent, however. When the researchers trained flies with a normal light/dark cycle at a constant temperature, then switched to a cold/warm cycle that was either aligned with or exactly out of phase with the light/dark cycle, the flies maintained or shifted their activity patterns to align with the new temperature cycle. The two cycles may not be driven by the same clock, but they apparently drive the same behaviors.

Young and his colleagues also uncovered some new mysteries. For example, mutant flies without a functioning circadian clock exhibit a peak in cold-stimulated gene expression in the middle of the 12-hour cold period the researchers administered. With broken circadian clocks, how do the flies know when the middle of the cycle is? One possibility is that a second, still undiscovered clock may be anticipating a specific temperature cycle.

Ask your nestmates about carbamazepine

Michael Rosbash of Brandeis University, continuing the discussion of fly sleep, began by detailing the striking consistency of slumber across evolutionary time. A sleeping fly, for example, has decreased activity, an increased arousal threshold, and a circadian clock timing its rest, just like a sleeping person. Sleep is also homeostatically regulated in flies, just as in humans: the longer it's been since the fly last slept, the stronger its sleep drive is.

For Rosbash, the next logical question was whether the gross similarities in fly and human sleep are reflected at the molecular level. In humans, for example, the inhibitory neurotransmitter γ-aminobutyric acid (GABA) is also the major promoter of sleep, and most sleep drugs are agonists of GABA. "Drosophila has been shown to have GABA, but the system in general, that is the inhibitory portion of the CNS, is poorly explored in flies, and from a sleep point of view essentially not at all," says Rosbash.

To remedy that, Rosbash and his colleagues looked at a fly strain called Rdl, which carries a mutation in a transmembrane protein involved in GABA activity. In sleep experiments, Rdl flies have much shorter sleep latency than wild-type flies, meaning they fall asleep faster after the lights go off.

Flies respond to sleep-disrupting drugs.

Next, the investigators gave the flies carbamazepine, a common epilepsy treatment whose mechanism of action has been difficult to probe. The drug lengthened sleep latency and decreased sleep duration in wild-type flies, but Rdl mutants on the drug still fell asleep quickly. "So the effect of the drug is significantly blocked by this single point mutation in this GABA receptor," says Rosbash. Interestingly, the mutant flies still had shorter sleep durations while on the drug, showing that sleep latency can be uncoupled from sleep duration.

Based on these data and additional biochemical studies, the researchers propose that the fly, like mammals, has both sleep-initiating and sleep-maintaining neurons. In this model, repeated rapid firing by the sleep-initiation neurons onto GABA-sensitive receptors eventually inhibits wake-promoting neurons, causing sleep to start. Carbamazepine could target this process through one receptor, and sleep maintenance through another, explaining why the two processes can be uncoupled.

For drug developers, this level of mechanistic detail is critical for developing more specific insomnia treatments. A drug that targets only sleep latency or only sleep maintenance, depending on a patient's specific complaint, could help manage the condition with fewer side-effects than current therapies.

Hungry for a snooze

Martha Gillette of the University of Illinois moved the discussion from flies to humans, and discussed both the signals that regulate sleep and the hormone changes that sleep causes. It's little surprise that both processes are complex. "The crescent of dawn has come over the world over a trillion times while life has evolved, and what we observe is ... sleep and wakefulness are very tightly organized over the course of day and night," says Gillette.

Besides being tightly controlled by our internal circadian clocks, sleep is enmeshed with a complex hormonal cycle. Cortisol, thyroid-stimulating hormone, growth hormone, prolactin, and parathyroid hormone, for example, all oscillate during a normal light/dark sleep/wake cycle. Sleep disorders derail these changes, affecting everything from metabolism and cardiovascular function to immune system activity.

In recent years, researchers have also uncovered two other hormones regulated by sleep, with potentially enormous consequences: leptin and ghrelin. Leptin, which inhibits hunger and food intake, is high at night and low during the day. Ghrelin, which opposes leptin and stimulates eating, rises at the end of the night and falls after meals.

The pattern becomes more ominous in sleep restriction studies. When healthy subjects are only given four hours of sleep in a night, their leptin levels do not rise as high as when they sleep a full eight hours. Indeed, subjects' leptin levels after sleep deprivation are similar to leptin levels following three days of dietary restriction.

In another study, researchers looked not only at leptin and ghrelin levels, but also hunger and food preferences. With only four hours of sleep, healthy young men were hungrier and more interested in carbohydrate-rich food than when they had a full night's sleep. The subjects also had a much lower glucose tolerance after sleep deprivation than when fully rested. "So here we have a change in an appetite regulator that's induced by restricted sleep, and it's leading to multiple changes, not only at the hormonal level but also at the metabolic level," says Gillette.

Epidemiological studies show that less sleep means more fat.

Epidemiological studies extend this result beyond the lab, showing that indiduals with self-reported sleep times less than seven hours per night had higher average body mass indices than longer sleepers. Less sleep means more fat.

So how much sleep does a person really need? In another study, subjects who claim they normally sleep less than six hours a night actually slept much longer when given the opportunity. "In other words, they aren't really six-hour sleepers—they choose to sleep for six hours," says Gillette. Furthermore, the short-duration sleepers score significantly lower than their long-sleeping colleagues on a wide range of cognitive and psychomotor tests, but their scores come back up when they're allowed to sleep longer.

The results have important public health implications. In a series of large surveys of American sleep habits, researchers have found self-scored sleep durations dropping, from more than eight hours in 1960 to seven hours in 1995, and below six hours in 2004. The precipitous decline in sleep in the past decade is particularly disturbing, as it aligns neatly with a dramatic rise in obesity. The link between the two phenomena is far from proven, but it's something to sleep on.

How do light-sensitive and temperature-sensitive signals interact in the circadian clock?

Can drugs targeting specific neural receptors affect sleep latency or sleep maintenance selectively?

What proportion of the national obesity epidemic is caused by a lack of sleep?

What is the smallest collection of neurons that can initiate sleep?

Is neuronal plasticity the primary purpose of sleep?

Is sleep disturbance a consequence or a cause of clinical depression?

Speakers:
James Krueger, Washington State University
Marcos Frank, University of Pennsylvania
Eric Nofzinger, University of Pittsburgh School of Medicine

Highlights

  • Sleep is an evolutionarily ancient behavior that seems to be related to learning.
  • Rather than being centrally controlled, sleep may originate in a decentralized, swarm-like pattern.
  • Nighttime overactivity in the prefrontal cortex characterizes sleep disorders and depression.

The four-billion-year nap

There is no doubt that sleep must be biologically essential. How else could such a maladaptive behavior be conserved across billions of years of evolution? Time spent snoozing is time lost from the critical tasks of eating and mating; energy spent finding a place to sleep is energy lost from other activities; and perhaps most troubling, sleeping animals are easy prey.

So why, exactly, do we sleep? Conversely, why do some of us sometimes fail to sleep? While scientists have been asking these questions for centuries, a new battery of tools is finally allowing them to uncover some tantalizing clues. Along the way, sleep science is pointing the way toward a new understanding of—and new treatments for—insomnia and other sleep disorders.

Think globally, sleep locally

James Krueger of Washington State University started his talk by highlighting a basic problem in the field: when an animal sleeps, we don't know exactly what it is that sleeps. While slumber is clearly a neural phenomenon, the brain continues to consume energy during sleep, and many neurons continue to fire. Indeed, humans with parasomnias such as sleepwalking can even behave as if they are sleeping and awake simultaneously, and researchers in the 1980s found that a dolphin can put half of its brain to sleep at a time, leaving the other half awake.

Clearly, sleep is a local process within brain regions, so what coordinates it across the whole brain? According to a longstanding model, sleep regulation follows a defined chain of command, originating with the circadian clock and proceeding hierarchically to the local areas that need to sleep.

Sleep may spread like a swarm through the brain.

While he concedes that this model is useful for explaining circadian rhythms, Krueger prefers a more decentralized explanation for other aspects of sleep regulation. Rather than requiring a sleep signal from a single control center, he argues, sleep originates in a decentralized, swarm-like pattern in clusters of neurons.

"It's a property of highly interconnected networks. I would argue that you could even get sleep in a culture dish, as long as you grew your neurons at high enough density that they interacted with each other," says Krueger. Besides fitting well with mathematical models of sleep, the theory aligns neatly with a number of empirical observations. For example, no survivable brain lesion, no matter how large, has ever been found to eliminate sleep in an animal, demonstrating that no single brain region is essential for organizing sleep.

At the molecular level, Krueger and his colleagues have found that in rats, the expression of the cytokine tumor necrosis factor (TNF) correlates with sleep, and also with neuronal activity in individual columns of cells in the cerebral cortex. Inhibiting TNF protein activity in various ways reduces the animals' sleep. Furthermore, injecting TNF into one side of a rat's brain causes that side to sleep more deeply than the other side, similar to the asymmetrical pattern dolphins exhibited in earlier studies.

TNF enhances local sleep intensity.

The researchers also explored the link between the waking and sleeping activities of a brain region. By twitching a whisker on one side of a rat's face twice as fast as a whisker on the opposite side, the team stimulated two different neuronal columns at different rates. The faster-twitched column was significantly more likely to go into a sleep-like state later. Because TNF is associated with both activity and sleep, the investigators suspect that the cytokine may form a critical part of this linkage.

Sleep, memory

Marcos Frank of the University of Pennsylvania also began with a fundamental and enduring puzzle: why do we sleep? "We as scientists don't have an answer to this question, but it's not for lack of trying," says Frank, adding that "over the last few decades, literally dozens of theories have been proposed to explain why it is that animals sleep. As a consequence the situation today is very untidy."

The question is not just philosophically important; humans spend nearly half their lives asleep, so the entire field of neuroscience is incomplete without an understanding of the function of this activity. Unfortunately, it's been difficult to dissect. As one of the most ancient behaviors in the animal lineage, sleep is regulated by a complex, highly interlinked set of signals, it affects nearly every aspect of an organism's physiology, and it interacts intimately with the circadian clock; designing clean experiments on such a system is a tall order.

There are some clues, though. One invariant ingredient of sleep, for instance, is a heightened arousal threshold: sleeping animals are less aware of their environment than when they're awake. "From frogs to flies to Flipper, when we go to sleep ... there is a change in the brain so that it's no longer responding as much to the external world, and it begins to look more inward," says Frank, adding that "maybe sleep's ultimate functions have to do with the brain."

The brain may use sleep to remodel neuronal connections.

More specifically, they appear to be related to learning. Though the evidence is still not conclusive, numerous studies have found a consistent, positive relationship between sleep and memory. A good night's sleep improves learning in adults of many species. Meanwhile infants, whose brains are undergoing their most rapid phase of growth and change, need substantially more sleep than their elders.

To probe this connection further, Frank and his colleagues focused on ocular dominance plasticity. In this system, blocking vision in one eye of an animal causes the neurons that would normally process that eye's input to rearrange, connecting instead to the other eye.

When the researchers blocked one of a cat's eyes, then played with the cat in a well-lit room for six hours, the animal shifted some of its cortical neurons to the open eye. Allowing a similarly treated cat to sleep in the dark during the experiment, however, caused it to rewire even more neurons to the open eye, underscoring the importance of sleep in plasticity. A third group of treated cats, which researchers played with in the dark, actually reversed its rewiring, as if the memory of the open eye's visual experience was being erased.

"In the absence of sleep, we not only do not get enhancement of this change, but in fact the cortex begins to revert ... the impact of the experience is being lost," says Frank. Injecting inhibitory drugs into the cats' visual cortices while they sleep blocks the rewiring in all three groups, showing that sleep's effect on plasticity requires neuronal activity.

The unquiet mind

Eric Nofzinger of the University of Pittsburgh School of Medicine gave an update on the current state of clinical sleep science. Few humans would be willing to submit to the kinds of biochemical and genetic experiments researchers can do in animals, so Nofzinger and his colleagues have pioneered a non-invasive technique for probing human sleep.

For a typical experiment, the scientists inject a sleeping patient with fluorodeoxyglucose (FDG) during the first non-REM period of sleep, and track the dye's uptake while the subject continues to sleep. After about 20 minutes, they wake the patient for a positron emission tomography (PET) scan, producing an image of the brain regions that were most active at the time of the injection. Software can superimpose an equivalent scan taken while the patient was awake, highlighting the areas with the greatest increases or decreases in activity between the two conditions.

In healthy subjects, the thalamus and prefrontal cortex undergo the deepest deactivation during sleep, pointing to these areas as important sleep centers. Depriving patients of sleep, then allowing them to sleep the next night, produces a more intense reduction in the brain's metabolic activity during the recovery sleep. "So driving down metabolic activity is essential for the recovery process of sleep, and especially in regions of the brain that we think [are] involved in waking executive function," says Nofzinger.

The prefrontal cortex is a major focus of sleep.

Having established a baseline for normal sleep, the researchers next looked at sleep in patients with various disorders, starting with depression. Depressed patients have a litany of sleep problems, ranging from difficulty falling asleep and staying asleep to a tendency to experience REM sleep earlier in the night. In about 30 patients with depression, FDG-PET reveals a weaker metabolic decrease in the frontal area, the same region that EEG studies have highlighted as malfunctioning in depression. "So the waking abnormality may be a function of what's happening during sleep," says Nofzinger.

The team has also studied the changes in sleep patterns during aging and in insomnia. In the aging study, they found that the decreased time and depth of sleep typically found in the elderly correlates with age-related atrophy in the midfrontal gyrus.

Meanwhile, patients with insomnia have physically normal brains, but FDG-PET reveals that they have much higher brain metabolic activity during sleep than their soundly sleeping peers. The problem seems to stem from an arousal network within the brain that fails to deactivate in insomnia. That validates a common complaint in this patient population. "When they tell us that they can't shut their brains off, that their brains seem to be too active, in fact neurologically that actually seems to be the case," says Nofzinger.

A panel discussion following the talks allowed the speakers to comment on a wide range of related topics, from the role of temperature in mammalian circadian rhythms to the function of REM sleep. While the field's progress has clearly accelerated in recent years, there's still no shortage of unanswered questions. Just don't let them keep you up at night.