Throughout the arc of human history, sleep has been a mysterious process that has captured the imaginations of both artists and scientists. Researchers are slowly deciphering how sleep restores us, and how its woeful absence makes us ill. Scientists now think a good night’s rest benefits the brain by clearing away cellular waste and laying down memories. Recent studies on how these processes work may have implications for neurodegenerative disease, particularly Alzheimer’s. “There is an increasing awareness that we need to know more about how sleep is implicated in disease etiologies,” said Maiken Nedergaard at the University of Rochester Medical Center, New York, who led one of the studies.
This story explains how sleep and osmotic pressure are intimately connected, how poor fluid flow in the brain correlates with disease, and how brain injury may disturb sleep. (For new insights into the role of sleep in memory, see Part 2 of this series.)
Sleeping by Osmosis
Both the volume and flow of extracellular fluid are known to surge when the brain catches some ZZZ's. This extra flow facilitates the clearance of solutes including Aβ, according to one theory.
Nedergaard wanted to know why there is more fluid in sleeping brains. Because osmotic forces control fluid movements, Nedergaard speculated that the answer might have something to do with the extracellular concentrations of ions and solutes. As reported in the April 29 Science, joint first authors Fengfei Ding and John O’Donnell and their colleagues tested this idea by adding a “wakeup” mix of neuromodulators to mouse cortical brain slices. The mix included norepinephrine, acetylcholine, and dopamine, which are produced by brainstem and basal brain regions and are known to stimulate waking patterns of electrical activity in the brain. The treatment induced a rapid and persistent rise in extracellular potassium in the slices. Neurons release this ion when they fire, but surprisingly, potassium spiked even when brain activity in the slices was silenced with tetrodotoxin. This demonstrated that the ion change occurred independently of electrical activity. It is not yet clear what caused the potassium efflux, Nedergaard said.
Intrigued, the authors measured ion concentrations in waking and sleeping mice via microdialysis. As in brain slices, potassium levels shot up within a few seconds of waking; they remained high until the animals nodded off, then plunged. These changes occurred even when electrical activity was dampened with an AMPA receptor antagonist. Meanwhile, calcium and magnesium ions waxed and waned in opposition to the potassium, rising during sleep and falling as animals awoke. Their transitions were a bit slower, taking about a minute apiece. The ion changes all occurred independently of each other, the scientists reported.
To tease out the effect of different ion concentrations, the authors infused ion cocktails into the brains of mice through a cranial window. When they added a “waking” mix of ions to one cortical region of a sleeping mouse, the neurons in that small region shifted into waking patterns of electrical activity, although the mouse remained asleep. Likewise, a “sleeping” mix added to awake animals induced slow-wave electrical activity characteristic of sleep in that teeny portion of cortex, even while the animals remained active. At the same time, the volume of local extracellular fluid shrank or expanded in keeping with the ion mix (see image above). This was determined by the classic method of infusing the cation tetramethylammonium and recording how quickly it diffused from the delivery site.
Then the authors went one step further. They changed the ion composition throughout the brain by infusing ion cocktails into the cerebrospinal fluid. With that, they found they could transition global brain activity, waking up sleeping mice and putting awake mice to sleep.
The findings suggest, at a simple level, that ion concentration itself controls sleep/wake in the brain, Nedergaard told Alzforum. The idea is not new; studies from the 1930s first proposed that calcium and magnesium promoted sleep, while potassium promoted wakefulness, noted Hans-Peter Landolt and Sebastian Holst at the University of Zurich, in an accompanying editorial. However, this idea fell into obscurity when subsequent research assumed that changes in electrical activity caused the ups and downs in ion concentrations during sleep and wakefulness. The new data imply the opposite, Nedergaard noted. As an animal falls asleep, extracellular potassium plummets first, hyperpolarizing and silencing neurons at the same time fluid volume swells to facilitate clearance of solutes. “When the brain needs to clean itself, it shuts itself off by lowering potassium,” Nedergaard suggested.
Brendan Lucey at Washington University in St. Louis found the data surprising. “I wouldn’t have thought that regulating ion concentrations in mouse CSF would result in state changes in the brain,” he told Alzforum. He noted that the study used careful controls and built its case well, moving from brain slices to live animals.
What Can Go Wrong
Nedergaard thinks the research may also provide clues to what goes awry in disease states. A number of studies have linked disrupted sleep to Alzheimer’s risk (see, e.g., Aug 2012 conference news; Oct 2013 news). Recent work indicates that Aβ exits the brain primarily during sleep, suggesting one possible reason for the association (see Sep 2009 news; Apr 2013 conference news; Jun 2014 news). Nedergaard and colleagues had previously proposed a mechanism for this clearance. They described a “glymphatic” flow that washes through the brain interstitial space, dragging Aβ and other solutes along with it. Specialized channels on astrocytes that pump water power the flow, which is much greater during sleep (see Aug 2012 news; May 2014 conference news). The new data suggest the increase in osmotic pressure that occurs during sleep may help power that current. “The data help establish a mechanism for Nedergaard’s prior observations that Aβ clearance occurs during sleep,” Lucey said.
Ion imbalance might underlie other symptoms as well. For example, Ding and colleagues found that in animals recovering from anesthesia, the ions lagged in returning to normal waking levels, with potassium taking a couple of minutes and calcium and magnesium requiring up to half an hour to stabilize. Mice in this state appeared disoriented, running into the walls of their cage. This slow transition may explain the grogginess common upon waking from anesthesia, Nedergaard speculated. Perhaps a similar ion perturbation causes some of the confusion seen in people with Alzheimer’s, particularly in the evenings, Nedergaard said. In ongoing work, she is measuring ion concentrations in the brains of aged AD model mice to test this idea. She is also interested in whether extracellular potassium levels continue to climb if animals are kept awake past their normal hour for shut-eye. High potassium is known to cause seizures, and might provide an explanation for why these occur after prolonged sleep deprivation, Nedergaard noted.
The findings might also point toward therapeutic targets, Landolt and Holst suggested. “Pumps and transporters that control ion flow across cell membranes may be promising new targets for treating sleep-wake disorders,” they wrote. For her part, Nedergaard noted that astrocytes express specific subtypes of sodium/potassium pumps, suggesting these could be selectively targeted.
Beyond Glymph—Sleep and Other Clearance Pathways
Glymphatic flow is but one of several complementary routes for clearing solutes from the brain. Researchers last year reported the discovery of lymphatic vessels in the dura mater that absorb fluid from interstitial fluid (ISF) and CSF and transport it to cervical lymph nodes (see Louveau et al., 2015; Aspelund et al., 2015). In addition, fluids can drain along the basement membranes of brain blood vessels, through the perivascular spaces. Enlargements in these Virchow-Robin spaces, as they are called, correlate with cardiovascular disease, stroke, cognitive problems, and dementia.
Researchers led by Joel Ramirez at the University of Toronto have associated low sleep quality with anatomical changes in these fluid-filled gaps. Along with Courtney Berezuk, Ramirez examined data from 26 adults with an average age of 60 who were evaluated for cardiovascular disease (see Berezuk et al., 2015). Participants with the largest Virchow-Robin spaces slept worst. They took longer to fall asleep, woke up more during the night, and spent less time in the third and deepest stage of non-REM sleep.
Why these spaces were enlarged remains unclear. The authors speculated that lack of sleep rendered drainage from perivascular spaces inefficient, which in turn could cause these channels to clog up with waste products such as Aβ and expand (see Weller et al., 2009). On the other hand, underlying cardiovascular disease by itself might be responsible for these spaces ballooning, regardless of sleep problems. One theory holds that as arteries harden, they beat more against surrounding glia and carve out a larger space, said Costantino Iadecola at Weill Cornell Medical Center, New York. The consequences of enlarged Virchow-Robin spaces are unknown. Berislav Zlokovic at the University of Southern California, Los Angeles, pointed out that it might influence the rate of solute clearance from the ISF to the CSF. However, Iadecola noted that remains to be proven (see full comment below).
Other studies support the idea that normal clearance mechanisms falter in AD. Researchers led by Tsutomu Nakada at the University of Niigata, Japan, reported that the flow of water from ISF into CSF slows down in older people, and lags even more in those with Alzheimer’s disease (see Suzuki et al., 2015). They compared fluid flow in 10 young volunteers, 10 healthy aged volunteers, and 10 people diagnosed with AD dementia, using PET imaging of water labeled with an oxygen isotope. The flow in AD patients was about half that in the younger people, with healthy elderly falling about halfway between the two groups.
John Cirrito at WashU noted that these findings fit with previous evidence from his WashU colleague Randy Bateman, who found that rather than spiking during sleep as they do in healthy adults, CSF Aβ levels remain steady throughout the circadian cycle in AD patients (see Dec 2010 news; Aug 2012 conference news; Huang et al., 2012). “Nakada’s study suggests the reason for flat Aβ levels is because the flow from ISF to CSF is impaired,” Cirrito said.
Perturbed sleep shows up in other brain disorders as well. In the April 27 Neurology, researchers led by Lukas Imbach and Christian Baumann at University Hospital Zurich, reported that people with traumatic brain injury sleep more than controls even 18 months after their injury. The findings extended data from a previous study, where the authors reported increased sleepiness in a cohort of 42 young adults six months after TBI (see Imbach et al., 2015). At 18 months, the authors were able to obtain additional polysomnography data from 31 of those participants. Compared with 42 age-matched controls, the TBI patients slept about one hour more per night, but nonetheless remained sleepier during the day, as measured by how quickly they fell asleep for naps. Overall, two-thirds of the TBI patients were diagnosed with excessive daytime sleepiness, as compared to one-fifth of controls. Other sleep parameters appeared normal. The findings match the data at six months, indicating the changes in sleep patterns persisted.
Unlike at six months after their injury, at 18 months no correlation emerged between sleep problems and the severity of the injury. It may be that the need for extra sleep soon after injury serves a purpose in healing the brain, but over time sleep disturbances transform into a chronic, maladaptive condition, the authors speculated. Intriguingly, a recent study from the same group found that when rats were induced to sleep more in the first two weeks after a traumatic brain injury, they maintained their ability to remember new objects, whereas controls did not. These power sleepers expressed less APP in the hippocampus and cortex than injured controls did, suggesting the extra sleep might mitigate axonal injury (see Morawska et al., 2016). However, it remains to be determined whether the extra sleep TBI patients get more than a year after injury helps or harms their brains.
Notably, these patients did not realize they had a sleep problem, as their self-reports and clinical interviews regarding sleep habits resembled those of controls. This suggests sleep disorders may go unrecognized in TBI patients, and therefore untreated. Such patients should be evaluated by objective sleep laboratory measures, the authors suggest.
“Imbach et al. make a compelling case that post-traumatic sleep-wake disorders may represent a silent epidemic,” wrote Brian Edlow at Massachusetts General Hospital, Boston, and Gert-Jan Lammers at Leiden University Medical Centre, the Netherlands, in an accompanying editorial. They noted that the study represents the most comprehensive longitudinal analysis of post-traumatic sleep-wake disturbances to date.
Would sleep therapy help TBI patients, or people with AD for that matter? No one knows. “We need very good controlled, prospective studies on how sleep therapy (not sleep medications) can reduce the progression of different diseases, including Alzheimer’s,” Nedergaard said.—Madolyn Bowman Rogers
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- While You Were Sleeping—Synapses Forged, Amyloid Purged
- Brain Drain—“Glymphatic” Pathway Clears Aβ, Requires Water Channel
- Glymphatic Flow, Sleep, microRNA Are Frontiers in Alzheimer’s Research
- Paper Alert: In Vivo Human Data Shows Reduced Aβ Clearance in AD
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