Scientists have uncovered that rhythmic oscillations of a specific neurotransmitter are crucial for the clearance of toxic proteins from the brain during non-rapid eye movement (non-REM) sleep. These oscillations facilitate the glymphatic system by enabling the coordinated movement of cerebrospinal fluid and blood. However, it has been observed that the commonly prescribed sleep aid zolpidem, often marketed as Ambien, interferes with this process, potentially hindering the brain’s ability to eliminate waste. .
The glymphatic system, a network within the brain, plays a key role in removing waste products such as amyloid and tau proteins, which are linked to neurodegenerative diseases like Alzheimer’s. Unlike other organs, the brain does not possess traditional lymphatic vessels for waste removal. Instead, it depends on cerebrospinal fluid (CSF) to flush out toxins through specialized spaces surrounding blood vessels.
“When this study was initiated, it was already understood that the glymphatic system is essential for brain cleansing, that it relies on brain fluid (CSF) circulating through the brain, and that it becomes active during sleep. However, the mechanism by which sleep drives waste removal from the brain was not yet known,” explained Natalie Hauglund, the study’s first author and a postdoctoral fellow at the University of Oxford.
A series of experiments were conducted on mice to observe the glymphatic system in action during sleep, shedding light on its intricate mechanisms.
The initial experiments focused on the interaction between norepinephrine, a neurotransmitter involved in regulating arousal and blood vessel constriction, and its effects on cerebral blood flow and cerebrospinal fluid (CSF) movement during sleep. Utilizing “flow fiber photometry,” a technique enabling real-time tracking of norepinephrine levels, blood flow, and CSF dynamics, it was discovered that norepinephrine levels displayed slow, rhythmic oscillations during non-REM sleep.
These oscillations were found to align with synchronized cycles of blood vessel constriction and relaxation, known as vasomotion, which generated a pumping mechanism to propel CSF through the brain. Notably, these oscillations were absent during wakefulness and disrupted during REM sleep, indicating that non-REM sleep creates a unique state conducive to optimal glymphatic activity.
To establish a direct link between norepinephrine oscillations and vasomotion, optogenetics was employed to manipulate the locus coeruleus, a brain region responsible for norepinephrine release. Through stimulation and inhibition of this region, it was demonstrated that norepinephrine release precisely regulated blood vessel dynamics and, consequently, CSF flow.
Further experiments were conducted to directly test whether vasomotion functions as a “pump” for CSF flow. Optogenetic techniques were applied to target smooth muscle cells in blood vessels, using light to induce rhythmic constriction and relaxation in naturally sleeping mice. This artificial increase in vasomotion frequency was observed to enhance CSF flow and glymphatic clearance in brain regions near the site of vascular manipulation.
“These findings provide direct evidence that cycles of arterial constriction and dilation are central to driving glymphatic activity,” the researchers noted, confirming the pivotal role of vasomotion in the glymphatic system.
Natalie Hauglund
“It was discovered that the driving force behind CSF flow through the brain, and consequently brain cleansing during sleep, is a slow pumping mechanism generated by the synchronous constriction and dilation of blood vessels in the brain. This process is regulated by a signaling molecule known as norepinephrine, which is released in the brain approximately every 50 seconds, creating slow oscillations in norepinephrine levels during sleep.”
Natalie Hauglund
Additional experiments explored how natural sleep microarchitecture, particularly the frequency of brief awakenings referred to as micro-arousals, influences glymphatic activity. By employing EEG and EMG recordings to monitor brain activity and sleep states, correlations were drawn between the frequency of micro-arousals and the efficiency of glymphatic clearance.
Mice exhibiting more frequent micro-arousals during non-REM sleep demonstrated enhanced glymphatic clearance of tracer molecules. This observation reinforced the notion that norepinephrine oscillations and the associated vascular dynamics, which often coincide with micro-arousals, play a critical role in driving CSF flow.
Interestingly, while micro-arousals were linked to increased glymphatic activity, they were not identified as the sole factor influencing clearance. It was concluded that the oscillatory release of norepinephrine during non-REM sleep serves as the primary driver of CSF flow, with micro-arousals acting as a secondary, parallel process.
“The study revealed that the frequency of micro-arousals, which are tiny awakenings occurring throughout the night without being perceived by the sleeper, positively correlates with glymphatic flow. This may appear surprising, as micro-arousals are often considered a sign of fragmented sleep.”
“However, growing evidence suggests that micro-arousals are a natural component of healthy sleep and may serve important functions in the beneficial effects of sleep. The correlation between glymphatic flow and micro-arousals arises because the norepinephrine waves controlling the ‘pump’ that drives CSF flow also trigger micro-arousals.”
Natalie Hauglund
Researchers examined the effects of zolpidem on sleep architecture and glymphatic activity by administering the drug to a group of mice while monitoring norepinephrine levels, blood vessel dynamics, and cerebrospinal fluid (CSF) flow. While zolpidem facilitated faster sleep onset, a significant disruption in infraslow norepinephrine oscillations and blood vessel vasomotion was observed, both of which play a crucial role in glymphatic clearance.
EEG recordings indicated an increase in micro-arousals among zolpidem-treated mice, accompanied by a reduction in norepinephrine peaks. This disturbance led to impaired synchronization of blood flow and CSF movement. Fluorescent tracer injections into the CSF further revealed diminished tracer inflow and clearance in zolpidem-treated mice, suggesting interference with the brain’s natural waste removal processes during sleep.
“We found that the sleep aid zolpidem disrupted the norepinephrine oscillations and thereby reduced the fluid flow. This suggests that the sleep you get while using sleep medication is not as beneficial as regular sleep in terms of restorative processes, such as brain cleaning.”
Natalie Hauglund
Since the experiments were conducted in mice, results may not fully reflect human sleep architecture or physiology. However, Hauglund noted similarities between animal and human studies, stating:
“Results from human studies indicate that the same mechanism exists. For example, MRI scans of people sleeping inside a scanner have shown that slow oscillations in blood volume and CSF volume are present in the brain during sleep.”
Natalie Hauglund
Further research may explore the impact of aging, vascular health, and neurodegenerative diseases on glymphatic efficiency. Potential strategies to optimize waste clearance—through pharmacological, lifestyle, or non-invasive methods—are of particular interest for future studies.
“Many questions are still waiting to be answered,” Hauglund said. “For example, it will be important to see how different disease states affect the CSF pumping, and if there are ways to enhance the ‘pump’ in order to boost the removal of waste from the brain.”
Natalie Hauglund
https://doi.org/10.1016/j.cell.2024.11.027
Summary
As the brain transitions from wakefulness to sleep, processing of external information diminishes while restorative processes, such as glymphatic removal of waste products, are activated. Yet, it is not known what drives brain clearance during sleep. We here employed an array of technologies and identified tightly synchronized oscillations in norepinephrine, cerebral blood volume, and cerebrospinal fluid (CSF) as the strongest predictors of glymphatic clearance during NREM sleep. Optogenetic stimulation of the locus coeruleus induced anti-correlated changes in vasomotion and CSF signal. Furthermore, stimulation of arterial oscillations enhanced CSF inflow, demonstrating that vasomotion acts as a pump driving CSF into the brain. On the contrary, the sleep aid zolpidem suppressed norepinephrine oscillations and glymphatic flow, highlighting the critical role of norepinephrine-driven vascular dynamics in brain clearance. Thus, the micro-architectural organization of NREM sleep, driven by norepinephrine fluctuations and vascular dynamics, is a key determinant for glymphatic clearance.