Norepinephrine-mediated slow vasomotion drives glymphatic clearance during sleep

doi:10.1016/j.cell.2024.11.027

Cell

Volume 188, Issue 3, 6 February 2025, Pages 606-622.e17

https://doi.org/10.1016/j.cell.2024.11.027 Get rights and content

Highlights

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Norepinephrine release from the locus coeruleus drives slow vasomotion in NREM sleep
•
Infraslow norepinephrine oscillations control opposing changes in blood and CSF volumes
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Norepinephrine oscillation frequency during NREM sleep predicts glymphatic clearance
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The sleep aid zolpidem suppresses norepinephrine oscillations and glymphatic flow

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.

Graphical abstract

Introduction

The glymphatic system is a highly organized, brain-wide network responsible for the transport of cerebrospinal fluid (CSF), which is crucial for the removal of protein waste, including amyloid and tau.¹^,²^,³ Glymphatic fluid transport also plays a critical function in water homeostasis,⁴^,⁵^,⁶ antigen presentation,⁷ immune cell trafficking,⁸^,⁹ and in the delivery of solutes, such as glucose.¹⁰ Due to the absence of lymphatic vessels, the brain relies on CSF circulation through the tissue to remove waste proteins. The glymphatic system uses the perivascular spaces (PVSs) as low-resistance routes for rapid CSF influx into deep brain regions.¹ Factors such as increased vascular stiffness, brain injury, or gliosis can impair glymphatic flow, thereby predisposing individuals to the development of neurodegenerative diseases.⁴^,⁵^,¹¹^,¹²^,¹³

Glymphatic fluid transport is enhanced during sleep.¹⁴^,¹⁵^,¹⁶^,¹⁷^,¹⁸^,¹⁹ In recent years, the link between sleep and the glymphatic system has received substantial attention, as poor sleep often precedes the onset of neurodegenerative diseases and is a predictor of early dementia.²⁰^,²¹^,²² Yet, the precise mechanisms by which sleep and wakefulness influence glymphatic flow remain unclear. Initial studies in anesthetized mice showed that cardiac pulsatility promotes glymphatic inflow,²³^,²⁴ but modeling studies have indicated that cardiac pulsatility might only serve a mixing effect.²⁵^,²⁶ Instead, slow vasomotion—spontaneous, rhythmic constriction and dilation of arteries²⁷^,²⁸^,²⁹^,³⁰^,³¹^,³²—has been proposed as a potential driver of solute clearance in NREM sleep, though direct evidence has been lacking.

In parallel, recent advances in biosensor imaging have revealed that NREM sleep is characterized by regular oscillations in brain norepinephrine (NE) levels, occurring at a frequency in the range of ∼0.02 Hz.³³^,³⁴^,³⁵ These infraslow oscillatory fluctuations in NE are critical for memory performance and the micro-architectural organization of sleep,³³^,³⁴^,³⁶ but their effects on the vasculature and CSF dynamics are unknown, despite NE being a potent vasoconstrictor.³⁷^,³⁸

Technical limitations have hampered progress in rodent glymphatic imaging studies, primarily due to the lack of methods to visualize CSF dynamics during natural, unrestrained sleep. As a result, most rodent studies have relied on anesthesia as a surrogate for sleep. Yet, anesthesia disrupts or eliminates many key characteristic features of natural sleep, such as oscillatory NE release, micro-arousals, vasomotion, and REM sleep, which renders anesthesia unsuitable for studying the natural drivers of glymphatic flow.³⁹^,⁴⁰^,⁴¹^,⁴² While neuroimaging in humans does not yet have the spatial resolution to quantify CSF flow in the brain parenchyma, several studies have shown that CSF movement in the 4th ventricle is temporally linked to neuronal slow wave activity (SWA) and vascular volume changes during NREM sleep.¹⁷^,⁴³ However, CSF flow in the 4th ventricle is not representative of glymphatic flow in the brain parenchyma,⁴⁴ leaving the fundamental question of what drives glymphatic flow during natural sleep unresolved.

We here developed a method, “flow fiber photometry,” that enabled recordings of blood and CSF dynamics during long, uninterrupted periods of wakefulness, NREM, and REM sleep by avoiding the need for head fixation and allowing mice to move freely in their home cage during recordings. We achieved chronic blood labeling by using liver-secreted, fluorescently tagged albumin,⁴⁵ in conjunction with tagging of CSF using a fluorescent tracer to investigate the impact of NE oscillations on both vascular and CSF dynamics. With simultaneous recordings of NE (using GRAB_NE2m),⁴⁶ blood,⁴⁵ CSF, and electroencephalogram (EEG)/electromyogram (EMG) activity, we uncovered a surprisingly intricate and tightly linked relationship between brain states and fluid dynamics.

Our findings identify locus coeruleus (LC)-induced NE oscillations as the key driver of slow vasomotion, which in turn facilitates glymphatic clearance during natural sleep. These insights have broad implications for understanding the components of restorative sleep, the functions of slow vasomotion, and the connection between vascular dysfunction and glymphatic failure, which predispose individuals to neurodegenerative diseases.

Section snippets

NE drives state-dependent vasomotion

Recent work has shown that NE release from the LC displays an infraslow (∼0.02 Hz) oscillatory pattern during NREM sleep.³³^,³⁴^,³⁵ Given that NE is a potent vasoconstrictor,³⁷^,³⁸ how does the LC-mediated NE release affect the cerebral vasculature? 4-month-old wild-type C57BL/6 mice expressing the fluorescent NE sensor GRAB_NE2m⁴⁶^,⁴⁷ were implanted with EEG, EMG, and a fiber optic cannula in the dorsal cortex. Furthermore, the mice were infected with AAV8/P3-alb-mScarlet, which targets the

Discussion

The motivation for this study was to understand what drives glymphatic flow during sleep. To explore this, we employed multiple approaches to examine the effects of NE dynamics on cerebral blood and CSF across wakefulness, NREM sleep, and REM sleep. First, we demonstrated that optogenetic stimulation of the LC leads to NE release, which decreases vascular volume in a dose-dependent manner. Pan-adrenergic receptor blockage eliminated vasoconstriction induced by LC activation, consistent with the

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Maiken Nedergaard (nedergaard@sund.ku.dk).

Materials availability

This study did not generate new unique reagents.

Data and code availability

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Source data and examples of raw EEG, EMG, and fiber photometry traces have been uploaded to Mendeley Data: https://doi.org/10.17632/jy7n6vpd9j.1.
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Key MATLAB code generated in this manuscript has been deposited on GitHub (https://github.com/NHauglund/Fiber-photometry-analysis) and can

Acknowledgments

We would like to thank Dan Xue for expert graphical support, the Department of Clinical Physiology and Nuclear Medicine, Rigshospitalet, for providing us with [^99mTc]-DTPA, Palle Koch for technical assistance with EEG recordings and SPECT imaging, Björn Sigurdsson for advice and support on data analysis, Felix Beinlich for technical advice on miniscope recordings, and Hashmat Ghanizada for guidance and help with preparation of pan-adrenergic blockers. This work was supported by Lundbeck

Author contributions

Conceptualization, N.L.H. and M.N.; fiber photometry, EEG, optogenetics, SPECT/CT, miniscope methodology, and investigation, N.L.H., F.L.S., M.G.K., T.R., P.W., C.K., M.A., K.T., Z.B., and V.U.; viral constructs, H.H.; analysis, N.L.H., F.L.S., S.B.B., and K.T.; visualization, N.L.H.; writing, N.L.H. and M.N. All authors contributed to manuscript editing and support the conclusions.

Declaration of interests

M.N. is a paid consultant of CNS2 for unrelated studies.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
DAPI	Thermo Fischer Scientific	Cat# D1306
Bacterial and virus strains
AAV9-hSyn-GRAB_NE2m	Feng et al.,⁴⁶ provided by Dr. Yulong Li, WZ Biosciences	YL003008-AV9
pAAV-hSyn-GRAB_NEmut	Addgene	123310-AAVrg
AAV9-CBh-mCherry	Provided by Dr. Ayumu Konno	N/A
AAV8/P3-Alb-mNeonGreen	Wang et al.,⁴⁵ provided by Dr. Hajime Hirase	Available at Zurich VVF: v820-8
AAV8/P3-Alb-mScarlet	Wang et al.,⁴⁵ provided by Dr. Hajime Hirase	Available at Zurich VVF: v821-8

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NE drives state-dependent vasomotion

Lead contact

Materials availability

Data and code availability

Key resources table

Cell Rep.

Curr. Opin. Physiol.

Prog. Neurobiol.

Sleep Med. Rev.

Neuron

Curr. Biol.

Curr. Opin. Neurobiol.

Cell Rep.

Neuroimage

Cell Rep. Methods

Neuron

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Cell Rep.

Neuron

Cell Rep. Methods

Curr. Biol.

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Exp. Gerontol.

Brain Res.

Neurosci. Lett.

Neuroimage

Neuropharmacology

Sleep Med. Rev.

Sci. Transl. Med.

J. Neurosci.

J. Exp. Med.

Science

Nature

Physiol. Rev.

J. Clin. Invest.

Nat. Neurosci.

Science

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Proc. Natl. Acad. Sci. USA

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