Circadian Mechanisms in Brain Fluid Biology

Abstract

The brain is a complex organ, fundamentally changing across the day to perform basic functions like sleep, thought, and regulating whole-body physiology. This requires a complex symphony of nutrients, hormones, ions, neurotransmitters and more to be properly distributed across the brain to maintain homeostasis throughout 24 hours. These solutes are distributed both by blood and by cerebrospinal fluid. Cerebrospinal fluid contents are distinct from the general circulation because of regulation at brain barriers including the choroid plexus, glymphatic system, and blood-brain barrier. In this review, we discuss the overlapping circadian (~24 hour) rhythms in brain fluid biology and at the brain barriers. Our goal is for the reader to gain both a fundamental understanding of brain barriers alongside an understanding of the interactions between these fluids and the circadian timing system. Ultimately, this review will provide new insight into how alterations in these finely tuned clocks may lead to pathology.

Basics of Circadian Rhythms

We live in a 24-hour world, with the clock dictating our activities, sleep, and all the physiology in between. Circadian rhythms are cellular, physiological and behavioral changes in biology that occur every 24 hours. Circadian rhythms are internally generated, i.e. they can persist without input from the environment, synchronize across the body, and entrain to environmental stimuli.¹ With the advent of shift work and globalization, understanding how our bodies predict and change in response to different time cues becomes critical in preventing pathology.

Most cells contain a negative feedback loop of transcription and translation called the “molecular clock”. This molecular clock can drive 24-hour gene expression, and has been implicated in most basic cell functions including metabolic homeostasis and maintenance of the cell cycle. Briefly, in mammals the circadian transcription factors BMAL and CLOCK bind to promoters for Period (Per) and Cryptochrome (CRY), driving expression of these proteins which then inhibit BMAL and CLOCK over the course of 24 hours² (Figure 1). There is a second negative feedback loop whereby BMAL is regulated by REV-ERB transcription factors^2,3, adding another layer of complexity to our intrinsic cellular timing system (Figure 1). The discovery of this molecular clock in drosophila won the Nobel Prize in 2017,⁴ enabling scientists to better understand the driving mechanisms of pathology after circadian disruption. Indeed, chronobiologists are now developing small-molecule modulators of the molecular clock that have shown some efficacy in improving preclinical models of disease including cardiovascular disease and obesity.^5–7

Figure 1: A model of the mammalian molecular clock.

In mammals, circadian timing is controlled by a negative feedback loop of circadian locomotor output cycles kaput (CLOCK) and brain and muscle ARNT-like 1 (BMAL1) driving expression of period (PER) and cryptochrome (CRY), which inhibit their own transcription and translation every ~24 hours. This molecular clock can control expression of other genes, potentially including the water channel aquaporin 4 (AQP4). There are other feedback loops of the molecular clock, including REV-ERB and ROR regulation of BMAL1.

These cellular clocks must synchronize within the body to produce coherent physiological rhythms. The suprachiasmatic nucleus (SCN) of the hypothalamus generates and synchronizes circadian rhythms throughout the body. The SCN receives direct light input from retinohypothalamic tract innervations⁸, as well as food and exercise input from several areas including the intergeniculate leaflet^9–11. The SCN itself is composed of self-perpetuating, imprecise oscillating neurons that must synchronize and encode these new time cues.^12,13 While the majority of neurons in the SCN utilize GABA as a primary neurotransmitter, there is a panoply of peptidergic signaling occurring simultaneously.⁸ For example, vasoactive intestinal peptide (VIP) signaling is a key driver of synchrony between neurons within the SCN.^14–16 After integration and synchronization of timing within the SCN, neurons from the SCN project to several centers within the hypothalamus including the paraventricular nucleus and dorsomedial hypothalamic nucleus, ultimately controlling glucocorticoid rhythms, a key synchronization cue to the periphery.^17,18

Beyond the SCN, many other physiological rhythms persist across the body including body temperature, heart rate, and blood pressure.^19,20 Alterations in these basic processes cause disease. For example, non-dipping blood pressure, where there is no nightly blood pressure decrease, correlates with increased risk of small vessel disease^21–23 and adverse cardiovascular events when accompanied by hypertension²⁴. Brain temperature also oscillates, and amplitude of these rhythms may predict survival after traumatic brain injury.^25,26 Here, we review and discuss evidence that these basic physiological rhythms may alter brain homeostasis.

We propose that cellular, neurocircuitry-controlled, and physiological circadian rhythms modulate cerebrospinal fluid (CSF), interstitial fluid (ISF), and blood homeostasis in the brain. We further hypothesize that these fluids are modulated by circadian rhythms in major barrier pathways in the brain including the choroid plexus, the perivascular spaces of the glymphatic system, and across the blood-brain barrier.

A broad overview of brain fluid pathways

In the periphery, interstitial fluid homeostasis and local immune responses are regulated by the lymphatic system. The lymphatic system is a specialized network of blind-ended capillaries connected to vessels with specialized valves, that directionally drive interstitial fluid and solutes from the tissue to the lymph nodes, ultimately emptying to the venous system.²⁷ During infection and injury, this network of capillaries and vessels directly deliver lymphocytes from lymph nodes to the tissue.²⁸ While the brain also requires fluid homeostasis, interstitial waste clearance, and immune surveillance, there is no traditional lymphatic tissue within the brain parenchyma. Instead, the brain has highly specialized fluid pathways and brain interfaces to perform similar functions as the peripheral lymphatic system.

Instead of a steady flow of interstitial fluid coming from the blood, the brain utilizes CSF to provide a buffer within the skull and a source for interstitial fluid within the parenchyma²⁹. The choroid plexus is regarded as the main source of CSF for the mature brain. Choroid plexus tissues extend into the brain ventricles and their structure is a specialized cuboidal epithelium surrounding fenestrated capillaries integrated with mesenchymal, neuronal, and immune cell populations (Figure 2). It is contiguous with the pia and distinct from the ependymal cell layer that lines brain ventricles. The choroid plexus is also the blood-CSF-barrier. It interfaces with blood and the immune system to regulate development, growth, and neuroimmune state of the CSF. From the choroid plexus, CSF circulates in dynamic fashion through the ventricles, ultimately exiting the fourth ventricle to enter the subarachnoid space. While bulk flow of CSF generally moves in the directions described above, CSF movement is also pulsatile enabling mixing throughout the system. From there, CSF can either exit the skull via perineuronal and lymphatic routes, or enter the glymphatic system.

Figure 2: A model of the choroid plexus, the blood-CSF barrier.

The choroid plexus is the blood-CSF barrier that makes CSF by transporting ions from interstitial fluid across the epithelial cells of the choroid plexus through a cohort of ion channels (left inset). It also modulates hormone transport including thyroid hormone (T4) in part by modulating transporters (MCT8, OAT1C) and transthyretin (TTR) expression in the epithelial cells (right inset). The choroid plexus is also a hub of immune cell communication and transport.

The glymphatic system is a network of perivascular spaces bordering blood vessels and wrapped in astrocyte endfeet. This system hijacks the vascular network, enabling directional flow of CSF entering the periarterial space, ISF mixing and traveling through the parenchyma, ultimately leaving, in part, along the perivenous space (Figure 3).^30–32 This model has been supported by several lines of evidence including in vivo and postmortem imaging^31,33, modeling of fluid dynamics in the periarterial space^34–37 and extracellular spaces^38–40, as well as datasets collected using magnetic resonance imaging^41,42. This system can clear solutes including lactate⁴³, lipids⁴⁴, and peptides such as amyloid-beta^31,45,46. Glymphatic fluid drains into the traditional lymphatic system in the meninges and is cleared to the cervical lymph nodes^47–51, but the exact connection between the perivenous space and the lymphatic network has not been identified. Similarly, whether the glymphatic system has analogous valve structures as the lymphatic system, or relies on dynamic changes in vasoreactivity to gate flow needs more exploration. There is an extensive literature on the modern interpretation of the glymphatic system, how it fits with historical analysis of perivascular spaces, and potential caveats of the current model.^{30,32,52–57} This review will focus on how perivascular fluid dynamics interact with the circadian timing system.

Figure 3: A model of the glymphatic system.

The glymphatic system is a network of perivascular spaces that enables the bulk flow of cerebrospinal fluid (CSF, light blue) down the periarterial space (arteries, red), through the tissue (grey), clearing waste to the perivenous space (veins, dark blue). Note: perivascular spaces have been described since the 1840s^268,269, with experiments in the 1920s suggesting they extend along arterioles, capillaries and venules²⁷⁰. Periarterial and perivenous spaces have been captured with in-vivo imaging^35,271–273. Pericapillary spaces have not been visualized in-vivo due to limits in resolution sensitivity of current in-vivo imaging techniques, but their existence is supported by ex-vivo studies^274–277. Because of this, pericapillary spaces are depicted as small and hypothesized flow is marked in grey in this model. The neurovascular unit (NVU, insets) is a fundamental component of the glymphatic system and the blood-brain barrier (BBB). It is composed of astrocytic endfeet enriched with water channel aquaporin 4 (AQP4) enwrapping the vasculature’s endothelial cells and pericytes in capillaries (bottom inset), or smooth muscle cells in arterioles (top inset). Microglia and perivascular macrophages survey the immune environment of the NVU.

The blood-brain barrier (BBB) is a dynamic system that functions to maintain central nervous system (CNS) homeostasis by regulating interactions of the brain parenchyma with peripheral circulation. Structurally, the BBB is part of the neurovascular unit (NVU): non-fenestrated brain endothelial cells bound tightly together through tight junctions, surrounded by pericytes, astrocytes, microglia, and neurons (Figure 3, insets). The proximity of these components enables crosstalk, facilitating coordinated efforts to meet metabolic requirements of the CNS and adjust to changes in environment. The BBB allows the passage of molecules through various mechanisms, including endocytosis, carrier-mediated transport systems, adenosine triphosphate (ATP)-binding cassette (ABC) transporters, and transcellular diffusion.

These systems:

CSF production by the choroid plexus, pulsatility of CSF in the ventricles and aqueduct, bulk flow of CSF and ISF through the glymphatic system, and transport of molecules and metabolites across the BBB, are all inextricably linked. However, evidence suggests each system may be governed independently. For example, CSF production is higher in female mice⁵⁸ but there is no difference in glymphatic function between healthy male and female mice⁵⁹. Similarly, CSF production is increased during the rest phase in humans and during the active phase in rats⁶⁰, while glymphatic function peaks only during the rest phase in humans⁶¹, rats⁶², and mice⁶³. Circadian timing may be a way to compartmentalize these functions. We will explore how circadian rhythms in brain fluid pathways may interact with other known rhythms in brain physiology to regulate basic circadian timing within the brain and throughout the body.

Rhythms at the interface of CSF and blood: the choroid plexus

In addition to serving as a fluid cushion, waste sink, and buffer for the brain, CSF plays critical roles in regulating the CNS throughout life, including distributing essential health and growth-promoting factors.⁶⁴ CSF production^60,65, composition^66–71, and glymphatic clearance/ distribution^46,72 change substantially throughout the 24-hour day. As a direct modulator of CSF volume and composition, the choroid plexus has been the subject of growing interest for its important roles in brain development and health.⁶⁴ Here we will elaborate evidence supporting the choroid plexus as an understudied functional component of the brain-circadian axis.

CSF contains biomarkers of circadian rhythmicity and plays key roles in relaying the output of diurnal clocks to target brain tissues. Historic CSF transplantation studies showed that CSF can carry cues for drowsiness^73,74 and satiety⁷⁵. Transplant studies of the SCN indicate that diffusible factors can mediate the circadian rhythmicity of locomotion through the CSF.^{67,68,76–78} This diffusible signal could include the peptides arginine–vasopressin (AVP)⁷⁹ and VIP which are expressed by the SCN and detectable in the CSF.^80,81 CSF orexins from the hypothalamus decrease in the inactive phase, directly coupling the sleep-wake-cycle with CSF.^82,83 CSF melatonin levels very closely reflect melatonin levels from the pineal gland^79,84, however direct effects of melatonin on choroid plexus output are unknown. Serotonin levels also cycle in the CSF⁸⁵ and can signal through 5-HT_2c receptors to increase choroid plexus calcium activity and protein secretion⁸⁶. The CSF may be a medium through which a multitude of circadian signals can be communicated to distant targets, with broad-ranging implications for CSF-based delivery of key factors to the brain.

The choroid plexus tissues are key sources of CSF and directly interact with systemic circulation through fenestrated capillaries. Fluid secretion at the choroid plexus is an active process (Figure 2, left inset).⁸⁷ Ions are moved from blood at the basal surface of choroid plexus epithelial cells to the CSF at the apical surface through pumps, channels, and transporters. These transporters are largely ion-selective co-transporters that depend on the Na⁺/K⁺ gradients generated by the Na⁺-K⁺-ATPase. A distinct complement of transporters populates the basal and apical surfaces, separated by apico-lateral tight junctions that form the blood-CSF-barrier. Actively generated osmotic gradients drive net water fluxes. The water channel aquaporin 1 is on the apical surface of choroid plexus epithelial cells, but to date, no basal aquaporin has been identified. Some evidence suggests the possibility of water transport through ion co-transporters^88,89 or paracellular water transport through the pore-forming claudin-2⁸⁷. However, water transport at the choroid plexus and contributions of extra-choroidal sources of CSF remain incompletely understood.

Choroid plexus epithelial cells display cell-autonomous circadian rhythmicity in gene expression of core components of the molecular clock including BMAL1, CLOCK, CRY, and PER2. ^90–95 The circadian rhythmicity in choroid plexus explants exhibit multi-day autonomous oscillations of Per2::Luciferase^90,93,94, that can be resynchronized with dexamethasone⁹⁴. Developmentally, choroid plexus rhythmicity is evident by birth, but the 24-hour period is not solidified in mice until P11, around the time of eye opening.⁹⁰ While circadian rhythmicity in choroid plexus has been reported in both male ⁹⁴ and female rodents⁹², studies from ovariectomized rats demonstrate that rhythmicity in females is dependent on estrogen and mediated through the estrogen receptor⁹¹. The interaction of choroid plexus rhythmicity and behavior, especially in conditions affecting estrogen availability (e.g., menopause, hormone replacement therapy, estrogen-blocking cancer treatments like tamoxifen) remains an open question for future studies.

Loss of Bmal1 from ciliated epithelial cells, including choroid plexus epithelial cells, lengthens circadian behavioral periodicity.⁹⁰ This suggests that choroid plexus output may fine-tune circadian timing, potentially by modulating melatonin in the CSF. While the pineal gland is the major melatonin source, the choroid plexus expresses melatonin biosynthetic pathway enzymes and conditioned media from porcine choroid plexus explants accumulated melatonin, although without rhythmicity.⁹⁶ Recently, ribosomal pulldown analyses showed choroid plexus translation and protein synthesis are regulated diurnally, with higher levels of translation taking place during the dark phase when rodents are more active.⁹⁴ These oscillations in translation may drive molecular cues to long-distance brain targets through choroid plexus output into the CSF. For example, choroid plexus transthyretin (TTR), the main transporter of thyroid hormone from the blood through choroid plexus epithelial cells into CSF (Figure 2, right inset), is higher during the active phase. This corresponds to higher levels of thyroid hormone T3 in the CSF. ⁹⁴ Why these day/night differences in TTR and CSF thyroid hormone exist, and how they may maintain circadian period, remain to be elucidated.

Choroid plexus metabolism is crucial to producing CSF and maintaining the blood-CSF barrier.⁹⁷ Mass spectrometry-based choroid plexus metabolomics in mice identified more oxidative phosphorylation in the choroid plexus during the active phase, substantially different metabolite profiles present in the tissue between day and night, with correspondently altered CSF metabolite profiles.⁹⁴ Functional consequences of these diurnal shifts remain to be investigated, but similar diurnal metabolic processes are broadly implicated in Parkinson’s Disease and Alzheimer’s disease.^98–100

In addition to the metabolic profile of the choroid plexus, gene expression of membrane transporters and tight junction components, together with transmission electron micrographs of the apico-lateral tight junctions suggest that the blood-CSF barrier may be more permeable during the rest-phase.⁹⁴ This could have large implications not only for drug transport across the barrier⁹⁷, but also for immune cell trafficking^101,102. For example, intravital 2-photon microscopy of the choroid plexus showed real time macrophage infiltration into the choroid plexus after immune activation in adult mice⁸⁶ and during development after maternal inflammation¹⁰³. Changes in barrier properties associated with immune cell trafficking are induced by brain and blood cytokines, and even breeches to the gut barrier affect choroid plexus permeability at the endothelium.¹⁰⁴ While immune responses have a well-studied circadian component^105–107, direct relationships between circadian rhythms, the blood-CSF-barrier, and immune cell trafficking remain to be investigated.

Beyond the choroid plexus, the ventricular system is lined by ependymal cells and tanycytes, which border circumventricular organs and mediate close associations between the CSF and certain brain structures. Rhythmic expression of circadian clock genes and key neurotransmitters, such as oxytocin and parvalbumin, occurs in ependymal cells that form the CSF-brain-barrier.^108,109 In rodents, this expression is independent of melatonin expression¹¹⁰, but does interact with the thyroid hormone axis in hypothalamus^111,112 raising the hypothesis that circadian changes in CSF thyroid hormone described above could also interact with ependyma. Tanycytes are responsive both to systemic and CSF glucose levels, and to SCN signals.¹¹³ Thus CSF-based signals within brain ventricles have the potential to influence a number of critical tissues.

In summary, composition and production of CSF varies by time of day. This may be driven by underlying circadian changes in choroid plexus metabolism, permeability, and secretion. How changes in permeability of the blood-CSF barrier, immune state of the blood-CSF barrier, composition of the CSF, and interactions at the CSF-brain barrier in the ventricles may be regulated by, and fine-tune circadian rhythms throughout the brain and body remain open for investigation.

Rhythms in CSF pathways: the glymphatic and lymphatic systems

Circadian rhythms maintain homeostasis of key biological processes in order to predict changes in resource availability across the day. Is it surprising, then, that the two main waste-clearance systems within the brain, the glymphatic system and the blood-brain barrier, are under circadian control? In this section, we explore the glymphatic system, what drives it, and why it might be critical for circadian homeostasis.

In both humans and rodents, CSF/ISF flow along the perivascular network, known as the glymphatic system (Figure 3), and through the parenchyma is upregulated during sleep.⁴⁶ This is supported by an underlying rhythm in glymphatic influx of CSF and clearance of intraparenchymal solutes during the rest phase, and a shunt of CSF from brain parenchyma directly to the cervical lymphatic system during the active phase.⁶³ There is some evidence in humans that clearance of CSF/ISF from the brain along the parasagittal dura can be regulated by sleep¹¹⁴, but whether there is an underlying timing mechanism remains unknown. How and why these rhythms in CSF distribution occur is an active area of research.

The perivascular space itself is bounded on one side by the cerebrovasculature, and the other by astrocytic endfeet (Figure 3). In large periarterial spaces, perivascular macrophages monitor the microenvironment. The cells lining the perivascular space, including astrocytes, vasculature, smooth muscle cells and immune cells, are likely to regulate rhythms in glymphatic function. Indeed, the water channel aquaporin 4 (AQP4) supports rhythmic glymphatic function by localizing to the astrocytic endfoot along the vasculature during the rest phase, without significant changes to total protein levels of AQP4.⁶³

The role of astrocytes in modulating circadian rhythms is a growing field. The molecular clock in astrocytes is not necessary to drive behavioral rhythmicity, but astrocytes play a large role in dictating circadian periodicity¹¹⁵ and can drive rhythmicity in a mouse in the absence of neuronal rhythms¹¹⁶. In the SCN, rhythms in astrocyte function regulate excitatory glutamatergic signaling^116,117, inhibitory GABAergic signaling^118,119, ultimately altering cellular metabolism and glucose homeostasis throughout the body^119,120. Beyond the SCN, loss of BMAL1 in astrocytes leads to reactive gliosis, endosomal dysfunction, and altered homeostasis of amyloid-beta within the CSF^121–124, and astrocytes may undergo daily morphological changes¹²⁵. Apart from genetic models of circadian clock disruption, the primary evidence for astrocytic regulation of circadian rhythms comes from studies of astrocytes in sleep. Astrocytes can dynamically increase their volume in response to arousal cues such as norepinephrine¹²⁶, decreasing extracellular volume and, thus, potentially ISF flow. Astrocytes also dynamically regulate intracellular calcium^127–134 and chloride¹³⁵ signaling depending on sleep/arousal state, which may change local osmotic gradients to drive fluid flow. There is also evidence that astrocytes may regulate local waste clearance during extended wakefulness via glycogen turnover and phagocytosis^136,137, and connexin 43-mediated cell-cell communication between astrocytes is necessary to maintain wakefulness¹³⁸.

So, how do these observations of astrocytes in terms of circadian rhythms and sleep fit together? The two-process model of sleep postulates that there is both a need and a time for sleep, and these two functions ultimately dictate the arousal state of the animal.^139–141 Sleep need keeps track of how long the organism has been awake, increasing sleep pressure exponentially until released to sleep. Timing for sleep runs underneath this, providing a long-term multi-day signal to tell humans to sleep at night. We speculate that astrocytes reflect both sides of the two-process model to regulate glymphatic flow. Fast, dynamic changes in volume and signaling may regulate the perivascular and extracellular spaces to compensate for rapid alterations in arousal state across a single sleep/wake cycle. The molecular clock in astrocytes may regulate glymphatic flow by altering water permeability of the parenchyma, increasing osmotic pressure of the interstitial fluid by altering neurotransmitter levels or ISF composition¹⁴², ultimately regulating distribution of metabolites throughout the brain via sustained, relatively unchanged daily cycles in gene transcription, translation, and protein localization.

Glymphatic flow is not only determined by astrocytes, because the vasculature provides a boundary that pushes fluid along the perivascular space. Influx of CSF along the perivascular space is driven by arterial pulsatility.^143,144 This effect can be reduced by inducing acute hypertension, limiting the full motion of the arterial pulse wave.³⁶ It is possible that the daily drop in blood pressure during the sleep phase may promote glymphatic flow, partially by promoting arterial pulsatility. In fact, most circadian-controlled parameters of physiology that occur during sleep including lowered heart rate¹⁴⁵ and respiration rate^36,146 increase glymphatic function. The exact mechanisms of how these rhythms regulate glymphatics is unclear. For example: whether respiration works on the periarterial side to promote influx by pushing CSF into the perivascular space, or the perivenous side to increase clearance from the brain by widening the outflow route of ISF, remains unexplored.

Clues for how circadian-regulated physiology might regulate glymphatic influx and clearance might be found in ventricular and aqueduct fluid dynamics, where sleep promotes pulsatility of CSF by altering respiration and cerebral blood volume.¹⁴⁷ Respiration can direct CSF towards the thoracic cavity away from the brain^148–151, and pulsatility can enhanced by visual stimuli (a key entrainment cue for circadian rhythms)¹⁵². More work needs to be done to understand how CSF pulsatility in large spaces within the brain correlate to glymphatic influx and clearance in small perivascular spaces and the brain parenchyma. Furthermore, there is little to no research on how alterations of these physiological parameters during circadian disruption, like shiftwork, may impact glymphatic function.

We’ve discussed how astrocytic rhythms and cerebrovascular physiology may contribute to glymphatic fluid flow. But there is another component of the glymphatic system we have not yet critically evaluated: immune cells. Perivascular macrophages are critical for glymphatic flow in the periarterial space, remodeling the extracellular matrix via regulating matrix metalloproteinase activity to reduce arterial stiffness.¹⁵³ Macrophages have intrinsic molecular clocks that respond to inflammatory stimuli independent of systemic corticosterone rhythms¹⁵⁴, and these autonomous molecular clocks temporally couple immunometabolism by increasing pro-inflammatory markers during the active phase¹⁵⁵. When paired with the rhythms in leukocyte recruitment to arteries and veins in the periphery¹⁵⁶, some of which differentiate into macrophages, leads to the interesting hypothesis that rhythms in the immune system may contribute to glymphatic function; promoting influx at the arteries and, though unexplored, promoting clearance at the perivenous side at distinct times-of-day.

In closing, the perivascular spaces of the glymphatic system are critical circadian interaction zones; integrating peripheral cardiovascular physiology, neuroimmunology, and brain state information across multiple cell types, priming the brain to remove waste during sleep.

Rhythms in the interface of interstitial fluid and the blood: the blood-brain barrier

The glymphatic system does not work in isolation, as one boundary of the perivascular space is the blood-brain barrier. The blood-brain barrier cannot properly form without astrocytes.^157,158 Similarly, glymphatic function doesn’t “turn on” until the blood-brain barrier is in place.¹⁵⁹ In the next section, we will discuss circadian control of the blood-brain barrier, how this might promote further waste clearance.

Function and integrity of the BBB is modulated by all components of the NVU – brain endothelial cells, astrocytes, neurons, vascular smooth muscle cells, pericytes, and microglia (Figure 3, inset) — which possess autonomous molecular clocks producing independent rhythms.^160–162 BBB permeability itself may also increase in response to circadian time cues like light.¹⁶³ Structurally, the BBB is formed through tight junctions binding non-fenestrated brain endothelial cells together. In the periphery, protein and mRNA of endothelial junctions occludin and claudin-5 fluctuate across the day, and these cycles are lost in CLOCK^Δ19/Δ19 mutant mice and BMAL1 KO mice.^164,165 But, whether these components are similarly regulated in the brain is unknown. In this section, we will expand on BBB physiology and its relationship with circadian rhythms within the brain and the periphery.

The barrier in brain endothelial cells share many similarities to that of the blood-CSF barrier in choroid plexus epithelial cells. Most efflux at both the blood-CSF barrier and the BBB is managed by ATP-binding cassette transporters. These utilize ATP to pump lipophilic molecules that would otherwise enter the CNS via passive diffusion, back into the lumen of blood vessels. A major transporter within this family, P-glycoprotein multidrug transporter (PGP), displays diurnal rhythms in activity and expression in brain endothelial cells.¹⁶⁶ Upstream of PGP, BMAL1 can regulate rhythmic transcription and translation of the magnesium transporter TRPM7¹⁶⁷, ultimately altering magnesium stabilization and the ability of PGP to bind ATP¹⁶⁸. In drosophila, the molecular clock drives decreased intracellular magnesium at night, decreasing PGP activity, increasing BBB permeability.¹⁶⁹ Suggestive of rhythmic metabolism, xenobiotic metabolic enzymes such as the cytochrome P450 peroxygenases that are rhythmic in the liver^170,171 also exhibit daily oscillation in brain endothelial cells¹⁷². Characterizing circadian rhythms in both blood-CSF and BBB brain endothelial cells may provide insight into both the conserved and specialized functions of these structures.

Pericytes function at the BBB by regulating endothelial BBB-specific gene expression^173,174, immune interactions¹⁷⁵, microvascular cerebral blood flow¹⁷⁵, and waste clearance¹⁷³. During development, they interact with astrocytes and brain endothelial cells to form the NVU and are essential to early BBB integrity.^176,177 Circadian gene expression in pericytes can also affect angiogenesis by influencing endothelial molecular clock synchrony, maturation, and structure.¹⁷⁸ How pericyte rhythms regulate homeostasis of the BBB or glymphatic system, perhaps via pericyte-NVU crosstalk, remains largely unexplored.

In the mature brain, astrocyte endfeet cover the majority of the parenchymal surface of the endothelium and are necessary for BBB maintenance.¹⁷⁹ In vitro, astrocytes regulate brain endothelial tight junction function¹⁸⁰, gene expression and localization of transporters^181,182 and transport related enzymatic systems^183,184. This suite of astrocyte-driven regulation may depend on the metabolic status of astrocytes. Glucose-deprived astrocytes increase endothelial GLUT1 expression and thereby boost glucose reuptake through the BBB.^185,186 As discussed above, astrocyte metabolism is tightly regulated by circadian timing, and may have unrecognized consequences on BBB function.

Transport at the BBB is a predominant mechanism by which the brain mediates waste clearance¹⁸⁷, yet why would the brain need both the BBB and glymphatic system? In vivo, permeability across the BBB¹⁸⁸ and glymphatic fluid movement is upregulated during the rest phase of the animal⁴⁶. The BBB and the glymphatic system are both encapsulated by the NVU and molecularly interact to determine function. For example, brain endothelial cell agrin expression, an extracellular heparan sulfate proteoglycan located in the basal lamina of brain endothelial cells, can localize the water channel AQP4 to the vascular endfeet of astrocytes.^189–191 Whether agrin drives the known circadian rhythm in AQP4 localization, and ultimately promoting glymphatic rhythms in vivo remains unknown. This BBB-glymphatic crosstalk may be key to understanding how the brain balances waste clearance and nutrient delivery within ISF.

Microglia are brain resident macrophages that sit both in the parenchyma and at the NVU. Microglia respond to neuroinflammation and tissue injury¹⁹², regulate satiety¹⁹³, promote memory¹⁹⁴, and modify pain responses^195,196. Loss of microglia in rats drives changes in temperature homeostasis, energy metabolism, and diurnal activity rhythms, as well as dysregulates clock genes and proteins in the SCN and hippocampus.¹⁹³ Similar to immune cells in the periphery, immune responses by microglia are also controlled by an intrinsic circadian clock, with increased response during the active phase and evidence of glucocorticoid entrainment.^197,198 While the role of microglial molecular clock rhythms interacting with the NVU has not been directly addressed, vessel-associated microglia maintain BBB integrity, modulating Claudin-5 expression.¹⁹⁹ In sustained inflammation, microglia phagocytose astrocytic endfeet and impair BBB function.¹⁹⁹ Understanding how microglial molecular clocks interact with the NVU may be essential for regulating both waste clearance and neuroimmune homeostasis.

Peripheral signals can modulate BBB and brain function. Higher diet-quality scores correlate with lower levels of biomarkers associated with inflammation and endothelial dysfunction, suggesting a healthy BBB.²⁰⁰ Blood-borne protein is taken up by brain endothelial cells, glia and neurons residing in the NVU, which drastically declines with age.²⁰¹ When young mice receive blood factors from aged mice there is decreased adult neurogenesis^202,203, increased inflammatory states of microglia²⁰⁴ and brain endothelial cells²⁰⁵, and cognitive functions²⁰², further demonstrating blood-brain crosstalk. Molecules such as leptin²⁰⁶, B-amyloid²⁰⁷, delta-sleep inducing peptide¹⁶³ and prostaglandin D2²⁰⁸ exhibit rhythmicity in the CNS, and may be rhythmically transported through the BBB¹⁸⁸. The role of these potential time cues from systemic circulation regulating BBB function, ultimately impacting circadian rhythmicity and neuronal function in the brain, is an active area of research.

Functionally, the spine has much more movement and contact with diverse tissues along its length compared to the brain. Similar to the BBB, the blood-spinal cord barrier (BSCB) shares the same NVU components and overall barrier properties with some key differences: lower expression of tight junctions, adherens junctions, and PGP,^209,210 higher cytokine permeability,²¹¹ and glycogen deposition²¹⁰. Interestingly, the BSCB exhibits rhythmic uptake of the inflammatory cytokine Tumor Necrosis Factor alpha while the brain does not²¹², perhaps suggesting that immune-BSCB interactions may be integral to the function of the CNS-peripheral nervous system interface. There is little to no research on how BSCB rhythms may differ from those of the BBB, and how those differences may alter crosstalk between spinal NVU components.

Brain fluids in health and disease: bringing the rhythms together

Having covered circadian rhythm interactions of CSF at the choroid plexus, perivascular spaces and fluid flow through the glymphatic system, and transport of molecules and waste across the BBB, a question remains—how do alterations to these finely tuned clocks lead to pathology? In this section, we bring these rhythms together and postulate how diseases associated with CSF or waste clearance may be diseases of timing-misalignment. We also speculate how these brain-fluid homeostasis rhythms may interact with peripheral rhythms in the cardiovascular system to promote health, or, inflict pathology.

Abnormal CSF components are associated with a number of neurologic conditions that co-present with circadian disruptions, including hydrocephalus.²¹³ In children with hydrocephalus, VIP may be elevated.⁸¹ In adults with normal pressure hydrocephalus (NPH), CSF vasopressin does not cycle as it does in healthy CSF even with appropriate daily plasma vasopressin cycling.²¹³ Intracranial pressure in NPH patients is no longer correlated with time of day, but rather only with blood pressure.²¹⁴ Shunt-mediated CSF drainage in NPH does not restore circadian intracranial pressure rhythms²¹⁵, indicating upstream mechanisms other than CSF accumulation as the cause of disputed CSF pressure cycles in NPH. Interestingly, sleep apnea in hydrocephalus has been associated exclusively with NPH and is also unaffected by therapeutic CSF shunting.²¹⁶ Finally, oscillations in intracranial pressure are strongly correlated with changes in sleep state in children with hydrocephalus.^217,218 We speculate that known oscillations in blood volume and CSF volume during sleep¹⁴⁷ may be dysregulated in hydrocephalus. Taken together, it is important to understand how these diseases of disrupted CSF homeostasis may also have circadian and sleep components.

In addition to CSF homeostasis, the cardiovascular system has its own rhythmic components, with peripheral clocks present in all cardiovascular cell types including endothelial cells, vascular smooth muscle cells, fibroblasts, cardiomyocytes and cardiac progenitor-like cells.²¹⁹ Physiological processes such as heart rate²²⁰, blood pressure¹⁹, vasodilation and vasoconstriction²²¹ all exhibit circadian rhythmicity. Desynchrony of circadian rhythms via an extended light cycle causes cardiac fibrosis, impaired cardiac contractility and cardiomyopathy, which can be attenuated by returning to a regular light-dark cycle.²²² Genetic manipulation of the molecular clock causes atherosclerosis, dampened blood pressure rhythms, cardiac arrhythmias, altered metabolism, dilated cardiomyopathy and overall reduced lifespan.¹⁰⁵ Yet, interactions between disrupted cardiovascular rhythms and brain fluid homeostasis remain to be studied.

The lack of research between cardiovascular and brain rhythms is surprising given how the blood-CSF barrier, the perivascular spaces of the glymphatic system, and the BBB at the NVU are all dependent on cerebrovascular function. For example, preservation of blood pressure rhythmicity is crucial to prevent hypertension^24,221,223. Disruption of the SCN completely ablates blood pressure rhythms.^224,225 In turn, hypertension can cause BBB breakage²²⁶, and fluid movement through perivascular spaces in the glymphatic system is reduced when hypertension is induced²²⁷. Similarly, hypercholesterolemia can disrupt circadian rhythms²²⁸, exponentially increasing the risk of atherosclerosis²²⁹, leading to arterial stiffening and increased systemic inflammation, both of which affect BBB and glymphatic function^230,231. This evidence suggests that peripheral dampening of endogenous cardiovascular rhythms may directly impair brain homeostatic waste clearance systems.

Dampened circadian rhythms may act as a bridge between the concerted disruptions in cerebrovasculature, choroid plexus, BBB and glymphatic function seen in dementia syndromes. Circadian rhythms dampen with age, both in the brain and in the periphery.^232–235 Some genes exhibit age-dependent rhythmicity or alterations in rhythmicity pattern, while others gain rhythmicity with age.²³⁶ Postmortem choroid plexus from patients with neurodegenerative disease showed upregulated metabolism and inflammation and downregulated barrier components.²³⁷ Additionally, the choroid plexus calcifies with age, as does the pineal gland.²³⁸ With aging, glymphatic function is decreased^45,239,240, which is exacerbated in Alzheimer’s disease^241–244. There is also age-dependent BBB breakdown in the hippocampus, with worsened phenotype in patients with mild cognitive impairment.²⁴⁵ These age-related changes may drive other age-related cerebrovascular pathology including small vessel disease, where glymphatic dysfunction has been implicated²⁴⁶.

Ischemic stroke is another condition intertwined with circadian rhythms. After an ischemic event, loss of blood flow triggers a spreading depolarization, vasoconstriction, and a rapid influx of CSF along the glymphatic system, causing edema and brain damage.²⁴⁷ Over time, the blood-brain barrier opens and immune cells from the periphery invade the lesion site. If stable, well-entrained cardiovascular rhythms prevent ischemic stroke, environmental disruption would drive pathology. Indeed, shift workers, who regularly experience circadian disruption, have increased risk for ischemic stroke, high blood pressure, obesity, and sleep disturbances.^248–251 In preclinical models, disruption of a normal rodent light cycle leads to worse stroke severity.^252–254 Not only do disrupted environmental cycles drive stroke severity, there is evidence that normal changes in physiological timing may prime the bodies for worse cardiovascular outcomes, with stroke and adverse cardiovascular events more likely to occur in the morning^219,255along with increased mortality independent of sex, stroke severity, or age.²⁵⁶ After stroke, 24h behavioral activity correlates to stroke severity^257,258, mood and rehabilitation^259–261, highlighting that it is not only disease onset but during recovery where timing of endogenous circadian rhythms are important. Perhaps interventions which reinforce circadian timing can improve stroke recovery.

Changes to circadian timing can be induced by as little as milliseconds of light in humans^262,263, or single doses of anesthesia in rodents and humans^264–266, driving phase shifts of minutes to hours in circadian timing. There is almost no information about phase-shifting responses in the choroid plexus, glymphatic system, or BBB; though glymphatic function may be altered by timing of pentobarbital administration in a sex-specific manner⁵⁹. Gaining this information could inform long-term drug administration. For example, high-blood pressure medication is most effective at night,²⁶⁷ reinforcing the anticipated nightly drop of blood pressure. What happens if we give pharmaceuticals at the wrong time of day, phase-shifting endogenous rhythms that are necessary for health? It is possible that the process of treating chronic health conditions may trigger a cascade of pathological circadian disruption if pharmaceutical interventions are given at the incorrect time.

Concluding thoughts

The connections between circadian timing and brain fluid homeostasis are only now coming into focus. Daily rhythms in CSF production, choroid plexus function, glymphatic fluid transport and BBB function have only just been discovered. Whether these oscillations are necessary for brain health, if they respond to environmental stimuli to change internal timing, and how they may propagate circadian information from the SCN to the brain and the body to fine-tune physiology are new questions that are largely unexplored. Furthermore, understanding how disruptions in these fundamental homeostatic systems may feed off one-another and interact with endogenous cardiovascular rhythms to drive pathology may provide new insight into neurological and cardiovascular diseases. We hope that this review provided you with a strong stepping-off point into the wide world of rhythms in brain fluid biology.