The circadian clock in the choroid plexus drives rhythms in multiple cellular processes under the control of the suprachiasmatic nucleus.

Choroid plexus (ChP), the brain structure primarily responsible for cerebrospinal fluid production, contains a robust circadian clock, whose role remains to be elucidated. The aim of our study was to [1] identify rhythmically controlled cellular processes in the mouse ChP and [2] assess the role and nature of signals derived from the master clock in the suprachiasmatic nuclei (SCN) that control ChP rhythms. To accomplish this goal, we used various mouse models (WT, mPer2Luc, ChP-specific Bmal1 knockout) and combined multiple experimental approaches, including surgical lesion of the SCN (SCNx), time-resolved transcriptomics, and single cell luminescence microscopy. In ChP of control (Ctrl) mice collected every 4 h over 2 circadian cycles in darkness, we found that the ChP clock regulates many processes, including the cerebrospinal fluid circadian secretome, precisely times endoplasmic reticulum stress response, and controls genes involved in neurodegenerative diseases (Alzheimer's disease, Huntington's disease, and frontotemporal dementia). In ChP of SCNx mice, the rhythmicity detected in vivo and ex vivo was severely dampened to a comparable extent as in mice with ChP-specific Bmal1 knockout, and the dampened cellular rhythms were restored by daily injections of dexamethasone in mice. Our data demonstrate that the ChP clock controls tissue-specific gene expression and is strongly dependent on the presence of a functional connection with the SCN. The results may contribute to the search for a novel link between ChP clock disruption and impaired brain health.

LocoBox: Modular Hardware and Open-Source Software for Circadian Entrainment and Behavioral Monitoring in Home Cages.

Day-night locomotor activities are the most readily observed outputs of the circadian (~24-h period) clock in many animals. Temporal patterns of the light-dark schedule serve as input to the clock. While circadian activity patterns under various lighting conditions have been observed and documented, the full extent of circadian locomotor activities by genotype and entrainment remains uncharacterized. To facilitate large-scale, parallel cataloging of circadian input-output patterns, we created the LocoBox, an easy-to-construct and easy-to-operate system that can control environmental light with flexible entrainment scenarios combined with the T-cycle and measure locomotor activities in individual home cages. The LocoBox is made using economical, common components, and normal breeding cages can be used for long-term recording. We provide details of the components and blueprints, along with software programs for Arduino and a Python-based graphical user interface (GUI), so that the system can be easily replicated in other laboratories.

Weak synchronization can alter circadian period length: implications for aging and disease conditions.

The synchronization of multiple oscillators serves as the central mechanism for maintaining stable circadian rhythms in physiology and behavior. Aging and disease can disrupt synchronization, leading to changes in the periodicity of circadian activities. While our understanding of the circadian clock under synchronization has advanced significantly, less is known about its behavior outside synchronization, which can also fall within a predictable domain. These states not only impact the stability of the rhythms but also modulate the period length. In C57BL/6 mice, aging, diseases, and removal of peripheral circadian oscillators often result in lengthened behavioral circadian periods. Here, we show that these changes can be explained by a surprisingly simple mathematical relationship: the frequency is the reciprocal of the period, and its distribution becomes skewed when the period distribution is symmetric. The synchronized frequency of a population in the skewed distribution and the macroscopic frequency of combined oscillators differ, accounting for some of the atypical circadian period outputs observed in networks without synchronization. Building on this finding, we investigate the dynamics of circadian outputs in the context of aging and disease, where synchronization is weakened.

From Fast Oscillations to Circadian Rhythms: Coupling at Multiscale Frequency Bands in the Rodent Subcortical Visual System.

The subcortical visual system (SVS) is a unique collection of brain structures localised in the thalamus, hypothalamus and midbrain. The SVS receives ambient light inputs from retinal ganglion cells and integrates this signal with internal homeostatic demands to influence physiology. During this processing, a multitude of oscillatory frequency bands coalesces, with some originating from the retinas, while others are intrinsically generated in the SVS. Collectively, these rhythms are further modulated by the day and night cycle. The multiplexing of these diverse frequency bands (from circadian to infra-slow and gamma oscillations) makes the SVS an interesting system to study coupling at multiscale frequencies. We review the functional organisation of the SVS, and the various frequencies generated and processed by its neurons. We propose a perspective on how these different frequency bands couple with one another to synchronise the activity of the SVS to control physiology and behaviour.

Intrinsic circadian timekeeping properties of the thalamic lateral geniculate nucleus.

Circadian rhythmicity in mammals is sustained by the central brain clock-the suprachiasmatic nucleus of the hypothalamus (SCN), entrained to the ambient light-dark conditions through a dense retinal input. However, recent discoveries of autonomous clock gene expression cast doubt on the supremacy of the SCN and suggest circadian timekeeping mechanisms devolve to local brain clocks. Here, we use a combination of molecular, electrophysiological, and optogenetic tools to evaluate intrinsic clock properties of the main retinorecipient thalamic center-the lateral geniculate nucleus (LGN) in male rats and mice. We identify the dorsolateral geniculate nucleus as a slave oscillator, which exhibits core clock gene expression exclusively in vivo. Additionally, we provide compelling evidence for intrinsic clock gene expression accompanied by circadian variation in neuronal activity in the intergeniculate leaflet and ventrolateral geniculate nucleus (VLG). Finally, our optogenetic experiments propose the VLG as a light-entrainable oscillator, whose phase may be advanced by retinal input at the beginning of the projected night. Altogether, this study for the first time demonstrates autonomous timekeeping mechanisms shaping circadian physiology of the LGN.

Daily coordination of orexinergic gating in the rat superior colliculus-Implications for intrinsic clock activities in the visual system.

The orexinergic system delivers excitation for multiple brain centers to facilitate behavioral arousal, with its malfunction resulting in narcolepsy, somnolence, and notably, visual hallucinations. Since the circadian clock underlies the daily arousal, a timed coordination is expected between the orexin system and its target subcortical visual system, including the superior colliculus (SC). Here, we use a combination of electrophysiological, immunohistochemical, and molecular approaches across 24 h, together with the neuronal tract-tracing methods to investigate the daily coordination between the orexin system and the rodent SC. Higher orexinergic input was found to occur nocturnally in the superficial layers of the SC, in time for nocturnal silencing of spontaneous firing in this visual brain area. We identify autonomous daily and circadian expression of clock genes in the SC, which may underlie these day-night changes. Additionally, we establish the lateral hypothalamic origin of the orexin innervation to the SC and that the SC neurons robustly respond to orexin A via OX2 receptor in both excitatory and GABAA receptor-dependent inhibitory manners. Together, our evidence elucidates the combination of intrinsic and extrinsic clock mechanisms that shape the daily function of the visual layers of the SC.

The role of circadian rhythm in choroid plexus functions.

For several years, a great effort has been devoted to understand how circadian oscillations in physiological processes are determined by the circadian clock system. This system is composed by the master clock at the suprachiasmatic nucleus which sets the pace and tunes peripheral clocks in several organs. It was recently demonstrated that the choroid plexus epithelial cells that compose the blood-cerebrospinal fluid barrier hold a circadian clock which might control their multiple functions with implications for the maintenance of brain homeostasis. However, the choroid plexus activities regulated by its inner clock are still largely unknown. In this review, we propose that several choroid plexus functions might be regulated by the circadian clock, alike in other tissues. We provide evidences that the timing of cerebrospinal fluid secretion, clearance of amyloid-beta peptides and xenobiotics, and the barrier function of the blood-cerebrospinal fluid barrier are regulated by the circadian clock. These data, highlight that the circadian regulation of the blood-cerebrospinal fluid barrier must be taken into consideration for enhancing drug delivery to central nervous system disorders.

Timekeeping in the hindbrain: a multi-oscillatory circadian centre in the mouse dorsal vagal complex.

Metabolic and cardiovascular processes controlled by the hindbrain exhibit 24 h rhythms, but the extent to which the hindbrain possesses endogenous circadian timekeeping is unresolved. Here we provide compelling evidence that genetic, neuronal, and vascular activities of the brainstem's dorsal vagal complex are subject to intrinsic circadian control with a crucial role for the connection between its components in regulating their rhythmic properties. Robust 24 h variation in clock gene expression in vivo and neuronal firing ex vivo were observed in the area postrema (AP) and nucleus of the solitary tract (NTS), together with enhanced nocturnal responsiveness to metabolic cues. Unexpectedly, we also find functional and molecular evidence for increased penetration of blood borne molecules into the NTS at night. Our findings reveal that the hindbrain houses a local network complex of neuronal and non-neuronal autonomous circadian oscillators, with clear implications for understanding local temporal control of physiology in the brainstem.

Clocks in the Wild: Entrainment to Natural Light.

Entrainment denotes a process of coordinating the internal circadian clock to external rhythmic time-cues (Zeitgeber), mainly light. It is facilitated by stronger Zeitgeber signals and smaller period differences between the internal clock and the external Zeitgeber. The phase of entrainment ψ is a result of this process on the side of the circadian clock. On Earth, the period of the day-night cycle is fixed to 24 h, while the periods of circadian clocks distribute widely due to natural variation within and between species. The strength and duration of light depend locally on season and geographic latitude. Therefore, entrainment characteristics of a circadian clock vary under a local light environment and distribute along geoecological settings. Using conceptual models of circadian clocks, we investigate how local conditions of natural light shape global patterning of entrainment through seasons. This clock-side entrainment paradigm enables us to predict systematic changes in the global distribution of chronotypes.

Author Correction: The choroid plexus is an important circadian clock component.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Weak coupling between intracellular feedback loops explains dissociation of clock gene dynamics.

Circadian rhythms are generated by interlocked transcriptional-translational negative feedback loops (TTFLs), the molecular process implemented within a cell. The contributions, weighting and balancing between the multiple feedback loops remain debated. Dissociated, free-running dynamics in the expression of distinct clock genes has been described in recent experimental studies that applied various perturbations such as slice preparations, light pulses, jet-lag, and culture medium exchange. In this paper, we provide evidence that this "presumably transient" dissociation of circadian gene expression oscillations may occur at the single-cell level. Conceptual and detailed mechanistic mathematical modeling suggests that such dissociation is due to a weak interaction between multiple feedback loops present within a single cell. The dissociable loops provide insights into underlying mechanisms and general design principles of the molecular circadian clock.

The Kidney Clock Contributes to Timekeeping by the Master Circadian Clock.

The kidney harbors one of the strongest circadian clocks in the body. Kidney failure has long been known to cause circadian sleep disturbances. Using an adenine-induced model of chronic kidney disease (CKD) in mice, we probe the possibility that such sleep disturbances originate from aberrant circadian rhythms in kidney. Under the CKD condition, mice developed unstable behavioral circadian rhythms. When observed in isolation in vitro, the pacing of the master clock, the suprachiasmatic nucleus (SCN), remained uncompromised, while the kidney clock became a less robust circadian oscillator with a longer period. We find this analogous to the silencing of a strong slave clock in the brain, the choroid plexus, which alters the pacing of the SCN. We propose that the kidney also contributes to overall circadian timekeeping at the whole-body level, through bottom-up feedback in the hierarchical structure of the mammalian circadian clocks.

Strong Circadian Rhythms in the Choroid Plexus: Implications for Sleep-Independent Brain Metabolite Clearance.

Cerebrospinal fluid (CSF) is a fluidic part of the brain's microenvironment that isolates the brain from the rest of the body. CSF dilutes metabolites from neuronal activities and removes them from the brain. Its production and resorption are regulated dynamically and are central to maintaining brain homeostasis. We discovered that the major CSF source, the choroid plexus (CP), harbors the brain's strongest circadian clock. Here, we consider some implications of the CP circadian clock for metabolite clearance in the brain. If the circadian clock contributes to timed production of the CSF, its synchronization with sleep timing can maximize clearance efficiency and help prevent neurodegenerative diseases such as Alzheimer's disease.

The choroid plexus is an important circadian clock component.

Mammalian circadian clocks have a hierarchical organization, governed by the suprachiasmatic nucleus (SCN) in the hypothalamus. The brain itself contains multiple loci that maintain autonomous circadian rhythmicity, but the contribution of the non-SCN clocks to this hierarchy remains unclear. We examine circadian oscillations of clock gene expression in various brain loci and discovered that in mouse, robust, higher amplitude, relatively faster oscillations occur in the choroid plexus (CP) compared to the SCN. Our computational analysis and modeling show that the CP achieves these properties by synchronization of "twist" circadian oscillators via gap-junctional connections. Using an in vitro tissue coculture model and in vivo targeted deletion of the Bmal1 gene to silence the CP circadian clock, we demonstrate that the CP clock adjusts the SCN clock likely via circulation of cerebrospinal fluid, thus finely tuning behavioral circadian rhythms.

Encoding seasonal information in a two-oscillator model of the multi-oscillator circadian clock.

The suprachiasmatic nucleus (SCN) is a collection of about 10 000 neurons, each of which functions as a circadian clock with slightly different periods and phases, that work in concert with form and maintain the master circadian clock for the organism. The diversity among neurons confers on the SCN the ability to robustly encode both the 24-h light pattern as well as the seasonal time. Cluster synchronization brings the different neurons into line and reduces the large population to essentially two oscillators, coordinated by a macroscopic network motif of asymmetric repulsive-attractive coupling. We recount the steps leading to this simplification and rigorously examine the two-oscillator case by seeking an analytical solution. Through these steps, we identify physiologically relevant parameters that shape the behaviour of the SCN network and delineate its ability to store past details of seasonal variation in photoperiod.

Moran's I quantifies spatio-temporal pattern formation in neural imaging data.

MOTIVATION: Neural activities of the brain occur through the formation of spatio-temporal patterns. In recent years, macroscopic neural imaging techniques have produced a large body of data on these patterned activities, yet a numerical measure of spatio-temporal coherence has often been reduced to the global order parameter, which does not uncover the degree of spatial correlation. Here, we propose to use the spatial autocorrelation measure Moran's I, which can be applied to capture dynamic signatures of spatial organization. We demonstrate the application of this technique to collective cellular circadian clock activities measured in the small network of the suprachiasmatic nucleus (SCN) in the hypothalamus. RESULTS: We found that Moran's I is a practical quantitative measure of the degree of spatial coherence in neural imaging data. Initially developed with a geographical context in mind, Moran's I accounts for the spatial organization of any interacting units. Moran's I can be modified in accordance with the characteristic length scale of a neural activity pattern. It allows a quantification of statistical significance levels for the observed patterns. We describe the technique applied to synthetic datasets and various experimental imaging time-series from cultured SCN explants. It is demonstrated that major characteristics of the collective state can be described by Moran's I and the traditional Kuramoto order parameter R in a complementary fashion. AVAILABILITY AND IMPLEMENTATION: Python 2.7 code of illustrative examples can be found in the Supplementary Material. CONTACT: christoph.schmal@charite.de or grigory.bordyugov@hu-berlin.de. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Network Dynamics Mediate Circadian Clock Plasticity.

A circadian clock governs most aspects of mammalian behavior. Although its properties are in part genetically determined, altered light-dark environment can change circadian period length through a mechanism requiring de novo DNA methylation. We show here that this mechanism is mediated not via cell-autonomous clock properties, but rather through altered networking within the suprachiasmatic nuclei (SCN), the circadian "master clock," which is DNA methylated in region-specific manner. DNA methylation is necessary to temporally reorganize circadian phasing among SCN neurons, which in turn changes the period length of the network as a whole. Interruption of neural communication by inhibiting neuronal firing or by physical cutting suppresses both SCN reorganization and period changes. Mathematical modeling suggests, and experiments confirm, that this SCN reorganization depends upon GABAergic signaling. Our results therefore show that basic circadian clock properties are governed by dynamic interactions among SCN neurons, with neuroadaptations in network function driven by the environment.