Neuron-Astrocyte Interactions and Circadian Timekeeping in Mammals.

Almost every facet of our behavior and physiology varies predictably over the course of day and night, anticipating and adapting us to their associated opportunities and challenges. These rhythms are driven by endogenous biological clocks that, when deprived of environmental cues, can continue to oscillate within a period of approximately 1 day, hence circa-dian. Normally, retinal signals synchronize them to the cycle of light and darkness, but disruption of circadian organization, a common feature of modern lifestyles, carries considerable costs to health. Circadian timekeeping pivots around a cell-autonomous molecular clock, widely expressed across tissues. These cellular timers are in turn synchronized by the principal circadian clock of the brain: the hypothalamic suprachiasmatic nucleus (SCN). Intercellular signals make the SCN network a very powerful pacemaker. Previously, neurons were considered the sole SCN timekeepers, with glial cells playing supportive roles. New discoveries have revealed, however, that astrocytes are active partners in SCN network timekeeping, with their cell-autonomous clock regulating extracellular glutamate and GABA concentrations to control circadian cycles of SCN neuronal activity. Here, we introduce circadian timekeeping at the cellular and SCN network levels before focusing on the contributions of astrocytes and their mutual interaction with neurons in circadian control in the brain.

The Mammalian Circadian Time-Keeping System.

Our physiology and behavior follow precise daily programs that adapt us to the alternating opportunities and challenges of day and night. Under experimental isolation, these rhythms persist with a period of approximately one day (circadian), demonstrating their control by an internal autonomous clock. Circadian time is created at the cellular level by a transcriptional/translational feedback loop (TTFL) in which the protein products of the Period and Cryptochrome genes inhibit their own transcription. Because the accumulation of protein is slow and delayed, the system oscillates spontaneously with a period of ∼24 hours. This cell-autonomous TTFL controls cycles of gene expression in all major tissues and these cycles underpin our daily metabolic programs. In turn, our innumerable cellular clocks are coordinated by a central pacemaker, the suprachiasmatic nucleus (SCN) of the hypothalamus. When isolated in slice culture, the SCN TTFL and its dependent cycles of neural activity persist indefinitely, operating as "a clock in a dish". In vivo, SCN time is synchronized to solar time by direct innervation from specialized retinal photoreceptors. In turn, the precise circadian cycle of action potential firing signals SCN-generated time to hypothalamic and brain stem targets, which co-ordinate downstream autonomic, endocrine, and behavioral (feeding) cues to synchronize and sustain the distributed cellular clock network. Circadian time therefore pervades every level of biological organization, from molecules to society. Understanding its mechanisms offers important opportunities to mitigate the consequences of circadian disruption, so prevalent in modern societies, that arise from shiftwork, aging, and neurodegenerative diseases, not least Huntington's disease.

Astrocytic control of extracellular GABA drives circadian timekeeping in the suprachiasmatic nucleus.

The hypothalamic suprachiasmatic nucleus (SCN) is the master mammalian circadian clock. Its cell-autonomous timing mechanism, a transcriptional/translational feedback loop (TTFL), drives daily peaks of neuronal electrical activity, which in turn control circadian behavior. Intercellular signals, mediated by neuropeptides, synchronize and amplify TTFL and electrical rhythms across the circuit. SCN neurons are GABAergic, but the role of GABA in circuit-level timekeeping is unclear. How can a GABAergic circuit sustain circadian cycles of electrical activity, when such increased neuronal firing should become inhibitory to the network? To explore this paradox, we show that SCN slices expressing the GABA sensor iGABASnFR demonstrate a circadian oscillation of extracellular GABA ([GABA]e) that, counterintuitively, runs in antiphase to neuronal activity, with a prolonged peak in circadian night and a pronounced trough in circadian day. Resolving this unexpected relationship, we found that [GABA]e is regulated by GABA transporters (GATs), with uptake peaking during circadian day, hence the daytime trough and nighttime peak. This uptake is mediated by the astrocytically expressed transporter GAT3 (Slc6a11), expression of which is circadian-regulated, being elevated in daytime. Clearance of [GABA]e in circadian day facilitates neuronal firing and is necessary for circadian release of the neuropeptide vasoactive intestinal peptide, a critical regulator of TTFL and circuit-level rhythmicity. Finally, we show that genetic complementation of the astrocytic TTFL alone, in otherwise clockless SCN, is sufficient to drive [GABA]e rhythms and control network timekeeping. Thus, astrocytic clocks maintain the SCN circadian clockwork by temporally controlling GABAergic inhibition of SCN neurons.

Cryptochrome 1 as a state variable of the circadian clockwork of the suprachiasmatic nucleus: Evidence from translational switching.

The suprachiasmatic nucleus (SCN) of the hypothalamus is the principal clock driving circadian rhythms of physiology and behavior that adapt mammals to environmental cycles. Disruption of SCN-dependent rhythms compromises health, and so understanding SCN time keeping will inform management of diseases associated with modern lifestyles. SCN time keeping is a self-sustaining transcriptional/translational delayed feedback loop (TTFL), whereby negative regulators inhibit their own transcription. Formally, the SCN clock is viewed as a limit-cycle oscillator, the simplest being a trajectory of successive phases that progresses through two-dimensional space defined by two state variables mapped along their respective axes. The TTFL motif is readily compatible with limit-cycle models, and in Neurospora and Drosophila the negative regulators Frequency (FRQ) and Period (Per) have been identified as state variables of their respective TTFLs. The identity of state variables of the SCN oscillator is, however, less clear. Experimental identification of state variables requires reversible and temporally specific control over their abundance. Translational switching (ts) provides this, the expression of a protein of interest relying on the provision of a noncanonical amino acid. We show that the negative regulator Cryptochrome 1 (CRY1) fulfills criteria defining a state variable: ts-CRY1 dose-dependently and reversibly suppresses the baseline, amplitude, and period of SCN rhythms, and its acute withdrawal releases the TTFL to oscillate from a defined phase. Its effect also depends on its temporal pattern of expression, although constitutive ts-CRY1 sustained (albeit less stable) oscillations. We conclude that CRY1 has properties of a state variable, but may operate among several state variables within a multidimensional limit cycle.

Astrocytes sustain circadian oscillation and bidirectionally determine circadian period, but do not regulate circadian phase in the suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is the master circadian clock of mammals, generating and transmitting an internal representation of environmental time that is produced by the cell-autonomous transcriptional/post-translational feedback loops (TTFL) of the 10,000 neurons and 3,500 glial cells. Recently, we showed that TTFL function in SCN astrocytes alone is sufficient to drive circadian timekeeping and behaviour, raising questions about the respective contributions of astrocytes and neurons within the SCN circuit. We compared their relative roles in circadian timekeeping in mouse SCN explants, of either sex. Treatment with the glial-specific toxin fluorocitrate revealed a requirement for metabolically competent astrocytes for circuit-level timekeeping. Recombinase-mediated genetically complemented Cryptochrome (Cry) proteins in Cry1- and/or Cry2-deficient SCN, were used to compare the influence of the TTFLs of neurons or astrocytes in the initiation of de novo oscillation or in pacemaking. While neurons and astrocytes both initiated de novo oscillation and lengthened period equally, their kinetics were different: astrocytes taking twice as long. Furthermore, astrocytes could shorten period, but not as potently as neurons. Chemogenetic manipulation of Gi- and Gq-coupled signalling pathways in neurons acutely advanced or delayed ensemble phase, respectively. In contrast, comparable manipulations in astrocytes were without effect. Thus, astrocytes can initiate SCN rhythms and bi-directionally control SCN period, albeit with lower potency than neurons. Nevertheless, their activation does not influence SCN phase. The emergent SCN properties of high amplitude oscillation, initiation of rhythmicity, pacemaking and phase are differentially regulated: astrocytes and neurons sustain the ongoing oscillation, but its phase is determined by neurons.Significance Statement:The hypothalamic suprachiasmatic nucleus (SCN) encodes and disseminates time-of-day information to allow mammals to adapt their physiology to daily environmental cycles. Recent investigations have revealed a role for astrocytes, in addition to neurons, in regulation of this rhythm. Using pharmacology, genetic complementation and chemogenetics, we compared the abilities of neurons and astrocytes in determining the emergent SCN properties of high amplitude oscillation, initiation of rhythmicity, pacemaking and determination of phase. These findings parameterise the circadian properties of the astrocyte population in the SCN, and reveal the types of circadian information astrocytes and neurons can contribute within their heterogeneous cellular network.

Single-cell transcriptomics of suprachiasmatic nuclei reveal a Prokineticin-driven circadian network.

Circadian rhythms in mammals are governed by the hypothalamic suprachiasmatic nucleus (SCN), in which 20,000 clock cells are connected together into a powerful time-keeping network. In the absence of network-level cellular interactions, the SCN fails as a clock. The topology and specific roles of its distinct cell populations (nodes) that direct network functions are, however, not understood. To characterise its component cells and network structure, we conducted single-cell sequencing of SCN organotypic slices and identified eleven distinct neuronal sub-populations across circadian day and night. We defined neuropeptidergic signalling axes between these nodes, and built neuropeptide-specific network topologies. This revealed their temporal plasticity, being up-regulated in circadian day. Through intersectional genetics and real-time imaging, we interrogated the contribution of the Prok2-ProkR2 neuropeptidergic axis to network-wide time-keeping. We showed that Prok2-ProkR2 signalling acts as a key regulator of SCN period and rhythmicity and contributes to defining the network-level properties that underpin robust circadian co-ordination. These results highlight the diverse and distinct contributions of neuropeptide-modulated communication of temporal information across the SCN.

The VIP-VPAC2 neuropeptidergic axis is a cellular pacemaking hub of the suprachiasmatic nucleus circadian circuit.

The hypothalamic suprachiasmatic nuclei (SCN) are the principal mammalian circadian timekeeper, co-ordinating organism-wide daily and seasonal rhythms. To achieve this, cell-autonomous circadian timing by the ~20,000 SCN cells is welded into a tight circuit-wide ensemble oscillation. This creates essential, network-level emergent properties of precise, high-amplitude oscillation with tightly defined ensemble period and phase. Although synchronised, regional cell groups exhibit differentially phased activity, creating stereotypical spatiotemporal circadian waves of cellular activation across the circuit. The cellular circuit pacemaking components that generate these critical emergent properties are unknown. Using intersectional genetics and real-time imaging, we show that SCN cells expressing vasoactive intestinal polypeptide (VIP) or its cognate receptor, VPAC2, are neurochemically and electrophysiologically distinct, but together they control de novo rhythmicity, setting ensemble period and phase with circuit-level spatiotemporal complexity. The VIP/VPAC2 cellular axis is therefore a neurochemically and topologically specific pacemaker hub that determines the emergent properties of the SCN timekeeper.

Molecular-genetic Manipulation of the Suprachiasmatic Nucleus Circadian Clock.

Circadian (approximately daily) rhythms of physiology and behaviour adapt organisms to the alternating environments of day and night. The suprachiasmatic nucleus (SCN) of the hypothalamus is the principal circadian timekeeper of mammals. The mammalian cell-autonomous circadian clock is built around a self-sustaining transcriptional-translational negative feedback loop (TTFL) in which the negative regulators Per and Cry suppress their own expression, which is driven by the positive regulators Clock and Bmal1. Importantly, such TTFL-based clocks are present in all major tissues across the organism, and the SCN is their central co-ordinator. First, we analyse SCN timekeeping at the cell-autonomous and the circuit-based levels of organisation. We consider how molecular-genetic manipulations have been used to probe cell-autonomous timing in the SCN, identifying the integral components of the clock. Second, we consider new approaches that enable real-time monitoring of the activity of these clock components and clock-driven cellular outputs. Finally, we review how intersectional genetic manipulations of the cell-autonomous clockwork can be used to determine how SCN cells interact to generate an ensemble circadian signal. Critically, it is these network-level interactions that confer on the SCN its emergent properties of robustness, light-entrained phase and precision- properties that are essential for its role as the central co-ordinator. Remaining gaps in knowledge include an understanding of how the TTFL proteins behave individually and in complexes: whether particular SCN neuronal populations act as pacemakers, and if so, by which signalling mechanisms, and finally the nature of the recently discovered role of astrocytes within the SCN network.

Cell-autonomous clock of astrocytes drives circadian behavior in mammals.

Circadian (~24-hour) rhythms depend on intracellular transcription-translation negative feedback loops (TTFLs). How these self-sustained cellular clocks achieve multicellular integration and thereby direct daily rhythms of behavior in animals is largely obscure. The suprachiasmatic nucleus (SCN) is the fulcrum of this pathway from gene to cell to circuit to behavior in mammals. We describe cell type-specific, functionally distinct TTFLs in neurons and astrocytes of the SCN and show that, in the absence of other cellular clocks, the cell-autonomous astrocytic TTFL alone can drive molecular oscillations in the SCN and circadian behavior in mice. Astrocytic clocks achieve this by reinstating clock gene expression and circadian function of SCN neurons via glutamatergic signals. Our results demonstrate that astrocytes can autonomously initiate and sustain complex mammalian behavior.

Translational switching of Cry1 protein expression confers reversible control of circadian behavior in arrhythmic Cry-deficient mice.

The suprachiasmatic nucleus (SCN) is the principal circadian clock of mammals, coordinating daily rhythms of physiology and behavior. Circadian timing pivots around self-sustaining transcriptional-translational negative feedback loops (TTFLs), whereby CLOCK and BMAL1 drive the expression of the negative regulators Period and Cryptochrome (Cry). Global deletion of Cry1 and Cry2 disables the TTFL, resulting in arrhythmicity in downstream behaviors. We used this highly tractable biology to further develop genetic code expansion (GCE) as a translational switch to achieve reversible control of a biologically relevant protein, Cry1, in the SCN. This employed an orthogonal aminoacyl-tRNA synthetase/tRNACUA pair delivered to the SCN by adeno-associated virus (AAV) vectors, allowing incorporation of a noncanonical amino acid (ncAA) into AAV-encoded Cry1 protein carrying an ectopic amber stop codon. Thus, translational readthrough and Cry1 expression were conditional on the supply of ncAA via culture medium or drinking water and were restricted to neurons by synapsin-dependent expression of aminoacyl tRNA-synthetase. Activation of Cry1 translation by ncAA in neurons of arrhythmic Cry-null SCN slices immediately and dose-dependently initiated TTFL circadian rhythms, which dissipated rapidly after ncAA withdrawal. Moreover, genetic activation of the TTFL in SCN neurons rapidly and reversibly initiated circadian behavior in otherwise arrhythmic Cry-null mice, with rhythm amplitude being determined by the number of transduced SCN neurons. Thus, Cry1 does not specify the development of circadian circuitry and competence but is essential for its labile and rapidly reversible activation. This demonstrates reversible control of mammalian behavior using GCE-based translational switching, a method of potentially broad neurobiological interest.

The suprachiasmatic nucleus.

Like it or not, your two suprachiasmatic nuclei (SCN) govern your life: from when you wake up and fall asleep, to when you feel hungry or can best concentrate. Each is composed of approximately 10,000 tightly interconnected neurons, and the pair sit astride the mid-line third ventricle of the hypothalamus, immediately dorsal to the optic chiasm (Figure 1A). Together, they constitute the master circadian clock of the mammalian brain. They generate an internal representation of solar time that is conveyed to every cell in our body and in this way they co-ordinate the daily cycles of physiology and behaviour that adapt us to the twenty-four hour world. The temporary discomfort associated with jetlag is a reminder of the importance of this daily programme, but there is growing recognition that its chronic disruption carries a cost for health of far greater scale. In this primer, we shall briefly review the historical identification of the SCN as the master circadian clock, and then discuss it on three different levels: the cell-autonomous SCN, the SCN as a cellular network and, finally, the SCN as circadian orchestrator. We shall focus on the intrinsic electrical and transcriptional properties of the SCN and how these properties are thought to form an input to, and an output from, its intrinsic cellular clockwork. Second, we shall describe the anatomical arrangement of the SCN, how its sub-regions are delineated by different neuropeptides, and how SCN neurons communicate with each other via these neuropeptides and the neurotransmitter γ-aminobutyric acid (GABA). Finally, we shall discuss how the SCN functions as a circadian oscillator that dictates behaviour, and how intersectional genetic approaches are being used to try to unravel the specific contributions to pacemaking of specific SCN cell populations.

Astrocytes Control Circadian Timekeeping in the Suprachiasmatic Nucleus via Glutamatergic Signaling.

The suprachiasmatic nucleus (SCN) of the hypothalamus orchestrates daily rhythms of physiology and behavior in mammals. Its circadian (∼24 hr) oscillations of gene expression and electrical activity are generated intrinsically and can persist indefinitely in temporal isolation. This robust and resilient timekeeping is generally regarded as a product of the intrinsic connectivity of its neurons. Here we show that neurons constitute only one "half" of the SCN clock, the one metabolically active during circadian daytime. In contrast, SCN astrocytes are active during circadian nighttime, when they suppress the activity of SCN neurons by regulating extracellular glutamate levels. This glutamatergic gliotransmission is sensed by neurons of the dorsal SCN via specific pre-synaptic NMDA receptor assemblies containing NR2C subunits. Remarkably, somatic genetic re-programming of intracellular clocks in SCN astrocytes was capable of remodeling circadian behavioral rhythms in adult mice. Thus, SCN circuit-level timekeeping arises from interdependent and mutually supportive astrocytic-neuronal signaling.

Combined Pharmacological and Genetic Manipulations Unlock Unprecedented Temporal Elasticity and Reveal Phase-Specific Modulation of the Molecular Circadian Clock of the Mouse Suprachiasmatic Nucleus.

UNLABELLED: The suprachiasmatic nucleus (SCN) is the master circadian oscillator encoding time-of-day information. SCN timekeeping is sustained by a cell-autonomous transcriptional-translational feedback loop, whereby expression of the Period and Cryptochrome genes is negatively regulated by their protein products. This loop in turn drives circadian oscillations in gene expression that direct SCN electrical activity and thence behavior. The robustness of SCN timekeeping is further enhanced by interneuronal, circuit-level coupling. The aim of this study was to combine pharmacological and genetic manipulations to push the SCN clockwork toward its limits and, by doing so, probe cell-autonomous and emergent, circuit-level properties. Circadian oscillation of mouse SCN organotypic slice cultures was monitored as PER2::LUC bioluminescence. SCN of three genetic backgrounds-wild-type, short-period CK1ε(Tau/Tau) mutant, and long-period Fbxl3(Afh/Afh) mutant-all responded reversibly to pharmacological manipulation with period-altering compounds: picrotoxin, PF-670462 (4-[1-Cyclohexyl-4-(4-fluorophenyl)-1H-imidazol-5-yl]-2-pyrimidinamine dihydrochloride), and KNK437 (N-Formyl-3,4-methylenedioxy-benzylidine-gamma-butyrolactam). This revealed a remarkably wide operating range of sustained periods extending across 25 h, from ≤17 h to >42 h. Moreover, this range was maintained at network and single-cell levels. Development of a new technique for formal analysis of circadian waveform, first derivative analysis (FDA), revealed internal phase patterning to the circadian oscillation at these extreme periods and differential phase sensitivity of the SCN to genetic and pharmacological manipulations. For example, FDA of the CK1ε(Tau/Tau) mutant SCN treated with the CK1ε-specific inhibitor PF-4800567 (3-[(3-Chlorophenoxy)methyl]-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine hydrochloride) revealed that period acceleration in the mutant is due to inappropriately phased activity of the CK1ε isoform. In conclusion, extreme period manipulation reveals unprecedented elasticity and temporal structure of the SCN circadian oscillation. SIGNIFICANCE STATEMENT: The master circadian clock of the suprachiasmatic nucleus (SCN) encodes time-of-day information that allows mammals to predict and thereby adapt to daily environmental cycles. Using combined genetic and pharmacological interventions, we assessed the temporal elasticity of the SCN network. Despite having evolved to generate a 24 h circadian period, we show that the molecular clock is surprisingly elastic, able to reversibly sustain coherent periods between ≤17 and >42 h at the levels of individual cells and the overall circuit. Using quantitative techniques to analyze these extreme periodicities, we reveal that the oscillator progresses as a sequence of distinct stages. These findings reveal new properties of how the SCN functions as a network and should inform biological and mathematical analyses of circadian timekeeping.

PICK1 inhibition of the Arp2/3 complex controls dendritic spine size and synaptic plasticity.

Activity-dependent remodelling of dendritic spines is essential for neural circuit development and synaptic plasticity, but the precise molecular mechanisms that regulate this process are unclear. Activators of Arp2/3-mediated actin polymerisation are required for spine enlargement; however, during long-term depression (LTD), spines shrink via actin depolymerisation and Arp2/3 inhibitors in this process have not yet been identified. Here, we show that PICK1 regulates spine size in hippocampal neurons via inhibition of the Arp2/3 complex. PICK1 knockdown increases spine size, whereas PICK1 overexpression reduces spine size. NMDA receptor activation results in spine shrinkage, which is blocked by PICK1 knockdown or overexpression of a PICK1 mutant that cannot bind Arp2/3. Furthermore, we show that PICK1-Arp2/3 interactions are required for functional hippocampal LTD. This work demonstrates that PICK1 is a novel regulator of spine dynamics. Via Arp2/3 inhibition, PICK1 has complementary yet distinct roles during LTD to regulate AMPA receptor trafficking and spine size, and therefore functions as a crucial factor in both structural and functional plasticity.