Molecular-genetic Manipulation of the Suprachiasmatic Nucleus Circadian Clock

Review

Molecular-genetic Manipulation of the Suprachiasmatic Nucleus Circadian Clock

Michael H. Hastings, Nicola J. Smyllie and Andrew P. Patton Division of Neurobiology, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, CB2 0QH, UK

Correspondence to Michael H. Hastings: [email protected] https://doi.org/10.1016/j.jmb.2020.01.019 Edited by Eva Wolf

Abstract 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 selfsustaining 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 cellautonomous 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 precisiond 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. © 2020 Elsevier Ltd. All rights reserved.

Introduction Circadian (approximately daily) rhythms of physiology and behaviour adapt organisms to the alternating environments of day and night [1]. They are driven by internal molecular oscillators that are synchronised to, and are thereby predictive of, solar time. The formal properties of these circadian timekeepers are highly conserved across taxa although their molecular and cellular components vary [2]. The focus of the current review is the suprachiasmatic nucleus (SCN) of the hypothalamus, the principal circadian timekeeper of mammals [3]. Each nucleus consists of approximately 10,000 neurons and 3500 astrocytes (along with other glial cell types) and, as a pair, the SCN is positioned bilaterally against the third ventricle, at the base of 0022-2836/© 2020 Elsevier Ltd. All rights reserved.

the hypothalamus and above the optic chiasm [4]. In vivo, the SCN exhibits precise, high-amplitude circadian cycles of metabolic and electrical activity that communicate its circadian time cues to the rest of the brain and body, and remarkably, these rhythms persist autonomously in ex vivo culture. To be effective, however, the internal representation of solar time generated by the SCN must be synchronised with, and therefore predict, the daily environmental cycle. This synchronisation is mediated by a direct and specific retinal innervation, the retinohypothalamic tract [5], which is derived from intrinsically photosensitive retinal ganglion cells and uses glutamate as its excitatory neurotransmitter. Light presented at dawn and/or dusk acutely induces electrical and metabolic activity in the SCN and thereby corrects its intrinsic programme to the local Journal of Molecular Biology (2020) 432, 3639e3660

3640 time. Alongside this afferent glutamatergic input, the neurons of the SCN principally use the neurotransmitter ɣ-aminobutyric acid (GABA). However, layered atop this homogeneity is a heterogeneous array of neuropeptides, including vasoactive intestinal polypeptide (Vip), arginine vasopressin (Avp), gastrin-releasing peptide (Grp), prokineticin-2 (Prok2) and neuromedin-S (Nms), along with neuropeptide and neurotransmitter receptors [6]. These neuropeptides and their receptors demarcate different, spatially restricted SCN sub-populations, representing a high degree of structural, cellular and neurochemical heterogeneity [7]. This complex architecture confers on the SCN its emergent network-level properties of highly robust oscillation, ensemble cellular phase and ensemble cellular perioddproperties which enable it to maintain coherent, organism-wide circadian rhythmicity in the absence of environmental input. In fact, the SCN is such a robust oscillator that when isolated as an ex vivo organotypic culture, its cellular activities can continue to oscillate almost indefinitely with an intrinsic period of ca. 24 h. Under genetic and pharmacological manipulations, it can even sustain stable periods ranging from <17 to >42 h, far beyond the normal condition [8]. The SCN therefore represents a remarkably stable and tractable system within which to study circadian rhythmicity. In this review, we analyse SCN timekeeping at both cellautonomous and circuit-based levels of organisation. We first consider how molecular-genetic manipulations have been used to probe cell-autonomous timing in the SCN, identifying the integral components of the circadian clock. Second, we consider new approaches that enable real-time monitoring of the activity of these clock components and clockdriven cellular output rhythms. Finally, we review how intersectional genetic manipulations of the cellautonomous clockwork can be used to determine how the cells of the SCN circuit interact to generate an ensemble circadian timing signal of sufficient robustness and fidelity to co-ordinate circadian rhythms across the body.

Initial Identification of Mammalian Circadian Clock Genes Includes Forward and Reverse Genetics by Mutagenesis and Homology Screens How are the cells of the mammalian SCN able to direct and define robust rhythms? As noted previously, the first layer is at the genetically specified cell-autonomous level. Forward mutagenesis screens in genetically tractable lower eukaryotes have identified the core components of the circadian system, including period (per), timeless (tim), clock (Clk/Jrk) and cycle (cyc) in Drosophila, and fre-

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quency (frq) in Neurospora. Subsequent forward and reverse genetics revealed that cryptochrome (cry) and white collar (wc) encode the molecular mediators of photic entrainment in Drosophila and Neurospora, respectively. A model thence emerged of circadian clocks as transcriptional-translational negative feedback loops (TTFLs) in which positive regulators (Clk, cyc, wc-1, wc-2) activate the expression of negative regulators (per, tim, frq), which in turn suppress the initial transactivation [9,10]. This generates a self-sustaining cycle, which is entrained by light-induced, cry-mediated degradation of tim and per in flies and by wc-dependent induction of frq in Neurospora [11,12]. Although the spontaneous period is typically ca. 24 h, it is sensitive to the trans-activational efficacy of the positive regulators, the intracellular processing of mRNA and protein components and the stability of the negative regulators. For example, doubletime (dbt) is a kinase that regulates the stability of Drosophila per and was identified in a mutagenesis screen in which different dbt alleles either shortened or lengthened the period of behavioural rhythms [13]. Strikingly, the first circadian gene identified in mammals was also in a per-regulatory kinase. The tau mutation arose spontaneously in a Syrian hamster colony and was shown to shorten the freerunning period of SCN and behavioural circadian rhythms by ca. 2 and 4 h in heterozygotes and homozygotes, respectively. Importantly, the availability of the tau allele made it possible to conduct cross-genotype, intracerebral grafting studies that showed definitively that the SCN is the central oscillator controlling circadian behavioural rhythms in mammals [14]. Subsequent cloning and sequencing showed that the tau locus encodes casein kinase 1 epsilon (Ck1e), a mammalian homologue of dbt in flies and that Ck1e t au is a gain-of-function mutation that destabilised mammalian Per [15,16], thereby accelerating the TTFL. Intriguingly, rare familial sleep disorders in humans are associated with heritable mutations in PER or CK1delta (CK1d), emphasising the generality of this clock-regulatory axis [17]. Beyond tau, the structure of the TTFL in mammals was primarily delineated by a candidate gene approach, searching for mammalian homologues of the fly components, especially Per, Cry and Cycle. It should be noted, however, that PER1 was also identified independently via a transcript arising from human chromosome 17 [18] and that the mammalian Timeless gene is in fact closer to the Drosophila timeout (tim-2) gene and does not appear to be a clock component in mammals [19]. The stand-out success in mammalian circadian genetics, however, came with the discovery of the Clock gene by a forward mutagenesis screen in mice, which generated an allele that lengthened the circadian period of behavioural and SCN rhythms in the heterozygote

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and further lengthened and ultimately destabilised rhythms in homozygotes [20]. In a heroic pregenome-era effort, the mutation was positionally mapped, and then the defective gene identified by a series of transgenic rescues using BACs to express candidate areas from those mapped loci [21]. Finally, sequencing revealed the mutation in a transcription factor subsequently named CLOCK (in humans), and biochemical analysis showed that deletion of exon 19 in the mutant compromised the transactivational efficacy of the protein [22]. The independent discoveries of CLOCK and Ck1e in rodents [22] and fly [23] and the candidate gene homology approach based on the fly work, including identification of Bmal1 (homologue of cyc) as the respective partners of CLOCK [24,25], enabled a draft mechanism for the mammalian TTFL to be assembled and subsequently validated by biochemical and cell biological assays. At the core of the human molecular oscillator, the transcription factors CLOCK and BMAL1 heterodimerise and activate the PER (PER1, 2) and CRY (CRY1, 2) genes via Ebox enhancer elements. The resultant protein products accumulate over the course of circadian day and translocate to the nucleus, where the PER/ CRY complexes repress the activation of their cognate (and other) genes. In the absence of de novo transcription, protein levels fall during circadian night, ultimately relieving the negative feedback and allowing the process to start anewd the entire cycle of activation and repression taking approximately 24 h to complete. Targetted mutations in mice have revealed a degree of redundancy in the TTFL, for example, null mutation of Per1 or Per2 alone does not compromise the TTFL (or its acceleration by Ck1e t au), whereas double null mutations render the SCN and circadian behaviour arrhythmic [26]. Equally, loss of Clock in the SCN can be compensated for by its paralogue Npas2, which can also heterodimerise with Bmal1 and sustain the TTFLd only double Clock- and Npas2-null mutants are arrhythmic [27] although loss of Clock does shorten the free-running period [28]. Individual loss of Cry1 or Cry2, respectively, accelerates or slows down the SCN [29] and behavioural rhythms [30] of mice, indicative of a potential antagonistic interaction, but loss of both leads to arrhythmia in adult, but possibly not neonatal, SCN [31]. Of the core TTFL, Bmal1 is the sole factor that is essential for clock functiond its singular loss, whether global or restricted to the SCN, disorganises SCN and behavioural rhythms [24,32,33], whereas inducible transgenic expression of Bmal1 can reversibly rescue the arrhythmic phenotypes [34]. Underpinning its metabolic and electrical cycles, significant proportions of the SCN transcriptome and proteome are under circadian regulation, including ion channels and synaptic vesicle proteins [35,36]. The cell-autonomous TTFL directs this temporal

programme by co-ordinating the circadian expression of clock-controlled genes, including transcription factors such as the E-box-dependent PAR leucine zipper factors Dbp, Hef and Tef. Although these genes have very high circadian amplitudes, results from null mutant mice suggest that, alone or in combination, they are more important to the molecular output of the clock rather than to core TTFL function [37]. Importantly, the cell-autonomous molecular clock is present in most, if not all nucleated cells [38], and in peripheral tissues; it is very clear that clock-controlled transcription factors, such as Dbp, contribute to the complex cellular programmes that underpin circadian physiology and behaviour. In the case of Dbp, this is the circadian capacity of the liver to detoxify xenobiotics [39]. As such, there is an additional level of complexity to circadian organisation in mammals; to ensure daily coherence, the TTFL clocks need to be synchronised with each other, and ultimately the environment, across the whole organism [40]. This internal synchronisation is understood in outlinedit is mediated by SCN-dependent endocrine, autonomic and behavioural (sleep/wake-related) cues [40]. However, local TTFLs are sensitive to a variety of intracellular signalling cascades, metabolic cues and nuclear hormone receptors that impinge on regulatory elements in the Per genes and the transactivational efficacy of Clock/Bmal1 [41]. This creates a diversity of potential pathways, and so the consequent functional redundancy currently mitigates against a detailed understanding of this dynamic, organism-wide network. It is nevertheless clear that the mammalian circadian system is a hierarchy, with the light-entrained SCN (and the cellular TTFLs contained within) as the principal co-ordinator of a literally innumerable population of tissue- and cellbased local clocks [3]. Thus, a combination of forward and reverse genetic screens has led to the identification of the molecular components that form the cell autonomous TTFL of the mammalian circadian clock.

Elaborating TTFL Function With the framework of the TTFL established, it has been possible to expand its components and better understand its properties. The expression of Bmal1 mRNA in the SCN is rhythmic, peaking at night and so sitting in antiphase to the peak of Per expression in circadian daytime. The expression of Bmal1 is driven, in part, by retinoic acid-related orphan receptor response-elements (ROREs), which are transactivated by Rora and inhibited by Rev-Erb alpha and beta nuclear receptors that are themselves expressed on a circadian basis by Clock/ Bmal1 transactivation. This adds a supplementary loop to the core TTFL that is thought to stabilise and

3642 boost its amplitude. In Rora mutant (staggerer) mice, the expression of Bmal1 in the SCN is reduced and circadian period is shortened [42], whereas in RevErb double-null mice circadian, behaviour is destabilised [43] (consistent with Bmal1 knockout models), suggesting a more important role for this accessory component. Indeed, the RORE transcriptional axis is critically important in conferring circadian regulation to a host of clock-controlled cellular outputs, not least metabolic cycles [43]. Another clock-regulated transcriptional axis in the SCN was revealed by the Short Circuit (Sci) mutation, discovered in a forward screen, which shortens the period of SCN TTFL and behavioural rhythms by ca. 0.5 h in the heterozygote (homozygosity is lethal) [44]. Sci is a point mutation in the transcription factor Zfhx3 and reduces the ability of Zfhx3 to activate transcription via its target AT-box regulatory elements. The Zfhx3 Sci phenotype is associated with decreased activation of the AT motif in neuropeptide promoters, consequently reducing neuropeptide expression in the SCN. It is this disruption of signalling within the SCN circuit that is thought to accelerate the TTFL. Thus, Zfhx3 lies “upstream” of the TTFL, albeit indirectly via its effect on neuropeptides. Equally, however, it is also downstream of the TTFL insofar as the transcriptional activity of AT boxes is under circadian regulation. This thereby establishes a re-entrant loop motif with serial, recurrent interplay between Zfhx3 and the cellautonomous clock [44]. The importance of this loop is emphasised by the disruption (acceleration and/or arrhythmia) of behavioural rhythms in adult mice after inducible deletion of Zfhx3 [45]. The interplay between circuit-level and cell-autonomous timekeeping will be considered further below, but to return to the cell-autonomous TTFL, a combination of genetic and pharmacological approaches in mice has made it possible to specify the relative contributions of Ck1e and Ck1d to the mammalian clock. As with hamsters, in mice, the Ck1e tau mutation shortens SCN circadian period by 2 h for each copy of the allele [16]. Deletion of the allele reverts period to wildtype, indicating that the mutation is a gain-of-function mutation, and that, under normal circumstances, wild type Ck1e does not contribute appreciably to period determination. In contrast, deletion of Ck1d leads to lengthening of SCN circadian period [46]. Similarly, pharmacological inhibition of Ck1e has no effect on the period of wildtype SCN, whereas inhibition of Ck1d lengthens period [47]. Thus, under normal circumstances, Ck1d is a core regulator of SCN circadian period through its effect on Per stability, although the precise molecular mechanisms of Ck1d and Ck1e tau actions on Per remain unclear [48]. Further, it should also be emphasised that the period of the TTFL is determined by a balance between the effects of Ck1 kinases and phosphatase (Ppa1) on Per [49].

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This central role of kinase-regulated Per stability in setting circadian period was further demonstrated by the Earlydoors (Edo) mutation in Per2, generated by a forward screen [50]. Per2 Edo accelerates SCN and behavioural rhythms by ca. 1.5 h in the homozygote, and in combination with the Ck1e tau alleles drives behavioural rhythms to an unprecedentedly short period of <19 h. The short period is thought to arise from destabilisation of Per2, which is a consequence of loose packing of the highly conserved linker sequence between the two PAS domains of Per2 that is caused by the point mutation therein. This still allows association with other TTFL proteins but nevertheless increases the vulnerability of Per2 to Ck1-mediated degradation by increasing kinase access to a presumed degron sequence in Per2. Thus, it appears that the Per2 protein can determine oscillation and set its period and phase. This has been interrogated further through reversible manipulation of Per2 levels in mice carrying a doxycycline (Dox)-inhibited Per2 transgene (TgPer2). In Doxtreated animals, when TgPer2 expression was absent, strongly circadian, wild type behaviour was observed. On subsequent withdrawal of Dox, constitutive, high-level, sustained expression of TgPer2 was initiated and consequently circadian behaviour became arrhythmic. It was concluded, therefore, that rhythmic Per levels are critical for effective negative feedback and thereby circadian clock function [51]. The proposed biochemical basis for this is that rhythmically available Per2 is an important scaffold in recruiting Cry proteins to the negative regulatory complex on E-boxes in a circadian manner. Constitutive expression, however, prevents the release of the Per/Cry-mediated inhibitory feedback, thereby arresting the TTFL. Consistent with this, in mice lacking the E0 -box in Per2, the circadian oscillation is compromised, and Per2 protein expression is constitutively high, leading to a loss of both SCN and behavioural rhythms [52]. There may, however, be an interaction between circadian expression, constitutive expression and overall levels, because in a different model expressing TgPer2 in Per1/Per2 double-null arrhythmic mice, Dox-on TgPer2 induced at non-saturating Per2 levels, initiated circadian behavioural rhythms [53] with a period that, as a function of TgPer2 expression, was tuneable to well beyond the normal circadian range (ca. 30 h). Within a certain level of expression therefore, constitutive Per2 may suffice for SCN rhythmicity (at least in a background lacking endogenous Per2 and Per1) if other components, presumably Cry proteins, are able to pivot their oscillations about it. Manipulations of the stability and relative levels of Per2, therefore, influence the functionality of the clock mechanism. Forward screens have, similarly, also highlighted the importance of Cry protein

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stability for TTFL functions. The ENU-induced mutations Afterhours (Afh) [54,55] and Overtime (Ovt) [56] lengthen the circadian periods of the SCN and mouse behaviour to ca. 26e27 h in homozygotes. Both mutations lie in the Fbxl3 ubiquitin ligase responsible for directing Cry proteins to proteasomal degradation after their phosphorylation by AMP kinase (loss of which shortens circadian period in mice [57]). The Fbxl3 mutations enhanced Cry stability, and thereby prolonged the phase of negative feedback, distorting the waveform of Per expression by extending the nadir, and increasing overall TTFL period. Fbxl3 Afh acts on both Cry1 and Cry2, extending the period of short period Cry1-null and long period Cry2-null SCN and mice, indicating that both Cry isoforms exert a negative regulatory effect in the TTFL and are sensitive to Fbxl3 [29]. Fbxl3 does not, however, act alone. A forward screen created the mutation Past-time, which accelerated behavioural and SCN rhythms by ca. 1.5 h in the homozygote and antagonised the period lengthening caused by Fbxl3 mutation [58]. Pasttime was mapped to a second SCF ubiquitin ligase Fbxl21, a paralogue of Fbxl3. Targetted deletion of Fbxl21 alone had no effect on freerunning period but did attenuate the period lengthening caused by a complete null of Fbxl3, and doublenull mice lost circadian amplitude with ca. 30% becoming behaviourally arrhythmic [58]. The apparently contradictory observation that loss-of-function mutations of the two Fbxl proteins had antagonistic actions was resolved by the discovery that Fbxl3 directs ubiquitination of Cry in the nucleus to promote degradation, whereas Fbxl21 acts on Cry in the cytoplasm (but at different ubiquitination sites) to stabilise Cry proteins and regulate their nuclear entry. Intriguingly, human mutations have been identified in CRY1 [59] and CRY2 [60] that are associated with delayed sleep phase and advanced sleep phase, respectively. Furthermore, when tested in cell lines, the mutation in human CRY1 (deletion of exon 11) enhanced its nuclear localisation and transcriptional repressor activity, which is consistent with a lengthened circadian period and delayed sleep in carriers. Conversely, the human CRY2 point mutation destabilised CRY2 by increasing accessibility of FBXL3, and hence, when recapitulated in mice, shortened behavioural period by ca. 0.5 h. Both elements of the negative feedback arm, Per and Cry proteins can, therefore, independently determine circadian period of the SCN as a function of their stabilities. This independence is emphasised by the observation that intercrosses between Ck1 tau/ tau and Fbxl3 Afh/Afh mice additively determine the period of SCN and behaviour [61] in combination with their respective acceleration and period lengthening summate to an intermediate period. Moreover, and contrary to the expectation that Per and Cry mutually stabilise each other when in complex, the

stabilities of Cry and Per were independently modulated by Ck1e tau and Fbxl3 Afh, results which argue for independent, additive biochemical actions of Per and Cry in circadian control. Such independence may arise in part by Per- and Cry-mediated transcriptional feedback occurring at different stages of the TTFL cycle, a suggestion that is consistent with the delayed occupancy of regulatory sites by Cry1 relative to Per1 and Per2 revealed by ChIP-seq in the mouse liver [62]. Exactly when Per and Cry proteins mediate negative feedback in the SCN is, however, not known, and the success of these genetic approaches to understand the SCN clockwork now poses a range of questions at the structural, biochemical and cellular levels. For example, period-setting properties of Cry and Per may depend on both their stability and their effects on transcription, as seen in mice carrying an allelic series of Cry1 where circadian period is determined through both degradation-dependent (i.e. stability) and degradation-independent pathways [63]. Understanding how Clock/Bmal1 dimers engage the E-box sequences [64] and activate transcription [65], and how Per and Cry proteins assemble onto them and recruit inhibitory complexes [66e68] are areas of active interest. Indeed, structural biology and biophysics have now become tractable approaches to understand in molecular, even atomic, detail how the mammalian TTFL functions [38]. It should be noted, however, that although molecular-genetic manipulations have proved useful to generate qualitative models of cell-autonomous timekeeping in the SCN, a robust quantitative understanding awaits. One approach to this is to directly measure the activities of the components of the TTFL.

Monitoring SCN TTFL and Cellular Functions: Genetically Encoded Reporters By its very nature, SCN circadian timing is a dynamic process, and so real-time imaging approaches, more so than “snapshots” of gene expression in tissue extracts, provide an invaluable means to explore its behaviour. Genetically encoded reporters exploit fluorescent proteins (FPs) and luciferase-dependent bioluminescence, which are better suited, respectively, to precise spatial localisation and long-term, high-throughput monitoring of circadian rhythms. Both offer dual-wavelength capacity, although there are few wavelength variants of luciferases, compared with the considerable range of FP spectral variants. In both cases, destabilised versions offer greater temporal resolution, although perhaps at a cost of reduced signal intensity. Aggregate bioluminescence can be detected by photon-multiplier tubes (PMTs), whereas CCD-

3644 based bioluminescent or fluorescent microscopy provides cellular resolution with consequently lower throughput. Some configurations also allow combined bioluminescence and fluorescence imaging, with several circadian reporters recorded in spatial and temporal register in the same specimen [69]. Reporters of circadian gene expression and SCN cellular activity can be genomically encoded, as a transgene or a knockin (for faithful report of the endogenous protein), but somatic transgenesis can also be achieved by biolistics [70,71] and by adenoassociated (AAV)- and lenti-viral (LV)-based vectors [44,72]. Differences in packaging capacity (LV larger than AAV) and efficiency of transduction (AAV greater than LV in the SCN) and cell type-specific tropisms are important considerations when using viral vectors, but they nevertheless offer considerable advantages of flexibility, rapid generation and validation, and economy of use, both in slice configurations [69] and in vivo [73] (when delivered by stereotaxic injection). Indeed, their advantages have seen them replace biolistic approaches as a means to deliver reporters (and other factors) into SCN cells, and in many cases, they are preferable to the generation of genomically modified mice, which face several practical limitations (reviewed in Ref. [74]).

Looking at the TTFL Components The development of genetically encoded reporters has opened a “window” for exploring the inner workings of the molecular clock within the SCN. The creation of transgenic Per1-Luciferase (Per1-Luc) reporters in rats [75] and mice [76] allowed real-time monitoring of Per1 transcription in primary mammalian tissues. Robust circadian rhythms of bioluminescence were detected in ex vivo SCN slices as well as through fibre photometry in the living animal [77]. Importantly, the peak phase of Per1-Luc bioluminescence matched the peak of endogenous Per1 mRNA in these mice (late circadian daytime) [78]. Furthermore, by monitoring individual cells of the mouse SCN ex vivo, it was not only clear that there was cellular synchrony of Per1 expression but also that the cells displayed a clear sequence of cellspecific peak phases of Per1 [79]. This key observation was the starting point for understanding the inter-relationships and organisation of the cellular pacemakers of the SCN, discussed later. Further transcriptional reporter mice, including Bmal1-Luc [80] and Cry1-Luc [81], have added to the circadian reporter toolkit, where the latter has provided insight into the interactions between Cry1 transcription with different elements of the circadian clock machinery such as cAMP, Per proteins and neuropeptides. Fluorescence-based transcriptional report has, in the form of transgenic Per1-Venus and

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Per2-DsRed, revealed distinct expression patterns of Per1 and Per2 in the SCN (albeit with limited temporal information, due to the long half-life of the encoded FPs obscuring temporal dynamics) [82]. Dual real-time bioluminescent imaging of greenemitting Bmal1-ELuc and red-emitting Per2-Slr2, however, captured the circadian dynamics of transcription more effectively, including the antiphase relationship between these positive and negative arms of the TTFL, respectively [83,84]. Interestingly, a more easily degradable form of Gfp has also been used as a fluorescence report of Per1 and was able to recapitulate broadly the circadian dynamics of Per1 gene expression in the SCN [85,86]. Alongside these transcriptional reporters, just as important, the first translational circadian reporter mouse, Per2:Luc, produced this time as a knockin fusion protein rather than as a transgene, showed that endogenous Per2 protein levels reported as bioluminescence also exhibited robust rhythms in SCN and peripheral tissues in culture [87]. Per2:Luc continues to be an important tool for exploring the spatial and temporal dynamics of the TTFL at the SCN circuit level. Similarly, the Per2:Venus knockin mouse has enabled further characterisation of SCN dynamics, not only at the cellular and network level but also at the subcellular level, exploiting the quantitative imaging techniques better suited to fluorescent fusion proteins [88]. In contrast to this cellular and subcellular focus, at the organismal scale, it has been almost 20 years since the first realtime recordings of circadian reporter in the SCN in vivo [77]. Technological limitations have meant that further studies [89e91] relied on photometry and thus lack the spatial information that can be extracted in ex vivo SCN slice recordings. The recent miniaturisation of microscopes and development of specially adapted optics [92] will make it possible to capture both fast, cellular events alongside slower spatio-temporal information in the SCN. The ability to then correlate this information with other physiological and behavioural measures will be very powerful. In other words, what molecular and cellular events in the SCN and the wider brain control circadian behaviour?

Reporters for SCN Cellular Functions In conjunction with the cell autonomous TTFL, there are complex layers of circadian regulation across different cellular compartments, notably between the cytosol, nucleus and plasma membrane. For SCN cells, the cycles of encoding, transmission and decoding of circadian time forms the basis of inter-cellular communication. Importantly, this is not a linear pathway because TTFLdependent cytosolic regulation links back into the TTFL. Indeed, many circadian clock genes, including

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Per, carry calcium/cAMP response elements (CREs) [93]. It is therefore important to be able to report/ sense signalling molecules and other circadian outputs of the SCN in register with the TTFL components. Calcium (Ca 2þ) is a key second messenger for the molecular clock and for many other cellular functions. Intracellular calcium ([Ca 2þ] i) dynamics are frequently used as a proxy for electrical activity of neurons in the SCN, owing to the approximately 10-fold changes in [Ca 2þ]i concentration during action potentials [70]. The development of genetically encoded Ca 2þ indicators (GECIs) such as Cameleon and GCaMP have been widely used to profile neuronal firing patterns on the millisecond timescale as well as slower networklevel dynamics [73,94]. Similar to other reporter systems, GCaMPs and other GECIs have different excitation/emission spectra, allowing for dual recordings of [Ca 2þ]i when conditionally expressed in different compartments or cell populations. It should be noted that [Ca 2þ]i dynamics, of course, are not a direct readout of neuronal electrical activity. Direct measurements by patch clamp or by multi-electrode arrays (MEAs) have shown that SCN neurons are more electrically active during circadian daytime than during night [95,96]. Simultaneous recordings from MEAs alongside Cameleon-based [Ca 2þ]i report showed that although both [Ca 2þ]i and spontaneous firing rate (SFR) peak during the circadian day, [Ca 2þ]i is phase advanced to the MEA rhythm in the SCN [97]. Numerous studies have described in detail the heterogeneous electrical characteristics of dispersed neuronal cultures [98] and acute [97] and organotypic [73] SCN slices, but many of these are limited in terms of circuit-level spatial mapping and the ability to do long-term recordings. Genetically encoded voltage indicators (GEVIs) such as ArcLight [98], therefore, offer great potential, although they have proved challenging to work with because of low fluorescence intensity and signal-to-noise ratio, as well as the requirement for sufficiently rapid scanning to reliably detect action potentials [99]. Nonetheless, over longer timescales, ArcLight can detect slower, circadian changes in SCN electrical activity, consistent with electrophysiological measures [73,100], with greater ease and provides more extensive spatial information. Electrical activity is by no means the only membrane property that should be considered in understanding the circadian clock mechanism. Neuropeptide and neurotransmitter release is another critical feature in the SCN where both synaptic communication [79] and paracrine signalling [31] are essential for maintaining spatio-temporal organisation and synchrony across the network. Similar to GEVIs, the development of genetically encoded neurotransmitter sensors is in its infancy, but one success is the glutamate sensor, iGluSnFR, which was used in SCN slices to reveal

circadian rhythms in extracellular glutamate. This rhythm is driven by astrocytes [73] and is necessary for their contribution to SCN circadian pacemaking (see the following sections) [101]. Future development of other neurotransmitter sensors would expand the toolbox and allow further insight into what is happening outside and between, SCN cells, probing the different levels of circadian regulation of the master clock, from transcriptional to cytosolic to membranes to extracellular space in the SCN (Fig. 1).

Functional Insights Revealed by Circadian Reporters In addition to mapping the canonical transcriptional axes of the SCN TTFL, it is important to consider new, auxiliary axes of transcriptional regulation. As previously mentioned, AT motifs, to which the transcription factor Zfhx3 binds, were identified as a novel point of transcriptional control of circadian function. Transduction of wild type SCN slices with an AT-Luc LV reporter revealed robust circadian activation of these motifs. Furthermore, in Zfhx3 Sci SCN slices, these oscillations were damped and less robust. This transcriptional dysregulation appeared to affect neuropeptide coding genes at the level of promoter activation, transcript and protein levels, and Per2:Luc rhythms were also less synchronous, probably as a result. Thus, AT motif-dependent transcription provides another recurrent level of control that ensures robust intercellular signalling within the SCN [44]. In contrast to the more diffuse bioluminescent signals, FP tags enable precise spatial localisation of circadian proteins, as well as the ability to quantify various parameters of their behaviour within the cell. One such highly regulated cellular behaviour is protein shuttling, an essential feature of the TTFL (discussed further in this issue by Yagita et al.[103]). Early studies on mammalian cells showed the importance of Cry proteins for nuclear localisation of Per proteins [102], wheras dual-fluorescence imaging of the Drosophila per and tim revealed that these proteins are in fact subject to an “interval timer” mechanism in the fly clock, that gates nuclear entry of the per/tim complexes formed in the cytosol [104]. The creation of the Per2:Venus knockin mouse has provided a useful tool to carry out equivalent studies of the cellular dynamics of a mammalian endogenous clock protein. Using a combination of real-time and quantitative imaging, Per2 was shown to be present in the nucleus of SCN neurons throughout the day, implying that, in contrast to the behaviour of per and tim in Drosophila, Per2 gradually and progressively translocates to the nucleus without prior accumulation in the cytoplasm [88]. Per2 protein was also found to be

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SCN Astrocyte Fig. 1. Probing circadian control of the SCN. A schematic diagram showing the “daily life” of SCN neurons (upper panel) and astrocytes (lower panel), including examples of where bioluminescence (yellow ovals)- or fluorescence (green ovals)-based reporters can be used to monitor different aspects of the clock. The encoding and decoding of circadian time transfers between three cellular levels: TTFL, cytosolic and membrane-associated (separated by dotted lines in the upper diagram). The cell autonomous TTFL interacts with the cytosolic environment via 2nd messengers, such as Ca 2þ. Activation of signalling cascades result in changes to membrane properties via ion channels and the release of neurotransmitters or peptides. The flow of information is not linear, nor unidirectional. Changes in signalling and cytosolic environment can then feedback on to the TTFL, for example through Ca 2þ/cAMP response elements (CREs) in the promoter regions of selected circadian genes. Examples of “snapshots” (unpublished) from real-time imaging of reporters in the SCN are included as insets (upper left and upper right). Representations of reporter activity across the circadian day for neurons (right) and astrocytes (left) illustrate that these two cell types have contrasting daily lives: neurons being “day active” and astrocytes are instead “night active.” Note that extracellular glutamate (orange dotted trace) peaks in the circadian night, irrespective of whether measured from astrocytes or neurons. SCN, suprachiasmatic nucleus; TTFL, transcriptional-translational negative feedback loop; GEVI, genetically encoded voltage indicator; GECI, genetically encoded calcium indicator.

SCN Molecular-genetic Manipulation

highly mobile and, interestingly, mobility within and between the nucleus and cytoplasm was not subject to circadian gating. Furthermore, pharmacological inhibition of Ck1d/e enzymes in fact accelerated this mobility, again in a circadian-independent manner. Given that Per2 mostly does not exist as monomeric protein, but rather, it is present in multiple different “mega complexes,” the uniformity of the molecular mobility of Per2 was remarkable. In particular, it might be expected that the smaller ~1.1 MDa cytoplasmic Per complex, identified by a modified electrophoretic assay, would be more mobile than the ~1.9 MDa mature cytoplasmic Per complex [105]. It is possible however, and quite likely, that these complexes are subject to different types of regulation within each compartment, which in turn could modulate their mobilities. Ck1d, however, was present in both cytoplasmic and nuclear Per complexes [105]. It can thus be postulated that CK1 enzymes regulate the mobility of both nuclear and cytoplasmic Per, as suggested by the bidirectional acceleration of FRAP recovery of Per2 in the presence of Ck1 pharmacological inhibition. In addition to quantifying mobility, circa 10,000 molecules of Per2 (at peak expression) were estimated to be present in the nucleus of primary fibroblasts, undergoing a 10-fold amplitude cycle across the circadian day [88]. Although similar measurements are required for SCN cells, it is interesting to note that the estimated number of molecules of Per2 does exceed the number required (>4000 molecules) for a stable virtual TTFL, as predicted by stochastic simulations [106]. With so few studies using endogenous circadian fusion proteins, there is still broad scope for probing a whole panel of clock proteins, with the potential to look at their behaviours relative to each other, in real-time, and thereby develop precise quantitative models of the molecular dynamics of the TTFL. For this, multi-channel or multi-modal recordings are required. At a cellular level, multi-modal recordings of reporters have enabled temporal “phase-mapping” of the circadian cell-autonomous programme of the TTFL of neurons, from metabolic through to electrical activity. By using peak of Per2 expression to define CT12 (consistent with in vivo analysis of Per2 protein in the SCN and the onset of mouse circadian activity), a circadian phase map [69,107] for the “daily life” of an SCN neuron can be assembled [108]. Starting in the subjective morning, the SCN neuron is most electrically active and depolarised, with an accompanying peak of GCaMP-reported peak neuronal calcium at CT7 [69] (this is also the phase of peak cytosolic cAMP levels [109]). GCaMP report has then been used to cross-register luciferase reporters other than Per2:Luc [69], showing that the activation of CRE-dependent transcription, as monitored by a LV CRE-Luc reporter, peaks at ~ CT9, shortly after the rises of cAMP and calcium

3647 [69]. E-box-regulated transcription and consequently translational rhythms of Per1, followed by Per2 and then Cry1, peak in the late subjective day and early subjective night [110]. The SCN neuron is less active during circadian night, where most rhythmic TTFL and cytosolic outputs are declining, with the exception of Bmal1 transcription, which peaks in the late subjective night and whose protein product is then ready to transactivate a new TTFL the subsequent day [84]. As previously alluded to, neurons are not the only cell types that exhibit pacemaking properties in the SCN. By combining circadian reporters with the Cre-lox system for conditional gene expression, comparative phase mapping between different cell types is possible. “Flexed” reporters restricted to astrocytes exposed robust circadian rhythms in [Ca 2þ]i, extracellular glutamate, Cry1-Luc [73] and Bmal1-Luc, [111] but importantly, they peaked at different circadian times to neurons, and so astrocytes can be considered “night-active” cells as opposed to their “day-active” neuronal neighbours [101]. Through multiplexed monitoring of these different reporters in astrocytes and neurons, and appropriate pharmacological manipulations, it was possible to conclude, first, that the extracellular glutamate rhythm originated from astrocytes and, second, that glutamate acts as a “gliotransmitter” to contribute to SCN synchrony [73]. This therefore increases the complexity of network-level pacemaking in the SCN, as it is now essential not only to consider neurons but also to consider astrocytes (and potentially other glial cell types) as active contributors to circuit functions. Not only do the reporters identify the peak timing of activities but they also reveal the circadian waveform, i.e., temporal dynamics of events within the SCN oscillator [8]. In contrast to the “classical” sinusoidal wave exhibited by TTFL reporters, CRELuc reports asymmetric “saw-tooth” shaped rhythms with a steeper rise than decline after the peak [69], indicative of a rapid increase in CRE activation and a more graded reduction. Importantly, waveform analysis of Per2:Luc rhythms from SCN subjected to different genetic and pharmacological manipulations of period revealed that these manipulations can subtly alter different aspects of the temporal structure to these waveforms, whereas at the same time, the SCN network overall remains capable of maintaining robust rhythmicity [8]. Indeed, this robustness is an emergent property of the inter-cellular communication that occurs within the network and is a critical aspect of what makes the SCN unique as the master mammalian pacemaker. An imprint of this network communication lies in the complex, stereotypical spatio-temporal waves of peak gene expression and cellular activity that originate at the dorsomedial edge of the SCN, moving sequentially across the SCN in the ventral direction [79]. These synchronous “waves” are dependent on action

3648 potential firing [79] and paracrine signalling [31] and are abolished when either of these inter-cellular signalling axes is blocked. In the absence of such intercellular communication, SCN neurons express their own intrinsic properties, where notably the cellautonomous periods span from ~21 h to ~27 h [112]. The spatio-temporal wave, in which cells share a common period but exhibit systematically different phases, therefore embodies the principal emergent property of the SCN network. Cluster analysis of Per2:Luc recordings from the individual cells of SCN slices suggests that the wave is a product of serial activation of different sub-regions of the SCN [113]. Furthermore, SCN slices from mutant animals lacking either a clock (Cry1 //Cry2 / (Cry-null)) or intercellular communication (Vipr2 /), were able to maintain either spatial or temporal organisation, respectively [114]. This indicates that at some level, these aspects of network structure are encoded independently. Despite this insight, however, it remains unclear what parameters of the network specify the wave, nor whether the same dynamics are maintained in vivo. A better understanding of the “wired” synaptic and “wireless” paracrine circuits of the SCN in conjunction with better defined genetic access to distinct cell types will bring us closer in identifying these parameters.

Dissecting the SCN Circuit: Networklevel Encoding of Circadian Time The combination of real-time imaging of TTFL components alongside markers of neuronal function has made it possible to define the cell-autonomous properties and network-level emergent properties of the SCN clock. The next level of question is how, in the absence of environmental cues, do neurons and astrocytes of the SCN [3] generate this internal proxy of solar time and transmit it to the rest of the animal? As has already been stated, although they are all GABAergic, SCN neurons also express a wide array of neuropeptides and their cognate receptors [115]. Indeed, recent bulk and single-cell transcriptomic analyses have revealed several neurochemically defined modules that mediate signalling between and within SCN cells [115e119]. This diversity raises the question of whether all SCN cells and their neuropeptidergic signalling pathways are equal in effect or is there a division of labour, possibly a hierarchy, in how they interact and signal time [3]? Put another way, does the intra-cellular circadian programme of one cell population override the programmes of other populations? One way to test this is to manipulate the cell-autonomous circadian properties of specific SCN populations and observe the effects on network timekeeping. Suitable manipulations include the disruption of a core TTFL gene, the excision of a clock-associated component that

SCN Molecular-genetic Manipulation

determines cellular period, and/or local restoration of a missing clock component on an otherwise null mutation background in which all cells are arrhythmic.

Conditional Disruption of Bmal1 Bmal1 is the only core clock gene sole knockout of which renders the SCN and animals arrhythmic [24] and so, it was an early choice for intersectional targetting, exploiting a floxed (flanked by LoxP) Bmal1 allele sensitive to Cre recombinases (Fig. 2). Putative pan-neuronal, brain-wide inactivation of Bmal1 using the Nestin-Cre [120,121] did not render animals behaviourally arrhythmic because approximately 70% of SCN cells retained Bmal1 expression [120]. The SCN can still, therefore, drive circadian rhythms in behaviour in the absence of functional cellular clocks in the rest of the brain [120]. CaM kinase (CamK) 2a-Cre more effectively targetted floxed Bmal1 across the forebrain and SCN (ca. 90% reduction) [122] and rendered behaviour and SCN explants arrhythmic, whereas TTFL rhythms in peripheral tissues remained intact. Similarly, two other neuron-specific Cre lines are vesicular GABA transporter (Vgat)-Cre, which targets all GABAergic neurons, and Synaptotagmin10-Cre that are also able to specifically and severely disrupt Bmal1 expression in the SCN, leading to behavioural arrhythmicity [33,123]. Furthermore, in the case of Synaptotagmin10-Cre, the severity of the circadian phenotype can be titrated by increasing the number of Cre alleles or manipulating the genetic background of the Bmal1 genotype [33]. Thus, complete conditional Bmal1 disruption is possible in the SCN and can be differentially manipulated dependening on the Cre-driver chosen. To date, however, there is no specific Cre-driver that will allow researchers to specifically target the complete SCN alone without hitting extra-SCN areas. This approach can be extended by targetting SCN sub-populations. Cre-mediated deletion of Bmal1 from Nms cells, which constitute ca. 40% of SCN cells and with little extra-SCN neuronal expression, results in behavioural arrhythmicity under constant conditions [124]. Conditional disruption of Bmal1 in the less numerous Avp cells (which co-express Nms [124]) did not cause arrhythmia, rather behavioural period (but not SCN period) lengthened [125]. A cell-autonomous clock in Avp cells is therefore not necessary for circuit-level oscillation, although in its absence, the computation of ensemble circadian period in vivo is modified. It is also important to reiterate recent work that has identified that astrocytes are not passive cellular constituents of the SCN [73,101,111,126]. Partly, this has been shown using glial glutamate and aspartate transporter (Glast)-Cre to target

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a)

Nestin-Cre;Bmalfl/fl

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Fig. 2. Targeting strategies of conditional Bmal1 knockout. Red hashed areas show Cre-targeted brain or SCN regions, whereas the consequences of conditional Bmal1 knockout on behaviour (actogram, upper) and SCN molecular rhythms (blue line, lower) are shown to the right. (a) e (d) SCN and extra-SCN targeting. (a) Nestin-Cre mediated Bmal1 knockout results in incomplete SCN knockout. (b) Vgat-Cre-mediated knockout abolishes Bmal1 expression in the SCN and across the rest of the brain. (c) Camk2a-Cre abolishes Bmal1 expression in the SCN and across the forebrain. (d) Synaptotagmin10-Cre abolishes Bmal1 expression in the olfactory bulb and SCN. (e) e (h) SCN targeting. (e) Nms-Cre targets an SCN-specific cellular population comprising 40% of the SCN and spanning the dorsal and ventral regions. (f) Avp-Cre targets Avp cells (a subset of Nms cells) in the ventral SCN, abolishing Bmal1 expression in a small proportion of the SCN without compromising behaviour or molecular rhythmicity. (g) Glast-Cre targets astrocytes across the brain, and specifically abolishes Bmal1 expression in SCN astrocytes. (h) AldhL1-Cre specifically abolishes Bmal1 in SCN astrocytes via a conditional AAV-mediated Crispr approach delivered specifically to the SCN by stereotaxic injection. SCN, suprachiasmatic nucleus; Avp, arginine vasopressin; AAV, adeno-associated vector.

Bmal1 excision exclusively to astrocytes [126] or through the use of AAV-mediated conditional (CRISPR) gene editing approaches to knock down Bmal1 targeted using the astrocyte-specific aldehyde dehydrogenase 1 family member L1 (Aldh1L1)-Cre [111]. Both manipulations modestly lengthened behavioural period [111,126] and, unlike the conditional Avp disruption, this persisted in the SCN explant [111] suggesting that astrocytes transfer circadian information at the circuit level more potently than Avp-expressing cells alone. In short, conditional disruption of Bmal1 expression has started to reveal a potential hierarchy in how different cellular and neurochemical modules

in the SCN are able to contribute to circadian pacemaking.

Conditional Manipulation of Other Core Clock Factors Bmal1 is not the only clock component that can be targetted to elicit circadian effects. Using Vgat-Cre, conditional knockout of Clock (which encodes the partner to Bmal1) shortened behavioural period [28,123]. Loss of Clock alone did not, however, cause arrhythmia unless it is paired with null mutation of Npas2 [27]: in the SCN, Clock and

3650 Npas2 are mutually redundant partners of Bmal1 [27]. In contrast to null mutations, the Clock D19 mutation acts as a competitive inhibitor for endogenous Clock and can be used reversibly to control cell-autonomous period using a Cre-dependent TetOff system [101] in which expression of a transgene can be toggled off or on through provision or removal of tetracycline [127]. Overexpression of Clock D19 in Nms cells (but not Vip cells) lengthened circadian period at both the behavioural and explant levels [124]. Moreover, Tet-Off over-expression of TgPer2 to reversibly disrupt the cell-autonomous clock of Nms cells caused arrhythmicity at the behavioural level and compromised cellular rhythmicity and synchrony in SCN explants [124]. Together, these results highlight the importance of Nms cells as SCN pacemakers. An alternative approach to probing how circadian information is passed between cell groups is to use genetic complementation by restoring a competent copy of a clock gene to a null-background, commonly via AAVs [101,125,128,129]. For example, Bmal1 expressed conditionally via AAVs in Bmal1null Avp cells reversed the lengthening of behavioural period, confirming that this output effect is a direct consequence of Bmal1 disruption in SCN Avp cells [125]. More dramatically, delivery of AAV with a minimal Cry1 promoter driving a fusion protein of Cry1 and Egfp (Cry1-Cry1:Egfp) can rescue the arrhythmic phenotype of Cry-null mice, at the levels of both the SCN slice and whole animal behaviour. Importantly, rescue with AAV-expressed Cry1 establishes a characteristic long period (>26 h), whereas rescue with Cry2, driven by the minimal Cry1 promoter, sustains a Cry2-specific short circadian period (ca. 22 h). Furthermore, expression of Cry1Cry1:Egfp can rescue the shortened period of rhythmic Cry1-null SCN and mice, from 22 to 25 h [101,128,129]. Thus, it is the Cry1 and Cry2 proteins themselves and not differences in their promoters that determine cell-autonomous SCN period [128]. Along with the emergence of competent aggregate oscillations by Cry1-complementation in Cry-null SCN explants, network level waves are suitably established [128,129], although at some level, this requires Avp-ergic signalling; pharmacological blockade of Avp-receptors during Cry1-complementation prevents this network-level reorganisation [128]. In addition, the contributions of different SCN cellular populations to these network properties can be targetted separately using AAVs that express Cre under cell type-specific promoters [3]: Synapsin for neurons and Glial fibrillary acidic protein (Gfap) for astrocytes. Unsurprisingly given the importance of Nms neurons as pacemakers, expression of Cry1:Egfp solely in SCN neurons can initiate, de novo, long-period rhythms in Cry-null SCN and drive behavioural rhythms in mice. More surprisingly, Cry1

SCN Molecular-genetic Manipulation

expression restricted solely to SCN astrocytes of Cry-null mice can also initiate de novo rhythms. Astrocytes can therefore act as pacemakers, albeit taking a few more days for complete initiation than do neurons and establishing a slightly shorter period. Furthermore, this initiation of rhythmicity by SCN astrocytes is dependent on the function of hemichannels and the activity of NMDA receptors [73,101]. This again highlights the complexity of the SCN network organisation. Not only are there many different types of cells with pacemaking activity but they also require different pathways to assert their clock properties on the entire circuit. Conditional rescue of SCN circadian timekeeping has also been developed at the translational level [129]. Although the Tet-On/Tet-Off approach is a powerful tool for reversible molecular manipulation of the SCN, it provides a binary switch at the level of transcription. A more flexible tool, therefore, would be to dose-dependently manipulate protein translation. Genetic code expansion (GCE) provides a means to achieve this, where amber stop codon (TAG) suppression allows the level of protein expression to be reversibly manipulated at the translational level through provision or removal of a non-canonical amino acid (ncAA) [129,130] (Fig. 3). Using a bipartite AAV system, Cry1-expressed Cry1:Egfp disrupted by the amber stop codon was introduced alongside an orthogonal tRNA-synthetase and tRNA pair restricted to neurons by the Synapsin promoter. In this way, the expression of Cry1 could be modulated in vivo or ex vivo by providing the ncAA alkyne lysine (AlkK) in culture medium or drinking water. This enabled circadian rhythmicity to be initiated or disrupted at will via reversible control, at the translational level, of the AAV-expressed Cry1 [129]. Further, supply of AlkK at different concentrations allowed a dose-dependent titration of Cry1 levels and a consequent dosedependent extension of period; higher levels of Cry1 expression lengthened the period of Cry-null SCN across a range of 26e31 h [129]. In summary, the conditional systems described previously show AAV-mediated expression of Cry1 is a powerful tool that can be used to reversibly and dosedependently study period setting and rhythm initiation side-by-side.

Manipulation of Casein Kinase Isoforms As well as targetting core clock components directly, they can be manipulated indirectly by affecting their stability; as previously detailed, Ck1d and Ck1e can determine TTFL period through their effects on Per stability. Conditional manipulation can, therefore, be used to create temporally chimeric models where cell-autonomous clocks in

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a)

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Fig. 3. Translational switching via genetic code expansion allows reversible expression of Cry1. (a) In the absence of the orthogonal amino acid, translation cannot proceed beyond the amber stop codon (UAG, orange). Upon supplementation of the orthogonal amino acid, an ectopically expressed tRNA synthetase can incorporate it into a new tRNA with a complementary anticodon to the amber stop codon. This allows read through of the amber stop codon and incorporation of the orthogonal amino acid resulting in the production of a functional protein. Withdrawal of the orthogonal amino acid reverses this, leading to translational disruption. (b) In vivo stereotaxic injection of AAVs to supply amber stop codon disrupted Cry1:Egfp along with tRNA components allows Cry1 expression and, therefore, rhythmic behaviour to be reversibly initiated or terminated upon provision or withdrawal of the orthogonal amino acid via the drinking water. (c) Ex vivo transduction of SCN with amber stop codon disrupted Cry1:Egfp along with tRNA components via AAVs allows Cry1 expression and, therefore, molecular oscillations to be initiated or terminated following provision or withdrawal of the orthogonal amino acid via the culture media. SCN, suprachiasmatic nucleus; AAV, adeno-associated vector.

targetted SCN cells exhibit periods different to nontargetted cells. For example, conditional neuronal excision of Ck1d using Vgat-Cre lengthens behavioural and explant SCN periods, whereas peripheral tissues retain wildtype 24 h rhythms [131]. Furthermore, these effects on period are isoformspecific; excision of the Ck1e isoform does not alter period unless it is simultaneously excised with Ck1d, which consequently lengthens behavioural and explant period further [131]. Thus, the Ck1 isoforms have an additive allelic dose-dependent effect on circadian period. Importantly, extending conditional Ck1d-excision to the Avp cells via AvpCre lengthens behavioural period and is reversed by AAV-mediated restoration of Ck1d [132]. However, as with Avp-Cre conditional Bmal1 disruption [125], although manipulation Ck1d in SCN Avp cells lengthened circadian behaviour in vivo, these changes did not persist in explants unless signalling between the ventral and dorsal SCN was surgically disrupted, which released the dorsal

Avp cells to run with their new longer cellautonomous period [132]. This suggests that Avp cells are lower in the circuit pacemaking hierarchy than other cells. Although loss of Ck1e alone does not alter circadian period, the Ck1e tau mutant allele dosedependently accelerates the circadian clock [16]. The redundancy between Ck1d and Ck1e can therefore be exploited by conditionally excising Ck1e tau to create temporal chimaeras wherein cellautonomous periods differ by up to 4 h. In Ck1e tau/tau mice, using the dopamine receptor D1 (Drd1a)-Cre to target 60% of SCN cells creates a chimaera where Drd1a and non-Drd1a cells have 24 and 20 h cellautonomous periods, respectively [133]. Importantly, as with the Nms-Cre model, targetted cells span the ventral and dorsal SCN, including Vip and Avp cells [112,124]. Interestingly, this temporal chimaera produces two phenotypes: revertant and non-revertant animals with 24 and 20 h periods, respectively, that are consistent between behaviour and SCN

3652 explants. This incomplete penetrance suggests that both Drd1a-positive (24 h) and Drd1a-negative (20 h) cells have the potential to set circuit-level properties. The interaction is competitive, and that is explicit in ca. 10% of mice, which alternate between 20 h and 24 h periods, as the two cell populations gain and lose ascendancy. It also confers plasticity on the SCN, insofar as mice can be dynamically reprogrammed between 20 and 24 h periodicity by entrainment to corresponding 20 or 24 h lighting cycles [133]. Conditional excision of Ck1e tau also provided the first evidence that SCN astrocytes can confer their intrinsic circadian period on behavioural and SCN explant rhythms [73,111]. Using AAVs to excise CK1e tau in astrocytes via Gfap-Cre (or AldhL1-Cre) or in neurons via Synapsin-Cre, in both cases, lengthened behavioural and explant periods [73,111]. Combined with Bmal1 excision and Crycomplementation approaches, these experiments provide strong evidence that astrocytes are active network components of the SCN. It is becoming increasingly clear, therefore, that the SCN dynamically integrates the cell-autonomous, TTFL programmes of its different cell populations, which presumably requires complex circuit-level computations. The nature of these computations is not yet known, but likely involves reciprocal interactions between the TTFL, transcriptional programmes and intercellular signalling.

Manipulation of Transcription Factors in the SCN Transcription factors outside the TTFL exert diverse roles in the SCN, especially developmental. Conditional deletion of Lim homeobox 1 (Lhx1) using Six3-Cre or Rora-Cre to target the SCN, causes mice to become behaviourally arrhythmic, display larger responses when phase shifting to light pulses and have less precise molecular and electrical rhythms in SCN slices [134,135]. Consistent with this, SCN from these mice have a marked reduction in a wide range of neuropeptides including Vip, Avp, Grp, Prok2 and Nms, while the core clock is unaffected. Thus, Lhx1 appears to specify SCN neuropeptide expression, its loss compromising circuit-level synchronisation and thereby affecting the cell-autonomous clock indirectly. As noted previously, the circadian role of Zfhx3 was originally identified via the gain-of-function mutation Zfhx3 Sci [44,45], which compromised the expression of SCN neuropeptides. Adult-specific Zfhx3 knockout using an inducible Cre (ubiquitin C promoter, UBC CreERT2) [136] significantly shortened behavioural period and, similar to Lhx1-null mutants, accelerated re-entrainment to altered lighting cycles, indicative of

SCN Molecular-genetic Manipulation

a less robust SCN clock [136]. Zfhx3 thus appears to act in a dual capacity in the SCN e directing terminal differentiation of the SCN (Zfhx3 Sci mice have a slightly smaller SCN volume) and regulating neuropeptide expression and thus circuit function [44,45,137]. In contrast to Lhx1 and Zfhx3, however, which do not influence core clock gene expression, the transcription factor sex-determining region Y-box 2 (Sox2) regulates Per2 expression alongside a range of SCN neuropeptides [138]. Vgat-Cre conditional excision of Sox2 compromises circadian behaviour and re-entrainment to photic phase shifts [138]. At the SCN level, the amplitude of molecular rhythms is reduced, consistent with loss of Sox2dependent Per2 expression [138] and, interestingly, although Sox2 excision affects largely the same neuropeptides as does loss of Lhx1 and Zfhx3, the responses to photic phase shifts are more severely compromised. This suggests that proper adjustment to these stimuli requires not only strong circuit-level synchrony but also a robust cell-autonomous clock.

Manipulation of Neurochemical Signalling in the SCN Rather than manipulating the clock in cells defined by their signalling identity, the signalling factors themselves can also be targetted. SCN neurons are almost entirely GABAergic, but knockout of GABAergic function is developmentally lethal and so, until recently, interventions have been pharmacological, and consequently very few SCN circuit-level properties have been ascribed to GABAergic signalling (reviewed in Ref. [139]). It is, however, surprising that complete pharmacological blockade of the major SCN neurotransmitter does not compromise ongoing oscillations, but consistent with this, SCN explants from mice deficient in either Vgat or glutamate decarboxylase (Gad) 65 and 67 isoforms, are indistinguishable from wild type [140]. However, depleting GABAergic signalling by conditional Vgat knockout in the adult SCN via Synapsin-Cre AAVs did result in less precise circadian behavioural output [140] suggesting that GABA-ergic SCN efferent signals may mediate SCN output, similar to the predicted function of Prok2 signalling (discussed in further sections). Moreover, in response to different lighting cycles, GABA may act in concert with Vip to control the phase dispersal of SCN neurons [141], whereas pharmacological blockade of GABAergic signalling can transiently synchronise Vip-null SCN [142]. Perhaps, therefore, the key to understanding any role for GABA in the SCN is to consider the context of accompanying neuropeptidergic signalling. Vip is the defining neuropeptide of the SCN core, and deletions of either Vip or Vipr2 (the gene

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encoding the Vpac2 receptor) both produce behaviourally arrhythmic mice [143e145] and desynchroVip Avp Grp

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Fig. 4. Ex vivo SCN grafting experiments reveal hierarchical neuropeptidergic signalling. Schematics showing co-culture grafting experiments where nonbioluminescent graft SCN can be used to test the ability of paracrine signalling to drive rhythmicity in host Per2:Luc SCN in the absence of specific neuropeptidergic signalling axes. (a) Wild type SCN can synchronise and drive rhythmicity in Vip-deficient SCN demonstrating that Vip, Avp and Grp together can sustain rhythmicity. (b) Wild type SCN can synchronise and drive rhythmicity in Vpac2deficient SCN. As these host slices cannot respond to Vip, these experiments demonstrate that coherent Avp and Grp signals synchronise the SCN. c) Wildtype SCN can initiate and drive rhythms in an arrhythmic Cry-null/Vpac2-null SCN. As these host slices cannot respond to Vip, these experiments show that even in the absence of a functional clock coherent Avp and Grp signals can synchronise and drive molecular rhythmicity in the SCN. d) Avp-null graft SCN cannot initiate and drive rhythms in an arrhythmic Cry-null/Vpac2-null SCN. As host slices cannot receive Avp or Vip signals, this experiment shows that Grpsignalling alone cannot synchronise and drive molecular rhythmicity in the SCN. SCN, suprachiasmatic nucleus; Avp, arginine vasopressin; Grp, gastrin-releasing peptide, Vip, vasoactive intestinal peptide; Vpac2, Vip receptor2.

nised cellular rhythms with damped amplitude in SCN explants [31,146,147]. Despite this, Vpac2-null SCN can be transiently synchronised through bolus application of the neuropeptide Grp or K þ-dependent depolarisation [146], and both null-genotypes can be entrained through grafting with wildtype SCN as a source of paracrine intercellular signals [31] (Fig. 4). Vip appears to be the strongest synchroniser followed within a potential hierarchy by Avp and then Grp [31]. This comparison echoes the relatively weak circuit-level effects of Avp-targetted clock manipulations [125,132]. Indeed in similar grafting experiments, Avp-null SCN explants fail to drive rhythmicity in Cry-null, Vpac2-null SCN [148]. Moreover, mice lacking the Avp receptors V1a and V1b, alone or in combination, are behaviourally indistinguishable from wild type under constant conditions [149]. In response to an 8-h phase delay, however, the V1a/V1b double-null mice re-entrain almost instantaneously, whereas wild type animals typically require 6e8 days to re-entrain [149]. In addition, after a phase delay, TTFL rhythms in wild type SCN are attenuated before gradually recovering. By contrast, this disruption is much less severe in V1a/V1b-deficient SCN, indicative of a more loosely coupled circuit that is more reactive to aberrant lighting schedules [149]. This phenotype is also seen in Lhx1-null mice, where neuropeptidergic signalling is similarly compromised [134,135]). Thus, Avp acts as an SCN coupling factor, possibly mediating within shell and/or shellto-core feedback that normally limits resetting during photic entrainment. Vip and Avp are therefore important signalling axes for maintaining SCN circuit-level synchrony, but what about Nms, which is enriched in SCN cells with significant pacemaking functions? When genes encoding Nms, its paralogue Nmu or both are deleted, circadian behaviour is unaffected, suggesting that Nms is dispensable for proper circadian activity [124]. Circadian phenotypes revealed by manipulation of the Nms cells, therefore, may be due to the fact that Nms cells encompass at least two signalling axes that are important for effective SCN circuit-level function: Avp and Vip. Unlike Nms and Nmu knockouts, deletion of Prok2 or the gene encoding its receptor, ProkR2, disrupts behavioural rhythms [150]. However, it is likely that Prok2 signalling is more important for SCN output, because molecular rhythmicity in ProkR2-null SCN explants is comparable with wild type [150]. On this theme, excision of the gene encoding the transcription factor zincfinger and BTB domain-containing protein 20 (Zbtb20), mediated by Nestin-Cre, specifically depletes ProkR2 in the SCN and produces the same behavioural phenotype as ProkR2-null. Furthermore, this can be rescued by AAV-mediated expression of ProkR2 [151]. Together, therefore,

3654 these experiments reveal that SCN neuropeptidergic signalling is highly complex with different neuropeptides and their receptors governing different circadian functions ranging from maintenance of circuit-level synchrony and core-shell interactions, to SCN output, whereas the circadian functions of other SCN-enriched neuropeptides await determination.

Molecular-genetic Manipulation of Cellular Activity and Signalling in the SCN Genetic depletion of signalling factors within the SCN is not the only means of specifically manipulating circuit-level communication. Optogenetics uses light to activate or inhibit neuronal activity (reviewed in Ref. [152]) and has been used to identify and characterise Vip neurons in dissociated culture [153] and also to map GABAergic output from Vip cells in acute SCN slices [154]. Furthermore, optogenetic activation and inhibition can reset both locomotor and molecular rhythms, revealing that SCN electrical activity is both an input and an output of the oscillator [91,153,155]. Alternatively, the output from neurons can be manipulated through reversible expression of the neurotoxin component tetanus toxin light chain (TeNT) [124]. Using Cre-conditional targetting of TeNT to Nms cells, signalling from this cellular population could be temporarily disrupted, resulting in reversible behavioural arrhythmicity and severely desynchronised SCN explants [124]. Thus, electrical activity in the SCN is not only important for network function, it is also an important input and output for neurons to allow them to modulate their circadian clocks and synchronise through intercellular communication. A final means of manipulating SCN signalling is to phenocopy the intracellular signalling cascades induced by intercellular communication. This can be achieved molecularly via chemogenetics using a designer receptor exclusively activated by designer drugs (DREADDs), wherein genetically engineered G-protein coupled receptors (GPCRs) can be activated by a synthetic ligand with no off-target effects (reviewed in Ref. [156]). This has been applied to the SCN to dissect GPCR-dependent intracellular signalling cascades, revealing that chronic activation of a Gq-mediated axis selectively re-programmes circuit level cytosolic calcium levels and, in turn, the TTFL. This results in SCN explants with elongated period, reduced amplitude and altered spatiotemporal dynamics, changes which can be traced to altered Vip-ergic signalling through grafting experiments [69]. Furthermore, targeting Gq-activation to just the Vipcells reveals that Gq-signalling in this cellular population alone is sufficient to recapitulate the circuit-level effects [69]. Thus, through careful and targeted

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application of new molecular and genetic tools, different aspects of SCN signalling can be effectively manipulated to dissect their effects on circuit-level properties and behavioural output.

Conclusion and prospect The successes of genetic screens and the subsequent cell biological analyses that allowed the skeletal framework of the TTFL to be assembled can be seen as a landmark achievement that revolutionised circadian biology. A great deal still needs to be performed, however, to put “flesh” onto the “bones” of the TTFL, not least a comprehensive and quantitative understanding of how the encoded “clock” proteins behave within cells, including SCN neurons and astrocytes. As a dynamic process, this will require sophisticated imaging techniques to examine individual proteins and how they relate to each other as complexes and how they engage the core regulatory elements that drive circadian transcription. Allied to this, biophysical and structural biological approaches are starting to give insights into the molecular basis of clock function, transporting clock biology into the burgeoning field of “molecular machines”. Beyond the intra-cellular TTFL, the influence of the SCN as the central pacemaker is remarkable because of its action at scale; this small neural structure controls innumerable facets of behaviour and physiology across the organism. More fundamentally, however, the SCN creates and encodes, autonomously, information that is an accurate proxy of an external dimension: time. How does it achieve this? Simultaneous measurement and manipulation of the activities of different cellular populations of the SCN, will allow us to unravel the cellular and signalling hierarchies that define this uniquely robust circuit-level timekeeping. Currently, however, we have no knowledge of the network topology of the SCN that confers and sustains this autonomy. Do pacemaker nodes exist and, if so, what are their attributes and how are they connected to each other and to other SCN cells? The standard dichotomy of core and shell needs to be developed into a more formal, quantitative and comprehensive model of cellular interactions and information flow along them. As for extra-SCN signalling, although the neuronal projections of the SCN to other brain regions are broadly mapped, the molecular and cellular output pathways that mediate the transfer of temporal information are poorly characterised. To address this, we need to unravel the various layers of SCN timekeeping further, simultaneously observing and manipulating the subcellular dynamics of clock components, the circuit-level propagation of these dynamics and their effects in vivo on different brain regions and peripheral tissues. Although the focus of this review

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has been the SCN, the tools and approaches we have described are applicable to study circadian timekeeping in any number of tissues, setting the base upon which an organism-wide model of interand extra-SCN timekeeping can be built. Furthermore, understanding the inputs and outputs at these different levels of circadian regulation will propel investigations into the links between the clock and pathology, providing future prospects for translation, bringing clocks and circadian time firmly into the clinic [157].

Acknowledgements This work was supported by core funding from the U.K. Medical Research Council (U105170643) and by grants from the U.K. Biotechnology and Biological Sciences Research Council (BB/P017347/1, BB/ R016658/1).

Conflict of interest statement None. Received 22 November 2019; Received in revised form 10 January 2020; Accepted 15 January 2020 Available online 26 January 2020 Keywords: neurons; cryptochrome; period; translational switching; astrocytes

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