Review
Molecular-genetic Manipulation of the Suprachiasmatic Nucleus Circadian Clock

https://doi.org/10.1016/j.jmb.2020.01.019Get rights and content

Highlights

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    The suprachiasmatic nucleus (SCN) is the principal circadian clock of mammals.

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    The SCN cell-autonomous clock is a transcription-translation negative feedback loop.

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    Real-time imaging provides a window onto cellular and circuit-level timekeeping.

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    Critical emergent properties of the SCN network arise by intercellular interactions.

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    We do not know how clock proteins behave or how SCN cells interact to define time.

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 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.

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 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 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 period—properties 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 cell-autonomous 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 clock-driven cellular output rhythms. Finally, we review how intersectional genetic manipulations of the cell-autonomous 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.

Section snippets

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 frequency (frq) in Neurospora. Subsequent forward and reverse genetics revealed that cryptochrome (cry)

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

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,

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

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 TTFL-dependent cytosolic regulation links back into the TTFL. Indeed, many circadian clock genes, including Per, carry

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 Zfhx3Sci SCN slices, these oscillations were

Dissecting the SCN Circuit: Network-level 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

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

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 Npas2 are mutually redundant partners of Bmal1 [27]. In contrast to null mutations, the ClockΔ19 mutation acts as a competitive inhibitor for endogenous

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, Ck1δ and Ck1ε 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 targetted SCN cells exhibit periods different to non-targetted cells. For example, conditional neuronal excision of Ck1δ using Vgat-Cre lengthens behavioural

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,

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 GABA-ergic 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

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

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

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).

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