Marco Brancaccio

A Gq-Ca2+ axis controls circuit-level encoding of circadian time in the suprachiasmatic nucleus.

Summary The role of intracellular transcriptional/post-translational feedback loops (TTFL) within the circadian pacemaker of the suprachiasmatic nucleus (SCN) is well established. In contrast, contributions from G-coupled pathways and cytosolic rhythms to the intercellular control of SCN pacemaking are poorly understood. We therefore combined viral transduction of SCN slices with fluorescence/bioluminescence imaging to visualize GCaMP3-reported circadian oscillations of intracellular calcium [Ca2+]i alongside activation of Ca2+/cAMP-responsive elements. We phase-mapped them to the TTFL, in time and SCN space, and demonstrated their dependence upon G-coupled vasoactive intestinal peptide (VIP) signaling. Pharmacogenetic manipulation revealed the individual contributions of Gq, Gs, and Gi to cytosolic and TTFL circadian rhythms. Importantly, activation of Gq-dependent (but not Gs or Gi) pathways in a minority of neurons reprogrammed [Ca2+]i and TTFL rhythms across the entire SCN. This reprogramming was mediated by intrinsic VIPergic signaling, thus revealing a Gq/[Ca2+]i-VIP leitmotif and unanticipated plasticity within network encoding of SCN circadian time.

Circadian Pacemaking in Cells and Circuits of the Suprachiasmatic Nucleus

The suprachiasmatic nucleus (SCN) of the hypothalamus is the principal circadian pacemaker of the brain. It co‐ordinates the daily rhythms of sleep and wakefulness, as well as physiology and behaviour, that set the tempo to our lives. Disturbance of this daily pattern, most acutely with jet‐lag but more insidiously with rotational shift‐work, can have severely deleterious effects for mental function and long‐term health. The present review considers recent developments in our understanding of the properties of the SCN that make it a robust circadian time‐keeper. It first focuses on the intracellular transcriptional/ translational feedback loops (TTFL) that constitute the cellular clockwork of the SCN neurone. Daily timing by these loops pivots around the negative regulation of the Period (Per) and Cryptochrome (Cry) genes by their protein products. The period of the circadian cycle is set by the relative stability of Per and Cry proteins, and this can be controlled by both genetic and pharmacological interventions. It then considers the function of these feedback loops in the context of cytosolic signalling by cAMP and intracellular calcium ([Ca2+]i), which are both outputs from, and inputs to, the TTFL, as well as the critical role of vasoactive intestinal peptide (VIP) signalling in synchronising cellular clocks across the SCN. Synchronisation by VIP in the SCN is paracrine, operating over an unconventionally long time frame (i.e. 24 h) and wide spatial domain, mediated via the cytosolic pathways upstream of the TTFL. Finally, we show how intersectional pharmacogenetics can be used to control G‐protein‐coupled signalling in individual SCN neurones, and how manipulation of Gq/[Ca2+]i‐signalling in VIP neurones can re‐programme the circuit‐level encoding of circadian time. Circadian pacemaking in the SCN therefore provides an unrivalled context in which to understand how a complex, adaptive behaviour can be organised by the dynamic activity of a relatively few gene products, operating in a clearly defined neuronal circuit, with both cell‐autonomous and emergent, circuit‐level properties.

Astrocytes Control Circadian Timekeeping in the Suprachiasmatic Nucleus via Glutamatergic Signaling

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

Emx2 and Foxg1 Inhibit Gliogenesis and Promote Neuronogenesis

Neural stem cells (NSCs) give rise to all cell types forming the cortex: neurons, astrocytes, and oligodendrocytes. The transition from the former to the latter ones takes place via lineage‐restricted progenitors in a highly regulated way. This process is mastered by large sets of genes, among which some implicated in central nervous system pattern formation. The aim of this study was to disentangle the kinetic and histogenetic roles exerted by two of these genes, Emx2 and Foxg1, in cortico‐cerebral precursors. For this purpose, we set up a new integrated in vitro assay design. Embryonic cortical progenitors were transduced with lentiviral vectors driving overexpression of Emx2 and Foxg1 in NSCs and neuronal progenitors. Cells belonging to different neuronogenic and gliogenic compartments were labeled by spectrally distinguishable fluoroproteins driven by cell type‐specific promoters and by cell type‐specific antibodies and were scored via multiplex cytofluorometry and immunocytofluorescence. A detailed picture of Emx2 and Foxg1 activities in cortico‐cerebral histogenesis resulted from this study. Unexpectedly, we found that both genes inhibit gliogenesis and promote neuronogenesis, through distinct mechanisms, and Foxg1 also dramatically stimulates neurite outgrowth. Remarkably, such activities, alone or combined, may be exploited to ameliorate the neuronal output obtainable from neural cultures, for purposes of cell‐based brain repair. STEM CELLS 2010;28:1206–1218

Analysis of core circadian feedback loop in suprachiasmatic nucleus of mCry1-luc transgenic reporter mouse

The suprachiasmatic nucleus (SCN) coordinates circadian rhythms that adapt the individual to solar time. SCN pacemaking revolves around feedback loops in which expression of Period (Per) and Cryptochrome (Cry) genes is periodically suppressed by their protein products. Specifically, PER/CRY complexes act at E-box sequences in Per and Cry to inhibit their transactivation by CLOCK/BMAL1 heterodimers. To function effectively, these closed intracellular loops need to be synchronized between SCN cells and to the light/dark cycle. For Per expression, this is mediated by neuropeptidergic and glutamatergic extracellular cues acting via cAMP/calcium-responsive elements (CREs) in Per genes. Cry genes, however, carry no CREs, and how CRY-dependent SCN pacemaking is synchronized remains unclear. Furthermore, whereas reporter lines are available to explore Per circadian expression in real time, no Cry equivalent exists. We therefore created a mouse, B6.Cg-Tg(Cry1-luc)01Ld, carrying a transgene (mCry1-luc) consisting of mCry1 elements containing an E-box and E′-box driving firefly luciferase. mCry1-luc organotypic SCN slices exhibited stable circadian bioluminescence rhythms with appropriate phase, period, profile, and spatial organization. In SCN lacking vasoactive intestinal peptide or its receptor, mCry1 expression was damped and desynchronized between cells. Despite the absence of CREs, mCry1-luc expression was nevertheless (indirectly) sensitive to manipulation of cAMP-dependent signaling. In mPer1/2-null SCN, mCry1-luc bioluminescence was arrhythmic and no longer suppressed by elevation of cAMP. Finally, an SCN graft procedure showed that PER-independent as well as PER-dependent mechanisms could sustain circadian expression of mCry1. The mCry1-luc mouse therefore reports circadian mCry1 expression and its interactions with vasoactive intestinal peptide, cAMP, and PER at the heart of the SCN pacemaker.

The Regulatory Factor ZFHX3 Modifies Circadian Function in SCN via an AT Motif-Driven Axis.

Summary We identified a dominant missense mutation in the SCN transcription factor Zfhx3, termed short circuit (Zfhx3Sci), which accelerates circadian locomotor rhythms in mice. ZFHX3 regulates transcription via direct interaction with predicted AT motifs in target genes. The mutant protein has a decreased ability to activate consensus AT motifs in vitro. Using RNA sequencing, we found minimal effects on core clock genes in Zfhx3Sci/+ SCN, whereas the expression of neuropeptides critical for SCN intercellular signaling was significantly disturbed. Moreover, mutant ZFHX3 had a decreased ability to activate AT motifs in the promoters of these neuropeptide genes. Lentiviral transduction of SCN slices showed that the ZFHX3-mediated activation of AT motifs is circadian, with decreased amplitude and robustness of these oscillations in Zfhx3Sci/+ SCN slices. In conclusion, by cloning Zfhx3Sci, we have uncovered a circadian transcriptional axis that determines the period and robustness of behavioral and SCN molecular rhythms.

Network-Mediated Encoding of Circadian Time: The Suprachiasmatic Nucleus (SCN) from Genes to Neurons to Circuits, and Back

The transcriptional architecture of intracellular circadian clocks is similar across phyla, but in mammals interneuronal mechanisms confer a higher level of circadian integration. The suprachiasmatic nucleus (SCN) is a unique model to study these mechanisms, as it operates as a ∼24 h clock not only in the living animal, but also when isolated in culture. This “clock in a dish” can be used to address fundamental questions, such as how intraneuronal mechanisms are translated by SCN neurons into circuit-level emergent properties and how the circuit decodes, and responds to, light input. This review addresses recent developments in understanding the relationship between electrical activity, [Ca2+]i, and intracellular clocks. Furthermore, optogenetic and chemogenetic approaches to investigate the distinct roles of neurons and glial cells in circuit encoding of circadian time will be discussed, as well as the epigenetic and circuit-level mechanisms that enable the SCN to translate light input into coherent daily rhythms.

Rhythmic expression of cryptochrome induces the circadian clock of arrhythmic suprachiasmatic nuclei through arginine vasopressin signaling.

Significance Daily cycles of behavior adapt us to the 24-h rhythm of day and night. These rhythms are coordinated by a central clock, the suprachiasmatic nucleus (SCN), in which self-sustaining transcriptional/posttranslational feedback loops define approximately 24-h (circadian) time. Cryptochrome1 (CRY1) and CRY2 are essential clock components, but how they sustain cell-autonomous and circuit-level circadian timing in the SCN is not clear. To explore this, we developed a genetic complementation assay, using virally expressed, fluorescently tagged CRY proteins to test whether molecular pacemaking can be induced in arrhythmic Cry1/2-deficient SCN. We demonstrate protein-specific and robust induction of molecular pacemaking, the efficacy of which depends on the circadian pattern and phase of Cry expression and functional signaling via neuropeptide (AVP) receptors. Circadian rhythms in mammals are coordinated by the suprachiasmatic nucleus (SCN). SCN neurons define circadian time using transcriptional/posttranslational feedback loops (TTFL) in which expression of Cryptochrome (Cry) and Period (Per) genes is inhibited by their protein products. Loss of Cry1 and Cry2 stops the SCN clock, whereas individual deletions accelerate and decelerate it, respectively. At the circuit level, neuronal interactions synchronize cellular TTFLs, creating a spatiotemporal wave of gene expression across the SCN that is lost in Cry1/2-deficient SCN. To interrogate the properties of CRY proteins required for circadian function, we expressed CRY in SCN of Cry-deficient mice using adeno-associated virus (AAV). Expression of CRY1::EGFP or CRY2::EGFP under a minimal Cry1 promoter was circadian and rapidly induced PER2-dependent bioluminescence rhythms in previously arrhythmic Cry1/2-deficient SCN, with periods appropriate to each isoform. CRY1::EGFP appropriately lengthened the behavioral period in Cry1-deficient mice. Thus, determination of specific circadian periods reflects properties of the respective proteins, independently of their phase of expression. Phase of CRY1::EGFP expression was critical, however, because constitutive or phase-delayed promoters failed to sustain coherent rhythms. At the circuit level, CRY1::EGFP induced the spatiotemporal wave of PER2 expression in Cry1/2-deficient SCN. This was dependent on the neuropeptide arginine vasopressin (AVP) because it was prevented by pharmacological blockade of AVP receptors. Thus, our genetic complementation assay reveals acute, protein-specific induction of cell-autonomous and network-level circadian rhythmicity in SCN never previously exposed to CRY. Specifically, Cry expression must be circadian and appropriately phased to support rhythms, and AVP receptor signaling is required to impose circuit-level circadian function.

In-depth Characterization of Firefly Luciferase as a Reporter of Circadian Gene Expression in Mammalian Cells

Firefly luciferase (Fluc) is frequently used to report circadian gene expression rhythms in mammalian cells and tissues. During longitudinal assays it is generally assumed that enzymatic substrates are in saturating excess, such that total bioluminescence is directly proportional to Fluc protein level. To test this assumption, we compared the enzyme kinetics of purified luciferase with its activity in mammalian cells. We found that Fluc activity in solution has a lower Michaelis constant (Km) for luciferin, lower temperature dependence, and lower catalytic half-life than Fluc in cells. In consequence, extracellular luciferin concentration significantly affects the apparent circadian amplitude and phase of the widely used PER2::LUC reporter in cultured fibroblasts, but not in SCN, and we suggest that this arises from differences in plasma membrane luciferin transporter activity. We found that at very high concentrations (>1 mM), luciferin lengthens circadian period, in both fibroblasts and organotypic SCN slices. We conclude that the amplitude and phase of circadian gene expression inferred from bioluminescence recordings should be treated with some caution, and we suggest that optimal luciferin concentration should be determined empirically for each luciferase reporter and cell type.

Generation of circadian rhythms in the suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) of the hypothalamus is remarkable. Despite numbering only about 10,000 neurons on each side of the third ventricle, the SCN is our principal circadian clock, directing the daily cycles of behaviour and physiology that set the tempo of our lives. When this nucleus is isolated in organotypic culture, its autonomous timing mechanism can persist indefinitely, with precision and robustness. The discovery of the cell-autonomous transcriptional and post-translational feedback loops that drive circadian activity in the SCN provided a powerful exemplar of the genetic specification of complex mammalian behaviours. However, the analysis of circadian time-keeping is moving beyond single cells. Technical and conceptual advances, including intersectional genetics, multidimensional imaging and network theory, are beginning to uncover the circuit-level mechanisms and emergent properties that make the SCN a uniquely precise and robust clock. However, much remains unknown about the SCN, not least the intrinsic properties of SCN neurons, its circuit topology and the neuronal computations that these circuits support. Moreover, the convention that the SCN is a neuronal clock has been overturned by the discovery that astrocytes are an integral part of the timepiece. As a test bed for examining the relationships between genes, cells and circuits in sculpting complex behaviours, the SCN continues to offer powerful lessons and opportunities for contemporary neuroscience.In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus acts as the principal pacemaker for circadian rhythms, which are powerful regulators of physiology and behaviour. In this Review, Hastings, Maywood and Brancaccio examine the molecular, cellular and circuit mechanisms underlying time-keeping in the SCN.