Circadian pacemaking in cells and circuits of the suprachiasmatic nucleus

Abstract

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 ([Ca(2+) ]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/[Ca(2+) ]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.

Keywords: DREADD; VIP; paracrine; pharmacogenetic; sleep.

Fig. 1

A schematic view of the core molecular feedback loop that sits at the heart of the mammalian pacemaker. The definition of circadian time pivots around the activation of Per and Cry genes by Clock/Bmal1 heterodimers (acting at E-box enhancer sequences), alternating with repression of the same genes by their protein products. Clock-controlled output genes carrying E-boxes are also subject to daily activation and repression, generating downstream transcriptional cascades that ultimately encode circadian cycles of physiology and behaviour.

Fig. 2

Circadian rhythms of cellular [Ca²⁺]_i and CRE-dependent transcription in the suprachiasmatic nucleus (SCN), reported by adeno-associated virus GCamp3 and LV CRE-luciferase, respectively. Note that both types of circadian rhythm are lost in SCN lacking the neuropeptide vasoactive intestinal peptide (VIP) (a, b) and this arises from loss of cellular synchrony across the slice, as revealed by Rayleigh plots of the phases of individual neurones (c, d). Redrawn with permission from Brancaccio et al. AU, arbitary units; KO, knockout.

Fig. 3

A schematic representation of the phase map of cytosolic and transcriptional events within the suprachiasmatic nucleus clockwork. Electrophysiological activity peaks in the circadian day, followed by peak cAMP, [Ca²⁺]_i and CRE-dependent transcription. The expression of Per genes, which carry CREs then follows, whereas Cry1 expression, mediated by via E-boxes but not CREs, lags behind. Circadian night is characterised by electrical quiescence and altered metabolic state, as revealed by the peak of peroxiredoxin (PRX) superoxidation. Redrawn with permission from Brancaccio et al. and Edgar et al. .

Fig. 4

Co-culture reveals the role of paracrine vasoactive intestinal peptide (VIP) signalling in maintaining coherent circadian gene expression in the suprachiasmatic nucleus (SCN). Upper panels depict VIP-null, Per2::luc SCN (left) and then addition of nonbioluminescent wild-type (WT) graft onto mutant host (right). Middle panels present raster plots of the bioluminescent gene expression in host SCN, before (left) and after grafting (right). The lower trace indicates the aggregate bioluminescent signal recorded from VIP-null host SCN before (left) and after (right) the addition of the graft. Note rapid re-organisation of circadian gene expression following grafting. Redrawn with permission from Maywood et al. .

Fig. 5

Schematic model to illustrate how vasoactive intestinal peptide (VIP) acting via VPAC2 receptors is the binding factor between suprachiasmatic nucleus neurones, linking the transcriptional/ translational feedback loops (TTFL) on one cell to cytosolic signalling pathways upstream of the TTFL in target neurones.

Fig. 6

DREADD-mediated activation of Gq signalling in vasoactive intestinal peptide (VIP) neurones re-programmes the circuit-level encoding of circadian time. (a) Upper panel: CRE recombinase-mediated, targetted expression of Gq DREADD to VIP neurones of a suprachiasmatic nucleus organotypic slice. Lower panel: combined bioluminescent and fluorescent imaging of a SCN slice in which Gq-active DREADD was expressed in VIP neurones. Scale bar = 50 μm. (b) X–Y plot of the wave of bioluminescent circadian gene expression rhythms (expressed as the centre of mass of bioluminescence) in an organotypic SCN slice for 3 days before (above) and 3 days after (below) activation of Gq signalling in VIP neurones. Note the change in trajectory to a more ventral location following Gq activation. D, dorsal; L, lateral; M, medial; V, ventral.Redrawn with permission from Maywood et al.