Hugh D. Piggins

The VPAC2 Receptor Is Essential for Circadian Function in the Mouse Suprachiasmatic Nuclei

The neuropeptides pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are implicated in the photic entrainment of circadian rhythms in the suprachiasmatic nuclei (SCN). We now report that mice carrying a null mutation of the VPAC(2) receptor for VIP and PACAP (Vipr2(-/-)) are incapable of sustaining normal circadian rhythms of rest/activity behavior. These mice also fail to exhibit circadian expression of the core clock genes mPer1, mPer2, and mCry1 and the clock-controlled gene arginine vasopressin (AVP) in the SCN. Moreover, the mutants fail to show acute induction of mPer1 and mPer2 by nocturnal illumination. This study highlights the role of intercellular neuropeptidergic signaling in maintenance of circadian function within the SCN.

Challenging the omnipotence of the suprachiasmatic timekeeper: Are circadian oscillators present throughout the mammalian brain?

The suprachiasmatic nucleus of the hypothalamus (SCN) is the master circadian pacemaker or clock in the mammalian brain. Canonical theory holds that the output from this single, dominant clock is responsible for driving most daily rhythms in physiology and behaviour. However, important recent findings challenge this uniclock model and reveal clock‐like activities in many neural and non‐neural tissues. Thus, in addition to the SCN, a number of areas of the mammalian brain including the olfactory bulb, amygdala, lateral habenula and a variety of nuclei in the hypothalamus, express circadian rhythms in core clock gene expression, hormone output and electrical activity. This review examines the evidence for extra‐SCN circadian oscillators in the mammalian brain and highlights some of the essential properties and key differences between brain oscillators. The demonstration of neural pacemakers outside the SCN has wide‐ranging implications for models of the circadian system at a whole‐organism level.

Setting clock speed in mammals: the CK1 epsilon tau mutation in mice accelerates circadian pacemakers by selectively destabilizing PERIOD proteins.

The intrinsic period of circadian clocks is their defining adaptive property. To identify the biochemical mechanisms whereby casein kinase1 (CK1) determines circadian period in mammals, we created mouse null and tau mutants of Ck1 epsilon. Circadian period lengthened in CK1epsilon-/-, whereas CK1epsilon(tau/tau) shortened circadian period of behavior in vivo and suprachiasmatic nucleus firing rates in vitro, by accelerating PERIOD-dependent molecular feedback loops. CK1epsilon(tau/tau) also accelerated molecular oscillations in peripheral tissues, revealing its global role in circadian pacemaking. CK1epsilon(tau) acted by promoting degradation of both nuclear and cytoplasmic PERIOD, but not CRYPTOCHROME, proteins. Together, these whole-animal and biochemical studies explain how tau, as a gain-of-function mutation, acts at a specific circadian phase to promote degradation of PERIOD proteins and thereby accelerate the mammalian clockwork in brain and periphery.

Melanopsin Contributions to Irradiance Coding in the Thalamo-Cortical Visual System

Neurophysiological and anatomical studies identify melanopsin expressing retinal ganglion cells (mRGCs) as a major source of information in the mouse visual system.

The mouse VPAC2 receptor confers suprachiasmatic nuclei cellular rhythmicity and responsiveness to vasoactive intestinal polypeptide in vitro

Expression of coherent and rhythmic circadian (≈ 24 h) variation of behaviour, metabolism and other physiological processes in mammals is governed by a dominant biological clock located in the hypothalamic suprachiasmatic nuclei (SCN). Photic entrainment of the SCN circadian clock is mediated, in part, by vasoactive intestinal polypeptide (VIP) acting through the VPAC2 receptor. Here we used mice lacking the VPAC2 receptor (Vipr2−/−) to examine the contribution of this receptor to the electrophysiological actions of VIP on SCN neurons, and to the generation of SCN electrical firing rate rhythms SCN in vitro. Compared with wild‐type controls, fewer SCN cells from Vipr2−/− mice responded to VIP and the VPAC2 receptor‐selective agonist Ro 25‐1553. By contrast, similar proportions of Vipr2−/− and wild‐type SCN cells responded to gastrin‐releasing peptide, arginine vasopressin or N‐methyl‐d‐aspartate. Moreover, VIP‐evoked responses from control SCN neurons were attenuated by the selective VPAC2 receptor antagonist PG 99‐465. In firing rate rhythm experiments, the midday peak in activity observed in control SCN cells was lost in Vipr2−/− mice. The loss of electrical activity rhythm in Vipr2−/− mice was mimicked in control SCN slices by chronic treatment with PG 99‐465. These results demonstrate that the VPAC2 receptor is necessary for the major part of the electrophysiological actions of VIP on SCN cells in vitro, and is of fundamental importance for the rhythmic and coherent expression of circadian rhythms governed by the SCN clock. These findings suggest a novel role of VPAC2 receptor signalling, and of cell‐to‐cell communication in general, in the maintenance of core clock function in mammals, impacting on the cellular physiology of SCN neurons.

Vasoactive intestinal polypeptide (VIP) phase-shifts the rat suprachiasmatic nucleus clock in vitro.

In mammals, the principal circadian pacemaker is housed in the hypothalamic suprachiasmatic nuclei (SCN). The SCN exhibit high levels of vasoactive intestinal polypeptide (VIP) immunoreactivity and two of the three VIP receptors, VPAC2 and PAC1, are found in the rat SCN. However, the role of VIP in the SCN remains unclear. In this study, we examined the phase‐resetting actions of VIP and selective VIP receptor agonists on the electrical activity rhythm of rat SCN neurons in vitro. Application of VIP during the subjective day did not shift the peak in the firing rate rhythm. However, VIP treatment during the early or late subjective night evoked a small phase delay or a large phase advance, respectively. The phase‐advancing effect of VIP was reproduced by the novel VPAC2 receptor agonist RO 25‐1553, but not by pituitary adenylate cyclase‐activating peptide (a potent PAC1 receptor agonist), or by [K15,R16,L27]VIP(1‐7)/GRF(8‐27), a novel, selective VPAC1 receptor agonist. These data show that VIP phase‐dependently phase‐resets the rodent SCN pacemaker in vitro, presumably via the VPAC2 receptor. As the pattern of phase‐shifting evoked by VIP and RO 25‐1553 resembles the phase‐resetting actions of light on rodent behavioural rhythms, these data support a role for VIP and the VPAC2 receptor in photic entrainment of the rodent circadian pacemaker.

Daily Electrical Silencing in the Mammalian Circadian Clock

Quiet Clock Many physiological processes have circadian rhythms driven by a biological clock in the suprachiasmatic nuclei (SCN) of the brain. Within the SCN, some neurons express the molecular components of the clock and others do not. Exactly how the clock mechanism is coupled to neuronal activity is not precisely understood. Investigation of the electrophysiological properties of SCN neurons by Belle et al. (p. 281) found that, contrary to the conventionally expected rapid firing rate of the cells during the day, clock-containing cells tended not to fire, despite being in an electrically excited state. Modeling and experimental characterization of changes in channel activity revealed unexpected electrophysiological properties of the SCN cells requiring a reassessment of how the circadian clock regulates activity of SCN neurons. Clock-containing neurons in the mouse brain display complex electrophysiology not seen in other brain cells. Neurons in the brain’s suprachiasmatic nuclei (SCNs), which control the timing of daily rhythms, are thought to encode time of day by changing their firing frequency, with high rates during the day and lower rates at night. Some SCN neurons express a key clock gene, period 1 (per1). We found that during the day, neurons containing per1 sustain an electrically excited state and do not fire, whereas non-per1 neurons show the previously reported daily variation in firing activity. Using a combined experimental and theoretical approach, we explain how ionic currents lead to the unusual electrophysiological behaviors of per1 cells, which unlike other mammalian brain cells can survive and function at depolarized states.

A riot of rhythms: neuronal and glial circadian oscillators in the mediobasal hypothalamus

BackgroundIn mammals, the synchronized activity of cell autonomous clocks in the suprachiasmatic nuclei (SCN) enables this structure to function as the master circadian clock, coordinating daily rhythms in physiology and behavior. However, the dominance of this clock has been challenged by the observations that metabolic duress can over-ride SCN controlled rhythms, and that clock genes are expressed in many brain areas, including those implicated in the regulation of appetite and feeding. The recent development of mice in which clock gene/protein activity is reported by bioluminescent constructs (luciferase or luc) now enables us to track molecular oscillations in numerous tissues ex vivo. Consequently we determined both clock activities and responsiveness to metabolic perturbations of cells and tissues within the mediobasal hypothalamus (MBH), a site pivotal for optimal internal homeostatic regulation.ResultsHere we demonstrate endogenous circadian rhythms of PER2::LUC expression in discrete subdivisions of the arcuate (Arc) and dorsomedial nuclei (DMH). Rhythms resolved to single cells did not maintain long-term synchrony with one-another, leading to a damping of oscillations at both cell and tissue levels. Complementary electrophysiology recordings revealed rhythms in neuronal activity in the Arc and DMH. Further, PER2::LUC rhythms were detected in the ependymal layer of the third ventricle and in the median eminence/pars tuberalis (ME/PT). A high-fat diet had no effect on the molecular oscillations in the MBH, whereas food deprivation resulted in an altered phase in the ME/PT.ConclusionOur results provide the first single cell resolution of endogenous circadian rhythms in clock gene expression in any intact tissue outside the SCN, reveal the cellular basis for tissue level damping in extra-SCN oscillators and demonstrate that an oscillator in the ME/PT is responsive to changes in metabolism.

Aberrant Gating of Photic Input to the Suprachiasmatic Circadian Pacemaker of Mice Lacking the VPAC2 Receptor

VIP acting via the VPAC2 receptor is implicated as a key signaling pathway in the maintenance and resetting of the hypothalamic suprachiasmatic nuclei (SCN) circadian pacemaker; circadian rhythms in SCN clock gene expression and wheel-running behavior are abolished in mice lacking the VPAC2 receptor (Vipr2–/–). Here, using immunohistochemical detection of pERK (phosphorylated extracellular signal-regulated kinases 1/2) and c-FOS, we tested whether the gating of photic input to the SCN is maintained in these apparently arrhythmic Vipr2–/– mice. Under light/dark and constant darkness, spontaneous expression of pERK and c-FOS in the wild-type mouse SCN was significantly elevated during subjective day compared with subjective night; no diurnal or circadian variation in pERK or c-FOS was detected in the SCN of Vipr2–/– mice. In constant darkness, light pulses given during the subjective night but not the subjective day significantly increased expression of pERK and c-FOS in the wild-type SCN. In contrast, light pulses given during both subjective day and subjective night robustly increased expression of pERK and c-FOS in the Vipr2–/– mouse SCN. Although photic stimuli activate intracellular pathways within the SCN of Vipr2–/– mice, they do not engage the core clock mechanisms. The absence of photic gating, together with the general lack of overt rhythms in circadian output, strongly suggests that the SCN circadian pacemaker is completely dysfunctional in the Vipr2–/– mouse.

Spatiotemporal Heterogeneity in the Electrical Activity of Suprachiasmatic Nuclei Neurons and their Response to Photoperiod

The coordinated activity of thousands of cellular oscillators in the suprachiasmatic nuclei (SCN) temporally regulates mammalian physiology to anticipate daily environmental changes across the seasons. The phasing of clock gene expression varies according to anatomical location in the SCN and is thought to encode photoperiodic information. However, it is unclear whether similar variations in phase occur in the electrical activity of SCN neurons, a measure of both intraSCN signaling and clock output. To address this, we recorded single-unit and multiunit activity (SUA/MUA) from dorsal and ventral subregions of the middle level of the rostrocaudal axis of the SCN in coronal brain slices prepared from mice housed under different photoperiods. We demonstrate that under a symmetrical (12 h light:12 h dark) photoperiod, cells in the dorsal SCN are less tightly synchronized than those in the ventral SCN. Comparison of recordings made from mice under short (8 h light:16 h dark) or long (16 h light:8 h dark) photoperiods shows that the phase distribution of ventral, but not dorsal, SCN neurons expands with increasing day length. Conversely, the duration that individual neurons are active increases in dorsal, but not ventral, SCN under long days. These data indicate that in the ventral SCN photoperiod is encoded at the network level, while this coding occurs at the level of individual cells in the dorsal SCN.